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WO2011011181A1 - Pharmacological chaperones for the treatment of alzheimer's disease - Google Patents

Pharmacological chaperones for the treatment of alzheimer's disease Download PDF

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Publication number
WO2011011181A1
WO2011011181A1 PCT/US2010/040774 US2010040774W WO2011011181A1 WO 2011011181 A1 WO2011011181 A1 WO 2011011181A1 US 2010040774 W US2010040774 W US 2010040774W WO 2011011181 A1 WO2011011181 A1 WO 2011011181A1
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Prior art keywords
secretase
protein
activity
cell
batimastat
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PCT/US2010/040774
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French (fr)
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Robert Boyd
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Amicus Therapeutics, Inc.
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Publication of WO2011011181A1 publication Critical patent/WO2011011181A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings

Definitions

  • the present invention relates to a method for treating an individual having a neurological disorder with an associated increase in the ⁇ -amyloidogenic metabolism of amyloid precursor protein (APP).
  • the individual is administered a pharmacological chaperone for ⁇ -secretase, which increases trafficking of the ⁇ -secretase protein from the ER to the cytoplasm in neural cells, concomitantly increasing ⁇ -secretase enzyme activity in neural cells.
  • APP amyloid precursor protein
  • AD Alzheimer's disease
  • a chronic and progressive neurodegenerative disorder characterized neuropathologically by the presence of amyloid- ⁇ plaques, neurofibrillary tangles, and gray matter loss.
  • multiple regions of brain gray matter have a profound neuronal loss, including basal forebrain, hippocampus, entorhinal, and temporal cortices.
  • Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called Tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability.
  • the extracellular amyloid- ⁇ plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid- ⁇ (A ⁇ l -42) peptide and have led to the foundation of the amyloid hypothesis. This hypothesis postulates that A ⁇ l-42 is one of the principal causative factors of neuronal death in the brains of Alzheimer's patients.
  • AD Alzheimer's disease
  • AD is set to become the developed world's largest socioeconomic healthcare burden over the coming decades. AD is thought to affect 4-8% of the population over 65 years of age, with the incidence continuing to increase with increasing age.
  • Antemortem clinical diagnosis of AD is difficult and requires a recorded decline in cognitive function as well as evidence of progressive deficits in other behavioral areas such as executive function and language skills. Unqualified diagnosis of AD can still only be made neuropathologically postmortem by examination of patients' brains and the detection of amyloid- ⁇ plaques and tangles.
  • AD cases The majority of Alzheimer cases seem to be sporadic or to result from complex interactions of several genes. Nevertheless, a minority of AD cases ( ⁇ 1%) result from autosomal dominant inheritance of an age-dependent trait with high penetrance (Tanzi, R. E., et al., Neuron 2001 ; 32: 181-184; St George-Hyslop, P.H., Ann NYAcad Sci 2000; 924: 1-7).
  • a ⁇ is a hydrophobic 39- to 42-amino acid peptide, found in all biological fluids, and derived from the enzymatic cleavage of a larger type I membrane protein, the amyloid precursor protein (APP) (Hardy, J., et al., Science 200; 297: 353-356).
  • APP amyloid precursor protein
  • APOE4 apolipoprotein E4
  • the ⁇ -amiloydogenic pathway involves the sequential proteolysis of APP by ⁇ - secretase (BACE) followed by ⁇ -secretase. Although this is a minor APP processing route, it is this pathway that generates A ⁇ fragments believed to give rise to AD.
  • BACE-I and BACE-2 two ⁇ - secretase genes have been identified, referred to as BACE-I and BACE-2, colocalized with APP in the endosomal compartment (Vassar, R. et al., Science 1999; 286: 735-741 ). Whereas both can process APP at the same site, only BACE-I is significantly expressed in brain, particularly in neurons, indicating that neurons are the major source of amyloid- ⁇ peptides in brain.
  • BACE-2 is expressed in heart, kidney, and placenta
  • drugs developed as ⁇ -secretase inhibitors may need to be selective against BACE-2 to prevent unwanted peripheral side effects in the clinic
  • the predominant pathway by which APP is processed does not give rise to A ⁇ fragments, and hence is referred to as the non- ⁇ -amyloidogenic pathway.
  • the initial APP processing involves the cleavage of APP by ⁇ -secretase.
  • the identification of proteins with ⁇ -secretase activity is ongoing, and currently includes three members of the A Disintegrin And Metal loproteinase (ADAM) protein family.
  • ADAM A Disintegrin And Metal loproteinase
  • ADAMl O also known as TACE (tumor necrosis factor- ⁇ converting enzyme)
  • ADAM9 Yamamoto S., et al., Proc Natl Acad Sci USA 1999; 96: 3922-3927; Zheng Y., et al., J Biol Chem 2004; 279: 42898 ⁇ 2906; Kowalska, A., Pol. J. Pharmacol 2004; 56: 171-178).
  • ⁇ -secretase cleavage site is within the A ⁇ sequence of APP, and none of the proteolytic fragments created by ⁇ -secretase cleavage have been associated with the generation of AD, enhanced cleavage at this site could represent a disease modifying strategy for AD.
  • AD amyloid- ⁇ fragment
  • a ⁇ amyloid- ⁇ fragment
  • AD Alzheimer's Disease
  • AD therapies As such, blockade of the NMDA receptor, one of the principal excitatory glutamate receptors in the brain, has been shown to have neuroprotective effects in a number of acute preclinical in vitro and in vivo models. Additional research to develop AD therapies has focused on inhibition of ⁇ -secretase and the metabolism of APP to form A ⁇ peptide.
  • An alternative approach to developing treatments for AD may focus on the shunting of APP into the non- ⁇ -amyloidogenic metabolism of APP into sAPP ⁇ by ⁇ -secretase.
  • the use of pharmacological inhibitors to function as pharmacological chaperones, increasing the enzymatic activity of misfolded mutant proteins, as well as normal wild type proteins has been demonstrated (see U.S. Patent Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; and 6,916,829, all incorporated herein by reference).
  • proteins are involved in almost every aspect of cellular function. Proteins are linear strings of amino acids that fold and twist into specific three-dimensional shapes in order to function properly. Certain human diseases result from mutations that cause changes in the amino acid sequence of a protein which reduce its stability and may prevent it from folding properly. The majority of genetic mutations that lead to the production of less stable or misfolded proteins are called missense mutations. These mutations result in the substitution of a single amino acid for another in the protein. Because of this error, missense mutations often result in proteins that have a reduced level of biological activity. In addition to missense mutations, there are also other types of mutations that can result in proteins with reduced biological activity.
  • Proteins generally fold in a specific region of the cell known as the endoplasmic reticulum, or ER.
  • the cell has quality control mechanisms that ensure that proteins are folded into their correct three-dimensional shape before they can move from the ER to the appropriate destination in the cell, a process generally referred to as protein trafficking. Misfolded proteins are often eliminated by the quality control mechanisms after initially being retained in the ER. In certain instances, misfolded proteins can accumulate in the ER before being eliminated.
  • misfolded proteins in the ER interrupts their proper trafficking, and the resulting reduced biological activity can lead to impaired cellular function and ultimately to disease.
  • the accumulation of misfolded proteins in the ER may lead to various types of stress on cells, which may also contribute to cellular dysfunction and disease.
  • Endogenous molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and permit cells to survive under stresses such as heat shock and glucose starvation (Gething et al., Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et al, MoI. Biol. Cell 1993; 4.109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128).
  • BiP immunoglobulin heavy-chain binding protein, Grp78
  • Grp78 is the best characterized chaperone of the ER (Haas, Curr. Top. Microbiol.
  • BiP Like other chaperones, BiP interacts with many secretory and membrane proteins within the ER throughout their maturation. When nascent protein folding proceeds smoothly, this interaction is normally weak and short-lived. Once the native protein conformation is achieved, the molecular chaperone no longer interacts with the protein. BiP binding to a protein that fails to fold, assemble, or be properly glycosylated becomes stable, and usually leads to degradation of the protein through the ER-associated degradation pathway.
  • the pharmacological chaperones enhance ER secretion and activity of wild-type enzymes (see Examples).
  • a compound that induces a stable molecular conformation of an enzyme during folding serves as a "chaperone" to stabilize the enzyme in a proper conformation for exit from the ER.
  • a compound that induces a stable molecular conformation of an enzyme during folding serves as a "chaperone" to stabilize the enzyme in a proper conformation for exit from the ER.
  • enzymes one such compound unexpectedly turned out to be a competitive inhibitor of the enzyme.
  • Stabilization of these proteins also may be achieved using pharmacological chaperones.
  • small synthetic compounds were found to stabilize the D ⁇ A binding domain of mutant forms of the tumor suppressor protein p53, thereby allowing the protein to maintain an active conformation (Foster et al., Science 1999; 286:2507-10).
  • Synthesis of receptors has been shown to be rescued by small molecule receptor antagonists and ligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja- Repo et al., EMBO J. 2002; 21 :1628-37).
  • the present invention provides a method for the treatment of a neurological disorder in an individual, wherein the neurological disorder is associated with the ⁇ -amyloidogenic processing of Amyloid Precursor Protein (APP), by administering an effective amount of a specific pharmacological chaperone to treat the neurological disorder.
  • the individual has been diagnosed or is at risk of developing Alzheimer's disease (AD), including Familial or Sporadic forms of AD.
  • AD Alzheimer's disease
  • the present invention provides a method for enhancing intracellular folding of an ⁇ -secretase polypeptide into a functional conformation by contacting an ⁇ -secretase -expressing cell with an effective amount of a specific pharmacological chaperone. Enhancing intracellular folding of ⁇ -secretase will lead to an increased proportion of a-secretase which exits the ER, resulting in enhanced enzymatic function in the cells of the central nervous system. Increased ⁇ -secretase activity would, in turn, increase the non- ⁇ -amyloidogenic metabolism of APP, and may be useful in the treatment of neurological disorders such as AD.
  • the ⁇ -secretase polypeptide is a wild-type ⁇ -secretase polypeptide, which, for example, is encoded by a nucleic acid of the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM).
  • ADAMs include, but are not limited to, ADAM 9, ADAM 10, and TACE/ADAM 17.
  • the ⁇ -secretase polypeptide is a mutant ⁇ -secretase polypeptide
  • the mutant polypeptide contains a mutation that results in reduced or improper intracellular folding of the ⁇ -secretase polypeptide, resulting in a shift of equilibrium to increased ⁇ -amyloidogenic processing of APP
  • a pharmacological chaperone specific for ⁇ -secretase can rescue the mutant ⁇ -secretase.
  • the pharmacological chaperone binds the ⁇ -secretase active site. In another embodiment, the pharmacological chaperone binds a non-active site of ⁇ -secretase, which can include an allosteric site.
  • the method of the invention comprises the administration of one or more pharmacological chaperones of ⁇ -secretase to an individual diagnosed, at risk, or suspected to have Alzheimer's Disease. Suitable pharmacological chaperones include any compound(s) which, following administration to an individual, will bind to ⁇ -secretase and increase ⁇ -secretase enzymatic activity. In one particular embodiments, the pharmacological chaperone is a reversible inhibitor of ⁇ -secretase.
  • the increase in ⁇ -secretase enzymatic activity may increase the non- ⁇ -amyloidogenic processing of Amyloid Precursor Protein, producing sAPP ⁇ , and decrease the ⁇ -amyloidogenic processing of Amyloid Precursor Protein, reducing the accumulation of amyloid- ⁇ fragment, particularly the A ⁇ l-42 peptide, within the central nervous system of an individual.
  • pharmacological inhibitors of ⁇ -secretase as ⁇ -secretase chaperones can increase the wild-type enzymatic activity of ⁇ -secretase.
  • Pharmacological inhibitors of ⁇ secretase include, but are not limited to, hydroxamic acid-based zinc metalloproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I , TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
  • Figure 1 shows mean ⁇ -galactosidase A activity in white blood cells from normal, healthy volunteers who received 50 mg 1-deoxygalactonojirimycin (DGJ) b.i.d. (triangles), 150 mg DGJ b.i.d. (squares), or placebo (open circles).
  • Figure 2 shows mean ⁇ -galactosidase A activity in white blood cells from 1 1 Fabry disease patients who were treated with DGJ.
  • DGJ 1-deoxygalactonojirimycin
  • Figure 3 shows the pharmacological chaperones TAPI-2, GM6001 and GW4023 stabilize purified ADAMlO in vitro and increases wild-type levels of ADAMlO (precursor and mature forms) by 2- to 3-fold in SY5Y neuroblastomas.
  • the invention is based, in part, on the discovery that administration of a pharmacological chaperone to a human resulted in a meaningful increase in the level of activity of a wild-type protein.
  • This discovery combined with an understanding of a pharmacological chaperone's ability to promote proper protein folding in the ER, leading to correct protein trafficking and, significantly increased protein activity, e.g., sufficient protein activity to reverse or ameliorate a disease, disorder, or condition in a human subject.
  • This phenomenon is highly specific to the protein specifically bound by the particular pharmacological chaperone, in contrast to methods using compounds that operate generally to increase expression of all proteins, called "chemical chaperones.”
  • pharmacological chaperones increased endogenous wild-type protein activity in humans to about 120% of normal, 130% of normal, and 145% of normal at a lower dose, and to 150% and 185% of normal at a higher dose after administration of a pharmacological chaperone (see Example 1 and Figure 1).
  • This level of increase in vivo was not predictable from results with cells in tissue culture which remain exposed to the pharmacological chaperone.
  • U.S. Patent No. 6,274,597 describes a 30% increase of ⁇ -galactosidase A ( ⁇ -GAL) activity in normal lymphoblasts cultured with deoxygalactonojirimycin (DGJ), a pharmacological chaperone.
  • ⁇ -GAL ⁇ -galactosidase A
  • Example 10 of U.S. Patent No. 6,274,597 describes an increase in activity of a mutant enzyme in transgenic mice treated for one week with a pharmacological chaperone.
  • these experiments involved mutant forms of the rescued protein, not wild-type, and were conducted in mice, so the results were not predictive or suggestive of the results observed for wild-type protein in humans.
  • a pharmacological chaperone could increase the level of activity of a wild-type protein in vivo by at least 20-25%, and particularly not by at least about 50%.
  • administration of DGJ to subjects resulted in a dose-dependent increase in ⁇ -GAL activity.
  • the level of increase in enzyme activity increased by at least 50% (at least 1.5-fold) to up to 100% (at least 2-fold).
  • This extraordinary effect results from titrating a pharmacological chaperone, which is already demonstrated in accordance with existing technology, to rescue a mutant form of the protein, in human patients, to achieve this level of increase, as described herein.
  • the invention provides for titrating a dose of a pharmacological chaperone that has been found to rescue activity of a mutant protein to increase the level of activity of a wild-type protein.
  • the term "pharmacological chaperone,” or sometimes “specific pharmacological chaperone” (“SPC”) refers to a molecule that specifically binds to ⁇ - secretase and has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of the protein; (ii) enhances proper trafficking of the protein from the ER to another cellular location, preferably a native cellular location, i.e., preventing ER- associated degradation of the protein; (iii) preventing aggregation of conformational Iy unstable, i.e., misfolded proteins; (iv) restoring or enhancing at least partial wild-type function, stability, and/or activity of the protein; and/or (v) improving the phenotype or function of the cell harboring ⁇ -secretase.
  • SPC specific pharmacological chaperone
  • a pharmacological chaperone for ⁇ -secretase is a molecule that binds to ⁇ -secretase, resulting in proper folding, trafficking, non- aggregation, and activity of ⁇ -secretase.
  • this term does not refer to endogenous chaperones, such as BiP, or to non-specific agents which have demonstrated nonspecific chaperone activity against various proteins, such as glycerol, DMSO or deuterated water, i.e., chemical chaperones (see Welch et al , Cell Stress and Chaperones 1996; l(2): 109-1 15; Welch et al., Journal of Bioenergetics and Biomembranes 1997; 29(5):491- 502; U.S. Patent No. 5,900,360; U.S. Patent No. 6,270,954; and U.S. Patent No. 6,541 ,195).
  • specific binding molecules e.g., active site-specific chaperones (discussed above), inhibitors or antagonists, and agonists.
  • specifically binds refers to the interaction of a pharmacological chaperone with ⁇ -secretase, specifically, an interaction with amino acid residues of ⁇ -secretase that directly participate in contacting the pharmacological chaperone.
  • a pharmacological chaperone specifically binds a target protein, e.g., ⁇ -secretase, to exert a chaperone effect on ⁇ -secretase and not a generic group of related or unrelated proteins.
  • amino acid residues of ⁇ -secretase that interact with any given pharmacological chaperone may or may not be within the protein's "active site.” Specific binding can be evaluated through routine binding assays or through structural studies, e.g., co-crystallization, NMR, and the like.
  • the pharmacological chaperone is an inhibitor or antagonist of ⁇ -secretase. In another non-limiting embodiment, the pharmacological chaperone is an agonist of ⁇ -secretase. In yet another embodiment, the pharmacological chaperone is a mixed agonist/antagonist.
  • the term "antagonist” refers to any molecule that binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes an activity of ⁇ -secretase.
  • agonist refers to any molecule that binds to a protein and at least partially increases, enhances, restores, or mimics an activity of ⁇ - secretase. As discussed below, such molecules are known for ⁇ -secretase.
  • the terms "enhance ⁇ -secretase conformational stability" or “increase ⁇ -secretase conformational stability” refer to increasing the amount or proportion of ⁇ - secretase that adopts a functional conformation in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • the cells do not express a conformation mutant ⁇ -secretase.
  • the cells do express a mutant ⁇ - secretase polynucleotide encoding a polypeptide e.g., a conformational mutant ⁇ -secretase.
  • the terms "enhance ⁇ -secretase trafficking" or “increase ⁇ -secretase trafficking” refer to increasing the efficiency of transport of ⁇ -secretase into the cytosol of a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the efficiency of transport of ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ - secretase.
  • the terms "enhance ⁇ -secretase activity" or “increase ⁇ -secretase activity” refer to increasing the activity of ⁇ -secretase, as described herein, in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the activity of ⁇ - secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • the terms "enhance ⁇ -secretase level" or “increase ⁇ -secretase level” refer to increasing the level of ⁇ -secretase in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the level of ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • stabilize a proper conformation refers to the ability of an ⁇ -secretase pharmacological chaperone to induce or stabilize a conformation of a mutated ⁇ -secretase protein that is functionally identical to the conformation of the wild-type ⁇ -secretase protein.
  • conformational flexibility does not result in (1) protein aggregation, (2) elimination through the endoplasmic reticulum-associated degradation pathway, (3) impairment of protein function, e.g., APP metabolic activity, and/or (4) improper transport within the cell, e.g., localization to the cytosol, to a greater or lesser degree than that of the wild-type protein.
  • stable molecular conformation refers to a conformation of a protein, i.e., ⁇ -secretase, induced by a pharmacological chaperone that provides at least partial wild-type function in the cell.
  • a stable molecular conformation of a mutant ⁇ -secretase would be one where ⁇ -secretase escapes from the ER and traffics to the cytosol as for a wild- type ⁇ -secretase, instead of misfolding and being degraded.
  • a stable molecular conformation of a mutated ⁇ -secretase may also possess full or partial ⁇ -secretase activity, e.g., APP metabolism.
  • it is not necessary that the stable molecular conformation have all of the functional attributes of the wild-type protein.
  • ⁇ -secretase activity refers to the normal physiological function of a wild- type ⁇ -secretase in a cell.
  • ⁇ -secretase activity includes metabolism of APP.
  • Such functionality can be tested by any means known to establish functionality.
  • wild-type ⁇ -secretase or " ⁇ -secretase” refer to a polypeptide encoded by a nucleic acid selected from the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM) proteins comprising ADAM 9 (human ADAM9, GenBank Accession Nos. NM 003816, and NM 001005845; murine ADAM9, GenBank Accession No. NM_007404; and predicted rat ADAM9, GenBank Accession No. NMJ)Ol 014772), ADAM 10 (human ADAMl O, GenBank Accession No. NMJ)Ol I l O; murine ADAMlO, GenBank Accession No.
  • ADAM 9 human ADAM9, GenBank Accession Nos. NM 003816, and NM 001005845
  • murine ADAM9 GenBank Accession No. NM_007404
  • predicted rat ADAM9 GenBank Accession No. NMJ
  • ⁇ -secretase is encoded by ADAMlO (Obregon et al., J. Biol. Chem. 2006; 281 : 16419-16427).
  • ⁇ -secretase polypeptide may be encoded for by any nucleic acid molecule exhibiting 50%, 60%, 70%, 80% and up to 100% homology to the nucleic acid molecules encoding human ⁇ -secretase, and any sequences which hybridize under standard conditions to these sequences.
  • any other nucleotide sequence that encodes ⁇ -secretase polypeptide such as allelic variants in normal individuals, that have the ability to achieve a functional conformation in the ER, achieve proper localization within the cell, and exhibit wild-type activity (e.g., APP metabolism).
  • ⁇ -secretase polypeptide also refers to an amino acid sequence depicted from the ADAM family of proteins comprised of: ADAM 9 (human ADAM9, GenBank Accession Nos. NP 001005845, and NP 003807; murine ADAM9, GenBank Accession No. NP 031430; and predicted rat ADAM9, GenBank Accession No. NPJ)Ol 014772), ADAMlO (human ADAMl O, GenBank Accession No. NPJ)OI lOl ; murine ADAMl O, GenBank Accession No. NP 031425, and predicted rat ADAMlO, GenBank Accession No.
  • TACE/ADAM 17 human ADAM 17, GenBank Accession No. NP_003174; murine ADAM17, GenBank Accession No. NP_033745; and rat ADAM17, GenBank Accession No. NP_064702
  • mutant ⁇ -secretase refers to an ⁇ -secretase polypeptide translated from a gene containing a genetic mutation that results in an altered ⁇ -secretase amino acid sequence.
  • the mutation results in an ⁇ -secretase protein that does not achieve a native conformation under the conditions normally present in the ER, when compared with wild-type ⁇ -secretase, or exhibits decreased stability or activity as compared with wild-type ⁇ -secretase.
  • a mutation may occur in a non-coding part of the gene encoding ⁇ -secretase that results in less efficient expression of the protein, e.g., a mutation that affects transcription efficiency, splicing efficiency, mRNA stability, and the like.
  • Certain tests may evaluate attributes of a protein that may or may not correspond to its actual in vivo activity, but nevertheless are appropriate surrogates of protein functionality, and wild-type behavior in such tests demonstrates evidence to support the protein folding rescue or enhancement techniques of the invention.
  • One such activity in accordance with the invention is appropriate transport of a functional ⁇ -secretase from the endoplasmic reticulum to the cytosol.
  • endogenous expression and “endogenously expressed” refers to the normal physiological expression of ⁇ -secretase in cells in an individual not having or suspected of having a disease or disorder associated with ⁇ -secretase deficiency, overexpression of a dominant negative mutant, or other defect, such as a mutation in ⁇ - secretase nucleic acid or polypeptide sequence that alters, e.g., inhibits, its expression, activity, or stability.
  • This term also refers to the expression of ⁇ -secretase in cells or cell types in which it is normally expressed in healthy individuals, and does not include expression of ⁇ -secretase in cells or cell types, e.g., tumor cells, in which ⁇ -secretase is not expressed in healthy individuals.
  • the term "efficiency of transport” refers to the ability of a protein to be transported out of the endoplasmic reticulum to its native location within the cell, cell membrane, or into the extracellular environment.
  • a “competitive inhibitor" of an enzyme can refer to a compound which structurally resembles the chemical structure and molecular geometry of the enzyme substrate to bind the enzyme in approximately the same location as the substrate.
  • the inhibitor competes for the same active site as the substrate molecule, thus increasing the Km.
  • Competitive inhibition is usually reversible if sufficient substrate molecules are available to displace the inhibitor, i.e., competitive inhibitors can bind reversibly. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.
  • Non-classical competitive inhibition occurs when the inhibitor binds remotely to the active site, creating a conformational change in the enzyme such that the substrate can no longer bind to it.
  • the binding of substrate at the active site prevents the binding of inhibitor at a separate site and vice versa. This includes allosteric inhibition.
  • a "linear mixed-type inhibitor" of an enzyme is a type of competitive inhibitor that allows the substrate to bind, but reduces its affinity, so the Km is increased and the Vmax is decreased.
  • non-competitive inhibitor refers to a compound that forms strong bonds with an enzyme and may not be displaced by the addition of excess substrate, i.e., non-competitive inhibitors may be irreversible.
  • a non-competitive inhibitor may bind at, near, or remote from the active site of an enzyme or protein, and in connection with enzymes, has no effect on the Km but decreases the Vmax.
  • Uncompetitive inhibition refers to a situation in which inhibitor binds only to the enzyme-substrate (ES) complex. The enzyme becomes inactive when inhibitor binds. This differs from non-classical competitive inhibitors which can bind to the enzyme in the absence of substrate.
  • Vmax refers to the maximum initial velocity of an enzyme catalyzed reaction, i.e., at saturating substrate levels.
  • Km is the substrate concentration required to achieve /2 Vmax.
  • An enzyme “enhancer” is a compound that binds to ⁇ -secretase and increases the enzymatic reaction rate.
  • terapéuticaally effective dose and “effective amount” refer to an amount sufficient to enhance protein processing in the ER (permitting a functional conformation), without inhibiting protein already expressed at the appropriate cellular location (in the case of an antagonist), or without inducing ligand-mediated receptor internalization of protein from the appropriate cellular location (in the case of an agonist), and enhance activity of the target protein, thus resulting in a therapeutic response in a subject.
  • a therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including the foregoing symptoms and surrogate clinical markers.
  • a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or disorder, e.g., Alzheimer's Disease.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition, or other editions.
  • the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • an isolated nucleic acid means that the referenced material is removed from the environment in which it is normally found.
  • an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced.
  • an isolated nucleic acid includes a PCR product, an mRNA band on a gel, a cDNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns.
  • Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material, such as a ⁇ -secretase nucleic acid or polypeptide that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by conventional means, e g , chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • AD Alzheimer's Disease
  • a ⁇ l-42 amyloid- ⁇
  • amyloid- ⁇ particularly A ⁇ l-42
  • peptide is formed from the ⁇ - amyloidogenic metabolism of APP.
  • AD may be heritable in a Familial manifestation, or may be sporadic.
  • AD includes Familial, Sporadic, as well as intermediates and subgroups thereof based on phenotypic manifestations. Familial AD typically has an early-onset (before age 65) while Sporadic AD typically is late-onset (age 65 and later).
  • Familial AD may be associated with mutations in one or more genes selected from the group comprising presenilin 1 (human presenilin 1, GenBank Accession Nos. NM 000021 , NM 007318, and NM 007319; murine presenilin 1, GenBank Accession No. NM 008943; and rat presenilin 1, GenBank Accession No. NM 019163), presenilin 2 (human presenilin 2, GenBank Accession Nos. NM 000447, and NM_012486; murine presenilin 2, GenBank Accession No. NM Ol 1 183; and rat presenilin 2, GenBank Accession No.
  • presenilin 1 human presenilin 1, GenBank Accession Nos. NM 000021 , NM 007318, and NM 007319
  • murine presenilin 1 GenBank Accession No. NM 008943 GenBank Accession No. NM 019163
  • presenilin 2 human presenilin
  • NM 031087) Amyloid Precursor Protein (APP) (human APP, GenBank Accession Nos. NM_201414, NM_201413, and NM_000484; murine APP, GenBank Accession No. NM_007471 ; and rat APP, GenBank Accession No. NM O 19288).
  • Sporadic AD can not be tested for directly, but certain risk factors may increase an individual's susceptibility to developing sporadic AD.
  • individuals with at least one copy of the e4 allele of Apolipoprotein E (APOE) (human APOE, GenBank Accession No. NM_000041 ; murine APOE, GenBank Accession No. NM_009696; and rat APOE, GenBank Accession No. NM l 38828) are at risk of developing late-onset sporadic AD.
  • APOE Apolipoprotein E
  • This term also includes individuals with Down syndrome (DS) who invariably develop (in their third or fourth decade) cerebral amyloid (A ⁇ ) plaques and neurofibrillary tangles (NFTs), the characteristic lesions of Alzheimer disease (AD).
  • DS Down syndrome
  • a ⁇ cerebral amyloid
  • NFTs neurofibrillary tangles
  • AD Alzheimer disease
  • Recent studies have shown that the A ⁇ 42 is the earliest form of this protein deposited in Down syndrome brains, and may be seen in subjects as young as 12 years of age, and that soluble A ⁇ can be detected in the brains of DS subjects as early as 21 gestational weeks of age, well preceding the formation of A ⁇ plaques (Gyure et al., Archives of Pathology and Laboratory Medicine. 2000; 125:. 489-492).
  • a "neurological disorder” refers to any central nervous system (CNS) or peripheral nervous system (PNS) disease that is associated with the ⁇ -amyloidogenic processing of Amyloid Precursor Protein. This may result in neuronal or glial cell defects including but not limited to neuronal loss, neuronal degeneration, neuronal demyelination, gliosis (i.e. , astrogliosis), or neuronal or extraneuronal accumulation of aberrant proteins or toxins (e.g., amyloid- ⁇ ).
  • CNS central nervous system
  • PNS peripheral nervous system
  • CAA congophilic angiopathy
  • amyloid angiopathy This disorder is a form of angiopathy in which the same amyloid protein that is associated with Alzheimer's disease, amyloid- ⁇ (A ⁇ ), deposits in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessels to failure, increasing the risk of a hemorrhagic stroke. Since it is the same amyloid protein that is associated with Alzheimer's dementia, such brain hemorrhages are more common in people who suffer from Alzheimer's, however they can also occur in those who have no history of dementia.
  • CAA cerebral hemorrhage
  • the hemorrhage within the brain is usually confined to a particular lobe and this is slightly different compared to brain hemorrhages which occur as a consequence of high blood pressure (hypertension) - a more common cause of a hemorrhagic stroke (or cerebral hemorrhage).
  • CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.
  • patient or “patient population” refers to individual(s) diagnosed as having Alzheimer's Disease or at risk of developing Alzheimer's Disease.
  • individuals are diagnosed, or at risk of developing Familial AD.
  • the individual is diagnosed as having, or at risk of developing, Sporadic AD.
  • Diagnosis of AD may be made based on genotypic or phenotypic characteristics displayed by the individual. For example, an individual with a mutant variant of presenilin 1, presenilin 2, or APP are at risk of developing familial AD. In another, non- limiting example, individuals with the E4 variant of APOE are at risk for developing Sporadic AD.
  • Phenotypes associated with AD may be cognitive or psychiatric.
  • cognitive phenotypes include, but are not limited to, amnesia, aphasia, apraxia and agnosia.
  • psychiatric symptoms include, but are not limited to, personality changes, depression, hallucinations and delusions.
  • DSM-IV- TR Diagnostic and Statistical Manual of Mental disorders, 4th Edition (DSM-IV- TR) (published by the American Psychiatric Association) contains the following set of criteria for dementia of the Alzheimer's type:
  • the cognitive deficits are not due to other central nervous system, systemic, or substance-induced conditions that cause progressive deficits in memory and cognition;
  • NINDS-ADRDA National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorder Association
  • A. Definite Alzheimer's disease meets the criteria for probable Alzheimer's disease and has histopathologic evidence of Alzheimer's disease via autopsy or biopsy
  • Probable Alzheimer's disease dementia established by clinical and neuropsychological examination and involves
  • Alzheimer's disease a dementia syndrome with an atypical onset, presentation, or progression and without a known etiology; any co-morbid diseases capable of producing dementia are not believed to be the cause
  • D Unlikely Alzheimer's disease: a dementia syndrome with any of the following: sudden onset, focal neurologic signs, or seizures or gait disturbance early in the course of the illness. Phenotypic manifestations of AD may also be physical, such as by the direct (imaging) or indirect (biochemical) detection of amyloid- ⁇ plaques. Quantitation of amyloid- ⁇ (1 -40) in the peripheral blood has been demonstrated using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap (Du et al., J Biomol Tech. 2005;16(4):356-63).
  • ⁇ -amyloid In vivo imaging of ⁇ -amyloid can be achieved using radioiodinated flavone derivatives as imaging agents (Ono et al., J Med Chem. 2005;48(23):7253-60) and with amyloid binding dyes such as putrescein conjugated to a 40-residue radioiodinated A peptide (yielding 125 I-PUT-A 1-40), which was shown to cross the blood-brain barrier and bind to ⁇ plaques (Wengenack et al., Nature Biotechnology. 2000; 18(8): 868-72).
  • Pharmacological inhibitors of ⁇ -secretase include, but are not limited to, hydroxamic acid-based zinc metal loproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I, TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
  • Batimastat also known as BB-94, is a low molecular weight synthetic inhibitor of metal loproteinase activity that functions by binding the zinc ion in the active site of MMP's.
  • the batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP.
  • Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J.A., et al., Clinical Cancer Research 1996; 2: 1207-1214).
  • Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for ⁇ -secretase.
  • ADAM A Disintegrin And Metalloproteinase
  • batimastat, and its analogs function as chaperones to traffic ⁇ -secretase out of the endoplasmic reticulum
  • the ⁇ -secretase protein stabilized in a proper conformation is transported from the endoplasmic reticulum to a site of APP metabolism.
  • the pharmacological chaperone may be an inhibitor, or structurally similar analog thereof, of ⁇ -secretase.
  • inhibitors of ⁇ -secretase include, but are not limited to, the group comprising hydroxamic acid-based zinc metalloproteinase inhibitors.
  • hydroxamic acid- based zinc metalloproteinase inhibitors include, but are not limited to, the group comprising hydroxamic acid-based zinc metalloproteinase inhibitors.
  • hydroxamic acid- based zinc metalloproteinase inhibitors include hydroxamic acid- based zinc metalloproteinase inhibitors:
  • Marimastat having the structure:
  • TAPI-O having the structure:
  • TAPI-2 having the structure:
  • Immunex compound 3 having the structure:
  • BB21 16 having the structure: GM6001, having the structure:
  • rNCB8765 having the structure (l R,3S,4S)-3-[(hydroxyamino)carbonyl]-4-[(4- phenylpiperidin-l -yl)carbonyl]cyclohexyl pyrrolidine- 1 -carboxy late.
  • BB3103 is another MMP inhibitor and is available from British Biosciences.
  • Batimastat also known as BB-94, is a low molecular weight synthetic inhibitor of metalloproteinase activity that functions by binding the zinc ion in the active site of MMP's.
  • the batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP.
  • Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J. A., et al., Clinical Cancer Research 1996; 2: 1207-1214).
  • Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for ⁇ -secretase.
  • ADAM A Disintegrin And Metalloproteinase
  • batimastat, and its analogs may function as chaperones to traffic ⁇ - secretase out of the endoplasmic reticulum.
  • ADAMl O inhibitors include GW280264X and GI254023X, which are described in Hundhausen et al., Blood. 2003; 102: 1 186-1 195, have the following structures:
  • ⁇ -secretase inhibitors include ADAMl O inhibitors as described in PCT publication WO03/106381 , to Bannen et al., owned by Exelixis, Inc. Such compounds are provided in the following Table:
  • TACE inhibitors include IK682, several hydroxamate-cyclopropyl compounds and series of spirocyclopropyl compounds, which are described in Guo et al., Bioorganic & Medicinal Chemistry Letters. 2009; 19: 54-57.
  • IK682 has the structure:
  • hydroxamate-cyclopropyl compounds had the structure:
  • TACE inhibitors are given in Becherer et al., book chapter "The Tumor Necrosis Factor-alpha Converting Enzyme" and are shown in the following Table:
  • EWG refers to an electron withdrawing group.
  • inhibitors disclosed are several methanesulfonyl-arginine and pyridylsulfonyl-arginine inhibitors according to the structure:
  • GI254023X having the structure:
  • the present invention also provides methods for treating a condition associated with reduced protein stability, activity, and/or trafficking, or a condition that would benefit by an increase of basal level protein stability, activity, and/or trafficking, by administering to a subject in need of such treatment a pharmacological chaperone to enhance protein stability, activity, and/or trafficking of the protein.
  • the subject to be treated can be a subject who does not exhibit a mutation in the protein that affects folding and processing of the protein, but who would benefit from increased protein stability, activity, and/or trafficking.
  • the subject to be treated can also have a mutation in the protein that affects protein function, other than a mutation that affects folding and processing of the protein, and exhibits reduced protein levels in cells that normally express the protein.
  • the subject is homozygous for the wild-type ⁇ -secretase protein. In another embodiment, the subject is heterozygous for the wild-type ⁇ -secretase protein and has a mutant genotype with a null phenotype for the other allele encoding the ⁇ -secretase protein.
  • a pharmacological chaperone i.e , an agonist or antagonist or other compound as described above or as identified through the screening methods of the invention as set forth below, is advantageously formulated in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmacological chaperone may be designated as an active ingredient or therapeutic agent for the treatment of a disease or disorder that would benefit from an increase in protein activity, conformational stability, and/or trafficking.
  • the concentration of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Exemplary dose ranges of the active ingredient are from about 0.01 mg/kg to about 250 mg/kg of body weight per day; from about 10 mg/kg to about 100 mg/kg per day; or from about 10 mg/kg to about 75 mg/kg per day.
  • Batimastat has been administered to cancer patients at doses of 600 to 1050mg/m 2 (intraperitoneal Iy).
  • Maramistat has been orally administered in pancreatic cancer patient at doses of 5, 10, or 25 mg b.i.d.
  • Therapeutically effective compounds can be provided to a subject in standard formulations, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavorants, colorants, buffers, and disintegrants.
  • Standard formulations are well known in the art. See e.g., Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, 2000.
  • the formulation may be produced in useful dosage units for administration by any route that will permit the therapeutic chaperone to cross the blood- brain barrier.
  • Exemplary routes include oral, parenteral, transmucosal, intranasal, inhalation, or transdermal routes.
  • Parenteral routes include intravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration.
  • a pharmacologic chaperone is formulated in a solid oral dosage form.
  • the pharmaceutical composition may take the form of a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents ⁇ e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants ⁇ e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents ⁇ e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stea
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • a pharmacological chaperone is formulated for parenteral administration.
  • the pharmacological chaperone may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the chaperone may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the pharmacological chaperone can be delivered in a vesicle, particularly a liposome.
  • the pharmacological chaperone can be delivered in a controlled release manner.
  • a therapeutic agent can be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly- lactic/glutamic acid (PLGA), in a pellet containing a mixture of cholesterol and the active ingredient (SilasticRTM; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601), by subcutaneous implantation, or by transdermal patch. Combination Therapy.
  • the pharmaceutical composition may also include other biologically active substances in combination with the candidate compound (pharmacological chaperone) or may be administered in combination with other biologically active substances.
  • the pharmacological chaperone can be used to treat patients with Alzheimer's Disease in combination with other drugs that are also used to treat the disorder.
  • exemplary non-limiting pharmacological agents approved in the United States for the treatment of Alzheimer's include cholinesterase inhibitors such as Cognex® (tacrine), Aricept® (donepezil), Exelon® (rivastigmine), Reminyl® (galantamine), and NMDA receptor antagonists such as Namenda® (memantine).
  • Other potential therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos. 5,863,902; 5,872,101 ; inhibitors of ⁇ -amyloid production such as described in U.S. Patent Nos.
  • the pharmacological chaperone can be used in combination with gene therapy.
  • Gene therapy is contemplated both with replacement genes such as nerve growth factor (NGF), or with inhibitory RNA (siRNA) for enzymes that are involved in the amyloidogenic cleavage of APP (BACE-I).
  • NGF nerve growth factor
  • siRNA inhibitory RNA
  • BACE-I amyloidogenic cleavage of APP
  • contemplated combination therapy includes combinations of specific pharmacological chaperones with vaccine therapy, such as described in U.S. Patent Nos. 6,866,860 and 6,761 ,888.
  • the present invention further provides a method for identifying a candidate pharmacological chaperone compound that modulates the stability, activity, and/or trafficking of an ⁇ -secretase polypeptide.
  • the present invention provides a method for identifying a chaperone for a target protein, which comprises bringing a labeled or unlabeled test compound in contact with the protein or a fragment thereof and measuring the amount of the test compound bound to the protein or to the fragment thereof. For example, this can be achieved as follows: (a) contacting a first cell with a test compound for a time period sufficient to allow the cell to respond to said contact with the test compound;
  • step (b) determining the conformational stability, activity, and/or trafficking of the target protein (or a fragment thereof comprising a ligand binding domain) in the cell (or on the cell surface) contacted in step (a);
  • step (c) comparing the conformational stability, activity, and/or trafficking of the protein determined in step (b) to that of a protein in a control cell that has not been contacted with the test compound;
  • a detectable change in the conformational stability, activity, and/or trafficking of the protein in the first cell in response to contact with the test compound compared to that same value of the protein in the control cell that has not been contacted with the test compound indicates that the test compound modulates the conformational stability, activity, and/or trafficking of the protein and is a candidate compound for the treatment of a disorder that would benefit from an increase in conformational stability, protein activity and/or proper trafficking of the target protein.
  • the ⁇ -secretase protein can either be expressed in a host cell transformed with a vector encoding a non-endogenous ⁇ -secretase protein or expressed from an endogenous gene in the cell.
  • test compounds can be, without limitation, a small organic or inorganic molecule (preferred), a peptide or a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule (such as an aptamer), a polynucleotide molecule, or a chimera or derivative thereof.
  • Test compounds which are candidate pharmacological chaperones that specifically bind to an ⁇ -secretase protein can be identified using cell-based and/or cell-free assays.
  • preferred classes of compounds that may be identified include, but are not limited to/small molecules (i.e., organic or inorganic molecules which are less than about 2 kilodaltons (kD) in molecular weight, and, more preferably, less than about 1 kD in molecular weight).
  • small molecules i.e., organic or inorganic molecules which are less than about 2 kilodaltons (kD) in molecular weight, and, more preferably, less than about 1 kD in molecular weight).
  • ICCB Institute of Chemistry and Cell Biology
  • Chem Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds); CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2 (704 compounds); Peakdale 1 (2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and International (28,864 compounds); Mixed Commercial Plate 1 (352 compounds); Mixed Commercial Plate 2 (320 compounds); Mixed Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331 compounds); ChemBridge Microformat (50,000 compounds); Commercial Diversity Set 1 (5,056 compounds); NCI Collections: Structural Diversity Set, version 2 (1,900 compounds); Mechanistic Diversity Set (879 compounds); Open Collection 1 (90,000 compounds); Open Collection 2 (10,240 compounds); Known Bioactives Collections: NTNDS Custom Collection (1 ,040 compounds); ICCB Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds); ICCB Discretes Collections.
  • ICCBl 190 compounds
  • ICCB2 352 compounds
  • ICCB3 352 compounds
  • ICCB4 352 compounds
  • Natural Product Extracts NCI Marine Extracts (352 wells); Organic fractions - NCI Plant and Fungal Extracts (1,408 wells); Philippines Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis (DOS) Collections; DDSl (DOS Diversity Set) (9600 wells).
  • ArQuIe, Inc. (Woburn, MA) also has integrated technologies to perform high-throughput, automated production of chemical compounds and to deliver these compounds of known structure and high purity in sufficient quantities for lead optimization. Its AMAPTM (Automated Molecular Assembly Plant) performs high-throughput chemical syntheses for each phase of compound discovery.
  • HTS screening for ⁇ -secretase protein pharmacological chaperones employs microarrays.
  • Protein arrays are solid-phase, binding assay systems using immobilized proteins on various surfaces that are selected for example from glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
  • the binding assays using these arrays are highly parallel and often miniaturized. Their advantages are that they are rapid, can be automated, are capable of high sensitivity, are economical in their use of reagents, and provide an abundance of data from a single experiment.
  • Automated multi-well formats are the best-developed HTS systems. Automated 96- or 384-well plate-based screening systems are the most widely used. The current trend in plate-based screening systems is to reduce the volume of the reaction wells even further, and increase the density of the wells per plate (96 wells to 384 wells to 1 ,536 wells per plate). The trend results in increased throughput, dramatically decreased bioreagent costs per compound screened, and a decrease in the number of plates that need to be managed by automation.
  • protein arrays that can be used for HTS, see e.g. : U.S. Patents No. 6,475,809; 6,406,921 ; and 6,197,599; and International Publication Nos. WO 00/04389 and WO 00/07024.
  • sources of proteins or fragments thereof can include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for making peptides.
  • cell-based expression systems for recombinant proteins purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for making peptides.
  • synthetic methods for making peptides For capture arrays and protein function analysis, it is often the case that ⁇ -secretase proteins are correctly folded and functional.
  • the immobilization method used is preferably applicable to proteins of different properties (e.g., wild-type, mutant, full-length, partial- length fragments, hydrophilic, hydrophobic, etc ⁇ ), amenable to high throughput and automation, and generally compatible with retention of chaperone-binding ability.
  • Both covalent or non-covalent methods of protein immobilization can be used.
  • Substrates for covalent attachment include, e.g., glass slides coated with amino- or aldehyde-containing silane reagents (Telechem).
  • VersalinxTM system Prolinx
  • reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface.
  • Covalent coupling methods providing a stable linkage can be applied to a range of proteins.
  • Non-covalent binding of unmodified protein occurs within porous structures such as HydroGelTM (PerkinElmer), based on a
  • Cell-Based Arrays combine the technique of cell culture in conjunction with the use of fluidic devices for measurement of cell response to test compounds in a sample of interest, screening of samples for identifying molecules that induce a desired effect in cultured cells, and selection and identification of cell populations with novel and desired characteristics.
  • High-throughput screening can be performed on fixed cells using fluorescent-labeled antibodies, biological ligands or candidate chaperones and/or nucleic acid hybridization probes, or on live cells using multicolor fluorescent indicators and biosensors. The choice of fixed or live cell screens depends on the specific cell-based assay required.
  • Transfected cell microarrays are a complementary technique in which array features comprise clusters of cells overexpressing defined cDNAs. Complementary DNAs cloned in expression vectors are printed on microscope slides, which become living arrays after the addition of a lipid transfection reagent and adherent mammalian cells (Bailey et al., Drug Discov. Today 2002; 7(18 Suppl): Sl 13-8).
  • Cell-based arrays are described in detail in, e.g., Beske, Drug Discov. Today 2002; 7(18 Suppl): Sl 31-5; Sundberg et al., Curr. Opin. Biotechnol. 2000; 1 1 : 47-53; Johnston et al., Drug Discov. Today 2002; 7: 353-63; U.S. Patent Nos. 6,406,840 and 6,103,479, and U.S. published patent application no. 2002/0197656.
  • Detectable labels For detection of molecules such as candidate ⁇ -secretase protein pharmacological chaperones using screening assays, a functional assay can be used to follow unlabeled molecules as described elsewhere herein.
  • a molecule-of-interest ⁇ e.g., a small molecule, an antibody, or a polynucleotide probe
  • a library of same can also be detectably labeled with an atom (e.g., a radionuclide), a detectable molecule ⁇ e.g., fluorescein), or a complex that, due to a physical or chemical property, serves to indicate the presence of the molecule of interest.
  • a molecule can also be detectably labeled when it is covalently bound to a "reporter" molecule (e.g., a biomolecule such as an enzyme) that acts on a substrate to produce a detectable product.
  • reporter molecules e.g., a biomolecule such as an enzyme
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • Labels useful in the present invention include, but are not limited to, biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX from Amersham, SyBR Green I & II from Molecular Probes, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactor
  • radiolabels and chemiluminescent labels can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photo-detector to detect emitted light (e.g., as in fluorescence-activated cell sorting, FACS); and enzymatic labels can be detected by providing the enzyme with a substrate and detecting, e.g., a colored reaction product produced by the action of the enzyme on the substrate.
  • lead compounds are evaluated in wild-type animals to determine whether protein activity, stability, or trafficking is increased in response to the administration of the test compound.
  • enhanced ⁇ -secretase activity, and/or cellular localization can be determined by measuring an increase in cellular polypeptide, by determining an increase in trafficking to the appropriate cellular location, and by detecting increased ⁇ -secretase protein activity.
  • Non-limiting exemplary methods for assessing each of the foregoing are described below.
  • Determining protein level Methods for determining intracellular protein levels are well-known in the art. Such methods include Western blotting, immunoprecipitation followed by Western blotting (IP Western), or immunofluorescence using a tagged protein.
  • Determining ⁇ -secretase protein trafficking and localization Assessing trafficking of proteins through the biosynthetic pathway can be achieved e.g., using pulse- chase experiments with 35 S-labeled receptor protein, in conjunction with glycosidases; or by indirect or direct immunofluorescence to determine protein modification during trafficking. These and other methods are described for example in Current Protocols in Cell Biology 2001 ; John Wiley & Sons.
  • Sensitive methods for visually detecting cellular localization also include fluorescent microscopy using fluorescent proteins or fluorescent antibodies.
  • ⁇ -secretase protein can be tagged with e.g., green fluorescent protein (GFP), cyan fluorescent protein, yellow fluorescent protein, and red fluorescent protein, followed by multicolor and time-lapse microscopy and electron microscopy to study the fate of these proteins in fixed cells and in living cells.
  • GFP green fluorescent protein
  • cyan fluorescent protein cyan fluorescent protein
  • yellow fluorescent protein yellow fluorescent protein
  • red fluorescent protein followed by multicolor and time-lapse microscopy and electron microscopy to study the fate of these proteins in fixed cells and in living cells.
  • Fluorescence correlation spectroscopy is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution (Vukojevic et al., Cell MoI Life Sci 2005; 62(5): 535-50).
  • SPFI single-particle fluorescence imaging
  • For localization of proteins within lipid rafts see Latif et al., Endocrinology 2003; 144(1 1): 4725-8).
  • live cell imaging see Hariguchi, Cell Struct Funct 2002; 27(5):333-4).
  • Fluorescence resonance energy transfer (FRET) microscopy is also used to study the structure and localization of proteins under physiological conditions (Periasamy, J Biomed Opt 2001 ; 6(3): 287-91).
  • less sensitive assays can be used to detect whether they are present on the membrane.
  • Such methods include immunohistochemistry of fixed cells, or whole-cell labeling using radiolabeled ligand (e.g., 125 I).
  • the next step is determining whether the candidate compound can enhance the amount of ⁇ -secretase protein trafficked to the appropriate cellular location.
  • Numerous assays can be used to evaluate protein levels quantitatively.
  • radioactive ligand binding assays using e.g., ' 5 I-MSH, can be used to determine binding to either whole cells expressing ⁇ -secretase protein or to cell membrane fractions. See U.S. published application 2003/0176425 for a description of one exemplary method; see also Chhajlani, Peptides. 1996; 17(2):349-51.
  • immunofluorescence staining using either labeled antibodies or labeled ⁇ -secretase protein ⁇ e.g., FLAG-tagged ⁇ -secretase protein
  • FACS fluorescence-activated cell sorting
  • Activity assays for proteins are generally well-known in the art, and can include substrate metabolism or modification (such as phosphorylation or dephosphorylation), signal transduction, and changes to cellular phenotype.
  • host cell means any cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way, for the production of a desired substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein, or an enzyme. According to the present invention, the host cell is modified to express ⁇ -secretase-type nucleic acid and polypeptide. Host cells can further be used for screening or other assays. Exemplary host cells for use in the present invention are HEK-293T and COS cells.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • Polynucleotides herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non- coding regions, and the like.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include: methylation, "caps,” substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • express when used in the context of producing an amino acid sequence from a nucleic acid sequence, means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of the corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product", i.e., protein.
  • the expression product itself e.g., the resulting protein, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • heterologous refers to a combination of elements not naturally occurring in combination.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., an E. coli cell.
  • recombinantly engineered cell refers to any prokaryotic or eukaryotic cell that has been manipulated to express or overexpress the nucleic acid of interest, i.e., a nucleic acid encoding a polypeptide, by any appropriate method, including transfection, transformation or transduction. This term also includes endogenous activation of a nucleic acid in a cell that does not normally express that gene product or that expresses the gene product at a sub-optimal level.
  • vector means the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.; they are well known in the art.
  • This example describes a randomized, blinded, Phase Ib study of twice daily oral doses of 1-deoxygalactonojirimycin (DGJ) to evaluate the affects of DGJ on safety, tolerability, pharmacokinetics, and ⁇ -Galatosidase A ( ⁇ -GAL) enzymantic activity in healthy volunteers. It demonstrates the power of the pharmacological chaperone approach to increase the levels and/or activity of a wild-type enzyme in the normal, healthy subject.
  • DGJ 1-deoxygalactonojirimycin
  • Study Design and Duration This study was first-in-man, single-center, Phase Ib, randomized, double-blind, twice daily-dose, placebo controlled study to evaluate the safety, tolerability, pharmacokinetics, and ⁇ -GAL enzymantic activity affects of DGJ following oral administration.
  • the study tested two groups of of 8 subjects (6 active and 2 placebo) who received a twice daily-dose of 50 or 150 mg b.i.d. of DGJ or placebo administered orally for seven consecutive days, accompanied by a seven day follow up visit. Subjects were housed in the treatment facility from 14 hours prior to dosing until 24 hours after dosing. Meals were controlled by schedule and subjects remained abulatory for 4 hours post drug administration
  • Safety and Tolerability Assessments were determined by evaluating vital signs, laboratory parameters (serum chemistry, hematology, and urinalysis), ECGs, physical examination and by recording adverse events during the Treatment Period.
  • WBC a-GAL A Enzymatic Activity Sampling Blood samples (10 itiL each) were collected in blood collection tubes containing EDTA and WBC extracted before dosing and at the following times thereafter: 100 hours, 150 hours, and 336 hours. Samples were treated as described above, and WBC ⁇ -GAL enzymatic activity levels were determined as described in Desnick, R.J. (ed) Enzyme therapy in genetic diseases. VoI 2. Alan R Liss, New York, pp 17-32. Statistical Analysis. Safety data including laboratory evaluations, physical exams, adverse events, ECG monitoring and vital signs assessments were summarized by treatment group and point of time of collection.
  • Descriptive statistics (arithmetic mean, standard deviation, median, minimum and maximum) were calculated for quantitative safety data as well as for the difference to baseline. Frequency counts were compiled for classification of qualitative safety data.
  • a shift table describing out of normal range shifts was provided for clinical laboratory results.
  • a normal-abnormal shift table was also presented for physical exam results and ECGs.
  • Adverse events were coded using the MedDRA version 7.0 dictionary and summarized by treatment for the number of subjects reporting the adverse event and the number of adverse events reported. A by-subject adverse event data listing including verbatim term, coded term, treatment group, severity, and relationship to treatment was provided. Concomitant medications and medical history were listed by treatment.
  • Pharmacokinetic parameters were summarised by treatment group using descriptive statistics (arithmetic means, standard deviations, coefficients of variation, sample size, minimum, maximum and median).
  • C m3x of DGJ increased in a dose-proportional manner when doses were increased from 50 mg to 150 mg.
  • the mean percentage of DGJ excreted over the 12-hour post dose period was 16% and 42% at dose levels of 50 and 150 mg, respectively, on Day 1, increasing to 48% and 60%, respectively, on Day 7.
  • a-Galactosidase A (a-Gal A) Enzymatic Activity.
  • the ⁇ -GAL enzymatic activity data obtained during the study is shown in Figure 1.
  • DGJ did not inhibit WBC ⁇ -GAL enzymatic activity in subjects at dosages of 50 mg b.i.d. or 150 mg b.i.d.
  • AT- 1001 produced a dose-dependent trend of increased WBC ⁇ -GAL activity in healthy volunteers.
  • ⁇ -GAL enzymatic levels were measured in WBC of subjects administered placebo, 50 mg b.i.d. DGJ, and 150 mg b.i.d. DGJ. Placebo had no affect on WBC ⁇ -GAL enzymatic levels.
  • Blood samples were collected for pharmacokinetic analysis before the initial drug administration on Day 1 , before the 5th, 6th and 7th doses (on Days 5, 6 and 7) (for Cmin determination), and at the following times after the 1st (Day 1 ) and 7th (Day 7) doses: 0.5, 1 , 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, and 24 hours.
  • a single blood sample was collected 48 hours after the last dose (Day 9) and assayed for the presence of IFG.
  • blood samples were collected for pharmacodynamic measurements, i.e., analysis of WBC GCase levels, before dosing on Day 1 , Day 3, Day 5, and Day 7, and at return visits on Day 9, Day 14 and Day 21.
  • Patients were administered DGJ according to the following dosing schedule: 25 mg b.i.d. two weeks; 100 mg b.i.d. 2-4 weeks; 250 mg b.i.d. 4-6 weeks; and 25 mg b.i.d. 6-12 weeks; blood was draw into an 8 mL Vacutainer CPT tube at the end of each dosing period and treated as described below.
  • WBCs were prepared substantially as described in Example 1 , with the exception that no FBS/DMSO is added to the pellet prior to freezing.
  • lysis buffer 26 mM citrate/46 mM phosphate, pH 5.5
  • Tubes were incubated at room temperature for about 15 minutes, but agitate the suspension by vortexing every couple of minutes
  • Tubes were sonicated for 2 minutes, then vortexed for about 10 seconds
  • Container was vortexed and pooled lysates were divided into 0.100 mL aliquots in pre-chilled labeled 0.5 mL screw-cap polypropylene microcentrifuge tubes. Pooled lysates were mixed while aliquoting by vortexing between every 10-20 aliquots.
  • EXAMPLE 4 In vitro Stabilization of purified ADAMlO by TAPI-2, GM6001 and GW4023
  • Figure 3 depicts a time course (96 hr) showing the effects of GM6001 (Figure 3A) and TAPI-2 (Figure 3B) on ADAMlO levels in SY5Y neuroblastomas.
  • Figure 3C depicts a dose-response showing the effects of TAPI-2 on ADAMlO precursor levels in SY5Y neuroblastomas.
  • ADAMlO has a T m of 52 0 C.
  • the ADAMlO inhibitors TAPI-2 and GW4023 shift the T n , by 9 0 C and 16 0 C, respectively, demonstrating their ability to stabilize wilt-type ADAMlO.
  • SY5Y neuroblastoma cells in 6-well plates were dosed with lOOuM of alpha secretase inhibitors GM6001 (AA) or TAPI-2 (AB) in complete growth media (DMEM+10%FBS) for up to 96hrs.
  • AA alpha secretase inhibitor
  • TAPI-2 TAPI-2
  • DMEM+10%FBS complete growth media
  • Ohrs no dose control
  • 15hrs, 24hrs, 48hrs, 72hrs and 96hrs cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem.
  • ADAMlO was probed using a rabbit anti- ADAMlO polyclonal antibody (1 : 1000, Abeam cat. No. Abl997) and anti-rabbit/alkaline- phosphatase secondary antibody.
  • ADAMl O bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
  • SY5Y neuroblastoma cells in 6-well plates were dosed with OuM, 0.01 uM, 0. IuM, IuM, l OuM and lOOuM of the alpha secretase inhibitor TAPI-2 in complete growth medium (DMEM+10% FBS). After 96hrs, cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem. Co.) and equal amounts of protein were electrophoresed on a 4-12% Bis-NuPage gel (Invitrogen) followed by transfer (25V, l hr) onto PVDF for Western blotting.
  • ADAM lO was probed using a rabbit anti-ADAMIO polyclonal antibody (1 : 1000, Abeam cat. No. Ab 1997) and anti-rabbit/alkaline-phosphatase secondary antibody.
  • ADAMlO bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
  • the environmentally sensitive dye SYPRO Orange and a Realplex Mastercycler qRT- PCR system were used to apply a temperature gradient while simultaneously monitoring fluorescence changes in a 96-well format.
  • ADAMlO in neutral buffer (50 mM sodium phosphate, 150 mM sodium chloride, 2 ⁇ M zinc chloride, pH 7.4) was combined with 2.5 ⁇ l 50X SYPRO Orange and 20 ⁇ M TAPI-2 in a reaction volume of 25 ml. Once assembled, the plate is heated at a rate of l°C/min, and fluorescence (Ex. 470 nm; Em. 520 nm) intensities are measured and plotted as a function of temperature.
  • ADAMl O demonstrates a typical thermal denaturation curve with a T m (melting temperature) of 52 0 C.
  • T m melting temperature
  • the T m is shifted to 61 0 C, a shift of 9 0 C (Fig. 2A), while another more potent inhibitor GW4023 shifted the T m by 16 0 C (data not shown).
  • GW4023 is also known as GI254023X

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Abstract

The invention relates to methods of enhancing normal α-secretase expression, and to enhancing expression of an α-secretase having a mutation which affects protein folding and/or processing of the α-secretase. The invention provides a method of treating an individual having a condition in which increased expression or activity of an α-secretase in the central nervous system would be beneficial, by administering an effective amount of a pharmacologic chaperone for the α-secretase. The invention provides α-secretase inhibitors which have been identified as specific pharmacologic chaperones which enhance expression or enzymatic activity of α-secretase in the central nervous system.

Description

PHARMACOLOGICAL CHAPERONES FOR THE TREATMENT OF
ALZHEIMER'S DISEASE
FIELD OF THE INVENTION
The present invention relates to a method for treating an individual having a neurological disorder with an associated increase in the β-amyloidogenic metabolism of amyloid precursor protein (APP). Specifically, the individual is administered a pharmacological chaperone for α-secretase, which increases trafficking of the α-secretase protein from the ER to the cytoplasm in neural cells, concomitantly increasing α-secretase enzyme activity in neural cells.
BACKGROUND
Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disorder characterized neuropathologically by the presence of amyloid-β plaques, neurofibrillary tangles, and gray matter loss. In AD, multiple regions of brain gray matter have a profound neuronal loss, including basal forebrain, hippocampus, entorhinal, and temporal cortices. Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called Tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability. The extracellular amyloid-β plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid-β (Aβl -42) peptide and have led to the foundation of the amyloid hypothesis. This hypothesis postulates that Aβl-42 is one of the principal causative factors of neuronal death in the brains of Alzheimer's patients.
AD is set to become the developed world's largest socioeconomic healthcare burden over the coming decades. AD is thought to affect 4-8% of the population over 65 years of age, with the incidence continuing to increase with increasing age. Current U.S. estimates on the numbers of patients suffering from the disease range from three to five million, with an annual estimated cost of approximately $100 billion dollars. It is estimated that by 2050 the number of patients with AD could be as high as 25 million (Brookmeyer R., et al., Am J Public Health 1998; 88: 1337-134). Antemortem clinical diagnosis of AD is difficult and requires a recorded decline in cognitive function as well as evidence of progressive deficits in other behavioral areas such as executive function and language skills. Unqualified diagnosis of AD can still only be made neuropathologically postmortem by examination of patients' brains and the detection of amyloid-β plaques and tangles.
The majority of Alzheimer cases seem to be sporadic or to result from complex interactions of several genes. Nevertheless, a minority of AD cases (<1%) result from autosomal dominant inheritance of an age-dependent trait with high penetrance (Tanzi, R. E., et al., Neuron 2001 ; 32: 181-184; St George-Hyslop, P.H., Ann NYAcad Sci 2000; 924: 1-7).
Aβ is a hydrophobic 39- to 42-amino acid peptide, found in all biological fluids, and derived from the enzymatic cleavage of a larger type I membrane protein, the amyloid precursor protein (APP) (Hardy, J., et al., Science 200; 297: 353-356). A number of key findings have led people to postulate a central role for this peptide in the etiology and pathogenesis of the disease. Linkage studies of familial AD patients identified a number of mutations in two genes, APP and presenilin, associated with aberrant metabolism of APP and an increased production of aggregating forms of Aβ. Furthermore, Down syndrome (trisomy 21) patients who have high levels of Aβ deposits in their brains and dementia from an early age have three copies of the APP gene (Robakis N. K., et al., Neurobiol Aging 1994; 15 [Suppl 2]: S127-S129). Individuals carrying the apolipoprotein E4 (APOE4) genotype also have an increased risk of developing AD compared with APOE2 or APOE3 individuals.
Mutations in APP do not account for all cases of familial AD. In the mid 1990s, further genetic linkage studies uncovered mutations in presenilin 1 on chromosome 14 (Sherrington R., et al., Nature 1995; 375: 754-760, 1995) and presenilin 2 on chromosome 1. Presenilin forms the active site of the γ-secretase complex involved in the production of Aβ, particularly the Aβl-42 form. Cleavage of APP by γ and β-secretases produces the Aβ peptide, which aggregates into plaques (De Strooper, B., Neuron 2003; 38: 9-12, 2003).
The β-amiloydogenic pathway involves the sequential proteolysis of APP by β- secretase (BACE) followed by γ-secretase. Although this is a minor APP processing route, it is this pathway that generates Aβ fragments believed to give rise to AD. In humans, two β- secretase genes have been identified, referred to as BACE-I and BACE-2, colocalized with APP in the endosomal compartment (Vassar, R. et al., Science 1999; 286: 735-741 ). Whereas both can process APP at the same site, only BACE-I is significantly expressed in brain, particularly in neurons, indicating that neurons are the major source of amyloid-β peptides in brain. Because BACE-2 is expressed in heart, kidney, and placenta, drugs developed as β-secretase inhibitors may need to be selective against BACE-2 to prevent unwanted peripheral side effects in the clinic The predominant pathway by which APP is processed does not give rise to Aβ fragments, and hence is referred to as the non-β-amyloidogenic pathway. The initial APP processing involves the cleavage of APP by α-secretase. The identification of proteins with α-secretase activity is ongoing, and currently includes three members of the A Disintegrin And Metal loproteinase (ADAM) protein family. These are ADAMl O, ADAM 17 (also known as TACE (tumor necrosis factor-α converting enzyme)), and ADAM9 (Lammich S., et al., Proc Natl Acad Sci USA 1999; 96: 3922-3927; Zheng Y., et al., J Biol Chem 2004; 279: 42898^2906; Kowalska, A., Pol. J. Pharmacol 2004; 56: 171-178). Since the α-secretase cleavage site is within the Aβ sequence of APP, and none of the proteolytic fragments created by α-secretase cleavage have been associated with the generation of AD, enhanced cleavage at this site could represent a disease modifying strategy for AD.
Currently there is no cure for AD, and past work in the area of amyloid research has focused on inhibition of enzymes that are responsible for generating the amyloid-β fragment (Aβ), namely β-secretase and χ-secretase. Present advances have only provided treatments such as cognitive exercises and medicaments designed to slow the progression of AD. There remains in the art a particular need to increase α-secretase activity, to shift the APP processing equilibrium away from production of Aβ.
Treatments
The majority of efforts aimed at treating Alzheimer's Disease (AD) have focused on reducing the symptoms of AD, or retarding the progression of the disease. In particular, identification of a lower concentration of choline acetyltransferase in affected neurons of the forebrains of AD patients has lead to treatments aimed at inhibiting the hydrolysis of acetylcholine in the synaptic cleft and prolonging the level of acetylcholine at the synapse. Although this strategy has resulted in at least a partial correction of neurotransmitter levels, the therapeutic benefits have been small (Doody, R.S., et al., Arch Neurol 2001 ; 58: 427-433; and Farlow, M., et al., Eur Neurol 2000; 44: 236-241). More recently, focus has been placed upon reducing oxidative stress and glutamate induced excitotoxicity, both of which are thought to play a critical role in the neurodegenerative process of AD (Greenamyre, J.T., et al., Neurobiol Aging 1989; 10: 593-602; Mattson, M.P., et al., Ann N Y Acad Sci 1999; 893: 154-175). As such, blockade of the NMDA receptor, one of the principal excitatory glutamate receptors in the brain, has been shown to have neuroprotective effects in a number of acute preclinical in vitro and in vivo models. Additional research to develop AD therapies has focused on inhibition of β-secretase and the metabolism of APP to form Aβ peptide. This therapeutic potential was demonstrated by the findings that BACE-I knockout mice develop normally, and appear to have completely abolished the production of Aβ, suggesting that BACE-I is the principal β- secretase in neurons (Chai, H., et al., Nat Neurosci 2001 ; 4: 233-234; Luo, Y., et al., Nat Neurosci 2001 ; 4: 231-232; Roberds, S.L., et al., Hum MoI Genet 2001 ; 10: 1317-1324). Such inhibition does not preclude normal processing of APP by the non-β-amyloidogenic major pathway, and is the first step in the β-amyloidogenic cascade. Developing specific β- secretase inhibitors has been difficult, in part because there appears to be a nonlinear relationship between decrease of β-secretase activity in vivo, and a reduction of Aβ peptides in brain. Studies using heterozygous BACE-I knockout animals have shown that a 50% decrease in BACE activity leads to a much smaller decrease (-15%) of brain Aβl -42 levels. A further difficulty is the low brain penetration of most inhibitors, likely due to the fact that many of these are substrates for P-glycoprotein, plasma membrane proteins that actively extrude a wide range of amphiphilic and hydrophobic drugs from cells, and important in preventing the accumulation of several drugs in brain.
An alternative approach to developing treatments for AD may focus on the shunting of APP into the non-β-amyloidogenic metabolism of APP into sAPPα by α-secretase. The use of pharmacological inhibitors to function as pharmacological chaperones, increasing the enzymatic activity of misfolded mutant proteins, as well as normal wild type proteins has been demonstrated (see U.S. Patent Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; and 6,916,829, all incorporated herein by reference).
Molecular Chaperones Stabilize Protein Conformation
In the human body, proteins are involved in almost every aspect of cellular function. Proteins are linear strings of amino acids that fold and twist into specific three-dimensional shapes in order to function properly. Certain human diseases result from mutations that cause changes in the amino acid sequence of a protein which reduce its stability and may prevent it from folding properly. The majority of genetic mutations that lead to the production of less stable or misfolded proteins are called missense mutations. These mutations result in the substitution of a single amino acid for another in the protein. Because of this error, missense mutations often result in proteins that have a reduced level of biological activity. In addition to missense mutations, there are also other types of mutations that can result in proteins with reduced biological activity.
Proteins generally fold in a specific region of the cell known as the endoplasmic reticulum, or ER. The cell has quality control mechanisms that ensure that proteins are folded into their correct three-dimensional shape before they can move from the ER to the appropriate destination in the cell, a process generally referred to as protein trafficking. Misfolded proteins are often eliminated by the quality control mechanisms after initially being retained in the ER. In certain instances, misfolded proteins can accumulate in the ER before being eliminated.
The retention of misfolded proteins in the ER interrupts their proper trafficking, and the resulting reduced biological activity can lead to impaired cellular function and ultimately to disease. In addition, the accumulation of misfolded proteins in the ER may lead to various types of stress on cells, which may also contribute to cellular dysfunction and disease.
Endogenous molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and permit cells to survive under stresses such as heat shock and glucose starvation (Gething et al., Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et al, MoI. Biol. Cell 1993; 4.109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128). Among the endogenous chaperones, BiP (immunoglobulin heavy-chain binding protein, Grp78) is the best characterized chaperone of the ER (Haas, Curr. Top. Microbiol. Immunol. 1991 ; 167:71- 82). Like other chaperones, BiP interacts with many secretory and membrane proteins within the ER throughout their maturation. When nascent protein folding proceeds smoothly, this interaction is normally weak and short-lived. Once the native protein conformation is achieved, the molecular chaperone no longer interacts with the protein. BiP binding to a protein that fails to fold, assemble, or be properly glycosylated becomes stable, and usually leads to degradation of the protein through the ER-associated degradation pathway. This process serves as a "quality control" system in the ER, ensuring that only those properly folded and assembled proteins are transported out of the ER for further maturation, and improperly folded proteins, or unstable proteins, are retained for subsequent degradation (Hurtley et al., Annu. Rev. Cell. Biol. 1989; 5:277-307). Due to the combined actions of the inefficiency of the thermodynamic protein folding process and the ER quality control system, only a fraction of some wild-type proteins become folded into a stable conformation and successfully exit the ER. Pharmacological Chaperones Derived From Specific Enzyme Inhibitors Rescue Mutant Enzymes and Enhance Wild-Type Enzymes
It has previously been shown that the binding of small molecule inhibitors of enzymes associated with lysosomal storage diseases (LSDs) can increase the stability of both mutant enzyme and the corresponding wild-type enzyme (see U.S. Patent Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829, and 7,141 ,582 all incorporated herein by reference). In particular, it was discovered that administration of small molecule derivatives of glucose and galactose, which are specific, selective competitive inhibitors for several target lysosomal enzymes, effectively increased the stability of the enzymes in cells in vitro and, thus, increased trafficking of the enzymes to the lysosome. Thus, by increasing the amount of enzyme in the lysosome, hydrolysis of the enzyme substrates is expected to increase. The original theory behind this strategy was as follows: since the mutant enzyme protein is unstable in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is retarded in the normal transport pathway (ER -→ Golgi apparatus -→ endosomes→ lysosome) and prematurely degraded. Therefore, a compound which binds to and increases the stability of a mutant enzyme, may serve as a "chaperone" for the enzyme and increase the amount that can exit the ER and move to the lysosomes.
Since some enzyme inhibitors are known to bind specifically to the catalytic center of the enzyme (the "active site"), resulting in stabilization of enzyme conformation in vitro, these inhibitors were proposed, somewhat paradoxically, to be effective chaperones that could help restore exit from the ER, trafficking to the lysosomes, hydrolytic activity. These specific pharmacological chaperones were designated "active site-specific chaperones (ASSCs)" or "specific pharmacological chaperones" since they bound in the active site of the enzyme in a specific fashion. Pharmacological chaperone therapy has potential advantages over ERT since a small molecule can be orally administered and may have superior biodistribution compared to protein-based therapies.
In addition to rescuing the mutant enzymes, the pharmacological chaperones enhance ER secretion and activity of wild-type enzymes (see Examples). Thus, a compound that induces a stable molecular conformation of an enzyme during folding serves as a "chaperone" to stabilize the enzyme in a proper conformation for exit from the ER. As noted above, for enzymes, one such compound unexpectedly turned out to be a competitive inhibitor of the enzyme.
Enhancement of Other Mutant and Wild-Type Proteins with Chaperones In addition to the LSDs, a large and diverse number of diseases are now recognized as "conformational diseases" that are caused by adoption of non-native, unstable, protein conformations, which may lead to retardation of the protein in the ER and premature degradation of the proteins (Kuznetsov et al., N. Engl. J. Med. 1998; 339:1688-1695; Thomas et al., Trends Biochem. Sci. 1995; 20:456-459; Bychkova et al., FEBS Lett. 1995; 359:6-8; Brooks, FEBS Lett. 1997; 409: 1 15-120). Stabilization of these proteins also may be achieved using pharmacological chaperones. For example, small synthetic compounds were found to stabilize the DΝA binding domain of mutant forms of the tumor suppressor protein p53, thereby allowing the protein to maintain an active conformation (Foster et al., Science 1999; 286:2507-10). Synthesis of receptors has been shown to be rescued by small molecule receptor antagonists and ligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja- Repo et al., EMBO J. 2002; 21 :1628-37). Even pharmacological rescue of membrane channel proteins and other plasma membrane transporters has been demonstrated using channel-blocking drugs or substrates (Rajamani et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem. 1999; 274:31 123-26; Loo et al., J. Biol. Chem. 1997; 272: 709-12). There remains a need to address deficiencies in protein function not related to mutation.
SUMMARY OF THE INVENTION
As described herein, the present invention provides a method for the treatment of a neurological disorder in an individual, wherein the neurological disorder is associated with the β-amyloidogenic processing of Amyloid Precursor Protein (APP), by administering an effective amount of a specific pharmacological chaperone to treat the neurological disorder. In a specific embodiment, the individual has been diagnosed or is at risk of developing Alzheimer's disease (AD), including Familial or Sporadic forms of AD.
In one embodiment, the present invention provides a method for enhancing intracellular folding of an α-secretase polypeptide into a functional conformation by contacting an α-secretase -expressing cell with an effective amount of a specific pharmacological chaperone. Enhancing intracellular folding of α-secretase will lead to an increased proportion of a-secretase which exits the ER, resulting in enhanced enzymatic function in the cells of the central nervous system. Increased α-secretase activity would, in turn, increase the non-β-amyloidogenic metabolism of APP, and may be useful in the treatment of neurological disorders such as AD.
In one embodiment, the α-secretase polypeptide is a wild-type α-secretase polypeptide, which, for example, is encoded by a nucleic acid of the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM). Examples of ADAMs include, but are not limited to, ADAM 9, ADAM 10, and TACE/ADAM 17.
In an embodiment in which the α-secretase polypeptide is a mutant α-secretase polypeptide, in which the mutant polypeptide contains a mutation that results in reduced or improper intracellular folding of the α-secretase polypeptide, resulting in a shift of equilibrium to increased β-amyloidogenic processing of APP, a pharmacological chaperone specific for α-secretase can rescue the mutant α-secretase.
In one embodiment, the pharmacological chaperone binds the α-secretase active site. In another embodiment, the pharmacological chaperone binds a non-active site of α-secretase, which can include an allosteric site. The method of the invention comprises the administration of one or more pharmacological chaperones of α-secretase to an individual diagnosed, at risk, or suspected to have Alzheimer's Disease. Suitable pharmacological chaperones include any compound(s) which, following administration to an individual, will bind to α-secretase and increase α-secretase enzymatic activity. In one particular embodiments, the pharmacological chaperone is a reversible inhibitor of α-secretase. In another particular embodiment, the increase in α-secretase enzymatic activity may increase the non-β-amyloidogenic processing of Amyloid Precursor Protein, producing sAPPα, and decrease the β-amyloidogenic processing of Amyloid Precursor Protein, reducing the accumulation of amyloid-β fragment, particularly the Aβl-42 peptide, within the central nervous system of an individual.
Utilizing pharmacological inhibitors of α-secretase as α-secretase chaperones can increase the wild-type enzymatic activity of α-secretase. Pharmacological inhibitors of α secretase include, but are not limited to, hydroxamic acid-based zinc metalloproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I , TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
The present invention will be further understood by reference to the Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows mean α-galactosidase A activity in white blood cells from normal, healthy volunteers who received 50 mg 1-deoxygalactonojirimycin (DGJ) b.i.d. (triangles), 150 mg DGJ b.i.d. (squares), or placebo (open circles). Figure 2 shows mean α-galactosidase A activity in white blood cells from 1 1 Fabry disease patients who were treated with DGJ.
Figure 3 shows the pharmacological chaperones TAPI-2, GM6001 and GW4023 stabilize purified ADAMlO in vitro and increases wild-type levels of ADAMlO (precursor and mature forms) by 2- to 3-fold in SY5Y neuroblastomas.
DETAILED DESCRIPTION
The invention is based, in part, on the discovery that administration of a pharmacological chaperone to a human resulted in a meaningful increase in the level of activity of a wild-type protein. This discovery, combined with an understanding of a pharmacological chaperone's ability to promote proper protein folding in the ER, leading to correct protein trafficking and, significantly increased protein activity, e.g., sufficient protein activity to reverse or ameliorate a disease, disorder, or condition in a human subject. This phenomenon is highly specific to the protein specifically bound by the particular pharmacological chaperone, in contrast to methods using compounds that operate generally to increase expression of all proteins, called "chemical chaperones."
Certain experimental results underlie the present invention: pharmacological chaperones increased endogenous wild-type protein activity in humans to about 120% of normal, 130% of normal, and 145% of normal at a lower dose, and to 150% and 185% of normal at a higher dose after administration of a pharmacological chaperone (see Example 1 and Figure 1). This level of increase in vivo was not predictable from results with cells in tissue culture which remain exposed to the pharmacological chaperone. For example, U.S. Patent No. 6,274,597 describes a 30% increase of α-galactosidase A (α-GAL) activity in normal lymphoblasts cultured with deoxygalactonojirimycin (DGJ), a pharmacological chaperone. Given the expectation that normal physiological processes would be expected to reduce the effects of pharmacological chaperones on normal proteins in vivo through normal clearance, it was not expected that a pharmacological chaperone would yield a significant increase in wild-type protein activity. Example 10 of U.S. Patent No. 6,274,597 describes an increase in activity of a mutant enzyme in transgenic mice treated for one week with a pharmacological chaperone. However, these experiments involved mutant forms of the rescued protein, not wild-type, and were conducted in mice, so the results were not predictive or suggestive of the results observed for wild-type protein in humans. There was no basis to expect that a pharmacological chaperone could increase the level of activity of a wild-type protein in vivo by at least 20-25%, and particularly not by at least about 50%. Yet, as exemplified herein, administration of DGJ to subjects resulted in a dose-dependent increase in α-GAL activity. At some concentrations, which were readily tested using routine dose- response testing, the level of increase in enzyme activity increased by at least 50% (at least 1.5-fold) to up to 100% (at least 2-fold). This extraordinary effect results from titrating a pharmacological chaperone, which is already demonstrated in accordance with existing technology, to rescue a mutant form of the protein, in human patients, to achieve this level of increase, as described herein. Accordingly, the invention provides for titrating a dose of a pharmacological chaperone that has been found to rescue activity of a mutant protein to increase the level of activity of a wild-type protein.
Definitions
The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.
As used herein, the term "pharmacological chaperone," or sometimes "specific pharmacological chaperone" ("SPC"), refers to a molecule that specifically binds to α- secretase and has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of the protein; (ii) enhances proper trafficking of the protein from the ER to another cellular location, preferably a native cellular location, i.e., preventing ER- associated degradation of the protein; (iii) preventing aggregation of conformational Iy unstable, i.e., misfolded proteins; (iv) restoring or enhancing at least partial wild-type function, stability, and/or activity of the protein; and/or (v) improving the phenotype or function of the cell harboring α-secretase. Thus, a pharmacological chaperone for α-secretase is a molecule that binds to α-secretase, resulting in proper folding, trafficking, non- aggregation, and activity of α-secretase. As used herein, this term does not refer to endogenous chaperones, such as BiP, or to non-specific agents which have demonstrated nonspecific chaperone activity against various proteins, such as glycerol, DMSO or deuterated water, i.e., chemical chaperones (see Welch et al , Cell Stress and Chaperones 1996; l(2): 109-1 15; Welch et al., Journal of Bioenergetics and Biomembranes 1997; 29(5):491- 502; U.S. Patent No. 5,900,360; U.S. Patent No. 6,270,954; and U.S. Patent No. 6,541 ,195). It includes specific binding molecules, e.g., active site-specific chaperones (discussed above), inhibitors or antagonists, and agonists. As used herein, the term "specifically binds" refers to the interaction of a pharmacological chaperone with α-secretase, specifically, an interaction with amino acid residues of α-secretase that directly participate in contacting the pharmacological chaperone. A pharmacological chaperone specifically binds a target protein, e.g., α-secretase, to exert a chaperone effect on α-secretase and not a generic group of related or unrelated proteins. The amino acid residues of α-secretase that interact with any given pharmacological chaperone may or may not be within the protein's "active site." Specific binding can be evaluated through routine binding assays or through structural studies, e.g., co-crystallization, NMR, and the like.
In one non-limiting embodiment, the pharmacological chaperone is an inhibitor or antagonist of α-secretase. In another non-limiting embodiment, the pharmacological chaperone is an agonist of α-secretase. In yet another embodiment, the pharmacological chaperone is a mixed agonist/antagonist. As used herein, the term "antagonist" refers to any molecule that binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes an activity of α-secretase. The term "agonist" refers to any molecule that binds to a protein and at least partially increases, enhances, restores, or mimics an activity of α- secretase. As discussed below, such molecules are known for α-secretase.
As used herein, the terms "enhance α-secretase conformational stability" or "increase α-secretase conformational stability" refer to increasing the amount or proportion of α- secretase that adopts a functional conformation in a cell contacted with a pharmacological chaperone specific for α-secretase, relative to α-secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for α-secretase. In one embodiment, the cells do not express a conformation mutant α-secretase. In another embodiment, the cells do express a mutant α- secretase polynucleotide encoding a polypeptide e.g., a conformational mutant α-secretase.
As used herein, the terms "enhance α-secretase trafficking" or "increase α-secretase trafficking" refer to increasing the efficiency of transport of α-secretase into the cytosol of a cell contacted with a pharmacological chaperone specific for α-secretase, relative to the efficiency of transport of α-secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for α- secretase.
As used herein, the terms "enhance α-secretase activity" or "increase α-secretase activity" refer to increasing the activity of α-secretase, as described herein, in a cell contacted with a pharmacological chaperone specific for α-secretase, relative to the activity of α- secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for α-secretase.
As used herein, the terms "enhance α-secretase level" or "increase α-secretase level" refer to increasing the level of α-secretase in a cell contacted with a pharmacological chaperone specific for α-secretase, relative to the level of α-secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for α-secretase.
The term "stabilize a proper conformation" refers to the ability of an α-secretase pharmacological chaperone to induce or stabilize a conformation of a mutated α-secretase protein that is functionally identical to the conformation of the wild-type α-secretase protein. The term "functionally identical" means that while there may be minor variations in the conformation (almost all proteins exhibit some conformational flexibility in their physiological state), conformational flexibility does not result in (1) protein aggregation, (2) elimination through the endoplasmic reticulum-associated degradation pathway, (3) impairment of protein function, e.g., APP metabolic activity, and/or (4) improper transport within the cell, e.g., localization to the cytosol, to a greater or lesser degree than that of the wild-type protein.
The term "stable molecular conformation" refers to a conformation of a protein, i.e., α-secretase, induced by a pharmacological chaperone that provides at least partial wild-type function in the cell. For example, a stable molecular conformation of a mutant α-secretase would be one where α-secretase escapes from the ER and traffics to the cytosol as for a wild- type α-secretase, instead of misfolding and being degraded. In addition, a stable molecular conformation of a mutated α-secretase may also possess full or partial α-secretase activity, e.g., APP metabolism. However, it is not necessary that the stable molecular conformation have all of the functional attributes of the wild-type protein.
The term "α-secretase activity" refers to the normal physiological function of a wild- type α-secretase in a cell. For example, α-secretase activity includes metabolism of APP. Such functionality can be tested by any means known to establish functionality.
The terms "wild-type α-secretase" or "α-secretase" refer to a polypeptide encoded by a nucleic acid selected from the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM) proteins comprising ADAM 9 (human ADAM9, GenBank Accession Nos. NM 003816, and NM 001005845; murine ADAM9, GenBank Accession No. NM_007404; and predicted rat ADAM9, GenBank Accession No. NMJ)Ol 014772), ADAM 10 (human ADAMl O, GenBank Accession No. NMJ)Ol I l O; murine ADAMlO, GenBank Accession No. NM_007399; and predicted rat ADAMl O, GenBank Accession No. XM_217197), and TACE/ADAM 17 (human ADAM 17, GenBank Accession No. NM_003183; murine ADAM 17, GenBank Accession No. NM_009615; and rat ADAM 17, GenBank Accession No. NM_020306). In a preferred embodiment, α-secretase is encoded by ADAMlO (Obregon et al., J. Biol. Chem. 2006; 281 : 16419-16427). In one non-limiting embodiment, α-secretase polypeptide may be encoded for by any nucleic acid molecule exhibiting 50%, 60%, 70%, 80% and up to 100% homology to the nucleic acid molecules encoding human α-secretase, and any sequences which hybridize under standard conditions to these sequences. In another non-limiting embodiment, any other nucleotide sequence that encodes α-secretase polypeptide (having the same functional properties and binding affinities as the aforementioned polypeptide sequences), such as allelic variants in normal individuals, that have the ability to achieve a functional conformation in the ER, achieve proper localization within the cell, and exhibit wild-type activity (e.g., APP metabolism).
An "α-secretase polypeptide" also refers to an amino acid sequence depicted from the ADAM family of proteins comprised of: ADAM 9 (human ADAM9, GenBank Accession Nos. NP 001005845, and NP 003807; murine ADAM9, GenBank Accession No. NP 031430; and predicted rat ADAM9, GenBank Accession No. NPJ)Ol 014772), ADAMlO (human ADAMl O, GenBank Accession No. NPJ)OI lOl ; murine ADAMl O, GenBank Accession No. NP 031425, and predicted rat ADAMlO, GenBank Accession No. XP_217197), and TACE/ADAM 17 (human ADAM 17, GenBank Accession No. NP_003174; murine ADAM17, GenBank Accession No. NP_033745; and rat ADAM17, GenBank Accession No. NP_064702), and any other amino acid sequence that encodes an α- secretase polypeptide having the same function and ligand binding affinity as any one of GenBank Accession No.'s.
As used herein the term "mutant α-secretase" refers to an α-secretase polypeptide translated from a gene containing a genetic mutation that results in an altered α-secretase amino acid sequence. In one embodiment, the mutation results in an α-secretase protein that does not achieve a native conformation under the conditions normally present in the ER, when compared with wild-type α-secretase, or exhibits decreased stability or activity as compared with wild-type α-secretase. This type of mutation is referred to herein as a "conformational mutation," and the protein bearing such a mutation is referred as a "conformational mutant." The failure to achieve this conformation results in α-secretase protein being degraded or aggregated, rather than being transported through a normal pathway in the protein transport system to its native location in the cell or into the extracellular environment. In some embodiments, a mutation may occur in a non-coding part of the gene encoding α-secretase that results in less efficient expression of the protein, e.g., a mutation that affects transcription efficiency, splicing efficiency, mRNA stability, and the like. By enhancing the level of expression of wild-type as well as conformational mutant variants of α-secretase, administration of an α-secretase pharmacological chaperone can ameliorate a deficit resulting from such inefficient protein expression.
Certain tests may evaluate attributes of a protein that may or may not correspond to its actual in vivo activity, but nevertheless are appropriate surrogates of protein functionality, and wild-type behavior in such tests demonstrates evidence to support the protein folding rescue or enhancement techniques of the invention. One such activity in accordance with the invention is appropriate transport of a functional α-secretase from the endoplasmic reticulum to the cytosol.
The terms "endogenous expression" and "endogenously expressed" refers to the normal physiological expression of α-secretase in cells in an individual not having or suspected of having a disease or disorder associated with α-secretase deficiency, overexpression of a dominant negative mutant, or other defect, such as a mutation in α- secretase nucleic acid or polypeptide sequence that alters, e.g., inhibits, its expression, activity, or stability. This term also refers to the expression of α-secretase in cells or cell types in which it is normally expressed in healthy individuals, and does not include expression of α-secretase in cells or cell types, e.g., tumor cells, in which α-secretase is not expressed in healthy individuals.
As used herein, the term "efficiency of transport" refers to the ability of a protein to be transported out of the endoplasmic reticulum to its native location within the cell, cell membrane, or into the extracellular environment.
A "competitive inhibitor" of an enzyme can refer to a compound which structurally resembles the chemical structure and molecular geometry of the enzyme substrate to bind the enzyme in approximately the same location as the substrate. Thus, the inhibitor competes for the same active site as the substrate molecule, thus increasing the Km. Competitive inhibition is usually reversible if sufficient substrate molecules are available to displace the inhibitor, i.e., competitive inhibitors can bind reversibly. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.
Non-classical competitive inhibition occurs when the inhibitor binds remotely to the active site, creating a conformational change in the enzyme such that the substrate can no longer bind to it. In non-classical competitive inhibition, the binding of substrate at the active site prevents the binding of inhibitor at a separate site and vice versa. This includes allosteric inhibition.
A "linear mixed-type inhibitor" of an enzyme is a type of competitive inhibitor that allows the substrate to bind, but reduces its affinity, so the Km is increased and the Vmax is decreased.
A "non-competitive inhibitor" refers to a compound that forms strong bonds with an enzyme and may not be displaced by the addition of excess substrate, i.e., non-competitive inhibitors may be irreversible. A non-competitive inhibitor may bind at, near, or remote from the active site of an enzyme or protein, and in connection with enzymes, has no effect on the Km but decreases the Vmax. Uncompetitive inhibition refers to a situation in which inhibitor binds only to the enzyme-substrate (ES) complex. The enzyme becomes inactive when inhibitor binds. This differs from non-classical competitive inhibitors which can bind to the enzyme in the absence of substrate.
The term "Vmax" refers to the maximum initial velocity of an enzyme catalyzed reaction, i.e., at saturating substrate levels. The term "Km" is the substrate concentration required to achieve /2 Vmax.
An enzyme "enhancer" is a compound that binds to α-secretase and increases the enzymatic reaction rate.
The terms "therapeutically effective dose" and "effective amount" refer to an amount sufficient to enhance protein processing in the ER (permitting a functional conformation), without inhibiting protein already expressed at the appropriate cellular location (in the case of an antagonist), or without inducing ligand-mediated receptor internalization of protein from the appropriate cellular location (in the case of an agonist), and enhance activity of the target protein, thus resulting in a therapeutic response in a subject. A therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including the foregoing symptoms and surrogate clinical markers. Thus, a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or disorder, e.g., Alzheimer's Disease.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.
The terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.
As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an mRNA band on a gel, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.
The term "purified" as used herein refers to material, such as a α-secretase nucleic acid or polypeptide that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by conventional means, e g , chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
The term "Alzheimer's Disease" or "AD" refers to any condition where amyloid-β (Aβl-42) deposition accumulates in the cells of the central nervous system. In one, non- limiting embodiment, amyloid-β, particularly Aβl-42, peptide is formed from the β- amyloidogenic metabolism of APP. AD may be heritable in a Familial manifestation, or may be sporadic. Herein, AD includes Familial, Sporadic, as well as intermediates and subgroups thereof based on phenotypic manifestations. Familial AD typically has an early-onset (before age 65) while Sporadic AD typically is late-onset (age 65 and later). In a non-limiting embodiment, Familial AD may be associated with mutations in one or more genes selected from the group comprising presenilin 1 (human presenilin 1, GenBank Accession Nos. NM 000021 , NM 007318, and NM 007319; murine presenilin 1, GenBank Accession No. NM 008943; and rat presenilin 1, GenBank Accession No. NM 019163), presenilin 2 (human presenilin 2, GenBank Accession Nos. NM 000447, and NM_012486; murine presenilin 2, GenBank Accession No. NM Ol 1 183; and rat presenilin 2, GenBank Accession No. NM 031087), and Amyloid Precursor Protein (APP) (human APP, GenBank Accession Nos. NM_201414, NM_201413, and NM_000484; murine APP, GenBank Accession No. NM_007471 ; and rat APP, GenBank Accession No. NM O 19288). Sporadic AD can not be tested for directly, but certain risk factors may increase an individual's susceptibility to developing sporadic AD. In one, non-limiting embodiment, individuals with at least one copy of the e4 allele of Apolipoprotein E (APOE) (human APOE, GenBank Accession No. NM_000041 ; murine APOE, GenBank Accession No. NM_009696; and rat APOE, GenBank Accession No. NM l 38828) are at risk of developing late-onset sporadic AD.
This term also includes individuals with Down syndrome (DS) who invariably develop (in their third or fourth decade) cerebral amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), the characteristic lesions of Alzheimer disease (AD). Recent studies have shown that the Aβ42 is the earliest form of this protein deposited in Down syndrome brains, and may be seen in subjects as young as 12 years of age, and that soluble Aβ can be detected in the brains of DS subjects as early as 21 gestational weeks of age, well preceding the formation of Aβ plaques (Gyure et al., Archives of Pathology and Laboratory Medicine. 2000; 125:. 489-492).
For puφoses of the present invention, a "neurological disorder" refers to any central nervous system (CNS) or peripheral nervous system (PNS) disease that is associated with the β-amyloidogenic processing of Amyloid Precursor Protein. This may result in neuronal or glial cell defects including but not limited to neuronal loss, neuronal degeneration, neuronal demyelination, gliosis (i.e. , astrogliosis), or neuronal or extraneuronal accumulation of aberrant proteins or toxins (e.g., amyloid-β).
One exemplary neurological disorder is congophilic angiopathy (CAA), also referred to as amyloid angiopathy. This disorder is a form of angiopathy in which the same amyloid protein that is associated with Alzheimer's disease, amyloid-β (Aβ), deposits in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessels to failure, increasing the risk of a hemorrhagic stroke. Since it is the same amyloid protein that is associated with Alzheimer's dementia, such brain hemorrhages are more common in people who suffer from Alzheimer's, however they can also occur in those who have no history of dementia. The hemorrhage within the brain is usually confined to a particular lobe and this is slightly different compared to brain hemorrhages which occur as a consequence of high blood pressure (hypertension) - a more common cause of a hemorrhagic stroke (or cerebral hemorrhage). CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.
The term "patient" or "patient population" refers to individual(s) diagnosed as having Alzheimer's Disease or at risk of developing Alzheimer's Disease. In one, non-limiting embodiment, individuals are diagnosed, or at risk of developing Familial AD. In another, non-limiting embodiment, the individual is diagnosed as having, or at risk of developing, Sporadic AD. Diagnosis of AD may be made based on genotypic or phenotypic characteristics displayed by the individual. For example, an individual with a mutant variant of presenilin 1, presenilin 2, or APP are at risk of developing familial AD. In another, non- limiting example, individuals with the E4 variant of APOE are at risk for developing Sporadic AD.
An individual may be diagnosed as having AD, or at risk of developing AD, by exhibiting phenotypes associated with AD. Phenotypes associated with AD may be cognitive or psychiatric. Examples of cognitive phenotypes include, but are not limited to, amnesia, aphasia, apraxia and agnosia. Examples of psychiatric symptoms include, but are not limited to, personality changes, depression, hallucinations and delusions. As one non-limiting example, the Diagnostic and Statistical Manual of Mental disorders, 4th Edition (DSM-IV- TR) (published by the American Psychiatric Association) contains the following set of criteria for dementia of the Alzheimer's type:
A. The development of multiple cognitive deficits manifested by both memory impairment and one or more of Aphasia, Apraxia, Agnosia and disturbances in executive functioning;
B. The cognitive deficits represent as decline from- previous functioning and cause significant impairment in social or occupational functioning;
C. The course is characterized by gradual onset and continuing decline;
D. The cognitive deficits are not due to other central nervous system, systemic, or substance-induced conditions that cause progressive deficits in memory and cognition; and
E. The disturbance is not better accounted for by another psychiatric disorder.
Another non-limiting example is The National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorder Association (NINDS-ADRDA) Criteria for Alzheimer's Disease as follows:
A. Definite Alzheimer's disease: meets the criteria for probable Alzheimer's disease and has histopathologic evidence of Alzheimer's disease via autopsy or biopsy
B. Probable Alzheimer's disease: dementia established by clinical and neuropsychological examination and involves
(a) progressive deficits in two or more areas of cognition, including memory,
(b) onset between the ages of 40 and 90 years, and
(c) absence of systemic or other brain diseases capable of producing a dementia syndrome, including delirium
C. Possible Alzheimer's disease: a dementia syndrome with an atypical onset, presentation, or progression and without a known etiology; any co-morbid diseases capable of producing dementia are not believed to be the cause
D. Unlikely Alzheimer's disease: a dementia syndrome with any of the following: sudden onset, focal neurologic signs, or seizures or gait disturbance early in the course of the illness. Phenotypic manifestations of AD may also be physical, such as by the direct (imaging) or indirect (biochemical) detection of amyloid-β plaques. Quantitation of amyloid- β (1 -40) in the peripheral blood has been demonstrated using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap (Du et al., J Biomol Tech. 2005;16(4):356-63). Detection of single β-amyloid protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy also has been described (Pitschke et al., Nature Medicine. 1998; 4: 832 - 834). U.S. Patent 5,593,846 describes a method for detecting soluble amyloid-β. Indirect detection of amyloid-β peptide and receptor for advanced glycation end products (RAGE) using antibodies also has been described. Lastly, biochemical detection of increased BACE-I activity in cerebrospinal fluid using chromogenic substrates also has been postulated as a diagnostic or prognostic indicator of AD (Verheijen et al., Clin Chem. 2006 Apr 13 [Epub.]).
In vivo imaging of β-amyloid can be achieved using radioiodinated flavone derivatives as imaging agents (Ono et al., J Med Chem. 2005;48(23):7253-60) and with amyloid binding dyes such as putrescein conjugated to a 40-residue radioiodinated A peptide (yielding 125I-PUT-A 1-40), which was shown to cross the blood-brain barrier and bind to αβ plaques (Wengenack et al., Nature Biotechnology. 2000; 18(8): 868-72). Imaging of β- amyloid was also shown using stilbene [nC]SB-13 and the benzothiazole [' 1C]O-OH-BTA-I (also known as [1 1C]PIB) (Nicholaas et &\., Am J Geriatr Psychiatry. 2004; 12:584-595).
Pharmacological inhibitors of α-secretase include, but are not limited to, hydroxamic acid-based zinc metal loproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I, TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
Batimastat, also known as BB-94, is a low molecular weight synthetic inhibitor of metal loproteinase activity that functions by binding the zinc ion in the active site of MMP's. The batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP. Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J.A., et al., Clinical Cancer Research 1996; 2: 1207-1214). Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for α-secretase. By binding the ADAM protein, and forcing the protein to adopt a stable conformation, batimastat, and its analogs, function as chaperones to traffic α-secretase out of the endoplasmic reticulum In a non-limiting embodiment, the α-secretase protein stabilized in a proper conformation is transported from the endoplasmic reticulum to a site of APP metabolism.
Pharmacological Chaperones for α-Secretase
In one, non-limiting embodiment, the pharmacological chaperone may be an inhibitor, or structurally similar analog thereof, of α-secretase. Examples of inhibitors of α-secretase include, but are not limited to, the group comprising hydroxamic acid-based zinc metalloproteinase inhibitors. The following are non-limiting examples of hydroxamic acid- based zinc metalloproteinase inhibitors:
Batimastat, (BB94), having the structure:
Figure imgf000022_0001
SB223820, having the structure:
Figure imgf000022_0002
Marimastat, having the structure:
Figure imgf000022_0003
BB3132, having the structure:
Figure imgf000023_0001
TAPI-O, having the structure:
Figure imgf000023_0002
TAPI-I
Figure imgf000023_0003
TAPI-2, having the structure:
Figure imgf000024_0001
Immunex compound 3 (IC3), having the structure:
Figure imgf000024_0002
KD-IX-73-4, having the structure:
Figure imgf000024_0003
BB21 16, having the structure:
Figure imgf000024_0004
GM6001, having the structure:
Figure imgf000025_0001
rNCB8765, having the structure (l R,3S,4S)-3-[(hydroxyamino)carbonyl]-4-[(4- phenylpiperidin-l -yl)carbonyl]cyclohexyl pyrrolidine- 1 -carboxy late.
BB3103 is another MMP inhibitor and is available from British Biosciences.
Batimastat, also known as BB-94, is a low molecular weight synthetic inhibitor of metalloproteinase activity that functions by binding the zinc ion in the active site of MMP's. The batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP. Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J. A., et al., Clinical Cancer Research 1996; 2: 1207-1214). Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for α-secretase. By binding the ADAM protein, and forcing the protein to adopt a stable conformation, batimastat, and its analogs, may function as chaperones to traffic α- secretase out of the endoplasmic reticulum.
Additional ADAMl O inhibitors include GW280264X and GI254023X, which are described in Hundhausen et al., Blood. 2003; 102: 1 186-1 195, have the following structures:
Figure imgf000026_0001
and
Other α-secretase inhibitors include ADAMl O inhibitors as described in PCT publication WO03/106381 , to Bannen et al., owned by Exelixis, Inc. Such compounds are provided in the following Table:
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Additional TACE inhibitors include IK682, several hydroxamate-cyclopropyl compounds and series of spirocyclopropyl compounds, which are described in Guo et al., Bioorganic & Medicinal Chemistry Letters. 2009; 19: 54-57.
IK682 has the structure:
Figure imgf000035_0001
The hydroxamate-cyclopropyl compounds had the structure:
Figure imgf000035_0002
with substituents according to the following table:
Figure imgf000035_0003
The spirocyclopropyl compounds series had the structure:
Figure imgf000036_0001
with substituents according to the following table:
Figure imgf000036_0002
Other α-secretase inhibitors are described in Parkin et al., Biochemistry. 2002; 41 (15): 4972-4981 , and are provided in the following Table:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Additional TACE inhibitors are given in Becherer et al., book chapter "The Tumor Necrosis Factor-alpha Converting Enzyme" and are shown in the following Table:
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Rabinowitz et al., Journal of Medicinal Chemistry. 2001 ; 44 (24): 4252-4267 discloses various GW3333 derivative TACE inhibitors. Several examples include:
Figure imgf000042_0002
Note that EWG refers to an electron withdrawing group. Among the inhibitors disclosed are several nitro-arginine inhibitors according to the structure:
Figure imgf000043_0001
With substituents according to the following table:
R R'
Figure imgf000044_0001
Also among the inhibitors disclosed are several methanesulfonyl-arginine and pyridylsulfonyl-arginine inhibitors according to the structure:
Figure imgf000045_0001
With substituents according to the following table:
Where Z = methanesulfonyl:
R R '
Figure imgf000045_0002
Where Z = 2-pyridylsulfonyl:
Figure imgf000045_0003
Ψ XX
Figure imgf000045_0004
Also among the inhibitors disclosed are several β-methyl methanesulfonylarginine inhibitors according to the structure:
Figure imgf000046_0001
With substituents according to the following table:
Where Z = methanesulfonyl:
R R'
Figure imgf000046_0002
"3 ><k
Figure imgf000046_0003
Where Z = Nitro:
CH3 *k
Figure imgf000046_0004
Ludwig et all., Combinational Chemistry & High Throughput Screening. 2005; 8: 161 -171 discloses two more inhibitors:
GI254023X, having the structure:
Figure imgf000047_0001
GW280264X, having the structure:
Figure imgf000047_0002
Methods of Treatment
The present invention also provides methods for treating a condition associated with reduced protein stability, activity, and/or trafficking, or a condition that would benefit by an increase of basal level protein stability, activity, and/or trafficking, by administering to a subject in need of such treatment a pharmacological chaperone to enhance protein stability, activity, and/or trafficking of the protein. The subject to be treated can be a subject who does not exhibit a mutation in the protein that affects folding and processing of the protein, but who would benefit from increased protein stability, activity, and/or trafficking. The subject to be treated can also have a mutation in the protein that affects protein function, other than a mutation that affects folding and processing of the protein, and exhibits reduced protein levels in cells that normally express the protein. In one embodiment, the subject is homozygous for the wild-type α-secretase protein. In another embodiment, the subject is heterozygous for the wild-type α-secretase protein and has a mutant genotype with a null phenotype for the other allele encoding the α-secretase protein. Formulations and Administration
A pharmacological chaperone, i.e , an agonist or antagonist or other compound as described above or as identified through the screening methods of the invention as set forth below, is advantageously formulated in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The pharmacological chaperone may be designated as an active ingredient or therapeutic agent for the treatment of a disease or disorder that would benefit from an increase in protein activity, conformational stability, and/or trafficking.
The concentration of the active ingredient (pharmacological chaperone) depends on the desired dosage and administration regimen, as discussed below. Exemplary dose ranges of the active ingredient are from about 0.01 mg/kg to about 250 mg/kg of body weight per day; from about 10 mg/kg to about 100 mg/kg per day; or from about 10 mg/kg to about 75 mg/kg per day.
As another example, Batimastat has been administered to cancer patients at doses of 600 to 1050mg/m2 (intraperitoneal Iy). Maramistat has been orally administered in pancreatic cancer patient at doses of 5, 10, or 25 mg b.i.d.
Therapeutically effective compounds can be provided to a subject in standard formulations, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavorants, colorants, buffers, and disintegrants. Standard formulations are well known in the art. See e.g., Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, 2000. The formulation may be produced in useful dosage units for administration by any route that will permit the therapeutic chaperone to cross the blood- brain barrier. Exemplary routes include oral, parenteral, transmucosal, intranasal, inhalation, or transdermal routes. Parenteral routes include intravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration.
In one embodiment, a pharmacologic chaperone is formulated in a solid oral dosage form. For oral administration, e.g., for a small molecule, the pharmaceutical composition may take the form of a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents {e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers {e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants {e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
In another embodiment, a pharmacological chaperone is formulated for parenteral administration. The pharmacological chaperone may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
In addition to the formulations described previously, the chaperone may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
In another embodiment, the pharmacological chaperone can be delivered in a vesicle, particularly a liposome.
In another embodiment, the pharmacological chaperone can be delivered in a controlled release manner. For example, a therapeutic agent can be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly- lactic/glutamic acid (PLGA), in a pellet containing a mixture of cholesterol and the active ingredient (SilasticR™; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601), by subcutaneous implantation, or by transdermal patch. Combination Therapy. The pharmaceutical composition may also include other biologically active substances in combination with the candidate compound (pharmacological chaperone) or may be administered in combination with other biologically active substances.
Combination Drug Therapy
The pharmacological chaperone can be used to treat patients with Alzheimer's Disease in combination with other drugs that are also used to treat the disorder. Exemplary non-limiting pharmacological agents approved in the United States for the treatment of Alzheimer's include cholinesterase inhibitors such as Cognex® (tacrine), Aricept® (donepezil), Exelon® (rivastigmine), Reminyl® (galantamine), and NMDA receptor antagonists such as Namenda® (memantine). Other potential therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos. 5,863,902; 5,872,101 ; inhibitors of β-amyloid production such as described in U.S. Patent Nos. 7,01 1,901 ; 6,495,540; 6,610,734; 6,632,812; 6,713,476; and 6,737,420; modulators of β -amyloid aggregation, described in 6,303,567; 6,689,752; and inhibitors of β-secreatase such as disclosed in U.S. Patent Nos. 6,982,264; 7,034,182; 7,030,239.
In addition, the pharmacological chaperone can be used in combination with gene therapy. Gene therapy is contemplated both with replacement genes such as nerve growth factor (NGF), or with inhibitory RNA (siRNA) for enzymes that are involved in the amyloidogenic cleavage of APP (BACE-I). Gene therapy is described in more detail below.
Other contemplated combination therapy includes combinations of specific pharmacological chaperones with vaccine therapy, such as described in U.S. Patent Nos. 6,866,860 and 6,761 ,888.
Screening Assays for α-secretase Pharmacological Chaperones
The present invention further provides a method for identifying a candidate pharmacological chaperone compound that modulates the stability, activity, and/or trafficking of an α-secretase polypeptide. In one embodiment, the present invention provides a method for identifying a chaperone for a target protein, which comprises bringing a labeled or unlabeled test compound in contact with the protein or a fragment thereof and measuring the amount of the test compound bound to the protein or to the fragment thereof. For example, this can be achieved as follows: (a) contacting a first cell with a test compound for a time period sufficient to allow the cell to respond to said contact with the test compound;
(b) determining the conformational stability, activity, and/or trafficking of the target protein (or a fragment thereof comprising a ligand binding domain) in the cell (or on the cell surface) contacted in step (a); and
(c) comparing the conformational stability, activity, and/or trafficking of the protein determined in step (b) to that of a protein in a control cell that has not been contacted with the test compound;
wherein a detectable change in the conformational stability, activity, and/or trafficking of the protein in the first cell in response to contact with the test compound compared to that same value of the protein in the control cell that has not been contacted with the test compound, indicates that the test compound modulates the conformational stability, activity, and/or trafficking of the protein and is a candidate compound for the treatment of a disorder that would benefit from an increase in conformational stability, protein activity and/or proper trafficking of the target protein.
The α-secretase protein can either be expressed in a host cell transformed with a vector encoding a non-endogenous α-secretase protein or expressed from an endogenous gene in the cell.
Numerous high-throughput screening (HTS) methods can be employed to screen large numbers (e.g., hundreds, thousands, tens of thousands) of test compounds simultaneously for binding to a specific protein. A test compound can be, without limitation, a small organic or inorganic molecule (preferred), a peptide or a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule (such as an aptamer), a polynucleotide molecule, or a chimera or derivative thereof. Test compounds which are candidate pharmacological chaperones that specifically bind to an α-secretase protein can be identified using cell-based and/or cell-free assays. Several methods of automated assays that have been developed in recent years enable the screening of tens of thousands of compounds in a short period of time (see, e.g., U.S. Patent Nos. 5,585,277, 5,679,582, and 6,020,141). For example, one group reported the identification of one arylpiperazine receptor agonist through iterative directed screening of nonpeptidyl G-protein- coupled receptor biased libraries (Richardson et al., J Med Chem. 2004; 47(3):744-55). Such HTS methods are particularly useful, e.g., in microarrays.
For screening, preferred classes of compounds that may be identified include, but are not limited to/small molecules (i.e., organic or inorganic molecules which are less than about 2 kilodaltons (kD) in molecular weight, and, more preferably, less than about 1 kD in molecular weight).
Compound libraries. Libraries of high-purity small organic ligands and peptide agonists that have well-documented pharmacological activities are available from Sigma- Aldrich (LOPAC LIBRARY™ and LIGAND-SETS™). Also available from Sigma-Aldrich is an Aldrich Library of Rare Chemicals, which is a diverse library of more than 100,000 small-molecule compounds, including plant extracts and microbial culture extracts. Other compound libraries are available from Tripos (LeadQuest®) and TimTech (including targeted libraries for kinase modulators).
Other companies that supply or have supplied compound libraries of the type suitable for screening according to the invention include the following: 3-Dimensional Pharmaceuticals, Inc.; Advanced ChemTech; Abinitio PharmaSciences; Albany Molecular; Aramed Inc.; Annovis, Inc. (formerly Bearsden Bio, Inc.); ASINEX; AVANT Immunotherapeutics; AXYS Pharmaceuticals; Bachem; Bentley Pharmaceuticals; Bicoll Group; Biofor Inc.; BioProspect Australia Limited; Biosepra Inc.; Cadus Pharmaceutical Corp.; Cambridge Research Biochemicals; Cetek Corporation; Charybdis Technologies, Inc.; ChemBridge Corporation; ChemDiv, Inc.; ChemGenics Pharmaceuticals Inc.; ChemOvation Ltd.; ChemStar, Ltd.; Chrysalon; ComGenex, Inc.; Compugen Inc.; Cytokinetics; Dextra Laboratories Ltd.; Discovery Partners International Inc.; Discovery Technologies Ltd.; Diversa Corporation; Dovetail Technologies, Inc.; Drug Discovery Ltd.; ECM Pharma; Galilaeus Oy; Janssen Pharmaceutica; Jerini Bio Tools; J-Star Research; KOSAN Biosciences, Inc.; KP Pharmaceutical Technology, Inc.; Lexicon Genetics Inc.; Libris Discovery; MicroBotanica, Inc.; MicroChemistry Ltd.; MicroSource Discovery Systems, Inc.; Midwest Bio-tech Inc.; Molecular Design & Discovery; MorphoSys AG; Nanosyn, Inc.; Ontogen Corporation; Organix, Inc.; Pharmacopeia, Inc.; Pherin Pharmaceuticals; Phytera, Inc.; PTRL East, Inc.; REPLICor Inc.; RSP Amino Acid Analogues, Inc.; Sanofi- Synthelabo Pharmaceuticals (now Sanofi-Avnetis); Sequitur, Inc.; Signature BioScience Inc.; Spectrum Info Ltd.; Talon Cheminformatics Inc.; Telik, Inc.; Tera Biotechnology Corporation; Tocris Cookson; Torrey Pines Institute for Molecular Studies; Trega Biosciences, Inc.; and WorldMolecules/MMD.
In addition, the Institute of Chemistry and Cell Biology (ICCB), maintained by Harvard Medical School, provides the following chemical libraries, including natural product libraries, for screening: Chem Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds); CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2 (704 compounds); Peakdale 1 (2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and International (28,864 compounds); Mixed Commercial Plate 1 (352 compounds); Mixed Commercial Plate 2 (320 compounds); Mixed Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331 compounds); ChemBridge Microformat (50,000 compounds); Commercial Diversity Set 1 (5,056 compounds); NCI Collections: Structural Diversity Set, version 2 (1,900 compounds); Mechanistic Diversity Set (879 compounds); Open Collection 1 (90,000 compounds); Open Collection 2 (10,240 compounds); Known Bioactives Collections: NTNDS Custom Collection (1 ,040 compounds); ICCB Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds); ICCB Discretes Collections. The following ICCB compounds were collected individually from chemists at the ICCB, Harvard, and other collaborating institutions: ICCBl (190 compounds); ICCB2 (352 compounds); ICCB3 (352 compounds); ICCB4 (352 compounds). Natural Product Extracts: NCI Marine Extracts (352 wells); Organic fractions - NCI Plant and Fungal Extracts (1,408 wells); Philippines Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis (DOS) Collections; DDSl (DOS Diversity Set) (9600 wells).
There are numerous techniques available for creating more focused compound libraries rather than large, diverse ones. Chemical Computing Group, Inc. (Montreal) has developed software with a new approach to high-throughput drug design. The company's method uses high-throughput screening (HTS) experimental data to create a probabilistic QSAR (Quantitative Structure Activity Relationship) model, which is subsequently used to select building blocks in a virtual combinatorial library. It is based on statistical estimation instead of the standard regression analysis.
In addition, ArQuIe, Inc. (Woburn, MA) also has integrated technologies to perform high-throughput, automated production of chemical compounds and to deliver these compounds of known structure and high purity in sufficient quantities for lead optimization. Its AMAP™ (Automated Molecular Assembly Plant) performs high-throughput chemical syntheses for each phase of compound discovery.
Similarly compounds are often provided on online databases or on CD-ROM's for selective "cherry picking" of compounds. See, e g., Ablnitio PharmaSciences; ActiMol; Aral Biosynthetics; ASDI Biosciences; Biotechnology Corporation of America; Chembridge; ChemDiv; Florida Center - Heterocyclic Compounds; Microsource /MSDI; NorthStar; Peakdale; Texas Retaining Group; Zelinsky Institute; Advanced ChemTech; Ambinter; AnalytiCon Discovery; Aurora Fine Chemicals; Biofocus; Bionet /Key; Comgenex; Key Organics; LaboTest; Polyphor; SPECS and Biospecs; and Bharavi Laboratories. Microarrays
In one embodiment, HTS screening for α-secretase protein pharmacological chaperones employs microarrays.
Protein arrays. Protein arrays are solid-phase, binding assay systems using immobilized proteins on various surfaces that are selected for example from glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The binding assays using these arrays are highly parallel and often miniaturized. Their advantages are that they are rapid, can be automated, are capable of high sensitivity, are economical in their use of reagents, and provide an abundance of data from a single experiment.
Automated multi-well formats are the best-developed HTS systems. Automated 96- or 384-well plate-based screening systems are the most widely used. The current trend in plate-based screening systems is to reduce the volume of the reaction wells even further, and increase the density of the wells per plate (96 wells to 384 wells to 1 ,536 wells per plate). The trend results in increased throughput, dramatically decreased bioreagent costs per compound screened, and a decrease in the number of plates that need to be managed by automation. For a description of protein arrays that can be used for HTS, see e.g. : U.S. Patents No. 6,475,809; 6,406,921 ; and 6,197,599; and International Publication Nos. WO 00/04389 and WO 00/07024.
For construction of arrays, sources of proteins or fragments thereof, whether in wild- type or mutant form, can include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for making peptides. For capture arrays and protein function analysis, it is often the case that α-secretase proteins are correctly folded and functional.
The immobilization method used is preferably applicable to proteins of different properties (e.g., wild-type, mutant, full-length, partial- length fragments, hydrophilic, hydrophobic, etc^), amenable to high throughput and automation, and generally compatible with retention of chaperone-binding ability. Both covalent or non-covalent methods of protein immobilization can be used. Substrates for covalent attachment include, e.g., glass slides coated with amino- or aldehyde-containing silane reagents (Telechem). In the Versalinx™ system (Prolinx), reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. Covalent coupling methods providing a stable linkage can be applied to a range of proteins. Non-covalent binding of unmodified protein occurs within porous structures such as HydroGel™ (PerkinElmer), based on a 3-dimensional polyacrylamide gel.
Cell-Based Arrays. Cell-based arrays combine the technique of cell culture in conjunction with the use of fluidic devices for measurement of cell response to test compounds in a sample of interest, screening of samples for identifying molecules that induce a desired effect in cultured cells, and selection and identification of cell populations with novel and desired characteristics. High-throughput screening (HTS) can be performed on fixed cells using fluorescent-labeled antibodies, biological ligands or candidate chaperones and/or nucleic acid hybridization probes, or on live cells using multicolor fluorescent indicators and biosensors. The choice of fixed or live cell screens depends on the specific cell-based assay required.
There are numerous single- and multi-cell-based array techniques known in the art. Techniques such as micro-patterned arrays (described, e.g., in International PCT Publications WO 97/45730 and WO 98/38490) and microfluidic arrays provide valuable tools for comparative cell-based analysis. Transfected cell microarrays are a complementary technique in which array features comprise clusters of cells overexpressing defined cDNAs. Complementary DNAs cloned in expression vectors are printed on microscope slides, which become living arrays after the addition of a lipid transfection reagent and adherent mammalian cells (Bailey et al., Drug Discov. Today 2002; 7(18 Suppl): Sl 13-8). Cell-based arrays are described in detail in, e.g., Beske, Drug Discov. Today 2002; 7(18 Suppl): Sl 31-5; Sundberg et al., Curr. Opin. Biotechnol. 2000; 1 1 : 47-53; Johnston et al., Drug Discov. Today 2002; 7: 353-63; U.S. Patent Nos. 6,406,840 and 6,103,479, and U.S. published patent application no. 2002/0197656. For cell-based assays specifically used to screen for modulators of ligand-gated ion channels, see Mattheakis et al., Curr. Opin. Drug Discov. Devel. 2001 ; 1 : 124-34; and Baxter et al., J. Biomol. Screen. 2002; 7: 79-85.
Detectable labels. For detection of molecules such as candidate α-secretase protein pharmacological chaperones using screening assays, a functional assay can be used to follow unlabeled molecules as described elsewhere herein. A molecule-of-interest {e.g., a small molecule, an antibody, or a polynucleotide probe) or a library of same can also be detectably labeled with an atom (e.g., a radionuclide), a detectable molecule {e.g., fluorescein), or a complex that, due to a physical or chemical property, serves to indicate the presence of the molecule of interest. A molecule can also be detectably labeled when it is covalently bound to a "reporter" molecule (e.g., a biomolecule such as an enzyme) that acts on a substrate to produce a detectable product. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the present invention include, but are not limited to, biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX from Amersham, SyBR Green I & II from Molecular Probes, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Examples of patents describing the use of such labels include U.S. Patent Nos.: 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
Means of detecting such labels are known to those of skill in the art. For example, radiolabels and chemiluminescent labels can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photo-detector to detect emitted light (e.g., as in fluorescence-activated cell sorting, FACS); and enzymatic labels can be detected by providing the enzyme with a substrate and detecting, e.g., a colored reaction product produced by the action of the enzyme on the substrate.
In Vivo Screening
Also contemplated is evaluating compounds, for example compounds identified using in vitro screening, in animal models, e.g., animal models for specific diseases that may be treated or prevented using the methods of the invention, e.g., Alzheimer's Disease. In addition, lead compounds are evaluated in wild-type animals to determine whether protein activity, stability, or trafficking is increased in response to the administration of the test compound.
Activity and Localization Assays
As indicated previously, enhanced α-secretase activity, and/or cellular localization can be determined by measuring an increase in cellular polypeptide, by determining an increase in trafficking to the appropriate cellular location, and by detecting increased α-secretase protein activity. Non-limiting exemplary methods for assessing each of the foregoing are described below.
Determining protein level. Methods for determining intracellular protein levels are well-known in the art. Such methods include Western blotting, immunoprecipitation followed by Western blotting (IP Western), or immunofluorescence using a tagged protein.
Determining α-secretase protein trafficking and localization. Assessing trafficking of proteins through the biosynthetic pathway can be achieved e.g., using pulse- chase experiments with 35S-labeled receptor protein, in conjunction with glycosidases; or by indirect or direct immunofluorescence to determine protein modification during trafficking. These and other methods are described for example in Current Protocols in Cell Biology 2001 ; John Wiley & Sons.
Methods for detecting impaired trafficking of proteins are well known in the art. For example, for proteins which are N- and/or O-glycosylated in the Golgi apparatus, pulse-chase metabolic labeling using radioactively labeled proteins, combined with glycosidase treatment and immunoprecipation, can be used to detect whether the proteins are undergoing full glycosylation in the Golgi, or whether they are being retained in the ER instead of trafficking to the Golgi for further glycosylation.
Sensitive methods for visually detecting cellular localization also include fluorescent microscopy using fluorescent proteins or fluorescent antibodies. For example, α-secretase protein can be tagged with e.g., green fluorescent protein (GFP), cyan fluorescent protein, yellow fluorescent protein, and red fluorescent protein, followed by multicolor and time-lapse microscopy and electron microscopy to study the fate of these proteins in fixed cells and in living cells. For a review of the use of fluorescent imaging in protein trafficking, see Watson et al., Adv Drug Deliv Rev 2005; 57(1):43-61. For a description of the use of confocal microscopy for intracellular co- localization of proteins, see Miyashita et al., Methods MoI Biol. 2004; 261 :399-410.
Fluorescence correlation spectroscopy (FCS) is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution (Vukojevic et al., Cell MoI Life Sci 2005; 62(5): 535-50). SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles (Cherry et al., Biochem Soc Trans 2003; 31(Pt 5): 1028-31 ). For localization of proteins within lipid rafts, see Latif et al., Endocrinology 2003; 144(1 1): 4725-8). For a review of live cell imaging, see Hariguchi, Cell Struct Funct 2002; 27(5):333-4).
Fluorescence resonance energy transfer (FRET) microscopy is also used to study the structure and localization of proteins under physiological conditions (Periasamy, J Biomed Opt 2001 ; 6(3): 287-91).
For plasma membrane resident proteins, less sensitive assays can be used to detect whether they are present on the membrane. Such methods include immunohistochemistry of fixed cells, or whole-cell labeling using radiolabeled ligand (e.g., 125I).
Once a candidate compound has been identified, the next step is determining whether the candidate compound can enhance the amount of α-secretase protein trafficked to the appropriate cellular location. Numerous assays can be used to evaluate protein levels quantitatively. For example, radioactive ligand binding assays, using e.g., ' 5I-MSH, can be used to determine binding to either whole cells expressing α-secretase protein or to cell membrane fractions. See U.S. published application 2003/0176425 for a description of one exemplary method; see also Chhajlani, Peptides. 1996; 17(2):349-51. In addition, immunofluorescence staining, using either labeled antibodies or labeled α-secretase protein {e.g., FLAG-tagged α-secretase protein), may also be used. Another well-known method is fluorescence-activated cell sorting (FACS), which sorts or distinguishes populations of cells using labeled antibodies against cell surface markers. See also, Nijenhuis et al., supra.
Determining an increase in α-secretase protein activity. Activity assays for proteins are generally well-known in the art, and can include substrate metabolism or modification (such as phosphorylation or dephosphorylation), signal transduction, and changes to cellular phenotype.
Molecular Biology Definitions
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. These techniques are generally useful for the production of recombinant cells expressing α-secretase proteins for use in screening assays. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"), and later editions; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & SJ. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way, for the production of a desired substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein, or an enzyme. According to the present invention, the host cell is modified to express α-secretase-type nucleic acid and polypeptide. Host cells can further be used for screening or other assays. Exemplary host cells for use in the present invention are HEK-293T and COS cells.
A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.
Polynucleotides herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non- coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include: methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
The terms "express" and "expression," when used in the context of producing an amino acid sequence from a nucleic acid sequence, means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product", i.e., protein. The expression product itself, e.g., the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular or secreted.
The term "heterologous" refers to a combination of elements not naturally occurring in combination. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is an element operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., an E. coli cell.
The term "recombinantly engineered cell" refers to any prokaryotic or eukaryotic cell that has been manipulated to express or overexpress the nucleic acid of interest, i.e., a nucleic acid encoding a polypeptide, by any appropriate method, including transfection, transformation or transduction. This term also includes endogenous activation of a nucleic acid in a cell that does not normally express that gene product or that expresses the gene product at a sub-optimal level.
The terms "vector," "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.; they are well known in the art.
EXAMPLES
The present invention is further described by means of the example, presented below. The use of such example is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled. EXAMPLE 1: Administration of DGJ to Evaluate Safety, Tolerability,
Pharmacokinetics, and Effect on α-Galatosidase A Enzymatic Activity
This example describes a randomized, blinded, Phase Ib study of twice daily oral doses of 1-deoxygalactonojirimycin (DGJ) to evaluate the affects of DGJ on safety, tolerability, pharmacokinetics, and α-Galatosidase A (α-GAL) enzymantic activity in healthy volunteers. It demonstrates the power of the pharmacological chaperone approach to increase the levels and/or activity of a wild-type enzyme in the normal, healthy subject.
Study Design and Duration. This study was first-in-man, single-center, Phase Ib, randomized, double-blind, twice daily-dose, placebo controlled study to evaluate the safety, tolerability, pharmacokinetics, and α-GAL enzymantic activity affects of DGJ following oral administration. The study tested two groups of of 8 subjects (6 active and 2 placebo) who received a twice daily-dose of 50 or 150 mg b.i.d. of DGJ or placebo administered orally for seven consecutive days, accompanied by a seven day follow up visit. Subjects were housed in the treatment facility from 14 hours prior to dosing until 24 hours after dosing. Meals were controlled by schedule and subjects remained abulatory for 4 hours post drug administration
Pharmacokinetic parameters were calculated for DGJ in plasma on Day 1 and Day 7. In addition, the cumulative percentage of DGJ excreted (12 hours post dose) in urine was calculated. α-GAL activity was calculated in white blood cells (WBC) before dosing began, and again at 100 hours, 150 hours, and 336 hours into the trial.
Study Population. Subjects were healthy, non-institutionalized, non-smoking male volunteers between 19 and 50 years of age (inclusive) consisting of members of the community at large.
Safety and Tolerability Assessments. Safety was determined by evaluating vital signs, laboratory parameters (serum chemistry, hematology, and urinalysis), ECGs, physical examination and by recording adverse events during the Treatment Period.
Pharmacokinetic Sampling. Blood samples (10 mL each) were collected in blood collection tubes containing EDTA before dosing and at the following times thereafter: 0.25, 0.5, 0.75, 1 , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 and 12 hours. Blood samples were cooled in an ice bath and centrifuged under refrigeration as soon as possible. Plasma samples were divided into two aliqiiots and stored at 20 ± 100C pending assay. At the end of the study, all samples were transferred to MDS Pharma Services Analytical Laboratories (Lincoln) for analysis. The complete urine output was collected from each subject for analysis of DGJ to determine renal clearance for the first 12 hours after adiministration of DGJ on days 1 and 7. WBC a-GAL A Enzymatic Activity Sampling. Blood samples (10 itiL each) were collected in blood collection tubes containing EDTA and WBC extracted before dosing and at the following times thereafter: 100 hours, 150 hours, and 336 hours. Samples were treated as described above, and WBC α-GAL enzymatic activity levels were determined as described in Desnick, R.J. (ed) Enzyme therapy in genetic diseases. VoI 2. Alan R Liss, New York, pp 17-32. Statistical Analysis. Safety data including laboratory evaluations, physical exams, adverse events, ECG monitoring and vital signs assessments were summarized by treatment group and point of time of collection. Descriptive statistics (arithmetic mean, standard deviation, median, minimum and maximum) were calculated for quantitative safety data as well as for the difference to baseline. Frequency counts were compiled for classification of qualitative safety data. In addition, a shift table describing out of normal range shifts was provided for clinical laboratory results. A normal-abnormal shift table was also presented for physical exam results and ECGs.
Adverse events were coded using the MedDRA version 7.0 dictionary and summarized by treatment for the number of subjects reporting the adverse event and the number of adverse events reported. A by-subject adverse event data listing including verbatim term, coded term, treatment group, severity, and relationship to treatment was provided. Concomitant medications and medical history were listed by treatment.
Pharmacokinetic parameters were summarised by treatment group using descriptive statistics (arithmetic means, standard deviations, coefficients of variation, sample size, minimum, maximum and median).
Results. No placebo-treated subjects had AEs and no subject presented with AEs after receiving 50 mg b.i.d. or 150 mg b.i.d. DGJ. DGJ appeared to be safe and well tolerated by this group of healthy male subjects as doses were administered at 50 mg b.i.d. and 150 mg b.i.d.
Laboratory deviations from normal ranges occurred after dosing, but none was judged clinically significant. There were no clinically relevant mean data shifts in any parameter investigated throughout the course of the study. No clinically relevant abnormality occurred in any vital sign, ECG, or physical examination parameter.
Pharmacokinetic Evaluation. The following table summarizes the pharmacokinetic data obtained during study.
Table 2.
50 mg bid dose 150 mg bid dose
Figure imgf000063_0001
a Cumulative percentage of DGJ excreted over the 12-hour post dose period.
The pharmacokinetics of DGJ were well characterized in all subjects and at all dose levels. On average, peak concentrations occurred at approximately 3 hours for all dose levels. Cm3x of DGJ increased in a dose-proportional manner when doses were increased from 50 mg to 150 mg.
The mean elimination half-lives (ti/2) were comparable at dose levels of 50 and 150 mg on Day 1 (2.5 vs. 2.4 hours).
The mean percentage of DGJ excreted over the 12-hour post dose period was 16% and 42% at dose levels of 50 and 150 mg, respectively, on Day 1, increasing to 48% and 60%, respectively, on Day 7.
a-Galactosidase A (a-Gal A) Enzymatic Activity. The α-GAL enzymatic activity data obtained during the study is shown in Figure 1. DGJ did not inhibit WBC α-GAL enzymatic activity in subjects at dosages of 50 mg b.i.d. or 150 mg b.i.d. Furthermore, AT- 1001 produced a dose-dependent trend of increased WBC α-GAL activity in healthy volunteers. α-GAL enzymatic levels were measured in WBC of subjects administered placebo, 50 mg b.i.d. DGJ, and 150 mg b.i.d. DGJ. Placebo had no affect on WBC α-GAL enzymatic levels. Variations in enzymatic levels in response to placebo were not clinically significant. Both 50 mg b.i.d. and 150 mg b.i.d. DGJ increased normalized WBC α-GAL enzymatic levels. In response to 50 mg b.i.d. DGJ, WBC α-GAL enzymatic activity increased to 120%, 130%, and 145% pre-dose levels at 100 hours, 150 hours, and 336 hours post-dose, respectively. In response to 150 mg b.i.d. DGJ, WBC α-GAL enzymatic activity increased to 150%, 185%, and 185% pre-dose levels at 100 hours, 150 hours, and 336 hours post-dose, respectively.
Discussion These results represent proof-of-principle for the efficacy of chaperone therapy. DGJ was shown to increase the activity of α-GAL to levels significantly higher in afflicted individuals that what would have been expected from previous in vitro and transgenic animal studies. Unexpectedly, enzyme activity remains elevated even when the dose was reduced after 6 weeks to the lowest dose (50 mg/day), which demonstrates the potency of the chaperone therapy.
Conclusion. Based on the foregoing, pharmacological chaperone DGJ has been shown to enhance activity of wild-type α-GAL.
EXAMPLE 2: Administration of IFG to Evaluate Safety, Tolerability,
Pharmacokinetics,
and Affect on β-Glucocerebrosidase Enzymatic Activity
In the Phase 1 trials to evaluate the safety of IFG in healthy adult subjects, single doses of up to 300 mg, and repeated doses of up to 225 mg/d for 7 days were administered orally in randomized, double-blind, placebo controlled, studies. In the multiple dose study, three cohorts of 8 subjects (6 active and 2 placebo per cohort) received daily oral doses of 25, 75, or 225 mg IFG or placebo for 7 days, with a treatment-free safety evaluation period of 7 days. Blood samples were collected for pharmacokinetic analysis before the initial drug administration on Day 1 , before the 5th, 6th and 7th doses (on Days 5, 6 and 7) (for Cmin determination), and at the following times after the 1st (Day 1 ) and 7th (Day 7) doses: 0.5, 1 , 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, and 24 hours. In addition, a single blood sample was collected 48 hours after the last dose (Day 9) and assayed for the presence of IFG. In addition, blood samples were collected for pharmacodynamic measurements, i.e., analysis of WBC GCase levels, before dosing on Day 1 , Day 3, Day 5, and Day 7, and at return visits on Day 9, Day 14 and Day 21.
In the multiple-dose study, after 7 days of oral administration, the pharmacokinetic behavior was found to be linear with dose, with no unexpected accumulation of IFG. Mean plasma levels (Tmax) peaked at about 3.4 hr. (SEM: 0.6 hr.) and the plasma elimination half- life was about 14 hr. (SEM: 2 hr.).
Importantly, healthy subjects receiving IFG showed a dose-dependent increase in GCase levels in white blood cells during the 7-day treatment period, in most cases peaking on day 7 of treatment, followed by a more gradual decrease in enzyme levels upon removal of the drug and a return to near baseline levels by 14 days after the last dose. The maximum increase in enzyme level achieved was approximately 3.5-fold above baseline levels. The lowest daily dose that achieved the maximum rate of GCase accumulation over 7 days was about 75 mg.
EXAMPLE 3: In vivo Evaluation of DGJ on Mutant α-Gal A Activity
Enhancement
This example describes results from an open label Phase II study of DGJ in Fabry patients (n = 1 1 ) with α-GAL folding mutants and supports the use of the in vivo assay.
Patients were administered DGJ according to the following dosing schedule: 25 mg b.i.d. two weeks; 100 mg b.i.d. 2-4 weeks; 250 mg b.i.d. 4-6 weeks; and 25 mg b.i.d. 6-12 weeks; blood was draw into an 8 mL Vacutainer CPT tube at the end of each dosing period and treated as described below.
A1I Preparation of Human WBC Pellets for Assay
WBCs were prepared substantially as described in Example 1 , with the exception that no FBS/DMSO is added to the pellet prior to freezing.
The preliminary data is summarized in the following table.
B. Preparation of Human WBC Lysates for Assay
• To the microtubes containing the WBC pellet, 0.6 ml of lysis buffer (26 mM citrate/46 mM phosphate, pH 5.5) were added
• Tubes were vortexed until the cells were re-suspended
• Tubes were incubated at room temperature for about 15 minutes, but agitate the suspension by vortexing every couple of minutes
• Tubes were sonicated for 2 minutes, then vortexed for about 10 seconds
• Lysates were incubated on ice until chilled, and then pooled into a pre-chilled polyproylene container (on ice)
• Container was vortexed and pooled lysates were divided into 0.100 mL aliquots in pre-chilled labeled 0.5 mL screw-cap polypropylene microcentrifuge tubes. Pooled lysates were mixed while aliquoting by vortexing between every 10-20 aliquots.
• Aliquots were stored at -80°C until use.
C. Human WBC Assay • Each tube containing lysate was thawed on ice, sonicated for 2 minutes, then vortexed for 1 minute.
• 50 μl of each standard, control, or clinical sample was added into appropriate wells of a black polystyrene microplate (use 50 μl of 0.5% BSA in WBC lysis buffer for a standard)
• 50 μl of 1 17 mM GaINAc was added to all wells, and the plate was incubated for 5 minutes at ambient temperature;
• 50 μl of 5 mM 4-MU Gal substrate was added to to all wells and the wells were mixed on a plate shaker for 30 seconds
• The plate was covered and incubated for about 1 hour at 37°C
• 100 μl of 0.2M NaOH/Glycine buffer, pH 10.7 was added to each well to stop the reaction
• The plate was read using a fluorescent plate reader
Results
The available data from the first eleven patients treated with DGJ for at least 12 weeks suggest that treatment with DGJ leads to an increase in the activity of the enzyme deficient in Fabry disease in 10 of the 1 1 patients (Figure 2) Eight patients in the study received an acending dose of 25, 100, and then 250 mg b.i.d. over 6 weeks, followed by 50 mg/day for the remainder of the study (represented by closed circles). Three patients in the study received 150 mg of DGJ every other day throughout the entire study. For purposes of calculating the percentage of normal in the table, the level of α-GAL that is normal was derived by using the average ofthe levels of α-GAL in white blood cells of 15 healthy volunteers from the multiple-dose Phase I study. The 1 1 patients represented 10 different genetic mutations and had baseline levels of α-GAL that ranged from zero to 30% of normal.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
EXAMPLE 4: In vitro Stabilization of purified ADAMlO by TAPI-2, GM6001 and GW4023
Figure 3 depicts a time course (96 hr) showing the effects of GM6001 (Figure 3A) and TAPI-2 (Figure 3B) on ADAMlO levels in SY5Y neuroblastomas. Figure 3C depictss a dose-response showing the effects of TAPI-2 on ADAMlO precursor levels in SY5Y neuroblastomas. ADAMl O thermal denaturation (melting) curves with and without TAPI-2 (Figure 3D) and GW4023 (Figure 3E). ADAMlO has a Tm of 52 0C. The ADAMlO inhibitors TAPI-2 and GW4023 shift the Tn, by 9 0C and 16 0C, respectively, demonstrating their ability to stabilize wilt-type ADAMlO.
A. Methods for Figure 3A-3B
SY5Y neuroblastoma cells in 6-well plates were dosed with lOOuM of alpha secretase inhibitors GM6001 (AA) or TAPI-2 (AB) in complete growth media (DMEM+10%FBS) for up to 96hrs. At time points of Ohrs (no dose control), 15hrs, 24hrs, 48hrs, 72hrs and 96hrs, cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem. Co.) and equal amounts of protein were electrophoresed on a 4-12% Bis-NuPage gel (Invitrogen) followed by transfer (25V, lhr) onto PVDF for Western blotting. ADAMlO was probed using a rabbit anti- ADAMlO polyclonal antibody (1 : 1000, Abeam cat. No. Abl997) and anti-rabbit/alkaline- phosphatase secondary antibody. ADAMl O bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
B. Methods for Figure 3C
SY5Y neuroblastoma cells in 6-well plates were dosed with OuM, 0.01 uM, 0. IuM, IuM, l OuM and lOOuM of the alpha secretase inhibitor TAPI-2 in complete growth medium (DMEM+10% FBS). After 96hrs, cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem. Co.) and equal amounts of protein were electrophoresed on a 4-12% Bis-NuPage gel (Invitrogen) followed by transfer (25V, l hr) onto PVDF for Western blotting. ADAM lO was probed using a rabbit anti-ADAMIO polyclonal antibody (1 : 1000, Abeam cat. No. Ab 1997) and anti-rabbit/alkaline-phosphatase secondary antibody. ADAMlO bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
C, Methods for Figure 3D and 3E
The environmentally sensitive dye SYPRO Orange and a Realplex Mastercycler qRT- PCR system were used to apply a temperature gradient while simultaneously monitoring fluorescence changes in a 96-well format. ADAMlO, in neutral buffer (50 mM sodium phosphate, 150 mM sodium chloride, 2 μM zinc chloride, pH 7.4) was combined with 2.5 μl 50X SYPRO Orange and 20 μM TAPI-2 in a reaction volume of 25 ml. Once assembled, the plate is heated at a rate of l°C/min, and fluorescence (Ex. 470 nm; Em. 520 nm) intensities are measured and plotted as a function of temperature. ADAMl O demonstrates a typical thermal denaturation curve with a Tm (melting temperature) of 52 0C. In the presence of 20 μM TAPI-2, the Tm is shifted to 61 0C, a shift of 9 0C (Fig. 2A), while another more potent inhibitor GW4023 shifted the Tm by 16 0C (data not shown).
Note: GW4023 is also known as GI254023X

Claims

WHAT IS CLAIMED IS:
1. A method for increasing the activity of α-secretase in an individual suspected of suffering from Alzheimer's Disease, which comprises administering to the individual a pharmacological chaperone that binds to the α-secretase in an amount effective to increase activity of the α-secretase in the individual.
2. The method of claim 1, which comprises administering the pharmacological chaperone in an amount effective to increase the activity of the protein by at least about 50%.
3. The method of claim 2, which comprises administering the pharmacological chaperone in an amount effective to increase the activity of the protein by at least about 90%.
4. The method of claim 3, which comprises administering the pharmacological chaperone in an amount effective to increase the activity of the protein by at least about 100%.
5. The method of claim 1, wherein the individual is homozygous for the wild type α- secretase.
6. The method of claim 1, wherein the individual is heterozygous for the wild type α- secretase and has a mutant genotype for the other allele encoding α-secretase.
7. The method of claim 6, wherein the pharmacological chaperone is an α-secretase inhibitor.
8. The method of claim 7, wherein the α-secretase inhibitor is a batimastat compound, or a batimastat analog.
9. The method of claim 8, wherein the batimastat analog is SB223820.
10. The method of claim 8, wherein the batimastat analog is marimastat.
11. The method of claim 8, wherein the batimastat analog is BB3132.
12. The method of claim 8, wherein the batimastat analog is TAPI-O.
13. The method of claim 8, wherein the batimastat analog is TAPI-I .
14. The method of claim 8, wherein the batimastat analog is TAPI-2.
15. The method of claim 8, wherein the batimastat analog is Immunex compound 3 (IC3).
16. The method of claim 8, wherein the batimastat analog is KD-IX-73-4.
17. The method of claim 8, wherein the batimastat analog is BB2116.
18. The method of claim 8, wherein the batimistat compound is batimistat.
19. The method of claim 1, wherein the individual has a risk factor for Alzheimer's Disease.
20. The method of claim 1, wherein the individual has been diagnosed with Alzheimer's Disease.
21. The method of claim 1, wherein the individual is at risk for developing Alzheimer's Disease
22. A method for increasing the activity of α-secretase in an individual at risk of developing a neurological disorder associated with pathologic αβ42, which comprises administering to the individual a pharmacological chaperone that binds to the α-secretase in an amount effective to increase activity of the α-secretase in the individual.
23. The method of claim 20, wherein the neurological disorder is Down syndrome or congophilic angiopathy.
24. The method of claim 20, wherein the α-secretase inhibitor is a batimastat compound, or a batimastat analog.
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