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WO2008100376A2 - Variants de troncature de sirt1 et procédés d'utilisation de ceux-ci - Google Patents

Variants de troncature de sirt1 et procédés d'utilisation de ceux-ci Download PDF

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Publication number
WO2008100376A2
WO2008100376A2 PCT/US2008/001032 US2008001032W WO2008100376A2 WO 2008100376 A2 WO2008100376 A2 WO 2008100376A2 US 2008001032 W US2008001032 W US 2008001032W WO 2008100376 A2 WO2008100376 A2 WO 2008100376A2
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Prior art keywords
sirtuin
variant
sirtl
nucleic acid
cell
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PCT/US2008/001032
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English (en)
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WO2008100376A3 (fr
Inventor
Lei Jin
Yakov Korkhin
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Sirtris Pharmaceuticals, Inc.
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Publication of WO2008100376A2 publication Critical patent/WO2008100376A2/fr
Publication of WO2008100376A3 publication Critical patent/WO2008100376A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the Silent Information Regulator (SIR) family of genes represents a highly conserved group of genes present in the genomes of organisms ranging from archaebacteria to a variety of eukaryotes (Frye, 2000).
  • the encoded SIR proteins are involved in diverse processes from regulation of gene silencing to DNA repair.
  • the proteins encoded by members of the SIR gene family show high sequence conservation in a 250 amino acid core domain.
  • a well-characterized gene in this family is S. cerevisiae SIR2, which is involved in silencing HM loci that contain information specifying yeast mating type, telomere position effects and cell aging (Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000).
  • the yeast Sir2 protein belongs to a family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000).
  • the Sir2 homolog, CobB in Salmonella typhimurium, functions as an NAD (nicotinamide adenine dinucleotide)-dependent ADP-ribosyl transferase (Tsang and Escalante-Semerena, 1998).
  • the Sir2 protein is a class III deacetylase which uses NAD as a cosubstrate (Imai et al., 2000; Moazed, 2001; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed, 2001). Unlike other deacetylases, many of which are involved in gene silencing, Sir2 is insensitive to class I and II histone deacetylase inhibitors like trichostatin A (TSA) (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000).
  • TSA trichostatin A
  • acetylation of acetyl-lysine by Sir2 is tightly coupled to NAD hydrolysis, producing nicotinamide and a novel acetyl-ADP ribose compound (Tanner et al., 2000; Landry et al., 2000b; Tanny and Moazed, 2001).
  • the NAD-dependent deacetylase activity of Sir2 is essential for its functions which can connect its biological role with cellular metabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al., 2000; Smith et al., 2000).
  • Mammalian Sir2 homologs have NAD-737 l .DOC dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000). Most information about Sir2 mediated functions comes from the studies in yeast (Gartenberg, 2000; Gottschling, 2000).
  • Caloric restriction has been known for over 70 years to improve the health ' and extend the lifespan of mammals (Masoro, 2000). Yeast life span, like that of metazoans, is also extended by interventions that resemble caloric restriction, such as low glucose. The discovery that both yeast and flies lacking the SIR2 gene do not live longer when calorically restricted provides evidence that SIR2 genes mediate the beneficial health effects of this diet (Anderson et al., 2003; Helfand and Rogina, 2004).
  • yeast glucose-responsive cAMP adenosine 3'5'-monophosphate-dependent (PKA) pathway
  • PKA adenosine 3'5'-monophosphate-dependent pathway
  • small molecule activators and inhibitors of the SIR proteins have been reported (see e.g., U.S. Patent Application Publication Nos. 2005/0136537 and 2005/0096256 and PCT Publication Nos. WO 2005/002555 and WO 2005/002672) and a number of uses for these compounds have been identified.
  • small molecule activators of SIR proteins were shown to extend life span in yeast and cultured human cells as well as activate SIR protein activity in human cells (supra).
  • the small molecule SIR activators were shown to mimic calorie restriction and extend lifespan in Caenorhabditis elegans and Drosophila melanogaster (supra). Activators of the SIR proteins may therefore be useful for mimicking the effects of calorie restriction in eukaryotic cells and treating
  • 73737 1 DOC aging-related diseases such as stroke, cardiovascular disease, arthritis, high blood pressure, or Alzheimer's disease (supra). Additionally, it has been shown that resveratrol, butein, fisetin, piceatannol, and quercetin, small molecule activators of SIR proteins, promote fat mobilization in C. elegans, prevent fat accumulation in C. elegans, stimulate fat mobilization in mammalian cells, and inhibit adipogenesis in mammalian cells (see e.g., U.S. Patent Publication No. 2005/0171027 and PCT Publication No. WO 2005/065667). Similarly, nicotinamide, an inhibitor of SIR proteins, was shown to promote fat accumulation (supra).
  • resveratrol was shown to at least partially restore insulin sensitivity in insulin resistant cells (supra).
  • Activators of SIR proteins may therefore also be useful for treating or preventing insulin resistance disorders and have been suggested for uses relating to reducing weight or preventing weight gain (supra).
  • the human ortholog of yeast Sir2 (silent mating type information regulation 2), SIRTl, is an NAD + -dependent deacetylase (Imai S et al. Cold Spring Harb Symp Quant Biol. 2000; 65: 297-302).
  • the SIRTl protein is localized in the nucleus (Luo J et al. Cell. 2001 ; 107(2): 137-48; Vaziri H et al. Cell. 2001 ; 107(2): 149-59) and interacts with and deacetylates a large number of proteins.
  • the present invention provides new and advantageous methods, compositions, cell constructs, polynucleotides and polypeptides, and animal models related to novel sirtuin variants.
  • the invention provides novel SIRTl variants, wherein the variant (i) may be expressed in E. coli at a concentration of at least 5 mg/L, (ii) has deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl, and (iii) the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the invention provides novel SIRTl polynucleotides that encode SIRTl variants, wherein the variants i) may be expressed in E. coli at a concentration of at least 5 mg/L, ii) have deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl, and iii) the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the present invention provides a transgenic mammal, a majority of whose cells harbor a transgene including a nucleic acid sequence encoding a variant of SIRTl .
  • the SIRTl activity level is higher in the cells of the transgenic mammal than in the cells of a nontransgenic mammal of the same species.
  • the life span of those cells in the transgenic mammal that express a SIRTl variant is increased with respect to a non-transgenic mammal of the same species.
  • the heterologous nucleic acid further includes one or more of an enhancer sequence, a promoter sequence, and a polyadenylation sequence each of which is operably linked to the SIRTl sequence.
  • One apect of the invention provides for a method for identifying a compound that modulates SIRTl activity, comprising: (a) contacting a peptide substrate pool with a SIRTl variant in the presence of a test compound, wherein members of said peptide substrate pool comprise at least one acetylated amino acid side chain and wherein the SIRTl variant (i) may be expressed in E.
  • coli at a concentration of at Ieast5 mg/L, (ii) has deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl, and (iii) the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound, and (b) determining the level of acetylation of the peptide substrate pool, wherein a change in the level of acetylation of the peptide substrate pool in the presence of the test compound as compared to a control is indicative of a compound that modulates SIRTl .
  • Another aspect relates to a method for determining SIRTl activity, comprising: contacting a peptide substrate pool with a SIRTl variant, wherein members of said peptide substrate pool comprise at least one acetylated amino acid side chain, and wherein the SIRTl variant (i) may be expressed in E.
  • coli at a737 I DOC concentration of at least 5 mg/L (ii) has deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl , and (iii) the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound, and determining if the acetylated amino acid side chain in the peptide substrate pool is deacetylated.
  • a further aspect of the invention provides for a method of deacetylating at least one amino acid residue in a polypeptide comprising the step of preparing a mixture by combining a polypeptide having at least one acetylated amino acid with a SIRTl variant, wherein the SIRTl variant (i) may be expressed in E. coli at a concentration of at least 5 mg/L, (ii) has deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl, and (iii) the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the invention provides methods for using SIRTl variants, or compostions comprising SIRTl variants.
  • SIRTl variants that increase the level and/or activity of a sirtuin protein may be used for a variety of therapeutic applications including, for example, increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, and/or flushing, etc.
  • SIRTl variants may also be used for treating a disease or disorder in a subject that would benefit from increased mitochondrial activity, for enhancing muscle performance, for increasing muscle ATP levels, or for treating or preventing muscle tissue damage associated with hypoxia or ischemia.
  • the methods comprise administering to a subject in need thereof a pharmaceutically effective amount of SIRTl variants.
  • the SIRTl variants may be administered alone or in combination with other compounds, including other SIRTl variants, or other therapeutic agents.
  • 737 l may be administered alone or in combination with other compounds, including other SIRTl variants, or other therapeutic agents.
  • Figure 1 shows a mass spectrometry isobologram.
  • Figure 2 shows a series of SIRTl N- and C-terminal truncations.
  • Figure 3 shows a depiction of SIRTl N-terminal truncations that define the allosteric compound binding site.
  • Figure 4 shows the nucleotide (SEQ ID NO: 2) and amino acid (SEQ ID NO: 1) sequences for human SIRTl. This amino acid seuqence corresponds to Genbank Accession No. NP_036370 and the nucleotide sequence corresponds to a portion of Genbank Accession No. NM 012238.
  • Figure 5 is an alignment of the amino acid sequences for human SIRTl
  • SIRT2 SEQ ID NO: 7
  • SIRT3 SEQ ID NO: 8
  • SIRT4 SEQ ID NO: 9
  • SIRT5 SEQ ID NO: 10
  • SIRT6 SEQ ID NO: 11
  • SIRT7 SEQ ID NO: 12
  • yeast Sir2 SEQ ID NO: 5
  • mouse SIRTl SEQ ID NO: 6
  • activation refers the ability of a compound to increase the deacetylation activity of SIRTl .
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • a biological macromolecule such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide
  • an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • the activity of such agents may render it suitable as a
  • therapeutic agent which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • ⁇ residue refers to an amino acid that is a member of a group of amino acids having certain common properties.
  • conservative amino acid substitution refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer- Verlag).
  • groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer- Verlag).
  • One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of GIu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of GIu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Tip, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of VaI, Leu and He, (vii) a slightly-polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, GIy, Ala, GIu, GIn and Pro, (ix) an aliphatic group consisting of VaI, Leu, He, Met and Cys, and (x) a small hydroxyl group consisting of Ser and
  • deacetylation activity refers to the NAD + dependent deacetylation enzymatic activity of SIRTl .
  • Diabetes refers to high blood sugar or ketoacidosis, as well as chronic, general metabolic abnormalities arising from a prolonged high blood sugar status or a decrease in glucose tolerance. “Diabetes” encompasses both the type I and type II (Non Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease.
  • the risk factors for diabetes include the following factors: waistline of more than 40 inches for men or 35 inches for women, blood pressure of 130/85 mmHg or higher, triglycerides above 150 mg/dl, fasting blood glucose greater than 100 mg/dl or high- density lipoprotein of less than 40 mg/dl in men or 50 mg/dl in women.
  • a "direct activator" of a sirtuin is a molecule that activates a sirtuin by binding to it.
  • a “direct inhibitor” of a sirtuin is a molecule inhibits a sirruin by binding to it.
  • a “fusion protein” or “fusion polypeptide” refers to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences may be linked in frame.
  • a fusion protein may include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion expressed by different kinds of organisms.
  • the fusion polypeptide may comprise one or more amino acid sequences linked to a first polypeptide.
  • the fusion sequences may be multiple copies of the same sequence, or alternatively, may be different amino acid sequences.
  • the fusion polypeptides may be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide.
  • Exemplary fusion proteins include polypeptides comprising a glutathione S-transferase tag (GST-tag), histidine tag (His-tag), an immunoglobulin domain, an immunoglobulin binding domain, or an amino acid sequence which promotes transcytosis.
  • GST-tag glutathione S-transferase tag
  • His-tag histidine tag
  • immunoglobulin domain an immunoglobulin binding domain
  • amino acid sequence which promotes transcytosis or an amino acid sequence which promotes transcytosis.
  • hyperinsulinemia refers to a state in an individual in which the level of insulin in the blood is higher than normal.
  • insulin resistance refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.
  • insulin resistance disorder refers to any disease or condition that is caused by or contributed to by insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease 737 I DOC including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular
  • isolated nucleic acid refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which (1) is not associated with the cell in which the "isolated nucleic acid” is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
  • livestock animals refers to domesticated quadrupeds, which includes those being raised for meat and various byproducts, e.g., a bovine animal including cattle and other members of the genus Bos, a porcine animal including domestic swine and other members of the genus Sus, an ovine animal including sheep and other members of the genus Ovis, domestic goats and other members of the genus Capra; domesticated quadrupeds being raised for specialized tasks such as use as a beast of burden, e.g., an equine animal including domestic horses and other members of the family Equidae, genus Equus.
  • mammals include humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
  • livestock animals including bovines, porcines, etc.
  • companion animals e.g., canines, felines, etc.
  • rodents e.g., mice and rats.
  • NAD-like compound is also within the scope of the invention and refers to a compound (e.g., a synthetic or naturally occurring chemical, drug, protein, peptide, small organic molecule) which possesses structural similarity (e.g., adenine, ribose and phosphate groups) or functional similarity (e.g., oxidation of substrates, NAD-dependent deacetylation of histone proteins).
  • structural similarity e.g., adenine, ribose and phosphate groups
  • functional similarity e.g., oxidation of substrates, NAD-dependent deacetylation of histone proteins.
  • DOC like compounds can be NADH, NADP, NADPH, non-hydrolyzable NAD and fluorescent analogs of NAD (e.g., 1, N6-etheno NAD).
  • Obese individuals or individuals suffering from obesity are generally individuals having a body mass index (BMI) of at least 25 or greater. Obesity may or may not be associated with insulin resistance.
  • BMI body mass index
  • parenteral administration and “administered parenterally” are art-recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra- articulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.
  • a "patient”, “subject”, “individual” or “host” refers to either a human or a non-human animal.
  • the term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
  • ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD.
  • the percent identity of two sequences737 l .DOC can be determined by the GCG program with a gap weight of 1 , e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof.
  • Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, and isolated RNA of any sequence.
  • mRNA messenger RNA
  • cDNA messenger RNA
  • recombinant polynucleotides branched polynucleotides
  • plasmids vectors, isolated DNA of any sequence, and isolated RNA of any sequence.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • the term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
  • prophylactic or therapeutic treatment refers to administration of a drug to a host. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).
  • pyrogen-free refers to a composition that does not contain a pyrogen in an amount that would lead to an adverse effect (e.g., irritation, fever, inflammation, diarrhea, respiratory distress, endotoxic shock, etc.) in a subject to which the composition has been administered.
  • the term is meant to encompass compositions that are free of, or substantially free of, an endotoxin such as, for example, a lipopolysaccharide (LPS). 737 l .DOC 12
  • LPS lipopolysaccharide
  • recombinant protein or “recombinant polypeptide” refer to a polypeptide which is produced by recombinant DNA techniques. An example of such techniques includes the case when DNA encoding the expressed protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the protein or polypeptide encoded by the DNA.
  • Replicative lifespan of a cell refers to the number of daughter cells produced by an individual "mother cell.”
  • Increasing the lifespan of a cell or “extending the lifespan of a cell,” as applied to cells or organisms, refers to increasing the number of daughter cells produced by one cell; increasing the ability of cells or organisms to cope with stresses and combat damage, e.g., to DNA, proteins; and/or increasing the ability of cells or organisms to survive and exist in a living state for longer under a particular condition, e.g., stress (for example, heatshock, osmotic stress, high energy radiation, chemically-induced stress, DNA damage, inadequate salt level, inadequate nitrogen level, or inadequate nutrient level). Lifespan can be increased by at least about 20%, 30%, 40%, 50%, 60% or between 20% and 70%, 30% and 60%, 40% and 60% or more using methods described herein.
  • sirtuin-activating compound refers to a compound that increases the level of deacetylase activity of a sirtuin protein.
  • a sirtuin-activating compound may increase deacetylase activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100%, or more.
  • Sirtuin deacetylation activity may be determined using a variety of substrates for sirtuin proteins. Examples of sirtuin substrates include, for example, histones and p53, or fragments thereof.
  • Exemplary sirtuin activating compounds include flavones, stilbenes, flavanones, isoflavanones, catechins, chalcones, tannins and 737 l .DOC ⁇ anthocyanidins.
  • Exemplary stilbenes include hydroxystilbenes, such as trihydroxystilbenes, e.g., 3,5,4'-trihydroxystilbene ("resveratrol"). Resveratrol is also known as 3,4',5-stilbenetriol. Tetrahydroxystilbenes, e.g., piceatannol, are also encompassed. Hydroxychalones including trihydroxychalones, such as isoliquiritigenin, and tetrahydroxychalones, such as butein, can also be used.
  • Hydroxyflavones including tetrahydroxyflavones, such as fisetin, and pentahydroxyflavones, such as quercetin, can also be used.
  • Other sirtuin activating compounds are described herein below and in U.S. Patent Application Publication No. 2005/0096256 and PCT Application Nos. PCT/US06/002092, PCT/US06/007746, PCT/US06/007744, PCT/US06/007745, PCT/US06/007778, PCT/US06/007656, PCT/US06/007655 and PCT/US06/007773.
  • sirtuin protein refers to a member of the sirtuin deacetylase protein family, or preferably to the sir2 family, which include yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), and human SIRTl (GenBank Accession No. NM_012238 and NP 036370 (or AF083106)) and SIRT2 (GenBank Accession No. NM_012237, NM 030593, NP_036369, NP_085096, and AF083107) proteins.
  • HST genes additional yeast Sir2-like genes termed "HST genes” ( ⁇ omologues of Sir two) HSTl, HST2, HST3 and HST4, and the six other human homologues hSIRT2, hSIRT3, hSIRT4, hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273).
  • sirtuins are those that share more similarities with SIRTl, i.e., hSIRTl, and/or Sir2 than with SIRT2, such as those members having at least part of the N-terminal sequence present in SIRTl and absent in SIRT2 such as SIRT3 has.
  • SIRTl protein refers to a member of the sir2 family of sirtuin deacetylases.
  • a SIRTl protein includes yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP 501912), human SIRTl (SEQ ID NO: 1), and human SIRT2 (GenBank Accession No. NM_012237, NM_030593, NP_036369, NP 085096, or AF083107) proteins, and equivalents thereof.
  • a SIRTl protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set 737 1 DOC 14 forth in SEQ ID NO: 1 or GenBank Accession Nos.
  • SIRTl proteins include polypeptides comprising all or a portion of the amino acid sequence set forth in SEQ ID NO: 1 or GenBank Accession Nos. NP 501912, NP_085096, NP_036369, or P53685; the amino acid sequence set forth in SEQ ID NO: 1 or GenBank Accession Nos.
  • NP 501912, NP_085096, NP_036369, or P53685 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or GenBank Accession Nos. NP 501912, NP_085096, NP_036369, or P53685
  • the term "specifically hybridizes" refers to detectable and specific nucleic acid binding.
  • Polynucleotides, oligonucleotides and nucleic acids of the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • Stringent conditions may be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and nucleic acids of the invention and a nucleic acid sequence of interest will be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or more.
  • hybridization and washing conditions are performed under stringent conditions according to conventional hybridization procedures and as described further herein.
  • stringent conditions or “stringent hybridization conditions” refer to conditions which promote specific hydribization between two complementary polynucleotide strands so as to form a duplex.
  • Stringent conditions may be selected to be about 5° C lower than the thermal melting point (Tm) for a given polynucleotide duplex at a defined ionic strength and pH.
  • Tm thermal melting point
  • the length of the complementary polynucleotide strands and their GC content will determine the Tm of the duplex, and thus the hybridization conditions necessary for obtaining a desired specificity of hybridization.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the a polynucleotide sequence hybridizes to a perfectly matched complementary strand.
  • Tm the stringency of the hybridization conditions
  • 737 l .DOC ⁇ A variety of techniques for estimating the Tm are available. Typically, G-C base pairs in a duplex are estimated to contribute about 3° C to the Tm, while A-T base pairs are estimated to contribute about 2° C, up to a theoretical maximum of about 80-100° C. However, more sophisticated models of Tm are available in which G-C stacking interactions, solvent effects, the desired assay temperature and the like are taken into account.
  • Td dissociation temperature
  • Hybridization may be carried out in 5xSSC, 4xSSC, 3xSSC, 2xSSC, IxSSC or 0.2xSSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24 hours.
  • the temperature of the hybridization may be increased to adjust the stringency of the reaction, for example, from about 25° C. (room temperature), to about 45° C, 50° C, 55° C, 60° C, or 65° C
  • the hybridization reaction may also include another agent affecting the stringency, for example, hybridization conducted in the presence of 50% formamide increases the stringency of hybridization at a defined temperature.
  • the hybridization reaction may be followed by a single wash step, or two or more wash steps, which may be at the same or a different salinity and temperature.
  • the temperature of the wash may be increased to adjust the stringency from about 25° C (room temperature), to about 45° C, 50° C, 55° C, 60° C, 65° C, or higher.
  • the wash step may be conducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS.
  • hybridization may be followed by two wash steps at 65° C each for about 20 minutes in 2xSSC, 0.1% SDS, and optionally two additional wash steps at 65° C each for about 20 minutes in 0.2xSSC, 0.1% SDS.
  • Exemplary stringent hybridization conditions include overnight hybridization at 65° C in a solution comprising, or consisting of, 50% formamide, 10x Denhardt (0.2% Ficoll, 0.2% Polyvinylpyrrolidone, 0.2% bovine serum albumin) and 200 ⁇ g/ml of denatured carrier DNA, e.g., sheared salmon sperm DNA, followed by two
  • Hybridization may consist of hybridizing two nucleic acids in solution, or a nucleic acid in solution to a nucleic acid attached to a solid support, e.g., a filter.
  • a prehybridization step may be conducted prior to hybridization. Prehybridization may be carried out for at least about 1 hour, 3 hours or 10 hours in the same solution and at the same temperature as the hybridization solution (without the complementary polynucleotide strand).
  • systemic administration refers to the administration of a subject composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • therapeutic effect is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.
  • therapeutically- effective amount means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • the therapeutically effective amount of such substance will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For737 l .DOC ' ' example, certain compositions described herein may be administered in a sufficient amount to produce a desired effect at a reasonable benefit/risk ratio applicable to such treatment.
  • transgene refers to a nucleic acid sequence, which is partly or entirely heterologous to a transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene may include one or more regulatory sequences and any other nucleic acids, such as introns, that may be necessary for optimal expression.
  • transgenic animal refers to any animal, for example, a mouse, rat or other non-human mammal, a bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgene causes cells to express a recombinant form of a protein.
  • transgenic animals in which the recombinant gene is silent are also contemplated.
  • Treating refers to curing as well as ameliorating at least one symptom of the condition or disease.
  • vision impairment refers to diminished vision, which is often only partially reversible or irreversible upon treatment (e.g., surgery). Particularly severe vision impairment is termed “blindness” or “vision loss”, which refers to a complete loss of vision, vision worse than 20/200 that cannot be improved with corrective lenses, or a visual field of less than 20 degrees diameter (10 degrees radius).
  • the invention provides sirtuin variant polypeptides that have a higher expression level than the equivalent wild-type sirtuin, have deacetylase activity that is substantially equivalent to the deacetylase activity of the equivalent wild-type sirtuin, and which have deacetylase activity that may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the sirtuin variants are fragments of a full-length sirtuin protein and have increased expression with respect to the full length protein while maintaining deacetylase activity that is substantially equivalent to the corresponding full length sirtuin and which deacetylase activity is activatable by a sirtuin activating compound
  • the invention provides SIRTl variants that may be expressed in E. coli at a concentration of at least 5 mg/L, have deacetylase activity that is substantially equivalent to the deacetylase activity of the equivalent wild-type SIRTl, and have deacetylase activity that may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the SIRTl protein is a human SIRTl protein.
  • sirtuin variants described herein have increased expression levels as compared to the equivalent wild-type sirtuin.
  • the sirtuin variant is a variant of human SIRTl then the variant will have increased expression with respect to wild-type human SIRTl.
  • Determination of the expression level of the variant as compared to the wild-type protein may be conducted by comparing the expression levels of the variant as compared to the wild-type under the same conditions, e.g., expression from the same expression vector, in the same host cell, at the same temperature, for the length of expression, and/or under the same buffer, temperature, shaking conditions, etc.
  • expression of the variant may be at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more greater than the expression of the wild-type sirtuin under equivalent conditions.
  • expression of the sirtuin variant as compared to the wild-type protein is determined in a bacterial host cell, such as, for example, E. coli.
  • a SIRTl variant may be expressed in E. coli at a concentration of at least 2 mg/L, at least 5 mg/L, at least 6 mg/L, at least 8 mg/L, at least 10 mg/L, at least 12 mg/L, or at least 15 mg/L.
  • expression of a SIRTl variant in E. coli may be determined by expressing the SIRTl variant under the control of a T7 promoter- based expression system when expressed in E. coli BL21 StarTM cells (D E3) (Invitrogen) under the following conditions: cells are grown in LB media at 37 0 C until the OD600 reaches 0.8, the temperature of the culture is cooled down to 16 0 C on ice and IPTG is added to 1 mM, the culture is then incubated at 16 0 C for 14-16 hrs and cells are harvest by centrifugation at 29,000 g for 30 min at 4 °C.
  • D E3 E3
  • the deacetylase activity of a sirtuin variant can be measured using any suitable deacetylation assay, including for example, any of the assays described herein.
  • the deactylase activity of the sirtuin variant may be substantially the same as the deacetylase activity of the wild-type sirtuin under equivalent assay conditions, e.g, the deacetylase activity of the sirtuin variant is at least 95%, 96%, 97%, 98%, 99% or greater of the sirtuin activity of the corresponding wild-type sirtuin under comparable conditions.
  • the sirtuin variant is a SIRTl variant which has substantially the same deacetylase activity as wild-type SIRTl .
  • Activation of sirtuin deacetylase activity can be measured using any suitable deacetylation assay, including the ones described herein, in the presence of a sirtuin activating compound.
  • the activatability of the sirtuin variant may be substantially equivalent to the activatability of the corresponding wild-type sirtuin protein, e.g., the deacetylase activity of the sirtuin variant may be activated by at least 95%, 96%, 97%, 98%, 99%, or more, of level of activation of the wild-type sirtuin protein under equivalent conditions, e.g., in the presence of the same sirtuin activating compound, using the same substrate, using the same readout, under the same assay conditions, etc.
  • the sirtuin variant may be activated by at 2-fold, 3 -fold, 4-fold, 5-fold, 10-fold or more in the presence of a sirtuin activating compound. In certain embodiments, the sirtuin variant is activated by at least 2-fold in the presence of resveratrol or Compound #2 or Compound #3 as described herein. In an exemplary embodiment, the sirtuin variant is a SIRTl variant.
  • the invention provides isolated or purified sirtuin variants, including isolated or purified SIRTl variants.
  • the737 l .DOC ⁇ sirtuin variant may be a recombinant variant, such as, for example, a recombinant SIRTl variant.
  • the sirtuin variant is a SIRTl variant comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: (i) a fragment of SEQ ID NO: 1 having an N-terminus that falls between amino acid residues 140 to 190, 149 to 169, 154 to 184, 154 to 169, 169 to 184 of SEQ ID NO: 1 and a C-terminus that falls between amino acid residues 663 to 704, 663 to 671, or 663 to 748 of SEQ ID NO:1, (ii) a fragment of SEQ ID NO: 1 having amino acids residues 183-664, 141-747, 183-664, 183-705, 183-724, 155- 664, 155-747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO:1, (iii) a polypeptide which is at least 90%, 95%, 96%, 97%, 9
  • the SIRTl variants of the invention comprise amino acids residues 183-664, 141-747, 183-664, 183-705, 183-724, 155-664, 155- 747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO:1.
  • the SIRTl variants of the invention comprise a fragment of SEQ ID NO:1 consisting essentially of, or consisting of, amino acid residues 183- 664, 141-747, 183-664, 183-705, 183-724, 155-664, 155-747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO: 1.
  • the SIRTl variants described herein do not include a polypeptide consisting of amino acid residues 261 to 447 of SEQ ID NO: 1, a fragment consisting of amino acid residuess 242 to 493 of SEQ ID NO: 1 , or a fragment consisting of amino acid residues 254 to 495 of SEQ ID NO: 1.
  • the invention provides a SIRTl variant that is not activatable by resveratrol or other sirtuin activating compounds that bind to the same location on SIRTl as resveratrol.
  • Such variants may be useful, for example, to identify sirtuin activating compounds that activate SIRTl by a different mechanism of action.
  • the unactivatable SIRTl variant comprises at least of fragment of SEQ ID NO:1, with the proviso that the variant excludes amino acid residues 183-225 of SEQ ID NO: 1.
  • a sirtuin variant of the invention is a fusion protein containing a domain which increases its solubility and/or facilitates its purification, identification, detection, structural characterization, and/or cellular uptake.
  • exemplary domains include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusion proteins and tags.
  • Additional exemplary domains include domains that alter protein localization in vivo, such as signal peptides, type III secretion system-targeting peptides, transcytosis domains, nuclear localization signals, etc.
  • a variant of the invention may comprise one or more heterologous fusions.
  • Variants may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains. The fusions may occur at the N-terminus of the variant, at the C-terminus of the variant, or at both the N- and C-terminus of the variant.
  • linker sequences between a variant of the invention and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein.
  • the variant may be constructed so as to contain protease cleavage sites between the fusion domain and sirtuin variant in order to remove the tag after protein expression or thereafter.
  • suitable endoproteases include, for example, Factor Xa, thrombin, enterokinase and TEV proteases.
  • a sirtuin variant may be modified so that its rate of traversing the cellular membrane is increased.
  • the variant may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells.
  • the peptide may be a portion of the HIV transactivator (TAT) protein, such as the fragment corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989) Cell 55:1 179-1188).
  • TAT HIV transactivator
  • the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof.
  • the 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled.
  • variants may be fused to a737 l .
  • the transcytosis polypeptide may also be a non-naturally- occurring membrane-translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Pat. No. 6,248,558.
  • MTS membrane-translocating sequence
  • modified variants of the invention may be expressed in E. coli at a concentration of at least 5 mg/L, may have deacetylase activity that is substantially equivalent to the deacetylase activity of full length SIRTl, and the deacetylase activity may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • modified variants may be produced, for instance, by amino acid substitution, deletion, or addition, which substitutions may consist in whole or part by conservative amino acid substitutions.
  • libraries of candidate sirtuin variants may be generated and tested for expression level, deacetylase activity and activatability. Suitable methods for conducting such test are described herein.
  • the libraries may comprise, for example, a variety of truncation mutants for one or more sirtuin proteins.
  • the truncation mutants may have sequences removed at the N-terminus, the C-terminus, or both, as compared to the corresponding full length sirtuin protein.
  • a library may comprise, for example, 10, 50, 100, 250, 500, 750, 1000 or more different members.
  • a candidate variant library may be produced, for example, using737 I .
  • the libraries may comprise truncation mutants of a human sirtuin protein, such as, for example, hSIRTl, hSIRT2, hSIRT3, hSIRT4, hSIRT5, hSIRT ⁇ , hSIRT7, or combinations thereof.
  • the libraries comprise truncation mutants of one or more sirtuin proteins that are activatable by a sirtuin activating compound, such as resveratrol, as a full length protein, such as, for example, human SIRTl, human SIRT2, human SIRT3, human SIRT5, mouse SIRTl, rat SIRTl, or yeast Sir2.
  • a sirtuin activating compound such as resveratrol
  • a full length protein such as, for example, human SIRTl, human SIRT2, human SIRT3, human SIRT5, mouse SIRTl, rat SIRTl, or yeast Sir2.
  • the candidate sirtuin variants may comprise the deacetylase domain of a sirtuin protein (e.g., about amino acid residues 247-661, 249-663, or 250-664 of human SIRTl) and at least a portion of the sequence flanking the deacetylase domain to the N-terminus of the sirtuin protein, for example, corresponding to at least a portion of the region comprising amino acid residues 150-220 of human SIRTl . See Figure 5 for an alignment of the human sirtuin proteins along with several SIRTl homologs (yeast Sir2, mouse SIRTl and rat SIRTl).
  • the sirtuin variant polypeptides of the invention may be purified, for example, to at 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% purity, or greater, with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • a purified sirtuin variant polypeptide may be substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Sirtuin variant polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse- phase high performance liquid chromatography.
  • Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like.
  • PEI, DEAE, QAE and Q derivatives are suitable, including, for example, DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ).
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl737 I .
  • DOC groups such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • sirtuin variant polypeptides described herein can also be isolated an affinity tag (e.g., polyhistidine, maltose-binding protein, an immunoglobulin domain) to facilitate purification as described further herein.
  • affinity tag e.g., polyhistidine, maltose-binding protein, an immunoglobulin domain
  • the invention relates to nucleic acids that encode the sirtuin variant polypeptides described herein.
  • the invention relates to nucleic acids that encode sirtuin variant polypeptides that have a higher expression level than the equivalent wild-type sirtuin, have deacetylase activity that is substantially equivalent to the deacetylase activity of the equivalent wild-type sirtuin, and which have deacetylase activity that may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the invention provides nucleic acids that encode SIRTl variants that may be expressed in E. coli at a concentration of at least 5 mg/L, have deacetylase activity that is substantially equivalent to the deacetylase activity of the equivalent wild-type
  • the nucleic acids encode SIRTl variants that are variants of a human SIRTl protein.
  • the nucleic acid encodes a SIRTl variant that comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of: (i) a fragment of SEQ ID NO: 1 having an N-terminus that falls between amino acid residues 140 to 190, 149 to 169, 154 to 184, 154 to 169, 169 to 184 of SEQ ID NO: 1 and a C-terminus that falls between amino acid residues 663 to 704, 663 to 671, or 663 to 748 of SEQ ID NO:1, (ii) a fragment of SEQ ID NO: 1 having amino acids residues 183-664, 141-747, 183-664, 183-705, 183-724, 155- 664, 155-747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO:1, (iii) a polypeptide which is at least 90%, 95%, 96%, 97%, 9
  • the nucleic acid encodes SIRTl variants comprising amino acids residues 183-664, 141-747, 183-664, 183-705, 183-724, 155-664, 155- 747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO:1.
  • the nucleic acid encodes SIRTl variants comprising a fragment of SEQ ID NO:1 consisting essentially of, or consisting of, amino acid residues 183-664, 141-747, 183-664, 183-705, 183-724, 155-664, 155-747, 164-664, 164-747, 172-664, 219-664, 150-670, or 170-670 of SEQ ID NO: 1.
  • the invention provides a nucleic acid that encodes a SIRTl variant that is not activatable by resveratrol or other sirtuin activating compounds that bind to the same location on SIRTl as resveratrol.
  • Such variants may be useful, for example, to identify sirtuin activating compounds that activate SIRTl by a different mechanism of action.
  • the unactivatable SIRTl variant comprises at least of fragment of SEQ ID NO:1 , with the proviso that the variant excludes amino acid residues 183-225 of SEQ ID NO:1.
  • the nucleic acid comprises, consists essentially of, or consists of, a nucleic acid sequence selected from the group consisting of: (i) a fragment of SEQ ID NO: 2 having a 5' end that falls between nucleic acid residues737 I .
  • a nucleic acid comprising nucleotide residues 547- 1992, 421-2241, 547-21 15, 547-2172, 463-1992, 463-2241, 490-1992, 490-2241, 514-1992, 655-1992, 448-2010, or 508-2010 of SEQ ID NO: 2;
  • the nucleic acid is a fragment of a full length sirtuin protein.
  • the nucleic acid encodes a sirtuin variant polypeptide that has a higher expression level than the equivalent wild-type sirtuin, has deacetylase activity that is substantially equivalent to the deacetylase activity of the equivalent wild-type sirtuin, and which has deacetylase activity that may be activated by at least 2-fold in the presence of a sirtuin activating compound.
  • the nucleic acid encoding the sirtuin variant may be a naked DNA molecule, or it may be a component of a plasmid, a cosmid, a phagemid, an artificial chromosome, a virus particle or virus-like particle, a liposome, or any similar or equivalent vector which effectively acts to introduce the sirtuin variant nucleotide sequence into the cell.
  • the sirtuin variant nucleic acid advantageously is operably linked to at least one element such as an enhancer, a promoter, or a polyadenylation site that serves to promote the de novo intracellular expression of the encoded sirtuin variant.
  • the nucleic acid is an isolated or purified nucleic acid.
  • Nucleic acids which differ from the nucleic acids of the invention due to degeneracy in the genetic code are also within the scope of the invention.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the variants of the invention will exist.
  • these variations in one or more nucleotides from less than 1% up to about 3 or 5% or possibly more737 I .
  • DOC 27 ' of the nucleotides) of the nucleic acids encoding a sirtuin variant of the invention may exist among a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Bias in codon choice within genes in a single species appears related to the level of expression of the protein encoded by that gene. Accordingly, the invention encompasses nucleic acid sequences which have been optimized for improved expression in a host cell by altering the frequency of codon usage in the nucleic acid sequence to approach the frequency of preferred codon usage of the host cell.
  • the instant invention relates to any nucleotide sequence that encodes a sirtuin variant amino acid sequence as set forth herein.
  • the expression level of a sirtuin variant is determined with respect to the corresponding full length sirtuin protein wherein both the variant and the full length protein have been similarly codon optimized.
  • Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant variants.
  • a nucleic acid encoding a variant of the invention may be obtained from mRNA or genomic DNA from any organism in accordance with protocols described herein, as well as those generally known to those skilled in the art.
  • a cDNA encoding a variant of the invention for example, may be obtained by isolating total mRNA from an organism, e.g. a bacteria, virus, mammal, etc.
  • Double stranded cDNAs may then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.
  • a gene encoding a variant of the invention may also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided herein.
  • a nucleic acid encoding a sirtuin variant is provided in an expression vector such that the nucleotide sequence encoding the sirtuin variant is operably linked to at least one regulatory sequence. It should be
  • J Q 737 l J Q 737 l .DOC understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of variant desired to be expressed.
  • the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered.
  • the subject nucleic acids may be used to cause expression and over- expression of a sirtuin variant in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins or polypeptides.
  • vectors preferably expression vectors, containing a nucleic acid encoding sirtuin variants, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA into which additional DNA segments can be incorporated.
  • viral vector is another type of vector, wherein additional DNA segments can be incorporated into the viral genome, or a portion thereof.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • DOC 29 Recombinant expression vectors may comprise a nucleic acid encoding a sirtuin variant in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell, those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences), and those that direct inducible expression upon exposure to an external factor such as a small molecule, temperature, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce sirtuin variant polypeptides, including fusion polypeptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a sirtuin variant in prokaryotic or eukaryotic cells.
  • the sirtuin variant can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.
  • telomeres Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.737 l .DOC ⁇ "
  • T7 promoter regulatory sequences and T7 polymerase 737 l .DOC ⁇
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus or carboxy terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • the expression level or activity of a sirtuin variant which is a fusion protein is compared to the expression level or activity of the corresponding wild-type sirtuin protein, wherein both proteins are fused to the same polypeptide sequence at either the N-terminus and/or the C- terminus.
  • E. coli expression vectors examples include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al.,737 l .DOC ⁇ l (1992) Nucleic Acids Res. 20: 21 11-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the sirtuin variant is expressed using a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al., (1987) EMBO J 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
  • the sirtuin variant can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) MoI Cell Biol 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • sirtuin variants may be expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells See, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • the recombinant mammalian expression vector is capable of directing expression of a sirtuin variant preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev 1 : 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv Immunol 43: 235-275), in particular promoters of T cell737 I .
  • DOC 32 receptors Winoto and Baltimore (1989) EMBO J 8: 729-733 and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477
  • pancreas-specific promoters Edlund et al. (1985) Science 230
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3 : 537-546).
  • host cells comprising a sirtuin variant polypeptide, host cells comprising a nucleic acid encoding a sirtuin variant polypeptide, host cells comprising an expression vector comprising a nucleic acid sequence that encodes a sirtuin variant, and host cells expressing a sirtuin variant from a nucleic acid.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a sirtuin variant may be expressed in bacterial cells, such as E. coli, insect cells (baculovirus), yeast, or mammalian cells including, for example, human cells.
  • the cell may be in a live subject or may be isolated from a subject, e.g., in a cell culture, tissue sample, cell suspension, etc.
  • suitable host cells are known to those skilled in the art.
  • the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide.
  • Other methods suitable for maximizing expression of the polypeptide will be known to those in the art.
  • in vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein.
  • An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F).
  • CBP cap-binding protein
  • eIF4F eukaryotic initiation factor 4F
  • in vitro translation systems examples include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, 111.; and GIBCO/BRL, Grand Island, N.Y. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used.
  • eukaryotic lysates such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La Jolla
  • Such systems typically comprise at least an RNA polymerase holoenzyme, ribonucleotides and any necessary transcription initiation, elongation and termination factors.
  • In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.
  • sirtuin variant having an N-terminal deletion When expression of sirtuin variant having an N-terminal deletion is desired, i.e. the sirtuin variant is a truncation mutant of a full length sirtuin, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed.
  • ATG start codon
  • a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP).
  • MAP methionine aminopeptidase
  • Coding sequences for a sirtuin variant may be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide.
  • the present invention contemplates a nucleic acid comprising a nucleic acid encoding a sirtuin variant and at least one heterologous sequence encoding a heterologous peptide linked in frame to the nucleotide sequence of the sirtuin varinat so as to encode a fusion protein comprising the heterologous polypeptide.
  • the heterologous polypeptide may be fused to (a) the C-terminus of the polypeptide encoded by the nucleic acid of the invention, (b) the N-terminus of the polypeptide, or (c) the C-
  • the heterologous sequence encodes a polypeptide permitting the detection, isolation, solubilization, transcytosis and/or stabilization of the sirtuin variant polypeptide to which it is fused.
  • the heterologous sequence encodes a polypeptide selected from the group consisting of a polyHis tag, myc, HA, GST, protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose-binding protein, poly arginine, poly His-Asp, FLAG, a portion of an immunoglobulin protein, and a transcytosis peptide.
  • Fusion proteins may facilitate the expression and/or purification of proteins.
  • a sirtuin variant polypeptide may be generated as a glutathione- S- transferase (GST) fusion protein.
  • GST fusion proteins may be used to simplify purification of a sirtuin variant polypeptide, such as through the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., (N. Y.: John Wiley & Sons, 1991)).
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, may allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence may then be subsequently removed by treatment with enterokinase to provide the purified protein (e.g., see Hochuli et al., (1987) J. Chromatography 411 : 177; and Janknecht et al., PNAS USA 88:8972).
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed to generate a chimeric gene sequence (see, for example,3737 l .DOC ⁇ S Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • non-human transgenic animals comprising a sirtuin variant as disclosed herein.
  • a non-human transgenic animal comprising a SIRTl variant.
  • a cell transfected with a nucleic acid encoding a sirtuin variant, or a sirtuin variant transfected cell can be used to produce nonhuman transgenic animals.
  • a sirtuin variant- transfected cell is a fertilized oocyte or an embryonic stem cell into which a sirtuin variant protein-coding sequence has been introduced.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is stably integrated into the genome of a cell from which a transgenic animal develops, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous SIRTl gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a sirtuin variant protein-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection or retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Sirtuin variant nucleic acids can be
  • transgenic founder animal can be identified based upon the presence of the sirtuin variant transgene in its genome and/or expression of sirtuin variant mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a sirtuin variant can further be bred to other transgenic animals carrying other transgenes.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage Pl .
  • Cre/loxP recombinase system of bacteriophage Pl .
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 : 181-185).
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385: 810-813.
  • a cell e.g., a somatic cell
  • a somatic cell from the transgenic animal can be737 I .
  • DOC ⁇ ' isolated and induced to exit the growth cycle and enter G 0 phase.
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • a transgenic non-human mammal a majority of whose cells harbor a transgene comprising a nucleic acid that encodes a sirtuin variant.
  • the sirtuin variant is a SIRTl variant.
  • the majority of cells which harbor a transgene have increased sirtuin deacetylation activity in respect to cells which do not harbor a transgene.
  • the life span of the transgenic non-human mammal is increased with respect to a nontransgenic mammal of the same species.
  • assays for measuring deacetylase activity using the sirtuin variants described herein as well as assays for identifying compounds or agents that modulate sirtuin activity.
  • the assays may be used to identify compounds that either activate sirtuin deacetylase activity or compounds that inhibit sirtuin deacetylase activity.
  • Assays may be conducted in a cell based or cell free format. The assays may be conducted under conditions which permit deacetylation of a substrate by the sirtuin variant. In certain embodiments, the assays are conducted in the presence of NAD + .
  • screening assays are conducted in vitro, it may be desirable to contact a cell with a candidate sirtuin modulating compound identified in the in vitro screen to determine if the candidate test compound increases the life span of the cell.
  • Assay methods may involve, for example, contacting at least one acetylated sirtuin substrate with a sirtuin variant polypeptide and determining the level of acetylation of the sirtuin substrate.
  • the assays may further include addition of a test agent to the assay in order to determine if the test agent modulates deacetylation of the substrate by the sirtuin variant as compared to a control (e.g., an assay without the test agent, an assay in the presence of an agent having know sirtuin modulating activity, an assay in the presence of an agent having no sirtuin modulating activity, or a value in a database).
  • a control e.g., an assay without the test agent, an assay in the presence of an agent having know sirtuin modulating activity, an assay in the presence of an agent having no sirtuin modulating activity, or a value in a database.
  • the sirtuin variants described herein may be used in association with various types of deacetylation assays to determine sirtuin activity and/or to identify compounds that modulate sirtuin activity.
  • the sirtuin variants may be used in association with a fluorescence based assay such as the assay commercially available from Biomol, e.g., the SIRTl Fluorimetric Drug Discovery Kit (AK-555), SIRT2 Fluorimetric Drug Discovery Kit (AK-556), or SIRT3 Fluorimetric Drug Discovery Kit (AK-557) (Biomol International, Plymouth Meeting, PA).
  • nicotinamide release assay Kaeberlein et al., J. Biol. Chem. 280(17): 17038 (2005)
  • FRET assay Marcotte et al., Anal. Biochem. 332: 90 (2004)
  • C 14 NAD boron resin binding assay McDonagh et al., Methods 36: 346 (2005)
  • sirtuin variants described herein include radioimmunoassays (RIA), scintillation proximity assays, HPLC based assays, and reporter gene assays (e.g., for transcription factor targets).
  • RIA radioimmunoassays
  • HPLC based assays e.g., HPLC based assays
  • reporter gene assays e.g., for transcription factor targets.
  • a sirtuin variant polypeptide may be substituted for the sirtuin protein used in the referenced assays.
  • the sirtuin variants described herein may be used in association with a fluorescence polarization assay. Examples of fluorescence polarization assays are described herein and are also described in PCT Publication No. WO 2006/094239.
  • the sirtuin variants described herein may be used in association with mass spectrometry based assays.
  • mass spectrometry based assays are described herein and are also described in PCT Application No. PCT/US06/046021. 737 l .DOC 39
  • the sirtuin substrate may comprise a fluorophore and a high molecular weight group or bulky group. The high molecular weight or bulky group is separated from the fluorophore by at least one lysine residue.
  • the sirtuin substrate When the lysine residue is in the non-acetylated state, the sirtuin substrate is susceptible to cleavage at or near the lysine residue by a cleavage reagent, such as a protease. When the lysine residue is in the acetylated state, the sirtuin substrate is resistant to cleavage and remains intact upon contact with a cleavage reagent. Upon cleavage, the fluorophore is separated from the high molecular weight or bulky group thereby increasing the fluorescent polarization value of the sample.
  • exemplary ccleavage reagents for use in accordance with the methods described herein include chemical and enzymatic reagents.
  • the cleavage reagent is a protease, such as, for example, a protease that cleaves at or near a lysine residue.
  • a protease included, for example, lysylendopeptidase, endoproteinase, Lys-C, plasmin, calpain, or trypsin.
  • FP is useful for high throughput screening (HTS) assays.
  • HTS high throughput screening
  • FP is also amenable to performing assays in real-time, directly in solution and without the need for an immobilized phase.
  • Polarization values can be measured repeatedly both before and after the addition of reagents since measuring the samples is rapid and does not destroy the sample.
  • the methods described herein for determining sirtuin activity and/or for identifying a compound that modulates sirtuin activity utilize mass spectrometry for determining the level of acetylation of a sirtuin substrate.
  • the presence of an acetyl group on a polypeptide may be determined by a +42 Da molecular weight shift (per acetyl group) as compared to the unmodified polypeptide.
  • Mass spectrometry or simply MS encompasses any spectrometric technique or process in which molecules are ionized and separated and/or analyzed based on their respective molecular weights.
  • mass spectrometry and MS encompass any type of ionization method, including without limitation electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI) and other forms of atmospheric pressure ionization (API), and laser irradiation.
  • Mass spectrometers737 l .DOC 40 w may be combined with separation methods such as gas chromatography (GC) and liquid chromatography (LC). GC or LC separates the components in a mixture, and the components are then individually introduced into the mass spectrometer; such techniques are generally called GC/MS and LC/MS, respectively.
  • the assays described herein utilize a sirtuin substrate pool that comprises a plurality of copies of one or more sirtuin substrate polypeptides.
  • a sirtuin substrate pool comprises a plurality of copies of the same polypeptide substrate.
  • Such sirtuin substrate pools may comprise the sirtuin substrate free floating in solution or attached to a solid surface such as a plate, bead, filter, etc. Combinations of free floating and anchored sirtuin substrate molecules may also be used in accordance with the methods described herein.
  • Substrates suitable for use in accordance with the methods described herein may be based on any polypeptide that can be deacetylated by a sirtuin protein, such as, for example, p53 or histones.
  • exemplary substrates include, for example, the Fluor de Lys-SIRTl substrate from BIOMOL (Plymouth Meeting, PA).
  • Suitable substrates include, for example, Ac-EE-K(biotin)-GQSTSSHSK( Ac)NIeSTEG-K(MRl 21)-EE-NH 2 (SEQ ID NO: 3) and Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE- NH2 (SEQ ID NO: 4) wherein K(biotin) is a biotinylated lysine residue, K(Ac) is an acetylated lysine residue, NIe is norleucine, K(MRl 21) is a lysine residue modified by an MR121 fluorophore (excitation 635 nm/emission 680 nm), and K(5TMR) is a lysine resiude modified by a 5TMR fluorophore (excitation 540 nm/emission 580 nm
  • the sequence of the peptide substrates are based on p53 with several modifications.
  • all arginine and leucine residues other than the acetylated lysine residues are replaced with serine so that the peptides are not susceptible to trypsin cleavage in the absence of deacetylation.
  • the methionine residues naturally present in the sequences are replaced with the norleucine because the methionine may be susceptible to oxidation during synthesis and purification.
  • the sirtuin assays described herein may be carried out in a single reaction vessel without the need to remove reagents from the reaction mixture (e.g., a homogenous assay).
  • the components of the737 l .DOC reactions described herein may be added sequentially or simultaneously.
  • a cleavage reagent concurrently with, or subsequent to, exposure of the sirtuin substrate to the sirtuin deacetylase.
  • the invention provides a method for identifying a compound that modulates the activity of a sirtuin deacetylase.
  • the methods may involve comparing the activity of a sirtuin protein in the presence of a test compound as compared to a control.
  • the control may be the activity of a sirtuin protein in a control reaction or a value in a database.
  • a control reaction may simply be a duplicate reaction in which the test compound is not included.
  • the control reaction may be a duplicate reaction in the presence of a compound having a known effect on the sirtuin protein activity (e.g., an activator, an inhibitor, or a compound having no effect on enzyme activity).
  • the invention provides methods for screening for compounds that modulate activity of a sirtuin deacetylases.
  • the methods described herein may be used to identify a test compound that decreases or increases sirtuin activity by at least about 10%, 25%, 50%, 75%, 80%, 90%, or
  • Test compounds to be tested for activity in the assays described herein can include proteins (including post-translationally modified proteins), peptides (including chemically or enzymatically modified peptides), or small molecules (including carbohydrates, steroids, lipids, anions or cations, drugs, small organic molecules, oligonucleotides, antibodies, and genes encoding proteins of the agents or antisense molecules), including libraries of compounds.
  • the test compounds can be naturally occurring (e.g., found in nature or isolated from nature) or can be non- naturally occurring (e.g., synthetic, chemically synthesized or man-made).
  • test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide737 l .DOC 42 oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des.
  • Test compounds can be screened for the ability to modulate acetyltransferase or deacetylase activity using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • Assays involving free formats are described, for example, in Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994);
  • Compounds that activate or inhibit the acetyltransferase or deacetylase activity are useful as candidate compounds for antimicrobial substances, anticancer agents, and a variety of other uses.
  • compounds that activate a sirtuin deacetylase protein may be useful for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, and/or flushing, etc.
  • sirtuin deacetylase inhibitors may be useful for a variety of therapeutic applications including, for example, increasing cellular sensitivity to stress, increasing apoptosis, treatment of cancer, stimulation of appetite, and/or stimulation of weight gain, etc.
  • the invention provides methods of treating a variety of diseases and disorders in which an increase in sirtuin activity is desirable.
  • the methods involve increasing sirtuin activity by administering to a subject in need thereof a sirtuin variant therapeutic agent, e.g., a sirtuin variant polypeptide or a nucleic acid encloding a sirtuin variant.
  • a sirtuin variant therapeutic agent e.g., a sirtuin variant polypeptide or a nucleic acid encloding a sirtuin variant.
  • sirtuin variant therapeutic agent encompasses a single sirtuin variant polypeptide, a combination of two or more sirtuin variant polypeptides, a single nucleic acid encoding a sirtuin variant polypeptide, a combination nucleic acids encoding two or more sirtuin variant polypeptides, as well as combinations of sirtuin variant polypeptides and nucleic acids encoding sirtuin variant polypeptides. Exemplary sirtuin variant polypeptides and nucleic acids encoding sirtuin variant polypeptides are described in detail above.
  • diseases and disorders that would benefit from an increase in sirtuin activity include, for example, aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, and/or flushing, etc. Exemplary diseases and disorders that would benefit from an increase in sirtuin activity are discussed in more detail below.
  • the therapeutic methods described herein involve increasing sirtuin activity in a subject in need thereof using a sirtuin variant therapeutic agent as described herein.
  • the methods may involve, for example, administering to a subject a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide.
  • the sirtuin variant polypeptide may be modified, for example, to increase its stability or to facilitate cellular uptake. Such modifications are described further herein.
  • a sirtuin variant polypeptide is a fused to a transcytosis peptide (such as, for example, TAT or a fragment thereof) to facilitate cellular uptake, to an Fc domain to increase serum stability, or both.
  • a transcytosis peptide such as, for example, TAT or a fragment thereof
  • the methods described herein may utilize a nucleic acid encoding a sirtuin variant polypeptide.
  • Sirtuin variant therapeutic agents may be administered systemically to a subject or may be adminsitered locally to a desired location in the body as appropriate for the specific disease or disorder being treated.
  • the methods When using a nucleic acid therapeutic, the methods also contemplate ex vivo gene therapy methods, e.g., wherein cells are removed from a subject, transfected in vitro with the desired nucleic acid, followed by reintroduction of the modified cells into the same or a different subject. Methods of administering protein and nucleic acid therapeutics are described further herein. In exemplary embodiments, the methods involve administering a human SIRTl variant polypeptide or a nucleic acid encoding a SIRTl variant polypeptide to a human patient in need of increased SIRTl activity.
  • administration of the sirtuin variant therapeutic agent results in an increase in sirtuin activity in the subject.
  • Such an increase in sirtuin activity may be achieved, for example, by increasing the number of active sirtuin proteins in the cell using the sirtuin variant therapeutic agent, by introducing a sirtuin variant therapeutic agent into a cell which does not typically have sirtuin activity, or by introducing a sirtuin variant therapeutic agent into a cell having regulated sirtuin activity thereby producing a cell having sirtuin activity on a constitutive basis.
  • a sirtuin variant When using a nucleic acid therapeutic it may be desirable to have expression of the sirtuin variant under the control of a consistutive promoter and/or a promoter that produces a higher level of sirtuin expression than the native sirtuin promoter.
  • a sirtuin variant may be under the control of a737 l .DOC tissue or cell specific promoter or under the control of the native sirtuin promoter so that sirtuin variant expression is confined to a specific cell or tissue type or limited to a native level of protein expression.
  • a nucleic acid encoding a sirtuin variant that does not contain its own promoter may also be introduced into the genome of a host cell so that expression is controlled by a promoter that is endogenous to the host cell.
  • a sirtuin variant therapeutic agent may be administered alone or in combination with other compounds.
  • a mixture of two or more sirtuin variant therapeutic agents may be administered to a subject in need thereof.
  • Such combination may comprise, for example, a mixture of two different sirtuin variant polypepetides, a sirtuin variant polypeptide and a nucleic acid encoding the same or a different sirtuin variant polypeptide, or a combination of two nucleic acids encoding different sirtuin variant polypeptides.
  • sirtuin variant therapeutic agents may be used in combination with one or more of the following compounds: resveratrol, butein, fisetin, piceatannol, or quercetin.
  • sirtuin variant therapeutic agents may be administered in combination with nicotinic acid.
  • one or more sirtuin variant therapeutic agents e.g., sirtuin variant polypeptides or nucleic acids encoding sirtuin variant polypeptides
  • combination therapies comprising a sirtuin variant therapeutic agent may refer to (1) pharmaceutical compositions that comprise one or more sirtuin variant therapeutic agents in combination with one or more additional therapeutic agents (e.g., one or more additional therapeutic agents described herein); and (2) co-administration of one or more sirtuin variant therapeutic agents with one or more additional therapeutic agents wherein the sirtuin variant therapeutic agent and the additional therapeutic agent have not been formulated in the same compositions (but may be present within the same kit or package, such as a blister pack or other multi-chamber package; connected, separately sealed containers (e.g., foil pouches) that can be separated by the user; or a kit where the sirtuin variant therapeutic agent(s) and other additional737 I .
  • additional therapeutic agents e.g., one or more additional therapeutic agents described herein
  • co-administration of one or more sirtuin variant therapeutic agents with one or more additional therapeutic agents wherein the sirtuin variant therapeutic agent and the additional therapeutic agent have not been formulated in the same compositions (
  • DOC ⁇ O therapeutic agent(s) are in separate vessels).
  • sirtuin variant therapeutic agents may be administered at the same, intermittent, staggered, prior to, subsequent to, or combinations thereof, with the administration of another therapeutic agent.
  • Methods contemplated by the present invention include methods of extending the life span of a eukaryotic cell.
  • a method of extending the life span of a eukaryotic cell comprises introducing into the cell a sirtuin variant therapeutic agent, e.g., a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide, such as, for example, a SIRTl variant.
  • the eukaryotic cell so transformed may be in an in vitro cell culture, or it may be in an ex vivo tissue or organ sample, or it may exist in vivo as a constituent of a living organism.
  • the transfected or transformed cell may be a vertebrate cell, a mammalian cell, or a human cell.
  • sirtuin variant therapeutic agent e.g., a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide.
  • the invention provides a method extending the lifespan of a cell, extending the proliferative capacity of a cell, slowing ageing of a cell, promoting the survival of a cell, delaying cellular senescence in a cell, mimicking the effects of calorie restriction, increasing the resistance of a cell to stress, or preventing apoptosis of a cell, by introducing into the cell a sirtuin variant therapeutic agent.
  • the methods described herein may be used to increase the amount of time that cells, particularly primary cells (i.e.,. cells obtained from an organism, e.g., a human), may be kept alive in a cell culture.
  • Embryonic stem (ES) cells and pluripotent cells, and cells differentiated therefrom may also be modified with a sirtuin variant therapeutic agent to keep the cells, or progeny thereof, in culture for 3737 I .
  • DOC longer periods of time.
  • Such cells can also be used for transplantation into a subject, e.g., after ex vivo modification.
  • cells that are intended to be preserved for long periods of time may be modified with a sirruin variant therapeutic agent.
  • the cells may be in suspension (e.g., blood cells, serum, biological growth media, etc.) or in tissues or organs.
  • blood collected from an individual for purposes of transfusion may be modified with sirruin variant therapeutic agent to preserve the blood cells for longer periods of time.
  • blood to be used for forensic purposes may also be preserved using a sirruin variant therapeutic agent.
  • Other cells that may be treated to extend their lifespan or protect against apoptosis include cells for consumption, e.g., cells from non-human mammals (such as meat) or plant cells (such as vegetables).
  • Sirruin variant therapeutic agents may also be introduced into cells during developmental and growth phases in mammals, plants, insects or microorganisms, in order to, e.g., alter, retard or accelerate the developmental and/or growth process.
  • sirruin variant therapeutic agents may be used to treat cells useful for transplantation or cell therapy, including, for example, solid tissue grafts, organ transplants, cell suspensions, stem cells, bone marrow cells, etc.
  • the cells or tissue may be an autograft, an allograft, a syngraft or a xenograft.
  • the cells or tissue may be treated with a sirruin variant therapeutic agent prior to administration/implantation, concurrently with administration/implantation, and/or post administration/implantation into a subject.
  • the cells or tissue may be treated prior to removal of the cells from the donor individual, ex vivo after removal of the cells or tissue from the donor individual, or post implantation into the recipient.
  • the donor or recipient individual may be treated systemically with a sirruin variant therapeutic agent or may have a subset of cells/tissue treated locally with a sirtuin variant therapeutic agent.
  • the cells or tissue may additionally be treated with another therapeutic agent useful for prolonging graft survival, such as, for example, an immunosuppressive agent, a cytokine, an angiogenic factor, etc.
  • another therapeutic agent useful for prolonging graft survival such as, for example, an immunosuppressive agent, a cytokine, an angiogenic factor, etc.
  • cells may be modified with a sirtuin variant therapeutic agent in vivo, e.g., to increase their lifespan or prevent apoptosis.
  • a sirtuin variant therapeutic agent in vivo, e.g., to increase their lifespan or prevent apoptosis.
  • skin can be protected from aging (e.g., developing wrinkles, loss of elasticity, etc.) by treating skin or epithelial cells with a sirtuin variant therapeutic agent.
  • skin is contacted with a pharmaceutical or cosmetic composition comprising a sirtuin variant therapeutic agent.
  • Exemplary skin afflictions or skin conditions that may be treated in accordance with the methods described herein include disorders or diseases associated with or caused by inflammation, sun damage or natural aging.
  • compositions find utility in the prevention or treatment of contact dermatitis (including irritant contact dermatitis and allergic contact dermatitis), atopic dermatitis (also known as allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including penfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas (including erythema multiforme and erythema nodosum), damage caused by the sun or other light sources, discoid lupus erythematosus, dermatomyositis, psoriasis, skin cancer and the effects of natural aging.
  • contact dermatitis including irritant contact dermatitis and allergic contact dermatitis
  • atopic dermatitis also known as allergic eczema
  • actinic keratosis also known as allergic eczema
  • sirtuin variant therapeutic agents may be used for the treatment of wounds and/or bums to promote healing, including, for example, first-, second- or third-degree burns and/or a thermal, chemical or electrical burns.
  • the formulations may be administered topically, to the skin or mucosal tissue, as an ointment, lotion, cream, microemulsion, gel, solution or the like, as further described herein, within the context of a dosing regimen effective to bring about the desired result.
  • Topical formulations comprising one or more sirtuin variant therapeutic agents may also be used as preventive, e.g., chemopreventive, compositions.
  • preventive e.g., chemopreventive
  • susceptible skin is treated prior to any visible condition in a particular individual.
  • Sirtuin variant therapeutic agents may be delivered locally or systemically to a subject.
  • a sirtuin variant therapeutic agent e.g., a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide, is delivered locally to a tissue or organ of a subject by injection, topical formulation, etc.
  • a sirtuin variant therapeutic agent may be used for treating or preventing a disease or condition induced or exacerbated by cellular737 l .
  • DOC 49 senescence in a subject methods for decreasing the rate of senescence of a subject, e.g., after onset of senescence; methods for extending the lifespan of a subject; methods for treating or preventing a disease or condition relating to lifespan; methods for treating or preventing a disease or condition relating to the proliferative capacity of cells; and methods for treating or preventing a disease or condition resulting from cell damage or death, hi certain embodiments, the method does not act by decreasing the rate of occurrence of diseases that shorten the lifespan of a subject, hi certain embodiments, a method does not act by reducing the lethality caused by a disease, such as cancer.
  • a sirtuin variant therapeutic agent may be administered to a subject in order to generally increase the lifespan of its cells and to protect its cells against stress and/or against apoptosis. It is believed that treating a subject with a sirtuin variant therapeutic agent described herein is similar to subjecting the subject to hormesis, i.e., mild stress that is beneficial to organisms and may extend their lifespan.
  • Sirtuin variant therapeutic agents may be administered to a subject to prevent aging and aging-related consequences or diseases, such as stroke, heart disease, heart failure, arthritis, high blood pressure, and Alzheimer's disease.
  • Other conditions that can be treated include ocular disorders, e.g., associated with the aging of the eye, such as cataracts, glaucoma, and macular degeneration.
  • Sirtuin variant therapeutic agents can also be administered to subjects for treatment of diseases, e.g., chronic diseases, associated with cell death, in order to protect the cells from cell death.
  • Exemplary diseases include those associated with neural cell death, neuronal dysfunction, or muscular cell death or dysfunction, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amniotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminant hepatitis; diseases linked to degeneration of the brain, such as Creutzfeld-Jakob disease, retinitis pigmentosa and cerebellar degeneration; myelodysplasis such as aplastic anemia; ischemic diseases such as myocardial infarction and stroke; hepatic diseases such as alcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such as osteoarthritis; atherosclerosis; alopecia; damage to the skin due to UV light; lichen planus; 737 l .DOC ⁇ " atrophy of the skin; cataract; and graft rejections.
  • Cell death can also be caused by surgery, drug therapy, chemical exposure or radiation exposure.
  • Sirtuin variant therapeutic agents can also be administered to a subject suffering from an acute disease, e.g., damage to an organ or tissue, e.g., a subject suffering from stroke or myocardial infarction or a subject suffering from a spinal cord injury. Sirtuin variant therapeutic agents may also be used to repair an alcoholic's liver. Cardiovascular Disease
  • the invention provides a method for treating and/or preventing a cardiovascular disease by administering to a subject in need thereof a sirtuin variant therapeutic agent.
  • Cardiovascular diseases that can be treated or prevented by inceasing sirtuin activity include cardiomyopathy or myocarditis; such as idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy.
  • Also treatable or preventable using sirtuin variant therapeutic agents are atheromatous disorders of the major blood vessels (macrovascular disease) such as the aorta, the coronary arteries, the carotid arteries, the cerebrovascular arteries, the renal arteries, the iliac arteries, the femoral arteries, and the popliteal arteries.
  • vascular diseases that can be treated or prevented include those related to platelet aggregation, the retinal arterioles, the glomerular arterioles, the vasa nervorum, cardiac arterioles, and associated capillary beds of the eye, the kidney, the heart, and the central and peripheral nervous systems.
  • the sirtuin variant therapeutic agents may also be used for increasing HDL levels in plasma of an individual.
  • sirtuin variant therapeutic agents include restenosis, e.g., following coronary intervention, and disorders relating to an abnormal level of high density and low density cholesterol.
  • a sirtuin variant therapeutic agent may be administered as part of a combination therapeutic with another cardiovascular agent including, for example, an anti-arrhythmic agent, an antihypertensive agent, a calcium channel blocker, a cardioplegic solution, a cardiotonic agent, a fibrinolytic agent, a737 l .DOC ⁇ sclerosing solution, a vasoconstrictor agent, a vasodilator agent, a nitric oxide donor, a potassium channel blocker, a sodium channel blocker, statins, or a naturiuretic agent.
  • another cardiovascular agent including, for example, an anti-arrhythmic agent, an antihypertensive agent, a calcium channel blocker, a cardioplegic solution, a cardiotonic agent, a fibrinolytic agent, a737 l .DOC ⁇ sclerosing solution, a vasoconstrictor agent, a vasodilator agent, a nitric oxide donor,
  • a sirtuin variant therapeutic agent e.g., a sirtuin variant polypeptide or nucleic acid encoding a sirtuin variant polypeptide
  • a combination therapeutic with an anti-arrhythmia agent may be administered as part of a combination therapeutic with an anti-arrhythmia agent.
  • Anti-arrhythmia agents are often organized into four main groups according to their mechanism of action: type I, sodium channel blockade; type II, beta-adrenergic blockade; type III, repolarization prolongation; and type IV, calcium channel blockade.
  • Type I anti-arrhythmic agents include lidocaine, moricizine, mexiletine, tocainide, procainamide, encainide, flecanide, tocainide, phenytoin, propafenone, quinidine, disopyramide, and flecainide.
  • Type II anti-arrhythmic agents include propranolol and esmolol.
  • Type III includes agents that act by prolonging the duration of the action potential, such as amiodarone, artilide, bretylium, clofilium, isobutilide, sotalol, azimilide, dofetilide, dronedarone, ersentilide, ibutilide, tedisamil, and tedilide.
  • Type IV anti-arrhythmic agents include verapamil, diltaizem, digitalis, adenosine, nickel chloride, and magnesium ions.
  • a sirtuin variant therapeutic agent may be administered as part of a combination therapeutic with another cardiovascular agent.
  • cardiovascular agents include vasodilators, for example, hydralazine; angiotensin converting enzyme inhibitors, for example, captopril; anti-anginal agents, for example, isosorbide nitrate, glyceryl trinitrate and pentaerythritol tetranitrate; anti-arrhythmic agents, for example, quinidine, procainaltide and lignocaine; cardioglycosides, for example, digoxin and digitoxin; calcium antagonists, for example, verapamil and nifedipine; diuretics, such as thiazides and related compounds, for example, bendrofluazide, chlorothiazide, chlorothalidone, hydrochlorothiazide and other diuretics, for example, fursemide and triamterene, and sedatives, for example,
  • cardiovascular agents include, for example, a cyclooxygenase inhibitor such as aspirin or indomethacin, a platelet aggregation inhibitor such as clopidogrel, ticlopidene or aspirin, fibrinogen antagonists or a diuretic such as chlorothiazide, hydrochlorothiazide, flumethiazide,737 1 DOC 52 ⁇ hydroflumethiazide, bendroflumethiazide, methylchlorthiazide, trichloromethiazide, polythiazide or benzthiazide as well as ethacrynic acid tricrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamterene, amiloride and spironolactone and salts of such compounds, angiotensin converting enzyme inhibitors such as captopril, zofenopril, fosinopril, enalapril,
  • cardiovascular agents include, for example, vasodilators, e.g., bencyclane, cinnarizine, citicoline, cyclandelate, cyclonicate, ebumamonine, phenoxezyl, flunarizine, ibudilast, ifenprodil, lomerizine, naphlole, nikamate, nosergoline, nimodipine, papaverine, pentifylline, nofedoline, vincamin, vinpocetine, vichizyl, pentoxifylline, prostacyclin derivatives (such as prostaglandin El and prostaglandin 12), an endothelin receptor blocking drug (such as bosentan), diltiazem, nicorandil, and nitroglycerin.
  • vasodilators e.g., bencyclane, cinnarizine, citicoline, cyclandelate, cyclonicate, e
  • Examples of the cerebral protecting drug include radical scavengers (such as edaravone, vitamin E, and vitamin C), glutamate antagonists, AMPA antagonists, kainate antagonists, NMDA antagonists, GABA agonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin agonists, Na 4 VCa 2+ channel inhibitory drugs, and K + channel opening drugs.
  • Examples of the brain metabolic stimulants include amantadine, tiapride, and gamma-aminobutyric acid.
  • anticoagulant examples include heparins (such as heparin sodium, heparin potassium, dalteparin sodium, dalteparin calcium, heparin calcium, parnaparin sodium, reviparin sodium, and danaparoid sodium), warfarin, enoxaparin, argatroban, batroxobin, and sodium citrate.
  • heparins such as heparin sodium, heparin potassium, dalteparin sodium, dalteparin calcium, heparin calcium, parnaparin sodium, reviparin sodium, and danaparoid sodium
  • warfarin warfarin
  • enoxaparin argatroban
  • batroxobin and sodium citrate.
  • Examples of the antiplatelet737 l .DOC ⁇ drug include ticlopidine hydrochloride, dipyridamole, cilostazol, ethyl icosapentate, sarpogrelate hydrochloride, dilazep hydrochloride, trapidil, a nonsteroidal antiinflammatory agent (such as aspirin), beraprostsodium, iloprost, and indobufene.
  • thrombolytic drug examples include urokinase, tissue-type plasminogen activators (such as alteplase, tisokinase, nateplase, pamiteplase, monteplase, and rateplase), and nasaruplase.
  • tissue-type plasminogen activators such as alteplase, tisokinase, nateplase, pamiteplase, monteplase, and rateplase
  • antihypertensive drug examples include angiotensin converting enzyme inhibitors (such as captopril, alacepril, lisinopril, imidapril, quinapril, temocapril, delapril, benazepril, cilazapril, trandolapril, enalapril, ceronapril, fosinopril, imadapril, mobertpril, perindopril, ramipril, spirapril, and randolapril), angiotensin II antagonists (such as losartan, candesartan, valsartan, eprosartan, and irbesartan), calcium channel blocking drugs (such as aranidipine, efonidipine, nicardipine, bamidipine, benidipine, manidipine, cilnidipine, nisoldipine, nitrendipin
  • antianginal drug examples include nitrate drugs (such as amyl nitrite, nitroglycerin, and isosorbide), ⁇ -adrenaline receptor blocking drugs (such as propranolol, pindolol, 737 l .DOC indenolol, carteolol, bunitrolol, atenolol, acebutolol, metoprolol, timolol, nipradilol, penbutolol, nadolol, tilisolol, carvedilol, bisoprolol, betaxolol, celiprolol, bopindolol, bevantolol, labetalol, alprenolol, amosulalol, arotinolol, befiinolol, bucumolol, bufetolol, buferalol, buprandol
  • diuretic examples include thiazide diuretics (such as hydrochlorothiazide, methyclothiazide, trichlormethiazide, benzylhydrochlorothiazide, and penflutizide), loop diuretics (such as furosemide, etacrynic acid, bumetanide, piretanide, azosemide, and torasemide), K + sparing diuretics (spironolactone, triamterene, andpotassiumcanrenoate), osmotic diuretics (such as isosorbide, D-mannitol, and glycerin), nonthiazide diuretics (such as meticrane, tripamide, chlorthalidone, and mefruside), and acetazolamide.
  • thiazide diuretics such as hydrochlorothiazide, methyclothiazide, trichlormethiazide, benzylhydr
  • cardiotonic examples include digitalis formulations (such as digitoxin, digoxin, methyldigoxin, deslanoside, vesnarinone, lanatoside C, and proscillaridin), xanthine formulations (such as aminophylline, choline theophylline, diprophylline, and proxyphylline), catecholamine formulations (such as dopamine, dobutamine, and docarpamine), PDE III inhibitors (such as amrinone, olprinone, and milrinone), denopamine, ubidecarenone, pimobendan, levosimendan, aminoethylsulfonic acid, vesnarinone, carperitide, and colforsin daropate.
  • digitalis formulations such as digitoxin, digoxin, methyldigoxin, deslanoside, vesnarinone, lanatoside C, and proscillaridin
  • xanthine formulations such
  • antiarrhythmic drug examples include ajmaline, pirmenol, procainamide, cibenzoline, disopyramide, quinidine, aprindine, mexiletine, lidocaine, phenyloin, pilsicainide, propafenone, flecainide, atenolol, acebutolol, sotalol, propranolol, metoprolol, pindolol, amiodarone, nifekalant, diltiazem, bepridil, and verapamil.
  • antihyperlipidemic drug examples include737 I DOC ->5 atorvastatin, simvastatin, pravastatin sodium, fluvastatin sodium, clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate, colestimide, and cholestyramine.
  • immunosuppressant examples include azathioprine, mizoribine, cyclosporine, tacrolimus, gusperimus, and methotrexate.
  • Sirtuin variant therapeutic agents may be administered to subjects who have recently received or are likely to receive a dose of radiation or toxin.
  • the dose of radiation or toxin is received as part of a work-related or medical procedure, e.g., working in a nuclear power plant, flying an airplane, an X- ray, CAT scan, or the administration of a radioactive dye for medical imaging; in such an embodiment, the sirtuin variant therapeutic agent is administered as a prophylactic measure.
  • the radiation or toxin exposure is received unintentionally, e.g., as a result of an industrial accident, habitation in a location of natural radiation, terrorist act, or act of war involving radioactive or toxic material.
  • the sirtuin variant therapeutic agent is preferably administered as soon as possible after the exposure to inhibit apoptosis and the subsequent development of acute radiation syndrome.
  • Sirtuin variant therapeutic agents may also be used for treating and/or preventing cancer. Calorie restriction has been linked to a reduction in the incidence of age-related disorders including cancer (see e.g., Bordone and Guarente, Nat. Rev. MoI. Cell Biol. (2005 epub); Guarente and Picard, Cell 120: 473-82 (2005); Berrigan, et al, Carcinogenesis 23: 817-822 (2002); and Heilbronn and Ravussin, Am. J. Clin. Nutr. 78: 361-369 (2003)).
  • the Sir2 protein from yeast has been shown to be required for lifespan extension by glucose restriction (see e.g., Lin et al., Science 289: 2126-2128 (2000); Anderson et al., Nature 423: 181-185 (2003)), a yeast model for calorie restriction. Accordingly, an increase in the level and/or activity of a sirtuin protein may be useful for treating and/or preventing the incidence of age-related disorders, such as, for example, cancer.
  • Exemplary cancers that may be treated by increasing sirtuin activity are those of the brain and kidney; hormone-dependent cancers including breast, prostate, testicular, and737 l .DOC ° ovarian cancers; lymphomas, and leukemias.
  • a sirtuin variant therapeutic agent may be administered directly into the tumor.
  • Cancer of blood cells e.g., leukemia, can be treated by administering a sirtuin variant therapeutic agent into the blood stream or into the bone marrow.
  • Benign cell growth can also be treated, e.g., warts.
  • Other diseases that can be treated include autoimmune diseases, e.g., systemic lupus erythematosus, scleroderma, and arthritis, in which autoimmune cells should be removed.
  • Viral infections such as herpes, HIV, adenovirus, and HTLV-I associated malignant and benign disorders can also be treated by administration of a sirtuin variant therapeutic agent.
  • cells can be obtained from a subject, treated ex vivo to remove certain undesirable cells, e.g., cancer cells, and administered back to the same or a different subject.
  • Neuronal Diseases/Disorders e.g., warts.
  • sirtuin variant therapeutic agents can be used to treat patients suffering from neurodegenerative diseases, and traumatic or mechanical injury to the central nervous system (CNS), spinal cord or peripheral nervous system (PNS).
  • Neurodegenerative disease typically involves reductions in the mass and volume of the human brain, which may be due to the atrophy and/or death of brain cells, which are far more profound than those in a healthy person that are attributable to aging.
  • Neurodegenerative diseases can evolve gradually, after a long period of normal brain function, due to progressive degeneration (e.g., nerve cell dysfunction and death) of specific brain regions.
  • neurodegenerative diseases can have a quick onset, such as those associated with trauma or toxins. The actual onset of brain degeneration may precede clinical expression by many years.
  • neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntingdon's disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewy body disease, chorea- acanthocytosis, primary lateral sclerosis, ocular diseases (ocular neuritis), chemotherapy-induced neuropathies (e.g., from vincristine, paclitaxel. bortezomib), diabetes-induced neuropathies and Friedreich's ataxia.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • HD Huntingdon's disease
  • ALS amyotrophic lateral sclerosis
  • ocular diseases ocular neuritis
  • chemotherapy-induced neuropathies e.g., from vincristine, paclitaxel. bortezomib
  • diabetes-induced neuropathies e.g., and others described below, may be treated by increasing sirtuin activity
  • DOC AD is a chronic, incurable, and unstoppable CNS disorder that occurs gradually, resulting in memory loss, unusual behavior, personality changes, and a decline in thinking abilities. These losses are related to the death of specific types of brain cells and the breakdown of connections and their supporting network (e.g. glial cells) between them. AD has been described as childhood development in reverse. In most people with AD, symptoms appear after the age 60. The earliest symptoms include loss of recent memory, faulty judgment, and changes in personality. Later in the disease, those with AD may forget how to do simple tasks like washing their hands. Eventually people with AD lose all reasoning abilities and become dependent on other people for their everyday care. Finally, the disease becomes so debilitating that patients are bedridden and typically develop coexisting illnesses.
  • PD is a chronic, incurable, and unstoppable CNS disorder that occurs gradually and results in uncontrolled body movements, rigidity, tremor, and dyskinesia.
  • These motor system problems are related to the death of brain cells in an area of the brain that produces dopamine, a chemical that helps control muscle activity.
  • symptoms appear after age 50.
  • the initial symptoms of PD are a pronounced tremor affecting the extremities, notably in the hands or lips.
  • Subsequent characteristic symptoms of PD are stiffness or slowness of movement, a shuffling walk, stooped posture, and impaired balance.
  • secondary symptoms such as memory loss, dementia, depression, emotional changes, swallowing difficulties, abnormal speech, sexual dysfunction, and bladder and bowel problems.
  • ALS motor neuron disease
  • ALS motor neuron disease
  • the motor neurons deteriorate and eventually die, and though a person's brain normally remains fully functioning and alert, the command to move never reaches the muscles.
  • Most people who get ALS are between 40 and 70 years old.
  • the first motor neurons that weaken are those controlling the arms or legs. Those with ALS may have trouble walking, they may drop things, fall, slur their speech, and laugh or cry uncontrollably.
  • Eventually the737 I DOC muscles in the limbs begin to atrophy from disuse. This muscle weakness will become debilitating and a person will need a wheel chair or become unable to function out of bed.
  • HD is another neurodegenerative disease resulting from genetically programmed degeneration of neurons in certain areas of the brain. This degeneration causes uncontrolled movements, loss of intellectual faculties, and emotional disturbance.
  • HD is a familial disease, passed from parent to child through a dominant mutation in the wild-type gene.
  • Tay-Sachs disease and Sandhoff disease are glycolipid storage diseases caused by the lack of lysosomal ⁇ -hexosaminidase (Gravel et al., in The Metabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill, New York, pp. 2839- 2879, 1995).
  • GM2 ganglioside and related glycolipidssubstrates for ⁇ -hexosaminidase accumulate in the nervous system and trigger acute neurodegeneration.
  • the onset of symptoms begins in early infancy.
  • a precipitous neurodegenerative course then ensues, with affected infants exhibiting motor dysfunction, seizure, visual loss, and deafness. Death usually occurs by 2-5 years of age. Neuronal loss through an apoptotic mechanism has been demonstrated (Huang et al., Hum. MoI. Genet. 6: 1879-1885, 1997).
  • HIV-I also induces neurological disease.
  • Shi et al. (J. Clin. Invest. 98: 1979- 1990, 1996) examined apoptosis induced by HIV-I infection of the CNS in an in vitro model and in brain tissue from AIDS patients, and found that HIV-I infection 737 l .DOC 59 of primary brain cultures induced apoptosis in neurons and astrocytes in vitro. Apoptosis of neurons and astrocytes was also detected in brain tissue from 10/11 AIDS patients, including 5/5 patients with HIV-I dementia and 4/5 nondemented patients.
  • DSPN distal symmetrical polyneuropathy
  • AIDP/CIDP acute or chronic inflammatory demyelinating polyneuropathy
  • AIDP/CIDP there is damage to the fatty membrane covering the nerve impulses.
  • This kind of neuropathy involves inflammation and resembles the muscle deterioration often identified with long-term use of AZT. It can be the first manifestation of HIV infection, where the patient may not complain of pain, but fails to respond to standard reflex tests.
  • This kind of neuropathy may be associated with seroconversion, in which case it can sometimes resolve spontaneously. It can serve as a sign of HIV infection and indicate that it might be time to consider antiviral therapy.
  • AIDP/CIDP may be auto-immune in origin.
  • Drug-induced, or toxic, neuropathies can be very painful. Antiviral drugs commonly cause peripheral neuropathy, as do other drugs e.g. vincristine, dilantin (an anti-seizure medication), high-dose vitamins, isoniazid, and folic acid antagonists. Peripheral neuropathy is often used in clinical trials for antivirals as a dose-limiting side effect, which means that more drugs should not be administered. Additionally, the use of such drugs can exacerbate otherwise minor neuropathies. Usually, these drug-induced neuropathies are reversible with the discontinuation of the drug. 737 l .DOC CMV causes several neurological syndromes in AIDS, including encephalitis, myelitis, and polyradiculopathy.
  • Neuronal loss is also a salient feature of prion diseases, such as Creutzfeldt-
  • sirtuin activity e.g., using a sirtuin variant therapeutic agent may be useful for treating or preventing neuronal loss due to these prior diseases.
  • sirtuin variant therapeutic agents may be used to treat or prevent any disease or disorder involving axonopathy.
  • Distal axonopathy is a type of peripheral neuropathy that results from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of nerves to metabolic or toxic disturbances, and as such may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.
  • PNS peripheral nervous system
  • axons The most distal portions of axons are usually the first to degenerate, and axonal atrophy advances slowly towards the nerve's cell body. If the noxious stimulus is removed, regeneration is possible, though prognosis decreases depending on the duration and severity of the stimulus.
  • Those with distal axonopathies usually present with symmetrical glove-stocking sensori-motor disturbances. Deep tendon reflexes and autonomic nervous system (ANS) functions are also lost or diminished in affected areas.
  • ANS autonomic nervous system
  • Diabetic neuropathies are neuropathic disorders that are associated with diabetes mellitus. These conditions usually result from diabetic microvascular injury involving small blood vessels that supply nerves (vasa nervorum). Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy. Clinical manifestations of diabetic neuropathy include, for example, sensorimotor polyneuropathy such as numbness, sensory loss, dysesthesia and nighttime pain; autonomic neuropathy such as delayed gastric emptying or gastroparesis; and cranial 737 I .
  • DOC neuropathy such as oculomotor (3rd) neuropathies or Mononeuropathies of the thoracic or lumbar spinal nerves.
  • Peripheral neuropathy is the medical term for damage to nerves of the peripheral nervous system, which may be caused either by diseases of the nerve or from the side-effects of systemic illness. Peripheral neuropathies vary in their presentation and origin, and may affect the nerve or the neuromuscular junction. Major causes of peripheral neuropathy include seizures, nutritional deficiencies, and HIV, though diabetes is the most likely cause. Mechanical pressure from staying in one position for too long, a tumor, intraneural hemorrhage, exposing the body to extreme conditions such as radiation, cold temperatures, or toxic substances can also cause peripheral neuropathy.
  • a sirtuin variant therapeutic agent may be used to treat or prevent multiple sclerosis (MS), including relapsing MS and monosymptomatic MS, and other demyelinating conditions, such as, for example, chromic inflammatory demyelinating polyneuropathy (CIDP), or symptoms associated therewith.
  • MS multiple sclerosis
  • CIDP chromic inflammatory demyelinating polyneuropathy
  • MS is a chronic, often disabling disease of the central nervous system.
  • Various and converging lines of evidence point to the possibility that the disease is caused by a disturbance in the immune function, although the cause of this disturbance has not been established.
  • This disturbance permits cells of the immune system to "attack" myelin, the fat containing insulating sheath that surrounds the nerve axons located in the central nervous system ("CNS").
  • CNS central nervous system
  • myelin When myelin is damaged, electrical pulses cannot travel quickly or normally along nerve fiber pathways in the brain and spinal cord. This results in disruption of normal electrical conductivity within the axons, fatigue and disturbances of vision, strength, coordination, balance, sensation, and bladder and bowel function.
  • MS is now a common and well-known neurological disorder that is characterized by episodic patches of inflammation and demyelination which can occur anywhere in the CNS.
  • Demyelination produces a situation analogous to that resulting from cracks or tears in an insulator surrounding an electrical cord. That is, when the insulating sheath is disrupted, the circuit is "short737 I . DOC circuited" and the electrical apparatus associated therewith will function intermittently or nor at all.
  • Such loss of myelin surrounding nerve fibers results in short circuits in nerves traversing the brain and the spinal cord that thereby result in symptoms of MS.
  • demyelination occurs in patches, as opposed to along the entire CNS.
  • demyelination may be intermittent. Therefore, such plaques are disseminated in both time and space.
  • MS exists in both sexes and can occur at any age. However, its most common presentation is in the relatively young adult, often with a single focal lesion such as a damage of the optic nerve, an area of anesthesia (loss of sensation), or paraesthesia (localize loss of feeling), or muscular weakness.
  • a single focal lesion such as a damage of the optic nerve, an area of anesthesia (loss of sensation), or paraesthesia (localize loss of feeling), or muscular weakness.
  • vertigo, double vision, localized pain, incontinence, and pain in the arms and legs may occur upon flexing of the neck, as well as a large variety of less common symptoms.
  • MS MS-induced neurotoxicity
  • a fever a fever
  • sirtuin variant therapeutic agents may be used to treat trauma to the nerves, including, trauma due to disease, injury (including surgical intervention), or environmental trauma (e.g., neurotoxins, alcoholism, etc.).
  • Sirtuin variant therapeutic agents may also be useful to prevent, treat, and alleviate symptoms of various PNS disorders, such as the ones described below.
  • the PNS is composed of the nerves that lead to or branch off from the spinal cord and CNS.
  • the peripheral nerves handle a diverse array of functions in the body, including sensory, motor, and autonomic functions.
  • nerves either afferent or efferent
  • Nerve damage can arise from a number of causes, such as -disease, physical injury, poisoning, or malnutrition.
  • the nerve cell axon, its protective myelin sheath, or both may be injured or destroyed.
  • peripheral neuropathy encompasses a wide range of disorders in which the nerves outside of the brain and spinal cord — peripheral nerves — have been damaged.
  • Peripheral neuropathy may also be referred to as peripheral neuritis, or if many nerves are involved, the terms polyneuropathy or polyneuritis may be used.
  • Peripheral neuropathy is a widespread disorder, and there are many underlying causes. Some of these causes are common, such as diabetes, and others are extremely rare, such as acrylamide poisoning and certain inherited disorders. The most common worldwide cause of peripheral neuropathy is leprosy. Leprosy is caused by the bacterium Mycobacterium leprae, which attacks the peripheral nerves of affected people.
  • Leprosy is extremely rare in the United States, where diabetes is the most commonly known cause of peripheral neuropathy. It has been estimated that more than 17 million people in the United States and Europe have diabetes-related polyneuropathy. Many neuropathies are idiopathic; no known cause can be found. The most common of the inherited peripheral neuropathies in the United States is Charcot-Marie-Tooth disease, which affects approximately 125,000 persons.
  • peripheral neuropathies Another of the better known peripheral neuropathies is Guillain-Barre syndrome, which arises from complications associated with viral illnesses, such as cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus (HIV), or bacterial infection, including Campylobacter jejuni and Lyme disease. The worldwide incidence rate is approximately 1.7 cases per 100,000 people annually.
  • Other well-known causes of peripheral neuropathies include chronic alcoholism, infection of the varicella-zoster virus, botulism, and poliomyelitis.
  • Peripheral neuropathy may develop as a primary symptom, or it may be due to another disease. For example, peripheral neuropathy is only one symptom of diseases such as amyloid neuropathy, certain cancers, or inherited neurologic disorders.
  • PNS diseases treatable with sirtuin variant therapeutic agents include: Brachial Plexus Neuropathies (diseases of the cervical and first thoracic roots, nerve trunks, cords, and peripheral nerve components of the brachial plexus. Clinical manifestations include regional pain, paresthesia; muscle weakness, and decreased sensation in the upper extremity. These disorders may be associated with trauma, including birth injuries; thoracic outlet syndrome; neoplasms, neuritis, radiotherapy; and other conditions.
  • Diabetic Neuropathies peripheral, autonomic, and cranial nerve disorders that are associated with diabetes mellitus. These conditions usually result from diabetic microvascular injury involving small blood vessels that supply nerves (vasa nervorum).
  • Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy (see Adams et al., Principles of Neurology, 6th ed, pi 325); mononeuropathies (disease or trauma involving a single peripheral nerve in isolation, or out of proportion to evidence of diffuse peripheral nerve dysfunction).
  • Mononeuritis multiplex refers to a condition characterized by multiple isolated nerve injuries.
  • Mononeuropathies may result from a wide variety of causes, including ischemia; traumatic injury; compression; connective tissue diseases; cumulative trauma disorders; and other conditions; Neuralgia (intense or aching pain that occurs along the course or distribution of a peripheral or cranial nerve); Peripheral Nervous System Neoplasms (neoplasms which arise from peripheral nerve tissue). This includes neurofibromas; Schwannomas; granular cell tumors; and malignant peripheral nerve sheath tumors (see DeVita Jr et al., Cancer: Principles and Practice of Oncology, 5th ed, ppl 750-1); and Nerve Compression Syndromes (mechanical compression of nerves or nerve roots from internal or external causes.
  • the nerve and nerve sheath injuries may be caused by ischemia; inflammation; or a direct mechanical effect; Neuritis (a general term indicating inflammation of a peripheral or cranial nerve).
  • Clinical manifestation may include pain; paresthesias; paresis; or hyperesthesia; Polyneuropathies (diseases of multiple peripheral nerves).
  • the various forms are categorized by the type of737 1 DOC nerve affected (e.g., sensory, motor, or autonomic), by the distribution of nerve injury (e.g., distal vs. proximal), by nerve component primarily affected (e.g., demyelinating vs. axonal), by etiology, or by pattern of inheritance.
  • sirtuin variant therapeutic agents may be used to treat or prevent chemotherapeutic induced neuropathy.
  • the sirtuin variant therapeutic agent may be administered prior to administration of the chemotherapeutic agent, concurrently with administration of the chemotherapeutic drug, and/or after initiation of administration of the chemotherapeutic drug. If the sirtuin variant therapeutic agent is administered after the initiation of administration of the chemotherapeutic drug, it is desirable that the sirtuin variant therapeutic agent be administered prior to, or at the first signs, of chemotherapeutic induced neuropathy.
  • Chemotherapy drugs can damage any part of the nervous system. Encephalopathy and myelopathy are notably very rare. Damage to peripheral nerves is much more common and can be a side effect of treatment experienced by people with cancers, such as lymphoma. Most of the neuropathy affects sensory rather than motor nerves. Thus, the common symptoms are tingling, numbness or a loss of balance. The longest nerves in the body seem to be most sensitive hence the fact that most patients will report numbness or pins and needles in their hands and feet.
  • the chemotherapy drugs which are most commonly associated with neuropathy are the Vinca alkaloids (anti-cancer drugs originally derived from a member of the periwinkle - the Vinca plant genus) and a platinum- containing drug called Cisplatin.
  • the Vinca alkaloids include the drugs vinblastine, vincristine and vindesine.
  • Many combination chemotherapy treatments for lymphoma for example CHOP and CVP contain vincristine, which is the drug known to cause this problem most frequently. Indeed, it is the risk of neuropathy that limits the dose of vincristine that can be administered.
  • a sirtuin variant therapeutic agent may be used to treat or prevent a polyglutamine disease.
  • Huntington's Disease (HD) and Spinocerebellar ataxia type 1 (SCAl) are just two examples of a class of genetic diseases caused by dynamic mutations involving the expansion of triplet sequence repeats. In reference to this common mechanism, these disorders are called trinucleotide repeat diseases. At least 14 such diseases are known to affect human beings. Nine of them, including SCAl and Huntington's disease, have CAG as the repeated sequence (see Table 2 below). Since CAG codes for an amino acid called glutamine, these nine trinucleotide repeat disorders are collectively known as polyglutamine diseases.
  • mice have generated genetically engineered mice expressing proteins with long polyglutamine tracts. Regardless of whether the mice express full-length proteins or only those portions of the proteins containing the polyglutamine tracts, they develop symptoms of polyglutamine diseases. This737 l .DOC suggests that a long polyglutamine tract by itself is damaging to cells and does not have to be part of a functional protein to cause its damage.
  • LANP is needed for nerve cells to communicate with one another and thus for their survival.
  • the mutant ataxin-1 protein accumulates inside nerve cells, it "traps" the LANP protein, interfering with its normal function. After a while, the absence of LANP function appears to cause nerve cells to malfunction.
  • HDAC I/II Class 1/11 Histone Deacetylase
  • the invention provides a method for treating or preventing neuropathy related to ischemic injuries or diseases, such as, for example, coronary heart disease (including congestive heart failure and myocardial infarctions), stroke, emphysema, hemorrhagic shock, peripheral vascular disease (upper and lower extremities) and transplant related injuries.
  • ischemic injuries or diseases such as, for example, coronary heart disease (including congestive heart failure and myocardial infarctions), stroke, emphysema, hemorrhagic shock, peripheral vascular disease (upper and lower extremities) and transplant related injuries.
  • the invention provides a method to treat a central nervous system cell to prevent damage in response to a decrease in blood flow to the cell.
  • the severity of damage that may be prevented will depend in large part on the degree of reduction in blood flow to the cell and the duration of the reduction.
  • the normal amount of perfusion to brain gray matter in humans is about 60 to 70 mL/100 g of brain tissue/min.
  • Death of central nervous system cells typically occurs when the flow of blood falls below approximately 8-10 mL/100 g of brain tissue/min, while at slightly higher levels (i.e. 20-35 mL/100 g of brain tissue/min) the tissue remains alive but not able to function.
  • apoptotic or necrotic cell death may be prevented.
  • ischemic-mediated damage such as cytoxic edema or central nervous system tissue anoxemia, may be prevented.
  • the central nervous system cell may be a spinal cell or a brain cell.
  • ischemic condition is a stroke that results in any type of ischemic central nervous system damage, such as apoptotic or necrotic cell death, cytoxic737 1 DOC " ⁇ edema or central nervous system tissue anoxia.
  • the stroke may impact any area of the brain or be caused by any etiology commonly known to result in the occurrence of a stroke.
  • the stroke is a brain stem stroke.
  • brain stem strokes strike the brain stem, which control involuntary life-support functions such as breathing, blood pressure, and heartbeat.
  • the stroke is a cerebellar stroke.
  • cerebellar strokes impact the cerebellum area of the brain, which controls balance and coordination.
  • the stroke is an embolic stroke.
  • embolic strokes may impact any region of the brain and typically result from the blockage of an artery by a vaso-occlusion.
  • the stroke may be a hemorrhagic stroke.
  • hemorrhagic stroke may impact any region of the brain, and typically result from a ruptured blood vessel characterized by a hemorrhage (bleeding) within or surrounding the brain.
  • the stroke is a thrombotic stroke.
  • thrombotic strokes result from the blockage of a blood vessel by accumulated deposits.
  • the ischemic condition may result from a disorder that occurs in a part of the subject's body outside of the central nervous system, but yet still causes a reduction in blood flow to the central nervous system.
  • disorders may include, but are not limited to a peripheral vascular disorder, a venous thrombosis, a pulmonary embolus, arrhythmia (e.g. atrial fibrillation), a myocardial infarction, a transient ischemic attack, unstable angina, or sickle cell anemia.
  • the central nervous system ischemic condition may occur as result of the subject undergoing a surgical procedure.
  • the subject may be undergoing heart surgery, lung surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery, or organ transplantation surgery.
  • the organ transplantation surgery may include heart, lung, pancreas, kidney or liver transplantation surgery.
  • the central nervous system ischemic condition may occur as a result of a trauma or injury to a part of the subject's body outside the central nervous system.
  • the trauma or injury may cause a degree of bleeding that significantly reduces the total volume of blood in the subject's body.
  • DOC 70 nervous system is concomitantly reduced.
  • the trauma or injury may also result in the formation of a vaso-occlusion that restricts blood flow to the central nervous system.
  • the sirtuin variant therapeutic agents may be employed to treat the central nervous system ischemic condition irrespective of the cause of the condition.
  • the ischemic condition results from a vaso-occlusion.
  • the vaso-occlusion may be any type of occlusion, but is typically a cerebral thrombosis or an embolism.
  • the ischemic condition may result from a hemorrhage.
  • the hemorrhage may be any type of hemorrhage, but is generally a cerebral hemorrhage or a subararachnoid hemorrhage.
  • the ischemic condition may result from the narrowing of a vessel. Generally speaking, the vessel may narrow as a result of a vasoconstriction such as occurs during vasospasms, or due to arteriosclerosis.
  • the ischemic condition results from an injury to the brain or spinal cord.
  • a sirtuin variant therapeutic agent may be administered to reduce infarct size of the ischemic core following a central nervous system ischemic condition. Moreover, a sirtuin variant therapeutic agent may also be beneficially administered to reduce the size of the ischemic penumbra or transitional zone following a central nervous system ischemic condition.
  • a combination drug regimen may include drugs or compounds for the treatment or prevention of neurodegenerative disorders or secondary conditions associated with these conditions.
  • a combination drug regimen may include one or more sirtuin variant therapeutic agents, e.g., one or more sirtuin variant polypeptides, one or more nucleic acids encoding a sirtuin variant polypeptide, or combinations thereof, and one or more anti- neurodegeneration agents.
  • one or more sirtuin variant therapeutic agents can be combined with an effective amount of one or more of: L-DOPA; a dopamine agonist; an adenosine A2A receptor antagonist; a COMT inhibitor; a MAO inhibitor; an N-NOS inhibitor; a sodium channel antagonist; a selective N- methyl D-aspartate (NMDA) receptor antagonist; an AMPA/kainate receptor antagonist; a calcium channel antagonist; a GABA-A receptor agonist; an acetyl-737 l .DOC 71 choline esterase inhibitor; a matrix metalloprotease inhibitor; a PARP inhibitor; an inhibitor of p38 MAP kinase or c-jun-N-terminal kinases; TPA; NDA antagonists; beta-interferons; growth factors; glutamate inhibitors; and/or as part of a cell therapy.
  • L-DOPA L-DOPA
  • a dopamine agonist an adenosine A2A receptor antagonist
  • N-NOS inhibitors include 4-(6-amino-pyridin-2-yl)-3- methoxyphenol 6-[4-(2-dimethylamino-ethoxy)-2-methoxy-phenyl]-pyridin-2-yl- amine, 6-[4-(2-dimethylamino-ethoxy)-2,3-dimet-hyl-phenyl]-pyridin-2-yl-amine, 6- [4-(2-pyrrolidinyl-ethoxy)-2,3-dimethyl-p-henyl]-pyridin-2-yl-amine, 6-[4-(4-(n- methyl)piperidinyloxy)-2,3-dimethyl-p-henyl]-pyridin-2-yl-amine, 6-[4-(2- dimethylamino-ethoxy)-3-methoxy-phenyl]-pyridin-2-yl-amine, 6-[4-(2- pyrrolidinyl-ethoxy
  • DOC Exemplary NMDA receptor antagonist include (+)-(lS, 2S)-l-(4-hydroxy- phenyl)-2-(4-hydroxy-4-phenylpiperidino)-l-pro-panol, (IS, 2S)-l-(4-hydroxy-3- methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-l-propanol, (3R, 4S)-3-(4-(4- fluorophenyl)-4-hydroxypiperidin-l-yl-)-chroman-4,7-diol, (IR*, 2R*)-l-(4- hydroxy-3 -methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin- 1 -yl)-propan- 1-ol-mesylate or a pharmaceutically acceptable acid addition salt thereof.
  • dopamine agonist examples include ropininole; L-dopa decarboxylase inhibitors such as carbidopa or benserazide, bromocriptine, dihydroergocryptine, etisulergine, AF- 14, alaptide, pergolide, piribedil; dopamine Dl receptor agonists such as A-68939, A-77636, dihydrexine, and SKF-38393; dopamine D2 receptor agonists such as carbergoline, lisuride, N-0434, naxagolide, PD-118440, pramipexole, quinpirole and ropinirole; dopamine/ ⁇ -adrenegeric receptor agonists such as DPDMS and dopexamine; dopamine/5-HT uptake inhibitor/5-HT-lA agonists such as roxindole; dopamine/opiate receptor agonists such as NIH- 10494; ⁇ 2-
  • Exemplary acetyl cholinesterase inhibitors include donepizil, l-(2-methyl- lH-benzimida-zol-5-yl)-3-[l-(phenylmethyl)-4-piperidinyl]-l-propanone; l-(2- phenyl-lH-benzimidazol-5-yl)-3-[l-(phenylmethyl)-4-piperidinyl]-l-pr-opanone; 1- (l-ethyl-2-methyl-lH-benzimidazol-5-yl)-3-[l-(phenylmethyl)-4-p-iperidinyl]-l- propanone; 1 -(2-methyl-6-benzothiazolyl)-3-[ 1 -(phenylmethyl)-4-piperidinyl]- 1 - propanone; l-(2-methyl-6-benzothiazolyl)-3-[l-[(2-methyl-4-thiazolyl)methyl]-4- piperidin
  • Exemplary calcium channel antagonists include diltiazem, omega-conotoxin GVIA, methoxyverapamil, amlodipine, felodipine, lacidipine, and mibefradil.
  • Exemplary GABA-A receptor modulators include clomethiazole; IDDB; gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol); ganaxolone (3 ⁇ -hydroxy-3 ⁇ - methyl-5 ⁇ -pregnan-20-one); fengabine (2-[(butylimino)-(2-chlorophenyl)methyl]-4- chlorophenol); 2-(4-methoxyphenyl)-2,5,6,7,8,9-hexahydro-pyrazolo[4,3-c]cinnolin- 3-one; 7-cyclobutyl-6-(2-methyl-2H-l ,2,4-triazol-3-ylmethoxy)-3-
  • Exemplary potassium channel openers include diazoxide, flupirtine, pinacidil, levcromakalim, rilmakalim, chromakalim, PCO-400 and SKP-450 (2- [2"(1 ", 3"-dioxolone)-2-methyl]-4-(2'-oxo-r-pyrrolidinyl)-6-nitro-2H-l-benzopyra- n).
  • AMPA/kainate receptor antagonists include 6-cyano-7-nitroquinoxalin- 2,3-di-one (CNQX); 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX); 6,7-dinitroquinoxaline-2,3-dione (DNQX); 1 -(4-aminophenyl)-4-methyl-7,8-m- ethylenedioxy-5H-2,3 -benzodiazepine hydrochloride; and 2,3-dihydroxy-6-nitro-7- sulfamoylbenzo-[f]quinoxaline.
  • CNQX 6-cyano-7-nitroquinoxalin- 2,3-di-one
  • NBQX 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione
  • DNQX 6,7-dinitroquinoxaline-2,3-dione
  • Exemplary sodium channel antagonists include ajmaline, procainamide, flecainide and riluzole.
  • Exemplary matrix-metalloprotease inhibitors include 4-[4-(4- fluorophenoxy)benzenesulfon-ylamino]tetrahydropyran-4-carboxylic acid hydroxyamide; 5-Methyl-5-(4-(4'-fluorophenoxy)-phenoxy)-pyrimidine-2,4,6- trione; 5-n-Butyl-5-(4-(4'-fluorophenoxy)-phenoxy)-pyrimidine-2,4,6-trione and prinomistat.
  • PARP PoIy(ADP ribose) polymerase
  • ADP ribose polymerase PARP
  • PARP is an abundant nuclear enzyme which is activated by DNA strand single breaks to synthesize poly (ADP ribose) from NAD.
  • PARP is involved in base excision repair caused by oxidative stress via the activation and recruitment of DNA repair enzymes in the nucleus.
  • PARP plays a role in cell necrosis and DNA repair.
  • PARP also participates in regulating cytokine expression that mediates inflammation.
  • PARP is over-activated, resulting in cell-based energetic failure characterized by NAD depletion and leading to ATP consumption, cellular necrosis, tissue injury, and organ damage/failure.
  • PARP is thought to contribute to neurodegeneration by depleting nicotinamide adenine dinucleotide (NAD+) which then reduces adenosine triphosphate (ATP; Cosi and Marien, Ann. N.Y. Acad. Sci., 890:227, 1999) contributing to cell death which can be prevented by PARP inhibitors.
  • NAD+ nicotinamide adenine dinucleotide
  • ATP adenosine triphosphate
  • Exemplory PARP inhibitors can be found in Southan and Szabo, Current Medicinal Chemistry, 10:321 , 2003.
  • a combination therapy for treating or preventing MS comprises a therapeutically effective amount of one or more sirtuin variant therapeutic agents and one or more of Avonex ® (interferon beta- Ia), Tysabri ® (natalizumab), or Fumaderm ® (BG-12/Oral Fumarate).
  • a combination therapy for treating or preventing diabetic neuropathy or conditions associated therewith comprises a therapeutically effective amount of one or more sirtuin variant therapeutic agents and one or more of tricyclic antidepressants (TCAs) (including, for example, imipramine, amytriptyline, desipramine and nortriptyline), serotonin reuptake inhibitors (SSRIs) (including, for example, fluoxetine, paroxetine, sertralene, and citalopram) and antiepileptic drugs (AEDs) (including, for example, gabapentin, carbamazepine, and topimirate).
  • TCAs tricyclic antidepressants
  • SSRIs serotonin reuptake inhibitors
  • AEDs antiepileptic drugs
  • the invention provides a method for treating or preventing a polyglutamine disease using a combination comprising at least one sirtuin variant therapeutic agent, e.g., a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide, and at least one HDAC I/II inhibitor.
  • sirtuin variant therapeutic agent e.g., a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide
  • HDAC I/II inhibitors include hydroxamic acids, cyclic peptides, benzamides, short-chain fatty acids, and depudecin.
  • hydroxamic acids and hydroxamic acid derivatives examples include trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), oxamflatin, suberic bishydroxamic acid (SBHA), m-carboxy-cinnamic acid bishydroxamic acid (CBHA), valproic acid and pyroxamide.
  • TSA was isolated as an antifungi antibiotic (Tsuji et al (1976) J. Antibiot (Tokyo) 29:1-6) and found to be a potent inhibitor of mammalian HDAC (Yoshida et al. (1990) J. Biol. Chem. 265:17174-17179).
  • hydroxamic acid-based HDAC inhibitors SAHA, SBHA, and CBHA are synthetic compounds that are able to inhibit HDAC at micromolar concentration or lower in vitro or in vivo. Glick et al. (1999) Cancer Res. 59:4392-4399.
  • SAHA, SBHA, and CBHA are synthetic compounds that are able to inhibit HDAC at micromolar concentration or lower in vitro or in vivo.
  • CBHA hydroxamic acid-based HDAC inhibitors
  • SAHA, SBHA, and CBHA are synthetic compounds that are able to inhibit HDAC at micromolar concentration or lower in vitro or in vivo. Glick et al. (1999) Cancer Res. 59:4392-4399.
  • These hydroxamic acid-based HDAC inhibitors all possess an essential structural feature: a polar hydroxamic terminal linked through a hydrophobic methylene spacer (e.g. 6 carbon at length) to another polar site which is attached to a terminal hydrophobic mo
  • Cyclic peptides used as HDAC inhibitors are mainly cyclic tetrapeptides.
  • cyclic peptides include, but are not limited to, trapoxin A, apicidin and depsipeptide.
  • Trapoxin A is a cyclic tetrapeptide that contains a 2-amino-8-oxo- 9,10-epoxy-decanoyl (AOE) moiety.
  • AOE 2-amino-8-oxo- 9,10-epoxy-decanoyl
  • Depsipeptide is isolated from Chromobacterium violaceum, and has been shown to inhibit HDAC activity at micromolar concentrations.
  • benzamides include but are not limited to MS-27-275. Saito et al. (1990) Proc. Natl. Acad. Sci. USA. 96:4592-4597.
  • short-chain fatty acids include but are not limited to butyrates (e.g., butyric acid, arginine butyrate and phenylbutyrate (PB)).
  • PB phenylbutyrate
  • depudecin which has been shown to inhibit HDAC at micromolar concentrations (Kwon et al. (1998) Proc. Natl. Acad. Sci. USA. 95:3356-3361) also falls within the scope of histone deacetylase inhibitor as described herein. Blood Coagulation Disorders
  • sirtuin variant therapeutic agents can be used to treat or prevent blood coagulation disorders (or hemostatic disorders).
  • blood coagulation disorders or hemostatic disorders
  • the terms “hemostasis”, “blood coagulation,” and “blood clotting” refer to the control of bleeding, including the physiological properties of vasoconstriction and coagulation. Blood coagulation assists in maintaining the integrity of mammalian circulation after injury, inflammation, disease, congenital defect, dysfunction or other disruption. After initiation of clotting, blood coagulation proceeds through the sequential activation of certain plasma proenzymes to their enzyme forms (see, for example, Coleman, R. W. et al. (eds.) Hemostasis and Thrombosis, Second Edition, (1987)).
  • Plasma glycoproteins including Factor XII, Factor XI, Factor IX, Factor X, Factor VII, and prothrombin, are zymogens of serine proteases. Most of these blood clotting enzymes are effective on
  • Activated protein C is a specific enzyme that inactivates procoagulant components. Calcium ions are involved in many of the component reactions. Blood coagulation follows either the intrinsic pathway, where all of the protein components are present in blood, or the extrinsic pathway, where the cell-membrane protein tissue factor plays a critical role. Clot formation occurs when fibrinogen is cleaved by thrombin to form fibrin. Blood clots are composed of activated platelets and fibrin.
  • blood clots does not only limit bleeding in the case of an injury (hemostasis), but may lead to serious organ damage and death in the context of atherosclerotic diseases by occlusion of an important artery or vein.
  • Thrombosis is thus blood clot formation at the wrong time and place. It involves a cascade of complicated and regulated biochemical reactions between circulating blood proteins (coagulation factors), blood cells (in particular platelets), and elements of an injured vessel wall.
  • coagulation factors circulating blood proteins
  • blood cells in particular platelets
  • the present invention provides anticoagulation and antithrombotic treatments aiming at inhibiting the formation of blood clots in order to prevent or treat blood coagulation disorders, such as myocardial infarction, stroke, loss of a limb by peripheral artery disease or pulmonary embolism.
  • blood coagulation disorders such as myocardial infarction, stroke, loss of a limb by peripheral artery disease or pulmonary embolism.
  • modulating or modulation of hemostasis and “regulating or regulation of hemostasis” includes the induction (e.g., stimulation or increase) of hemostasis, as well as the inhibition (e.g., reduction or decrease) of hemostasis.
  • the invention provides a method for reducing or inhibiting hemostasis in a subject by administering a sirtuin variant therapeutic agent.
  • the compositions and methods disclosed herein are useful for the treatment or prevention of thrombotic disorders.
  • thrombotic disorder includes any disorder or condition characterized by excessive or unwanted coagulation or hemostatic activity, or a hypercoagulable state.
  • Thrombotic disorders include diseases or disorders involving platelet adhesion and thrombus formation, and may manifest as an increased propensity to form thromboses, e.g., an increased737 I .
  • thrombotic disorders include, but are not limited to, thromboembolism, deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, miscarriage, thrombophilia associated with anti-thrombin III deficiency, protein C deficiency, protein S deficiency, resistance to activated protein C, dysf ⁇ brinogenemia, fibrinolytic disorders, homocystinuria, pregnancy, inflammatory disorders, myeloproliferative disorders, arteriosclerosis, angina, e.g., unstable angina, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, cancer metastasis, sickle cell disease, glomerular nephritis, and drug induced thrombocytopenia (including, for example, heparin induced thrombocytopenia).
  • sirtuin variant therapeutic agents may be administered to prevent thrombotic events or to prevent re-occlusion during or
  • a combination drug regimen may include drugs or compounds for the treatment or prevention of blood coagulation disorders or secondary conditions associated with these conditions.
  • a combination drug regimen may include one or more sirtuin variant therapeutic agents, e.g., one or more sirtuin variant polypeptides, one or more nucleic acids encoding sirtuin variant polypeptides, or combinations thereof, and one or more anti-coagulation or anti- thrombosis agents.
  • one or more sirtuin variant therapeutic agents can be combined with an effective amount of one or more of: aspirin, heparin, and oral Warfarin that inhibits Vit K-dependent factors, low molecular weight heparins that inhibit factors X and II, thrombin inhibitors, inhibitors of platelet GP HbIIIa receptors, inhibitors of tissue factor (TF), inhibitors of human von Willebrand factor, inhibitors of one or more factors involved in hemostasis (in particular in the coagulation cascade).
  • sirtuin variant therapeutic agents can be combined with thrombolytic agents, such as t-PA, streptokinase, reptilase, TNK-t- PA, and staphylokinase.
  • sirtuin variant therapeutic agents may be used for treating or preventing weight gain or obesity in a subject.
  • sirtuin variant therapeutic agents may be used, for example, to treat or prevent hereditary obesity,737 l .DOC OJ dietary obesity, hormone related obesity, obesity related to the administration of medication, to reduce the weight of a subject, or to reduce or prevent weight gain in a subject.
  • a subject in need of such a treatment may be a subject who is obese, likely to become obese, overweight, or likely to become overweight.
  • Subjects who are likely to become obese or overweight can be identified, for example, based on family history, genetics, diet, activity level, medication intake, or various combinations thereof.
  • sirtuin variant therapeutic agents may be administered to subjects suffering from a variety of other diseases and conditions that may be treated or prevented by promoting weight loss in the subject.
  • diseases include, for example, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, type 2 diabetes, insulin resistance, glucose intolerance, hyperinsulinemia, coronary heart disease, angina pectoris, congestive heart failure, stroke, gallstones, cholescystitis and cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and respiratory problems, some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation), bladder control problems (such as stress incontinence); uric acid nephrolithiasis; psychological disorders (such as depression, eating disorders, distorted body image, and low self esteem). Stunkard AJ, Wadden TA. (
  • sirtuin variant therapeutic agents may be used for inhibiting adipogenesis or fat cell differentiation, whether in vitro or in vivo.
  • high circulating levels of insulin and/or insulin like growth factor (IGF) 1 will be prevented from recruiting preadipocytes to differentiate into adipocytes.
  • IGF insulin like growth factor
  • sirtuin variant therapeutic agents may be used for reducing appetite and/or increasing satiety, thereby causing weight loss or avoidance of weight gain.
  • a subject in need of such a treatment may be a subject who is overweight, obese or a subject likely to become overweight or obese.
  • the method737 l .DOC ° may comprise administering daily or, every other day, or once a week, a dose, e.g., in the form of a pill, to a subject.
  • the dose may be an "appetite reducing dose.”
  • a method for modulating weight may further comprise monitoring the weight of the subject and/or the level of sirtuin activity, for example, in adipose tissue.
  • sirtuin variant therapeutic agents may be administered as a combination therapy for treating or preventing weight gain or obesity.
  • one or more sirtuin variant therapeutic agents may be administered in combination with one or more anti-obesity agents.
  • Exemplary anti- obesity agents include, for example, phenylpropanolamine, ephedrine, pseudoephedrine, phentermine, a cholecystokinin-A agonist, a monoamine reuptake inhibitor (such as sibutramine), a sympathomimetic agent, a serotonergic agent (such as dexfenfluramine or fenfluramine), a dopamine agonist (such as bromocriptine), a melanocyte-stimulating hormone receptor agonist or mimetic, a melanocyte- stimulating hormone analog, a cannabinoid receptor antagonist, a melanin concentrating hormone antagonist, the OB protein (leptin), a leptin analog, a lep
  • anorectic agents include bombesin agonists, dehydroepiandrosterone or analogs thereof, glucocorticoid receptor agonists and antagonists, orexin receptor antagonists, urocortin binding protein antagonists, agonists of the glucagon-like peptide- 1 receptor such as Exendin and ciliary neurotrophic factors such as Axokine.
  • sirtuin variant therapeutic agents may be administered to reduce drug-induced weight gain.
  • sirtuin variant therapeutic agents may be administered as a combination therapy with medications that may stimulate appetite or cause weight gain, in particular, weight gain due to factors other than water retention.
  • Examples of medications that may cause weight gain include for example, diabetes treatments, including, for example, sulfonylureas (such as glipizide and glyburide), thiazolidinediones (such as pioglitazone and rosiglitazone), meglitinides, nateglinide, repaglinide, sulphonylurea medicines, and insulin; anti-depressants, including, for example, tricyclic antidepressants (such as amitriptyline and imipramine), irreversible monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), bupropion, paroxetine, and
  • diabetes treatments including, for example, sulfonylureas (such as glipizide and glyburide), thiazolidinediones (such as pioglitazone and rosiglitazone), meglitinides, nateglinide
  • sirtuin variant therapeutic agents may be administered as part of a smoking cessation program to prevent weight gain or reduce weight already gained.
  • sirtuin variant therapeutic agents may be used for treating or preventing a metabolic disorder, such as insulin-resistance, a pre-diabetic state, type II diabetes, and/or complications thereof.
  • Administration of a sirtuin variant therapeutic agent may increase insulin sensitivity and/or decrease insulin levels in a subject.
  • a subject in need of such a treatment may be a subject who has insulin resistance or other precursor symptom of type II diabetes, who has type II diabetes, or who is likely to develop any of these conditions.
  • the subject may be a subject having insulin resistance, e.g., having high circulating levels of insulin and/or associated conditions, such as hyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucose tolerance, high blood glucose sugar level, other manifestations of syndrome X, hypertension, atherosclerosis and lipodystrophy.
  • insulin resistance e.g., having high circulating levels of insulin and/or associated conditions, such as hyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucose tolerance, high blood glucose sugar level, other manifestations of syndrome X, hypertension, atherosclerosis and lipodystrophy.
  • sirtuin variant therapeutic agents may be administered as a combination therapy for treating or preventing a metabolic disorder.
  • one or more sirtuin variant therapeutic agents may be administered in combination with one or more anti-diabetic agents.
  • Exemplary antidiabetic agents include, for example, an aldose reductase inhibitor, a glycogen phosphorylase inhibitor, a sorbitol dehydrogenase inhibitor, a protein tyrosine phosphatase IB inhibitor, a dipeptidyl protease inhibitor, insulin (including orally bioavailable insulin preparations), an insulin mimetic, metformin, acarbose, a peroxisome proliferator-activated receptor- ⁇ (PPAR- ⁇ ) ligand such as troglitazone, rosaglitazone, pioglitazone or GW- 1929, a sulfonylurea, glipazide, glyburide, or chlorpropamide wherein the amounts of the first and second compounds result in a737 I .
  • PPAR- ⁇ peroxisome proliferator-activated receptor- ⁇
  • DOC therapeutic effect DOC therapeutic effect.
  • Other anti-diabetic agents include a glucosidase inhibitor, a glucagon-like peptide- 1 (GLP-I), insulin, a PPAR ⁇ / ⁇ dual agonist, a meglitimide and an ⁇ P2 inhibitor.
  • an anti-diabetic agent may be a dipeptidyl peptidase IV (DP-IV or DPP-IV) inhibitor, such as, for example LAF237 from Novartis (NVP DPP728; l-[[[2-[(5-cyanopyridin-2-yl)amino] ethyl]amino]acetyl]-2- cyano-(S)- pyrrolidine) or MK-04301 from Merck (see e.g., Hughes et al., Biochemistry 38: 11597-603 (1999)). Inflammatory Diseases
  • DP-IV or DPP-IV dipeptidyl peptidase IV
  • sirtuin variant therapeutic agents can be used to treat or prevent a disease or disorder associated with inflammation.
  • Sirtuin variant therapeutic agents may be administered prior to the onset of, at, or after the initiation of inflammation.
  • the compounds are preferably provided in advance of any inflammatory response or symptom. Administration of the compounds may prevent or attenuate inflammatory responses or symptoms.
  • Exemplary inflammatory conditions include, for example, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, degenerative joint disease, spondouloarthropathies, gouty arthritis, systemic lupus erythematosus, juvenile arthritis, rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes (e.g., insulin dependent diabetes mellitus or juvenile onset diabetes), menstrual cramps, cystic fibrosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucous colitis, ulcerative colitis, gastritis, esophagitis, pancreatitis, peritonitis, Alzheimer's disease, shock, ankylosing spondylitis, gastritis, conjunctivitis, pancreatis (acute or chronic), multiple organ injury syndrome (e.g., secondary to septicemia or trauma), myocardial infarction, atherosclerosis, stroke, reperfusion
  • Exemplary inflammatory conditions of the skin include, for example, eczema, atopic dermatitis, contact dermatitis, urticaria, schleroderma, psoriasis, and dermatosis with acute inflammatory components.
  • sirtuin variant therapeutic agents may be used to treat or prevent allergies and respiratory conditions, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • the compounds may be used to treat chronic hepatitis infection, including hepatitis B and hepatitis C.
  • sirtuin variant therapeutic agents may be used to treat autoimmune diseases and/or inflammation associated with autoimmune diseases such as organ-tissue autoimmune diseases (e.g., Raynaud's syndrome), scleroderma, myasthenia gravis, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosis, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), and Grave's disease.
  • organ-tissue autoimmune diseases e.g., Raynaud's syndrome
  • scleroderma myasthenia gravis
  • transplant rejection transplant rejection
  • endotoxin shock sepsis
  • psoriasis psoriasis
  • eczema dermatitis
  • dermatitis e.
  • one or more sirtuin variant therapeutic agents may be taken alone or in combination with other compounds useful for treating or preventing inflammation.
  • exemplary anti-inflammatory agents include, for example, steroids (e.g., Cortisol, cortisone, fludrocortisone, prednisone, 6 ⁇ - methylprednisone, triamcinolone, betamethasone or dexamethasone), nonsteroidal antiinflammatory drugs (NSAIDS (e.g., aspirin, acetaminophen, tolmetin, ibuprofen, mefenamic acid, piroxicam, nabumetone, rofecoxib, celecoxib, etodolac or nimesulide).
  • steroids e.g., Cortisol, cortisone, fludrocortisone, prednisone, 6 ⁇ - methylprednisone, triamcinolone, betamethasone or dexamethasone
  • NSAIDS
  • the other therapeutic agent is an antibiotic (e.g., vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, cefixime, rifampinmetronidazole, doxycycline or streptomycin).
  • an antibiotic e.g., vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, cefixime, rifampinmetronidazole, doxycycline or streptomycin.
  • the other therapeutic agent is a PDE4 inhibitor (e.g., roflumilast or rolipram).
  • the other therapeutic agent is an antihistamine (e.g., cyclizine, hydroxyzine, promethazine or diphenhydramine).
  • the other therapeutic agent is an antimalarial (e.g., artemisinin, artemether, artsunate, chloroquine phosphate, mefloquine hydrochloride, doxycycline hyclate, proguanil hydrochloride, atovaquone or halofantrine).
  • the other therapeutic agent is drotrecogin alfa.
  • anti-inflammatory agents include, for example, aceclofenac, acemetacin, e-acetamidocaproic acid, acetaminophen, acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine, alclofenac, alclometasone, alfentanil, algestone, allylprodine, alminoprofen, aloxiprin, alphaprodine, aluminum bis(acetylsalicylate), amcinonide, amfenac, aminochlorthenoxazin, 3- amino-4-hydroxybutyric acid, 2-amino-4-picoline, aminopropylon, aminopyrine, amixetrine, ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine, antipyrine, antrafenine, apazone, beclomethasone, bendazac, benory
  • sirtuin variant therapeutic agents may be administered with a selective COX-2 inhibitor for treating or preventing inflammation.
  • selective COX-2 inhibitors include, for example, deracoxib, parecoxib, celecoxib, valdecoxib, rofecoxib, etoricoxib, lumiracoxib, 2- (3,5-difluorophenyl)-3 ⁇ [4-(methylsulfonyl)phenyl]-2-cyclopenten-l-one, (S)-6,8- dichloro-2-(triflu- oromethyl)-2H-l-benzopyran-3-carboxylic acid, 2-(3,4- difluorophenyl)-4-(3— hydroxy-3-methyl-l-butoxy)-5-[4-(methylsulfonyl)phenyl]- 3-(2H)-pyridazinone, 4-[5-(4-fluorophenyl)-3-(trifluoromethyl)-lH-pyr
  • sirtuin variant therapeutic agents may be used for reducing the incidence or severity of flushing and/or hot flashes which are symptoms of a disorder.
  • the subject method includes the use of sirtuin variant therapeutic agents, alone or in combination with other agents, for reducing incidence or severity of flushing and/or hot flashes in cancer patients.
  • the method provides for the use of sirtuin variant therapeutic agents to reduce the incidence or severity of flushing and/or hot flashes in menopausal and postmenopausal woman.
  • sirtuin variant therapeutic agents may be used as a therapy for reducing the incidence or severity of flushing and/or hot flashes which are side- effects of another drug therapy, e.g., drug-induced flushing.
  • a method for treating and/or preventing drug-induced flushing comprises administering to a patient in need thereof a formulation comprising at least one flushing inducing compound and at least one sirtuin variant therapeutic agent.
  • a method for treating drug induced flushing comprises separately administering one or more compounds that induce flushing and one or737 I DOC more sirtuin variant therapeutic agents, e.g., wherein the sirtuin variant therapeutic agents and flushing inducing agent have not been formulated in the same compositions.
  • the sirtuin variant therapeutic agents may be administered (1) at the same as administration of the flushing inducing agent, (2) intermittently with the flushing inducing agent, (3) staggered relative to administration of the flushing inducing agent, (4) prior to administration of the flushing inducing agent, (5) subsequent to administration of the flushing inducing agent, and (6) various combination thereof.
  • Exemplary flushing inducing agents include, for example, niacin, faloxifene, antidepressants, anti-psychotics, chemotherapeutics, calcium channel blockers, and antibiotics.
  • sirtuin variant therapeutic agents may be used to reduce flushing side effects of a vasodilator or an antilipemic agent (including anticholesteremic agents and lipotropic agents).
  • a sirtuin variant therapeutic agent may be used to reduce flushing associated with the administration of niacin.
  • Nicotinic acid 3-pyridinecarboxylic acid or niacin
  • Nicotinic acid is an antilipidemic agent that is marketed under, for example, the trade names Nicolar ® , SloNiacin ® , Nicobid ® and Time Release Niacin ® .
  • Nicotinic acid has been used for many years in the treatment of lipidemic disorders such as hyperlipidemia, hypercholesterolemia and atherosclerosis. This compound has long been known to exhibit the beneficial effects of reducing total cholesterol, low density lipoproteins or "LDL cholesterol," triglycerides and apolipoprotein a (Lp(a)) in the human body, while increasing desirable high density lipoproteins or "HDL cholesterol".
  • Typical doses range from about 1 gram to about 3 grams daily. Nicotinic acid is normally administered two to four times per day after meals, depending upon the dosage form selected. Nicotinic acid is currently commercially available in two dosage forms. One dosage form is an immediate or rapid release tablet which should be administered three or four times per day. Immediate release (“IR”) nicotinic acid formulations generally release nearly all of their nicotinic acid within about 30 to 60 minutes following ingestion. The other dosage form is a sustained release form which is suitable for administration two to four times per day.
  • IR immediate release
  • sustained release (“SR") nicotinic acid formulations are designed to737 1 DOC 92 release significant quantities of drug for absorption into the blood stream over specific timed intervals in order to maintain therapeutic levels of nicotinic acid over an extended period such as 12 or 24 hours after ingestion.
  • nicotinic acid is meant to encompass nicotinic acid or a compound other than nicotinic acid itself which the body metabolizes into nicotinic acid, thus producing essentially the same effect as nicotinic acid.
  • exemplary compounds that produce an effect similar to that of nicotinic acid include, for example, nicotinyl alcohol tartrate, d-glucitol hexanicotinate, aluminum nicotinate, niceritrol and d,l-alpha-tocopheryl nicotinate. Each such compound will be collectively referred to herein as "nicotinic acid.”
  • the invention provides a method for treating and/or preventing hyperlipidemia with reduced flushing side effects.
  • the method comprises the steps of administering to a subject in need thereof a therapeutically effective amount of nicotinic acid and a sirtuin variant therapeutic agent in an amount sufficient to reduce flushing.
  • the nicotinic acid and/or sirtuin variant therapeutic agent may be administered nocturnally.
  • the method involves the use of sirtuin variant therapeutic agents to reduce flushing side effects of raloxifene.
  • Raloxifene acts like estrogen in certain places in the body, but is not a hormone. It helps prevent osteoporosis in women who have reached menopause. Osteoporosis causes bones to gradually grow thin, fragile, and more likely to break. Evista slows down the loss of bone mass that occurs with menopause, lowering the risk of spine fractures due to osteoporosis.
  • a common side effect of raloxifene is hot flashes (sweating and flushing). This can be uncomfortable for women who already have hot flashes due to menopause.
  • the method involves the use of sirtuin variant therapeutic agents to reduce flushing side effects of antidepressants or antipsychotic agent.
  • sirtuin variant therapeutic agents can be used in conjunction (administered separately or together) with a serotonin reuptake inhibitor, a 5HT2 receptor antagonist, an anticonvulsant, a norepinephrine reuptake inhibitor, an ⁇ -adrenoreceptor antagonist, an NK-3 antagonist, an NK-I receptor 737 I .
  • DOC 93 antagonist a PDE4 inhibitor, an Neuropeptide Y5 Receptor Antagonists, a D4 receptor antagonist, a 5HT1A receptor antagonist, a 5HT1D receptor antagonist, a CRF antagonist, a monoamine oxidase inhibitor, or a sedative-hypnotic drug.
  • sirtuin variant therapeutic agents may be used as part of a treatment with a serotonin reuptake inhibitor (SRI) to reduce flushing.
  • SRI serotonin reuptake inhibitor
  • the SRI is a selective serotonin reuptake inhibitor (SSRI), such as a fluoxetinoid (fluoxetine, norfluoxetine) or a nefazodonoid (nefazodone, hydroxynefazodone, oxonefazodone).
  • SSRI selective serotonin reuptake inhibitor
  • Other exemplary SSRI's include duloxetine, venlafaxine, milnacipran, citalopram, fluvoxamine, paroxetine and sertraline.
  • the sirtuin variant therapeutic agent can also be used as part of a treatment with sedative-hypnotic drug, such as selected from the group consisting of a benzodiazepine (such as alprazolam, chlordiazepoxide, clonazepam, chlorazepate, clobazam, diazepam, halazepam, lorazepam, oxazepam and prazepam), Zolpidem, and barbiturates.
  • a benzodiazepine such as alprazolam, chlordiazepoxide, clonazepam, chlorazepate, clobazam, diazepam, halazepam, lorazepam, oxazepam and prazepam
  • Zolpidem such as barbiturates.
  • a sirtuin variant therapeutic agent may be used as part of a treatment with a 5-HT1A receptor partial agonist, such as, for example, buspirone, flesinoxan, gepirone ot ipsapirone.
  • a 5-HT1A receptor partial agonist such as, for example, buspirone, flesinoxan, gepirone ot ipsapirone.
  • Sirtuin variant therapeutic agents can also used as part of a treatment with a norepinephrine reuptake inhibitor, such as, for example, tertiary amine tricyclics and secondary amine tricyclics.
  • exemplary tertiary amine tricyclic include amitriptyline, clomipramine, doxepin, imipramine and trimipramine.
  • sirtuin variant therapeutic agents may be used as part of a treatment with a monoamine oxidase inhibitor, such as, for example, isocarboxazid, phenelzine, tranylcypromine, selegiline or moclobemide.
  • sirtuin variant therapeutic agents may be used to reduce flushing side effects of chemotherapeutic agents, such as cyclophosphamide, tamoxifen.
  • sirtuin variant therapeutic agents may be used to reduce flushing side effects of calcium channel blockers, such as amlodipine.
  • sirtuin variant therapeutic agents may be used to reduce flushing side effects of antibiotics.
  • sirtuin variant therapeutic agents can be used in combination with levofloxacin.
  • Levofloxacin is used to treat737 l .DOC 94 infections of the sinuses, skin, lungs, ears, airways, bones, and joints caused by susceptible bacteria.
  • Levofloxacin also is frequently used to treat urinary infections, including those resistant to other antibiotics, as well as prostatitis.
  • Levofloxacin is effective in treating infectious diarrheas caused by E. coli, Campylobacter jejuni, and shigella bacteria.
  • Levofloxacin also can be used to treat various obstetric infections, including mastitis. Ocular Disorders
  • One aspect of the present invention is a method for inhibiting, reducing or otherwise treating vision impairment by administering to a patient a therapeutic dosage of a sirtuin variant therapeutic agent.
  • the vision impairment is caused by damage to the optic nerve or central nervous system
  • optic nerve damage is caused by high intraocular pressure, such as that created by glaucoma.
  • optic nerve damage is caused by swelling of the nerve, which is often associated with an infection or an immune (e.g., autoimmune) response such as in optic neuritis.
  • Glaucoma describes a group of disorders which are associated with a visual field defect, cupping of the optic disc, and optic nerve damage. These are commonly referred to as glaucomatous optic neuropathies. Most glaucomas are usually, but not always, associated with a rise in intraocular pressure.
  • Exemplary forms of glaucoma include Glaucoma and Penetrating Keratoplasty, Acute Angle Closure, Chronic Angle Closure, Chronic Open Angle, Angle Recession, Aphakic and Pseudophakic, Drug-Induced, Hyphema, Intraocular Tumors, Juvenile, Lens-Particle, Low Tension, Malignant, Neovascular, Phacolytic, Phacomorphic, Pigmentary, Plateau Iris, Primary Congenital, Primary Open Angle, Pseudoexfoliation, Secondary Congenital, Adult Suspect, Unilateral, Uveitic, Ocular Hypertension, Ocular Hypotony, Posner-Schlossman Syndrome and Scleral Expansion Procedure in Ocular Hypertension & Primary Open-angle Glaucoma.
  • Intraocular pressure can also be increased by various surgical procedures, such as phacoemulsification (i.e., cataract surgery) and implanation of structures such as an artificial lens.
  • phacoemulsification i.e., cataract surgery
  • implanation of structures such as an artificial lens.
  • spinal surgeries in particular, or any surgery in 737 l .DOC " ⁇ which the patient is prone for an extended period of time can lead to increased interoccular pressure.
  • Optic neuritis is inflammation of the optic nerve and causes acute loss of vision. It is highly associated with multiple sclerosis (MS) as 15-25% of MS patients initially present with ON, and 50-75% of ON patients are diagnosed with
  • MS. ON is also associated with infection (e.g., viral infection, meningitis, syphilis), inflammation (e.g., from a vaccine), infiltration and ischemia.
  • infection e.g., viral infection, meningitis, syphilis
  • inflammation e.g., from a vaccine
  • infiltration and ischemia e.g., infiltration and ischemia.
  • AION anterior ischemic optic neuropathy
  • Arteritic AION is due to giant cell arteritis (vasculitis) and leads to acute vision loss.
  • Non-arteritic AION encompasses all cases of ischemic optic neuropathy other than those due to giant cell arteritis.
  • the pathophysiology of AION is unclear although it appears to incorporate both inflammatory and ischemic mechanisms.
  • optic nerve damage typically associated with demyleination, inflammation, ischemia, toxins, or trauma to the optic nerve.
  • exemplary conditions where the optic nerve is damaged include Demyelinating Optic Neuropathy (Optic Neuritis, Retrobulbar Optic Neuritis), Optic Nerve Sheath Meningioma, Adult Optic Neuritis, Childhood Optic Neuritis, Anterior Ischemic Optic Neuropathy, Posterior Ischemic Optic Neuropathy, Compressive Optic Neuropathy, Papilledema, Pseudopapilledema and Toxic/Nutritional Optic Neuropathy.
  • Demyelinating Optic Neuropathy Optic Neuritis, Retrobulbar Optic Neuritis
  • Optic Nerve Sheath Meningioma Meningioma
  • Adult Optic Neuritis Childhood Optic Neuritis
  • Anterior Ischemic Optic Neuropathy Posterior Ischemic Optic Neuropathy
  • Compressive Optic Neuropathy Papilledema,
  • vision impairment is caused by retinal damage.
  • retinal damage is caused by disturbances in blood flow to the eye (e.g., arteriosclerosis, vasculitis).
  • retinal damage is caused by disrupton of the macula (e.g., exudative or non- exudative macular degeneration).
  • Exemplary retinal diseases include Exudative Age Related Macular
  • exemplary diseases include ocular bacterial infections (e.g. conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viral infections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus, Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as well as progressive outer retinal necrosis secondary to HIV or other HIV-associated and other immunodeficiency-associated ocular diseases.
  • ocular diseases include fungal infections (e.g. Candida choroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis) and others such as ocular toxocariasis and sarcoidosis.
  • One aspect of the invention is a method for inhibiting, reducing or treating vision impairment in a subject undergoing treatment with a chemotherapeutic drug (e.g., a neurotoxic drug, a drug that raises intraocular pressure such as a steroid), by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin variant therapeutic agent.
  • a chemotherapeutic drug e.g., a neurotoxic drug, a drug that raises intraocular pressure such as a steroid
  • Another aspect of the invention is a method for inhibiting, reducing or treating vision impairment in a subject undergoing surgery, including ocular or other surgeries performed in the prone position such as spinal cord surgery, by administering to the subject in need of such treatment a therapeutic dosage of a737 I .
  • DOC sirtuin variant therapeutic agent Ocular surgeries include cataract, iridotomy and lens replacements.
  • Another aspect of the invention is the treatment, including inhibition and prophylactic treatment, of age related ocular diseases include cataracts, dry eye, retinal damage and the like, by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin variant therapeutic agent.
  • cataracts are associated with several biochemical changes in the lens of the eye, such as decreased levels of antioxidants ascorbic acid and glutathione, increased lipid, amino acid and protein oxidation, increased sodium and calcium, loss of amino acids and decreased lens metabolism.
  • the lens which lacks blood vessels, is suspended in extracellular fluids in the anterior part of the eye.
  • Nutrients such as ascorbic acid, glutathione, vitamin E, selenium, bioflavonoids and carotenoids are required to maintain the transparency of the lens.
  • Low levels of selenium results in an increase of free radical-inducing hydrogen peroxide, which is neutralized by the selenium-dependent antioxidant enzyme glutathione peroxidase.
  • Lens-protective glutathione peroxidase is also dependent on the amino acids methionine, cysteine, glycine and glutamic acid.
  • Cataracts can also develop due to an inability to properly metabolize galactose found in dairy products that contain lactose, a disaccharide composed of the monosaccharide galactose and glucose. Cataracts can be prevented, delayed, slowed and possibly even reversed if detected early and metabolically corrected.
  • Retinal damage is attributed, inter alia, to free radical initiated reactions in glaucoma, diabetic retinopathy and age-related macular degeneration (AMD).
  • the eye is a part of the central nervous system and has limited regenerative capability.
  • the retina is composed of numerous nerve cells which contain the highest concentration of polyunsaturated fatty acids (PFA) and subject to oxidation.
  • PFA polyunsaturated fatty acids
  • Free radicals are generated by UV light entering the eye and mitochondria in the rods and cones, which generate the energy necessary to transform light into visual impulses.
  • Free radicals cause peroxidation of the PFA by hydroxyl or superoxide radicals which in turn propagate additional free radicals.
  • the free radicals cause temporary or permanent damage to retinal tissue.
  • 737 l .DOC Glaucoma is usually viewed as a disorder that causes an elevated intraocular pressure (IOP) that results in permanent damage to the retinal nerve fibers, but a sixth of all glaucoma cases do not develop an elevated IOP.
  • IOP intraocular pressure
  • This disorder is now perceived as one of reduced vascular perfusion and an increase in neurotoxic factors.
  • Recent studies have implicated elevated levels of glutamate, nitric oxide and peroxynitirite in the eye as the causes of the death of retinal ganglion cells.
  • Neuroprotective agents may be the future of glaucoma care.
  • nitric oxide synthase inhibitors block the formation of peroxynitrite from nitric oxide and superoxide.
  • animals treated with aminoguanidine, a nitric oxide synthase inhibitor had a reduction in the loss of retinal ganglion cells. It was concluded that nitric oxide in the eye caused cytotoxicity in many tissues and neurotoxicity in the central nervous system.
  • Diabetic retinopathy occurs when the underlying blood vessels develop microvascular abnormalities consisting primarily of microaneurysms and intraretinal hemorrhages. Oxidative metabolites are directly involved with the pathogenesis of diabetic retinopathy and free radicals augment the generation of growth factors that lead to enhanced proliferative activity. Nitric oxide produced by endothelial cells of the vessels may also cause smooth muscle cells to relax and result in vasodilation of segments of the vessel. Ischemia and hypoxia of the retina occur after thickening of the arterial basement membrane, endothelial proliferation and loss of pericytes.
  • the inadequate oxygenation causes capillary obliteration or nonperfusion, arteriolar- venular shunts, sluggish blood flow and an impaired ability of RBCs to release oxygen.
  • Lipid peroxidation of the retinal tissues also occurs as a result of free radical damage.
  • the macula is responsible for our acute central vision and composed of light- sensing cells (cones) while the underlying retinal pigment epithelium (RPE) and choroid nourish and help remove waste materials.
  • the RPE nourishes the cones with the vitamin A substrate for the photosensitive pigments and digests the cones shed outer tips.
  • RPE is exposed to high levels of UV radiation, and secretes factors that inhibit angiogenesis.
  • the choroid contains a dense vascular network that provides nutrients and removes the waste materials. 737 I . DOC 9 y 9 y In AMD, the shed cone tips become indigestible by the RPE, where the cells swell and die after collecting too much undigested material. Collections of undigested waste material, called drusen, form under the RPE. Photoxic damage also causes the accumulation of lipofuscin in RPE cells. The intracellular lipofuscin and accumulation of drusen in Bruch's membrane interferes with the transport of oxygen and nutrients to the retinal tissues, and ultimately leads to RPE and photoreceptor dysfunction.
  • Macular pigment one of the protective factors that prevent sunlight from damaging the retina, is formed by the accumulation of nutritionally derived carotenoids, such as lutein, the fatty yellow pigment that serves as a delivery vehicle for other important nutrients and zeaxanthin.
  • nutritionally derived carotenoids such as lutein, the fatty yellow pigment that serves as a delivery vehicle for other important nutrients and zeaxanthin.
  • Antioxidants such as vitamins C and E, beta-carotene and lutein, as well as zinc, selenium and copper, are all found in the healthy macula. In addition to providing nourishment, these antioxidants protect against free radical damage that initiates macular degeneration.
  • Another aspect of the invention is the prevention or treatment of damage to the eye caused by stress, chemical insult or radiation, by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin variant therapeutic agent.
  • Radiation or electromagnetic damage to the eye can include that caused by CRT's or exposure to sunlight or UV.
  • a combination drug regimen may include drugs or compounds for the treatment or prevention of ocular disorders or secondary conditions associated with these conditions.
  • a combination drug regimen may include one or more sirtuin variant therapeutic agents and one or more therapeutic agents for the treatment of an ocular disorder.
  • one or more sirtuin variant therapeutic agents can be combined with an effective amount of one or more of: an agent that reduces intraocular pressure, an agent for treating glaucoma, an agent for treating optic neuritis, an agent for treating CMV Retinopathy, an agent for treating multiple sclerosis, and/or an antibiotic, etc.
  • a sirtuin variant therapeutic agent can be administered in conjunction with a therapy for reducing intraocular pressure.
  • One group of therapies737 l .DOC UU involves blocking aqueous production.
  • topical beta-adrenergic antagonists timolol and betaxolol
  • Topical timolol causes IOP to fall in 30 minutes with peak effects in 1-2 hours.
  • Timoptic 0.5% one drop every 30 minutes for 2 doses.
  • the carbonic anhydrase inhibitor, acetazolamide also decreases aqueous production and should be given in conjunction with topical beta-antagonists.
  • An initial dose of 500 mg is administered followed by 250 mg every 6 hours.
  • alpha 2-agonists e.g., Apraclonidine
  • aqueous production e.g., 1,3-bis(trimethyl)-2-agonists
  • e.g., 1,3-bis(trimethyl)-2-agonists act by decreasing aqueous production.
  • Their effects are additive to topically administered beta-blockers. They have been approved for use in controlling an acute rise in pressure following anterior chamber laser procedures, but has been reported effective in treating acute closed-angle glaucoma. A reasonable regimen is 1 drop every 30 minutes for 2 doses.
  • a second group of therapies for reducing intraocular pressure involve reducing vitreous volume.
  • Hyperosmotic agents can be used to treat an acute attack. These agents draw water out of the globe by making the blood hyperosmolar.
  • Oral glycerol in a dose of 1 mL/kg in a cold 50% solution (mixed with lemon juice to make it more palatable) often is used. Glycerol is converted to glucose in the liver; persons with diabetes may need additional insulin if they become hyperglycemic after receiving glycerol.
  • Oral isosorbide is a metabolically inert alcohol that also can be used as an osmotic agent for patients with acute angle-closure glaucoma. Usual dose is 100 g taken p.o.
  • a third group of therapies involve facilitating aqueous outflow from the eye.
  • Miotic agents pull the iris from the iridocorneal angle and may help to relieve the obstruction of the trabecular meshwork by the peripheral iris.
  • Pilocarpine 2% blue eyes
  • Pilocarpine 4% brown eyes
  • NSAIDS are sometimes used to reduce inflammation.
  • Exemplary therapeutic agents for reducing intraocular pressure include ALPHAGAN® P (Allergan) (brimonidine tartrate ophthalmic solution), AZOPT® (Alcon) (brinzolamide ophthalmic suspension), BETAGAN® (Allergan) (levobunolol hydrochloride ophthalmic solution, USP), BETIMOL® (Vistakon) (timolol ophthalmic solution), BETOPTIC S® (Alcon) (betaxolol HCl), BRIMONIDINE TARTRATE (Bausch & Lomb), CARTEOLOL HYDROCHLORIDE (Bausch & Lomb), COSOPT® (Merck) (dorzolamide hydrochloride-timolol maleate ophthalmic solution), LUMIGAN® (Allergan) (bimatoprost ophthalmic solution), OPTIPRANOLOL® (Bausch & Lomb) (metipranolol ophthalmic solution),
  • a sirtuin variant therapeutic agent can be administered in conjunction with a therapy for treating and/or preventing glaucoma.
  • a therapy for treating and/or preventing glaucoma is DARANIDE® Tablets (Merck) (Dichlorphenamide).
  • a sirtuin variant therapeutic agent can be administered in conjunction with a therapy for treating and/or preventing optic neuritis.
  • drugs for optic neuritis include DECADRON® Phosphate Injection (Merck) (Dexamethasone Sodium Phosphate), DEPO-MEDROL® (Pharmacia & Upjohn)(methylprednisolone acetate), HYDROCORTONE® Tablets (Merck) (Hydrocortisone), ORAPRED® (Biomarin) (prednisolone sodium phosphate oral solution) and PEDIAPRED® (Celltech) (prednisolone sodium phosphate, USP).
  • a sirtuin variant therapeutic agent can be administered in conjunction with a therapy for treating and/or preventing CMV Retinopathy.
  • Treatments for CMV retinopathy include CYTOVENE® (ganciclovir capsules) and VALCYTE® (Roche Laboratories) (valganciclovir hydrochloride tablets).
  • a sirtuin variant therapeutic agent can be administered in conjunction with a therapy for treating and/or preventing multiple sclerosis.737 l .DOC 102
  • examples of such drugs include DANTRIUM® (Procter & Gamble Pharmaceuticals) (dantrolene sodium), NOVANTRONE® (Serono) (mitoxantrone), AVONEX® (Biogen pie) (Interferon beta- Ia), BETASERON® (Berlex) (Interferon beta- Ib), COPAXONE® (Teva Neuroscience) (glatiramer acetate injection) and REB IF® (Pfizer) (interferon beta- 1 a).
  • Macrolide antibiotics include tacrolimus, cyclosporine, sirolimus, everolimus, ascomycin, erythromycin, azithromycin, clarithromycin, clindamycin, lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin, tylosin, roxithromycin, ABT-773, telithromycin, leucomycins, and lincosamide.
  • Mitochondrial-Associated Diseases and Disorders include tacrolimus, cyclosporine, sirolimus, everolimus, ascomycin, erythromycin, azithromycin, clarithromycin, clindamycin, lincomycin, dirithromycin, josamycin, spiramycin, diacetyl-midecamycin, tylosin, roxithromycin, ABT-773, telithromycin, leucomycins, and lincosamide.
  • the invention provides methods for treating diseases or disorders that would benefit from increased mitochondrial activity.
  • the methods involve administering to a subject in need thereof a therapeutically effective amount of a sirtuin variant therapeutic agent.
  • Increased mitochondrial activity refers to increasing activity of the mitochondria while maintaining the overall numbers of mitochondria (e.g., mitochondrial mass), increasing the numbers of mitochondria thereby increasing mitochondrial activity (e.g., by stimulating mitochondrial biogenesis), or combinations thereof.
  • diseases and disorders that would benefit from increased mitochondrial activity include diseases or disorders associated with mitochondrial dysfunction.
  • methods for treating diseases or disorders that would benefit from increased mitochondrial activity may comprise identifying a subject suffering from a mitochondrial dysfunction.
  • Methods for diagnosing a mitochondrial dysfunction may involve molecular genetic, pathologic and/or biochemical analysis are summarized in Cohen and Gold, Cleveland Clinic Journal of Medicine, 68: 625-642 (2001).
  • One method for diagnosing a mitochondrial dysfunction is the Thor-Byrne-ier scale (see e.g., Cohen and Gold, supra; Collin S. et al., Eur Neurol. 36: 260-267 (1996)).
  • enzymatic assays e.g., a mitochondrial enzyme or an ATP biosynthesis factor such as an ETC enzyme or a3737 1 DOC Krebs cycle enzyme
  • determination or mitochondrial mass, mitochondrial volume, and/or mitochondrial number quantification of mitochondrial DNA
  • monitoring intracellular calcium homeostasis and/or cellular responses to perturbations of this homeostasis evaluation of response to an apoptogenic stimulus, determination of free radical production.
  • enzymatic assays e.g., a mitochondrial enzyme or an ATP biosynthesis factor such as an ETC enzyme or a3737 1 DOC Krebs cycle enzyme
  • determination or mitochondrial mass, mitochondrial volume, and/or mitochondrial number quantification of mitochondrial DNA
  • monitoring intracellular calcium homeostasis and/or cellular responses to perturbations of this homeostasis evaluation of response to an apoptogenic stimulus
  • determination of free radical production determination of free radical production.
  • Mitochondria are critical for the survival and proper function of almost all types of eukaryotic cells. Mitochondria in virtually any cell type can have congenital or acquired defects that affect their function. Thus, the clinically significant signs and symptoms of mitochondrial defects affecting respiratory chain function are heterogeneous and variable depending on the distribution of defective mitochondria among cells and the severity of their deficits, and upon physiological demands upon the affected cells. Nondividing tissues with high energy requirements, e.g. nervous tissue, skeletal muscle and cardiac muscle are particularly susceptible to mitochondrial respiratory chain dysfunction, but any organ system can be affected.
  • Diseases and disorders associated with mitochondrial dysfunction include diseases and disorders in which deficits in mitochondrial respiratory chain activity contribute to the development of pathophysiology of such diseases or disorders in a mammal. This includes 1) congenital genetic deficiencies in activity of one or more components of the mitochondrial respiratory chain; and 2) acquired deficiencies in the activity of one or more components of the mitochondrial respiratory chain, wherein such deficiencies are caused by a) oxidative damage during aging; b) elevated intracellular calcium; c) exposure of affected cells to nitric oxide; d) hypoxia or ischemia; e) microtubule-associated deficits in axonal transport of mitochondria, or f) expression of mitochondrial uncoupling proteins.
  • Diseases or disorders that would benefit from increased mitochondrial activity generally include for example, diseases in which free radical mediated oxidative injury leads to tissue degeneration, diseases in which cells inappropriately undergo apoptosis, and diseases in which cells fail to undergo apoptosis.
  • Exemplary diseases or disorders that would benefit from increased mitochondrial activity include, for example, AD (Alzheimer's Disease), ADPD (Alzheimer's Disease and Parkinsons's Disease), AMDF (Ataxia, Myoclonus and Deafness), auto-immune737 1 DOC l ⁇ ⁇ disease, cancer, CIPO (Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia), congenital muscular dystrophy, CPEO (Chronic Progressive External Ophthalmoplegia), DEAF (Maternally inherited DEAFness or aminoglycoside-induced DEAFness), DEMCHO (Dementia and Chorea), diabetes mellirus (Type I or Type II),
  • ALS amyotrophic lateral sclerosis
  • macular degeneration epilepsy, Alpers syndrome, Multiple mitochondrial DNA deletion syndrome, MtDNA depletion syndrome, Complex I deficiency, Complex II (SDH) deficiency, Complex III deficiency, Cytochrome c oxidase (COX, Complex IV) deficiency, Complex V deficiency, Adenine Nucleotide Translocator (ANT) deficiency, Pyruvate dehydrogenase (PDH) deficiency, Ethylmalonic aciduria with lactic acidemia, 3 -Methyl glutaconic aciduria with lactic acidemia, Refractory epilepsy with declines during infection, Asperger syndrome with declines during infection, Autism with declines during infection, Attention deficit hyperactivity disorder (ADHD), Cerebral palsy with decline
  • ADHD Attention deficit hyperactivity disorder
  • the invention provides methods for treating a subject suffering from mitochondrial disorders arising from, but not limited to, posttraumatic head injury and cerebral edema, stroke (invention methods useful for preventing or preventing reperfusion injury), Lewy body dementia, hepatorenal syndrome, acute liver failure, NASH (non-alcoholic steatohepatitis), Anti- metastasis/prodifferentiation therapy of cancer, idiopathic congestive heart failure, atrial fibrilation (non-valvular), Wolff-Parkinson- White Syndrome, idiopathic heart block, prevention of reperfusion injury in acute myocardial infarctions, familial migraines, irritable bowel syndrome, secondary prevention of non-Q wave myocardial infarctions, Premenstrual syndrome, Prevention of renal failure in hepatorenal syndrome, anti-phospholipid antibody syndrome, eclampsia/pre-737 l .DOC eclampsia, oopause infertility, ischemic heart
  • Types of pharmaceutical agents that are associated with mitochondrial disorders include reverse transcriptase inhibitors, protease inhibitors, inhibitors of DHOD, and the like.
  • reverse transcriptase inhibitors include, for example, Azidothymidine (AZT), Stavudine (D4T), Zalcitabine (ddC), Didanosine (DDI), Fluoroiodoarauracil (FIAU), Lamivudine (3TC), Abacavir and the like.
  • Examples of protease inhibitors include, for example, Ritonavir, Indinavir, Saquinavir, Nelfinavir and the like.
  • inhibitors of dihydroorotate dehydrogenase (DHOD) include, for example, Leflunomide, Brequinar, and the like.
  • Reverse transcriptase inhibitors not only inhibit reverse transcriptase but also polymerase gamma which is required for mitochondrial function. Inhibition of polymerase gamma activity (e.g., with a reverse transcriptase inhibitor) therefore leads to mitochondrial dysfunction and/or a reduced mitochondrial mass which manifests itself in patients as hyperlactatemia. This type of condition may benefit from an increase in the number of mitochondria and/or an improvement in mitochondrial function, e.g., by administration of a sirtuin variant therapeutic agent.
  • mitochondrial diseases include cardiomyopathy, muscle weakness and atrophy, developmental delays (involving motor, language, cognitive or executive function), ataxia, epilepsy, renal tubular acidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, sensorineural deafness, neurogenic bladder dysfunction, dilating cardiomyopathy, migraine, hepatic failure, lactic acidemia, and diabetes mellitus.
  • the invention provides methods for treating a disease or disorder that would benefit from increased mitochondrial activity that involves administering to a subject in need thereof one or more sirtuin variant therapeutic agents in combination with another therapeutic agent such as, for example, an agent useful for treating mitochondrial dysfunction (such as antioxidants, vitamins, or respiratory chain cofactors), an agent useful for reducing a symptom associated with a disease or disorder involving mitochondrial dysfunction737 l .
  • DOC 1 ⁇ ' such as, an anti-seizure agent, an agent useful for alleviating neuropathic pain, an agent for treating cardiac dysfunction), a cardiovascular agent (as described further below), a chemotherapeutic agent (as described further below), or an anti- neurodegeneration agent (as described further below).
  • the invention provides methods for treating a disease or disorder that would benefit from increased mitochondrial activity that involves administering to a subject in need thereof one or more sirtuin variant therapeutic agents in combination with one or more of the following: coenzyme Qi 0 , L-carnitine, thiamine, riboflavin, niacinamide, folate, vitamin E, selenium, lipoic acid, or prednisone.
  • sirtuin variant therapeutic agents in combination with one or more of the following: coenzyme Qi 0 , L-carnitine, thiamine, riboflavin, niacinamide, folate, vitamin E, selenium, lipoic acid, or prednisone.
  • Compositions comprising such combinations are also provided herein.
  • the invention provides methods for treating diseases or disorders that would benefit from increased mitochondrial acitivty by administering to a subject a therapeutically effective amount of a sirtuin variant therapeutic agent.
  • diseases or disorders include, for example, neuromuscular disorders (e.g., Friedreich's Ataxia, muscular dystrophy, multiple sclerosis, etc.), disorders of neuronal instability (e.g., seizure disorders, migrane, etc.), developmental delay, neurodegenerative disorders (e.g., Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.), ischemia, renal tubular acidosis, age-related neurodegeneration and cognitive decline, chemotherapy fatigue, age-related or chemotherapy-induced menopause or irregularities of menstrual cycling or ovulation, mitochondrial myopathies, mitochondrial damage (e.g., calcium accumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), and mitochondrial deregulation.
  • mitochondrial myopathies e.g., calcium accumulation
  • FA Friedreich's Ataxia
  • Frataxin is involved in regulation of mitochondrial iron content. When cellular frataxin content is subnormal, excess iron accumulates in mitochondria, promoting oxidative damage and consequent mitochondrial degeneration and dysfunction. When intermediate numbers of GAA repeats are present in the frataxin gene intron, the severe clinical phenotype of ataxia may not develop.
  • sirtuin variant therapeutic agents may be used for treating patients with disorders related to deficiencies or defects in frataxin, including Friedreich's Ataxia, myocardial dysfunction, diabetes mellitus and complications of diabetes like peripheral neuropathy.
  • Muscular dystrophy refers to a family of diseases involving deterioration of neuromuscular structure and function, often resulting in atrophy of skeletal muscle and myocardial dysfunction, hi the case of Duchenne muscular dystrophy, mutations or deficits in a specific protein, dystrophin, are implicated in its etiology. Mice with their dystrophin genes inactivated display some characteristics of muscular dystrophy, and have an approximately 50% deficit in mitochondrial respiratory chain activity. A final common pathway for neuromuscular degeneration in most cases is calcium-mediated impairment of mitochondrial function.
  • sirtuin variant therapeutic agents may be used for reducing the rate of decline in muscular functional capacities and for improving muscular functional status in patients with muscular dystrophy.
  • MS Multiple sclerosis
  • Epilepsy is often present in patients with mitochondrial cytopathies, involving a range of seizure severity and frequency, e.g. absence, tonic, atonic, myoclonic, and status epilepticus, occurring in isolated episodes or many times daily.
  • sirruin variant therapeutic agents may be used for treating patients with seizures secondary to mitochondrial dysfunction, including reducing frequency and severity of seizure activity.
  • Uridine nucleotides are involved inactivation and transfer of sugars to glycolipids and glycoproteins. Cytidine nucleotides are derived from uridine nucleotides, and are crucial for synthesis of major membrane phospholipid constituents like phosphatidylcholine, which receives its choline moiety from cytidine diphosphocholine.
  • disorders characterized by developmental delay include Rett's Syndrome, pervasive developmental delay (or PDD-NOS "pervasive developmental delay not otherwise specified” to distinguish it from specific subcategories like autism), autism, Asperger's Syndrome, and Attention Deficit/Hyperactivity Disorder (ADHD), which is becoming recognized as a delay or lag in development of neural circuitry underlying executive functions, hi certain embodiments, sirtuin variant therapeutic agents may be useful for treating treating patients with neurodevelopmental delays (e.g., involving motor, language, executive function, and cognitive skills), or other delays or arrests of neurological and neuropsychological development in the nervous system and somatic development in non-neural tissues like muscle and endocrine glands.
  • neurodevelopmental delays e.g., involving motor, language, executive function, and cognitive skills
  • AD Alzheimer's Disease
  • PD Parkinson's Disease
  • Complex I deficiencies in particular are frequently found not only in the nigrostriatal neurons that degenerate in Parkinson's disease, but also in peripheral tissues and cells like muscle and platelets of Parkinson's Disease patients.
  • mitochondrial respiratory chain activity is often depressed, especially Complex IV (Cytochrome c Oxidase).
  • mitochondrial respiratory function altogether is depressed as a consequence of aging, further amplifying the deleterious sequelae of additional molecular lesions affecting respiratory chain function.
  • Other factors in addition to primary mitochondrial dysfunction underlie neurodegeneration in AD, PD, and related disorders.
  • Excitotoxic stimulation and nitric oxide are implicated in both diseases, factors which both exacerbate mitochondrial respiratory chain deficits and whose deleterious actions are exaggerated on a background of respiratory chain dysfunction.
  • Huntington's Disease also involves mitochondrial dysfunction in affected brain regions, with cooperative interactions of excitotoxic stimulation and737 1 DOC mitochondrial dysfunction contributing to neuronal degeneration.
  • sirtuin variant therapeutic agents may be useful for treating and attenuating progression of age-related neurodegenerative diseases including AD and PD.
  • AD Amyotrophic Lateral
  • Sclerosis is mutation or deficiency in Copper-Zinc Superoxide Dismutase (SOD 1), an antioxidant enzyme. Mitochondria both produce and are primary targets for reactive oxygen species. Inefficient transfer of electrons to oxygen in mitochondria is the most significant physiological source of free radicals in mammalian systems. Deficiencies in antioxidants or antioxidant enzymes can result in or exacerbate mitochondrial degeneration. Mice transgenic for mutated SODl develop symptoms and pathology similar to those in human ALS. The development of the disease in these animals has been shown to involve oxidative destruction of mitochondria followed by functional decline of motor neurons and onset of clinical symptoms. Skeletal muscle from ALS patients has low mitochondrial Complex I activity. In certain embodiments, sirtuin variant therapeutic agents may be useful for treating ALS, for reversing or slowing the progression of clinical symptoms.
  • Oxygen deficiency results in both direct inhibition of mitochondrial respiratory chain activity by depriving cells of a terminal electron acceptor for Cytochrome c reoxidation at Complex IV, and indirectly, especially in the nervous system, via secondary post-anoxic excitotoxicity and nitric oxide formation.
  • tissues are relatively hypoxic.
  • treatments that increase mitochondrial activity provide protection of affected tissues from deleterious effects of hypoxia, attenuate secondary delayed cell death, and accelerate recovery from hypoxic tissue stress and injury.
  • sirtuin variant therapeutic agents may be useful for preventing delayed cell death (apoptosis in regions like the hippocampus or cortex occurring about 2 to 5 days after an episode of cerebral ischemia) after ischemic or hypoxic insult to the brain.
  • Acidosis due to renal dysfunction is often observed in patients with mitochondrial disease, whether the underlying respiratory chain dysfunction is737 1 DOC 1 12 congenital or induced by ischemia or cytotoxic agents like cisplatin.
  • Renal tubular acidosis often requires administration of exogenous sodium bicarbonate to maintain blood and tissue pH.
  • sirtuin variant therapeutic agents may be useful for treating renal tubular acidosis and other forms of renal dysfunction caused by mitochondrial respiratory chain deficits.
  • mitochondrial respiratory chain function During normal aging, there is a progressive decline in mitochondrial respiratory chain function. Beginning about age 40, there is an exponential rise in accumulation of mitochondrial DNA defects in humans, and a concurrent decline in nuclear-regulated elements of mitochondrial respiratory activity. Many mitochondrial DNA lesions have a selection advantage during mitochondrial turnover, especially in postmitotic cells.
  • mitochondria with a defective respiratory chain produce less oxidative damage to themselves than do mitochondria with intact functional respiratory chains (mitochondrial respiration is the primary source of free radicals in the body). Therefore, normally- functioning mitochondria accumulate oxidative damage to membrane lipids more rapidly than do defective mitochondria, and are therefore "tagged" for degradation by lysosomes.
  • Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in cells subjected to oxidative stress or cancer chemotherapy agents like cisplatin due to both greater vulnerability and less efficient repair of mitochondrial DNA.
  • mitochondrial DNA may be more sensitive to damage than nuclear DNA, it is relatively resistant, in some situations, to mutagenesis by chemical carcinogens. This is because mitochondria respond to some types of mitochondrial DNA damage by destroying their defective genomes rather than attempting to repair them. This results in global mitochondrial dysfunction for a period after cytotoxic chemotherapy.
  • sirtuin variant therapeutic agents may be useful for treatment and prevention of side effects of cancer chemotherapy related to mitochondrial dysfunction.
  • a crucial function of the ovary is to maintain integrity of the mitochondrial genome in oocytes, since mitochondria passed onto a fetus are all derived from those present in oocytes at the time of conception. Deletions in mitochondrial DNA become detectable around the age of menopause, and are also associated with abnormal menstrual cycles. Since cells cannot directly detect and respond to defects in mitochondrial DNA, but can only detect secondary effects that affect the cytoplasm, like impaired respiration, redox status, or deficits in pyrimidine synthesis, such products of mitochondrial function participate as a signal for oocyte selection and follicular atresia, ultimately triggering menopause when maintenance737 I DOC 114 of mitochondrial genomic fidelity and functional activity can no longer be guaranteed.
  • Inhibitors of mitochondrial respiration or protein synthesis inhibit hormone-induced ovulation, and furthermore inhibit production of ovarian steroid hormones in response to pituitary gonadotropins.
  • Women with Down's syndrome typically undergo menopause prematurely, and also are subject to early onset of Alzheimer- like dementia.
  • Low activity of cytochrome oxidase is consistently found in tissues of Down's patients and in late-onset Alzheimer's Disease.
  • Appropriate support of mitochondrial function or compensation for mitochondrial dysfunction therefore is useful for protecting against age-related or chemotherapy-induced menopause or irregularities of menstrual cycling or ovulation.
  • sirtuin variant therapeutic agents may be useful for treating and preventing amenorrhea, irregular ovulation, menopause, or secondary consequences of menopause.
  • sirtuin variant therapeutic agents may be useful for treatment mitochondrial myopathies.
  • Mitochondrial myopathies range from mild, slowly progressive weakness of the extraocular muscles to severe, fatal infantile myopathies and multisystem encephalomyopathies. Some syndromes have been defined, with some overlap between them.
  • Established syndromes affecting muscle include progressive external ophthalmoplegia, the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects, cerebellar ataxia, and sensorineural deafness), the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), the MERFF syndrome (myoclonic epilepsy and ragged red fibers), limb-girdle distribution weakness, and infantile myopathy (benign or severe and fatal).
  • Muscle biopsy737 I . DOC ⁇ specimens stained with modified Gomori's trichrome stain show ragged red fibers due to excessive accumulation of mitochondria.
  • sirtuin variant therapeutic agents may be useful for treating patients suffering from toxic damage to mitochondria, such as, toxic damage due to calcium accumulation, excitotoxicity, nitric oxide exposure, drug induced toxic damage, or hypoxia.
  • Excessive stimulation of neurons with excitatory amino acids is a common mechanism of cell death or injury in the central nervous system.
  • Activation of glutamate receptors especially of the subtype designated NMDA receptors, results in mitochondrial dysfunction, in part through elevation of intracellular calcium during excitotoxic stimulation.
  • deficits in mitochondrial respiration and oxidative phosphorylation sensitizes cells to excitotoxic stimuli, resulting in cell death or injury during exposure to levels of excitotoxic neurotransmitters or toxins that would be innocuous to normal cells.
  • Nitric oxide (about 1 micromolar) inhibits cytochrome oxidase (Complex IV) and thereby inhibits mitochondrial respiration; moreover, prolonged exposure to nitric oxide (NO) irreversibly reduces Complex I activity. Physiological or pathophysiological concentrations of NO thereby inhibit pyrimidine biosynthesis. Nitric oxide is implicated in a variety of neurodegenerative disorders including inflammatory and autoimmune diseases of the central nervous system, and is involved in mediation of excitotoxic and post-hypoxic damage to neurons. 737 I . DOC Oxygen is the terminal electron acceptor in the respiratory chain. Oxygen deficiency impairs electron transport chain activity, resulting in diminished pyrimidine synthesis as well as diminished ATP synthesis via oxidative phosphorylation. Human cells proliferate and retain viability under virtually anaerobic conditions if provided with uridine and pyruvate (or a similarly effective agent for oxidizing NADH to optimize glycolytic ATP production).
  • sirtuin variant therapeutic agents may be useful for treating diseases or disorders associated with mitochondrial deregulation.
  • mitochondrial DNA encoding respiratory chain components requires nuclear factors. In neuronal axons, mitochondria must shuttle back and forth to the nucleus in order to maintain respiratory chain activity. If axonal transport is impaired by hypoxia or by drugs like taxol which affect microtubule stability, mitochondria distant from the nucleus undergo loss of cytochrome oxidase activity. Accordingly, treatment with a sirtuin variant therapeutic agent may be useful for promoting nuclear-mitochondrial interactions.
  • Mitochondria are the primary source of free radicals and reactive oxygen species, due to spillover from the mitochondrial respiratory chain, especially when defects in one or more respiratory chain components impairs orderly transfer of electrons from metabolic intermediates to molecular oxygen.
  • cells can compensate by expressing mitochondrial uncoupling proteins (UCP), of which several have been identified.
  • UCP-2 is transcribed in response to oxidative damage, inflammatory cytokines, or excess lipid loads, e.g. fatty liver and steatohepatitis.
  • UCPs reduce spillover of reactive oxygen species from mitochondria by discharging proton gradients across the mitochondrial inner membrane, in effect wasting energy produced by metabolism and rendering cells vulnerable to energy stress as a trade-off for reduced oxidative injury.
  • Muscle Performance Muscle Performance
  • the invention provides methods for enhancing muscle performance by administering a therapeutically effective amount of a sirtuin variant therapeutic agent.
  • sirtuin variant therapeutic agents may be useful for improving physical endurance (e.g., ability to perform a physical task such as exercise, physical labor, sports activities, etc.), inhibiting or retarding physical737 I .
  • DOC 1 17 fatigues enhancing blood oxygen levels, enhancing energy in healthy individuals, enhance working capacity and endurance, reducing muscle fatigue, reducing stress, enhancing cardiac and cardiovascular function, improving sexual ability, increasing muscle ATP levels, and/or reducing lactic acid in blood.
  • the methods involve administering an amount of a sirtuin variant therapeutic agent that increase mitochondrial activity, increase mitochondrial biogenesis, and/or increase mitochondrial mass.
  • Sports performance refers to the ability of the athlete's muscles to perform when participating in sports activities. Enhanced sports performance, strength, speed and endurance are measured by an increase in muscular contraction strength, increase in amplitude of muscle contraction, shortening of muscle reaction time between stimulation and contraction. Athlete refers to an individual who participates in sports at any level and who seeks to achieve an improved level of strength, speed and endurance in their performance, such as, for example, body builders, bicyclists, long distance runners, short distance runners, etc. An athlete may be hard training, that is, performs sports activities intensely more than three days a week or for competition. An athlete may also be a fitness enthusiast who seeks to improve general health and well-being, improve energy levels, who works out for about 1-2 hours about 3 times a week. Enhanced sports performance in manifested by the ability to overcome muscle fatigue, ability to maintain activity for longer periods of time, and have a more effective workout.
  • sirtuin variant therapeutic agents will also be effective in the treatment of muscle related pathological conditions.
  • Other Uses Sirtuin variant therapeutic agents may be used for treating or preventing viral infections (such as infections by influenza, herpes or papilloma virus) or as antifungal agents.
  • sirtuin variant therapeutic agents may be administered as part of a combination drug therapy with another therapeutic agent for the treatment of viral diseases, including, for example, acyclovir, ganciclovir and zidovudine.
  • sirtuin variant therapeutic agents may be administered as part of a combination drug therapy with another anti-fungal agent including, for example, topical anti-fungals such as ciclopirox, clotrimazole, econazole, miconazole, nystatin, oxiconazole, terconazole, and tolnaftate, or systemic anti-fungal such as fluconazole (Diflucan), itraconazole (Sporanox), ketoconazole (Nizoral), and miconazole (Monistat I.V.).
  • topical anti-fungals such as ciclopirox, clotrimazole, econazole, miconazole, nystatin, oxiconazole, terconazole, and tolnaftate
  • systemic anti-fungal such as fluconazole (Diflucan), itraconazole (Sporanox), ketoconazole (Nizoral), and miconazole (Monistat I.V.).
  • Subjects that may be treated as described herein include eukaryotes, such as mammals, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-737 I .
  • Cells that may be treated include eukaryotic cells, e.g., from a subject described above, or plant cells, yeast cells and prokaryotic cells, e.g., bacterial cells.
  • sirtuin variant therapeutic agents may be administered to farm animals to improve their ability to withstand farming conditions longer.
  • Sirtuin variant therapeutic agents may also be used to increase lifespan, stress resistance, and resistance to apoptosis in plants.
  • a sirtuin variant therapeutic agent is introduced into to plants or to fungi.
  • plants are genetically modified to produce a sirtuin variant.
  • plants and fruits are treated with a sirtuin variant prior to picking and shipping to increase resistance to damage during shipping.
  • Plant seeds may also be contacted with sirtuin variants described herein, e.g., to preserve them.
  • sirtuin variant therapeutic agents may be used for modulating lifespan in yeast cells.
  • Situations in which it may be desirable to extend the lifespan of yeast cells include any process in which yeast is used, e.g., the making of beer, yogurt, and bakery items, e.g., bread.
  • Use of yeast having an extended lifespan can result in using less yeast or in having the yeast be active for longer periods of time.
  • Yeast or other mammalian cells used for recombinantly producing proteins may also be treated as described herein.
  • Sirtuin variant therapeutic agents may also be used to increase lifespan, stress resistance and resistance to apoptosis in insects.
  • sirtuin variants would be applied to useful insects, e.g., bees and other insects that are involved in pollination of plants.
  • a sirtuin variant would be applied to bees involved in the production of honey.
  • the methods described herein may be applied to any organism, e.g., eukaryote, that may have commercial importance. For example, they can be applied to fish (aquaculture) and birds (e.g., chicken and fowl).
  • sirtuin variant therapeutic agents can be applied to affect the reproduction of organisms such as insects, animals and microorganisms.
  • sirtuin variant therapeutic agents described herein may be used alone, or as part of a conjoint therapy with other compounds/pharmaceutical compositions.
  • Sirtuin variant therapeutic agents may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the optimum concentration of the sirtuin variant therapeutic agent(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists.
  • biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art.
  • compositions of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of sirtuin variant therapeutic agents suitable for veterinary uses, e.g., for the treatment of live stock (cow, sheep, goat, pig, and horse, etc.) or domestic animals, e.g., cats and dogs.
  • Sirtuin variant therapeutic agents may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a therapeutic at a particular target site.
  • Sustained-release preparations are also provided herein. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing a sirtuin variant therapeutic agent, which matrices are in the
  • DOC 122 form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • compositions according to the present invention may be administered as either a single dose or in multiple doses.
  • the pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents.
  • the treatments of the present invention may be combined with conventional therapies, which may be administered sequentially or simultaneously.
  • the pharmaceutical compositions of the present invention may be administered by any means that enables the sirtuin variant therapeutic agents to reach the targeted cells/tissues/organs.
  • routes of administration include those selected from the group consisting of oral, intravesically, intravenous, intraarterial, intraperitoneal, local administration into the blood supply of the organ in which the targeted cells reside or directly into the cells.
  • Intravenous administration is the preferred mode of administration. It may be accomplished with the aid of an infusion pump.
  • Sirtuin variant therapeutic agents may be administered to humans and other animals for therapy by any suitable route of administration, including orally, intravesically, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the sirtuin variant therapeutic agents of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are 737 l .
  • DOC * formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.
  • Actual dosage levels of the sirtuin variant therapeutic agents in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular sirtuin variant therapeutic agent employed, the route of administration, the time of administration, the rate of excretion of the particular polypeptide being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the sirtuin variant therapeutic agent employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a sirtuin variant therapeutic agent will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebrovenitricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
  • the effective daily dose of the sirtuin variant therapeutic agent may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the patient receiving this treatment may be any animal in need, including primates, in particular humans, and other non-human mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • Sirtuin variant therapeutic agents can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other therapeutic agents as described herein. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active agents in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • Cells e.g., treated ex vivo with a sirtuin variant therapeutic agent, can be administered according to methods for administering a graft to a subject, which may be accompanied, e.g., by administration of an immunosuppressant drug, e.g., cyclosporin A.
  • the methods described herein involve administering to a subject nucleic acid encoding a sirtuin variant. Delivery of nucleic acids my achieved using in vivo or ex vivo gene therapy methods.
  • expression constructs of the therapeutic sirtuin variants may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively transfecting cells in vivo with a recombinant fusion gene.
  • Approaches include insertion of the subject fusion gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized liposomes (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo.
  • lipofectin lipofectin
  • derivatized liposomes e.g. antibody conjugated
  • polylysine conjugates e.g. antibody conjugated
  • gramacidin S e.g. antibody conjugated
  • artificial viral envelopes e.g. antibody conjugated
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication- defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a CKI polypeptide, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1 ; Danos and 737 l .DOC 1 2 ° Mulligan (1988) Proc.
  • retroviral -based vectors by modifying the viral packaging polypeptides on the surface of the viral particle.
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env polypeptide (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J. Gen Virol 73:3251- 3255; and Goud et al.
  • Coupling can be in the form of the chemical cross-linking with a polypeptide or other variety ⁇ e.g. lactose to convert the env polypeptide to an asialoglycopolypeptide), as well as by generating fusion polypeptides ⁇ e.g. single- chain antibody/env fusion polypeptides).
  • This technique while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
  • non- viral methods can also be employed to cause expression of the subject sirtuin variants in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the gene by the targeted cell.
  • Exemplary gene delivery systems of this type include737 I .
  • a gene encoding one of the SIRTl variants can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • lipofection of neurogliofha cells can be carried out using liposomes tagged with monoclonal antibodies against glioma- associated antigen (Mizuno et al. (1992) Neurol. Med. Chir.
  • the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection ⁇ e.g. Chen et al. (1994) PNAS 91 : 3054- 3057).
  • Toxicity and therapeutic efficacy of sirtuin variant therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the LD 50 is the dose lethal to 50% of the population.
  • the ED 50 is the dose of a drug which produces 50% maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations.
  • the dose ratio between toxic and therapeutic effects (LDso/EDso) is the therapeutic index.
  • Sirtuin variant therapeutic agents that exhibit large therapeutic indexes are preferred. While sirtuin variant therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such agents may lie within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • kits e.g., kits for therapeutic purposes or kits for modulating the lifespan of cells or modulating apoptosis.
  • a kit may comprise one or more sirtuin variant therapeutic agents, e.g., in premeasured doses.
  • a kit may optionally comprise devices for contacting cells with the compounds and instructions for use. Devices include syringes, stents and other devices for introducing a sirtuin variant therapeutic agent into a subject (e.g., the blood vessel of a subject) or applying it to the skin of a subject.
  • kits for identifying sirtuin-modulating compounds contain (1) a sirtuin variant polypeptide or a nucleic acid encoding a sirtuin variant polypeptide and, optionally, (2) a sirtuin-modulating compound, for use as a control.
  • the reagents may be in separate vessels.
  • Such kits can be used, for example, to perform assays to test other compounds (typically provided by the user) for sirtuin-modulating activity.
  • these kits further comprise means for determining sirtuin activity (e.g., a peptide with an appropriate indicator, such as those disclosed herein).
  • Human SIRTl constructs were expressed from the vectors which place expression under the control of the T7 promoter.
  • the protein was expressed in E. coli BL21 Star (DE3) (Invitrogen) for both His and GST tagged proteins in LB media at 37 0 C until OD600 reaching 0.8.
  • the temperature of the culture was cooled down to 16 °C on ice and IPTG was added to 1 mM.
  • the culture was incubated at 16 0 C for 14-16 hrs and cells were harvest by centrifugation at 29,000 g for 30 min at 4 0 C. For higher purity, protein was purified by Ni2+-chelate chromatography.
  • This material was taken up CH 3 CN (4 mL) along with Et 3 N (0.51 mL, 3.64 mmol) and Boc-piperazine (680 mg, 3.64 mmol) and stirred at room temperature for 1 day. The reaction mixture was concentrated and the resulting residue was partitioned between CH 2 Cl 2 and water. The organic layer was dried (Na 2 SO 4 ) and concentrated to afford essentially quantitative yield of the product. This material was taken up in MeOH (6 mL) and water (1 mL) along with sodium hydrosulfide hydrate (200 mg). The resulting reaction mixture was stirred under reflux for 24 hours. It was then cooled to room temperature and concentrated. The resulting residue was diluted with water (2 mL) and extracted with CH 2 Cl 2 .
  • the diethyl-dithiocarbamic acid 4- ⁇ [2-(3,4-dimethoxy-phenyl)-3H-benzoimidazole- 4-carbonyl]-amino ⁇ -pyridin-3-yl ester (0.1 g, 0.2 mmol) was suspended in a 2 N HCl solution (2 mL). The reaction was stirred with heating (100 0 C x 30 min). Upon cooling, the solution was basified (1 N NaOH, pH 7.5). The resulting oil was solidified with the addition of EtOAc, and the solid was filtered, washed (MeOH) 737 I .
  • the mass spectrometry based assay utilizes a peptide having 20 amino acid residues as follows: Ac-Glu-Glu-Lys(Biotin)-Gly-Gln-Ser-Thr-Ser-Ser-His-Ser- Lys(Ac)-Nle-Ser-Thr-Glu-Gly-Lys(5TMR)-Glu-Glu-NH2 wherein K(Ac) is an acetylated lysine residue and NIe is a norleucine.
  • the peptide is labelled with the fluorophore 5TMR (excitation 540 nm/emission 580 ran) at the C-terminus.
  • the sequence of the peptide substrate is based on p53 with several modifications.
  • the mass spectrometry assay was conducted as follows: 1/10 of Km value for peptide substrate and 2/3 of the Km value for ⁇ NAD + were incubated with SIRTl concentration giving less than 10% product conversion at 30 min reaction time for a time course (0, 3, 6, 9, 12, 15, 20, 30 minutes) at 25 0 C in a reaction buffer (50 mM Tris-acetate pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 5 mM DTT, 0.05% BSA). Test compounds were added at 100 uM to the reaction or vehicle control, DMSO. After the incubation with SIRTl, 10% formic acid with 50 mM nicotinamide was added to stop the reaction. Determination of the mass of the substrate peptide allows for precise determination of the degree of acetylation (i.e. starting material) as compared to deacetylated peptide (product).
  • Resveratrol and Compound #1 were chosen to determine if when combined, activate SIRTl enzyme activity in an antagonistic, additive or synergistic manner.
  • the study was run using the cell-free SIRTl Mass Spectrometry Assay in which resveratrol and Compound #1 were combined and tested in a concentration matrix.
  • concentrations of resveratrol tested were 300 ⁇ M, 100 ⁇ M, 33.3 ⁇ M, 11.1 ⁇ M, 3.7 ⁇ M, 1.23 ⁇ M, and 0.41 ⁇ M.
  • the concentrations of Compound #1 tested in the matrix were 300 ⁇ M, 100 ⁇ iM, 33.3 ⁇ M, 1 1.1 ⁇ M, 3.7 ⁇ M, 1.23 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, and 0.015 ⁇ M.
  • the Isobologram mass spectrometry assay was run as an end point assay as described
  • the concentration of both compounds which corresponds to the respective ECl .25 value is used as an intercept on both the X and Y axes. Using these two intercepts, a hypothetical line ( Figure 1 dotted line) called the line of additivity is drawn between the two points. Experimental data obtained by the logarithmic titration of the two compounds mixed as a dose pair in a matrix a, which yield the same effect level (ECl.25), is plotted on the Isobologram. Statistical comparison of the line of additivity and the curve arising from experimental two drug dose combinations indicates if an effect is additive. Points falling below and above the line of additivity are subjected to regression analysis.
  • SIRTl coli are quantified as 'low' for less than 5 mg/L of purified SIRTl variant/starting L of culture, 'medium' for 5-15 mg/L, and 'high' for greater than 15 mg/L.
  • the full-length construct of SIRTl expresses poorly in E. coli.
  • Construct SIRTl -D4 demonstrates that deletion of a portion of the N-terminus permits higher levels of expression.
  • Deacetylation activity is measured for the deletion mutants using the mass spec assay. Deacetylation activity is quantified as 'high' for 100% activity when compared to the activity of full-length SIRTl, 'medium' when the activity is at least 25%, and 'low' when the activity of the deletion mutants is less than 5% of the full- length SIRTl deacetylation activity.
  • the ability of the deletion mutants to be activated by sirtuin modulating compounds was measured in the mass spec assay. Resveratrol, Compound #2 and Compound #3 were used in the assays. Activation is quantified as 'high' when a compound results in a two-fold activation or greater and 'low' when the activation is less than a 1.5-fold increase.
  • FIG. 3 depicts graphically a series of the SIRTl variants.
  • Enzyme activity refers to deacetylation activity of the polypeptides in the presence of DMSO alone (DMSO) (e.g., in the absence of a sirtuin modulating compound) as well activatability of the deacetylase activity of the polypeptides in the presence of a sirtuin modulating compound (Comp. #2).
  • DMSO DMSO alone
  • sirtuin modulating compound Comp. #2.
  • the SIRTl -E5c construct retains specific activity and activation properties essentially identical to full length SIRTl but is expressed at high levels in E. coli.
  • SIRT1-A8 amino acid residues 150-670 of SEQ ID NO: 1 and B8 (amino acid residues 170-670 of SEQ ID NO: 1) retain similar activity and activation as the E5c construct
  • SIRTl -C8 amino acid residues 190-670 of SEQ ID NO: 1
  • D8 amino acid residues 210-670 of SEQ ID NO: 1
  • the latter two fragments express poorly in E. coli and may lose activity simply due to misfolding.
  • the SIRTl -E8 (amino acid residues 225-670 of SEQ ID NO:1) construct exhibits enzymatic activity comparable to E5c, but loses the ability to be activated by Compound #2 suggesting that the stretch of amino acids 183-225 is important in defining the binding site for small molecule activators of the Compound #1 class of compounds.
  • 737 1 DOC ⁇ The SIRTl deletion experiments indicate that the region of amino acids 183- 225 of SEQ ID NO: 1 is critical for maintaining activation of SIRTl by these compounds and is involved in defining the allosteric binding site. It is possible that acetylated peptide substrate binding to SIRTl induces a conformational change that exposes an allosteric site in this region of the enzyme.
  • An endogenous regulator of SIRTl has yet to be identified and it is believed to speculate that an endogenous activator of SIRTl exists and may be increased following calorie restriction and other mild physiological stresses.

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Abstract

La présente invention concerne de nouveaux variants de la sirtuine et des procédés d'utilisation de ceux-ci. Les variants de la sirtuine peuvent être utilisés pour faire augmenter la durée de vie d'une cellule, et traiter et/ou prévenir une vaste variété de maladies et de troubles, comprenant, par exemple, des maladies ou des troubles liés au vieillissement ou au stress, le diabète, l'obésité, des maladies neurodégénératives, une maladie cardiovasculaire, des troubles de la coagulation sanguine, une inflammation, un cancer, et/ou des bouffées congestives ainsi que des maladies ou des troubles qui pourraient bénéficier d'une activité mitochondriale accrue. La présente invention propose également des compositions comprenant des variants de la sirtuine, facultativement en combinaison avec d'autres agents thérapeutiques.
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WO2011036450A3 (fr) * 2009-09-28 2011-07-28 The University Of York Variants d'épissage
JP2012514607A (ja) * 2009-01-08 2012-06-28 シャンハイ ジアオ トン ユニバーシティ ベンズイミダゾール−４−カルボキサミド誘導体及びその製造方法、薬物混合物及びその用途
EP2671614A1 (fr) * 2007-12-21 2013-12-11 University of Rochester Cibles moléculaires pour le traitement d'inflammation
US8685970B2 (en) 2008-05-01 2014-04-01 GlaxoSmithKline, LLC Quinolines and related analogs as sirtuin modulators
US20140234287A1 (en) * 2011-07-13 2014-08-21 Icahn School Of Medicine At Mount Sinai SUMOYLATION OF SERCA2a AND CARDIOVASCULAR DISEASE
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US9303034B2 (en) 2013-12-19 2016-04-05 Novartis Ag Compounds and compositions for the treatment of parasitic diseases
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US8685970B2 (en) 2008-05-01 2014-04-01 GlaxoSmithKline, LLC Quinolines and related analogs as sirtuin modulators
US8846947B2 (en) 2008-07-03 2014-09-30 Glaxosmithkline Llc Benzimidazoles and related analogs as sirtuin modulators
US9326986B2 (en) 2008-09-29 2016-05-03 Glaxosmithkline Llc Quinazolinone, quinolone and related analogs as sirtuin modulators
US8987258B2 (en) 2008-09-29 2015-03-24 Christopher Oalmann Chromenone analogs as sirtuin modulators
JP2012514607A (ja) * 2009-01-08 2012-06-28 シャンハイ ジアオ トン ユニバーシティ ベンズイミダゾール−４−カルボキサミド誘導体及びその製造方法、薬物混合物及びその用途
WO2011036450A3 (fr) * 2009-09-28 2011-07-28 The University Of York Variants d'épissage
US9556201B2 (en) 2009-10-29 2017-01-31 Glaxosmithkline Llc Bicyclic pyridines and analogs as sirtuin modulators
US20140234287A1 (en) * 2011-07-13 2014-08-21 Icahn School Of Medicine At Mount Sinai SUMOYLATION OF SERCA2a AND CARDIOVASCULAR DISEASE
US10105422B2 (en) 2011-07-13 2018-10-23 Icahn School Of Medicine At Mount Sinai Sumoylation of SERCA2a and cardiovascular disease
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US9233961B2 (en) 2013-03-15 2016-01-12 Novartis Ag Compounds and compositions for the treatment of parasitic diseases
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US9303034B2 (en) 2013-12-19 2016-04-05 Novartis Ag Compounds and compositions for the treatment of parasitic diseases
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