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EP1592707A2 - Hmgb1 modulator binding domain - Google Patents

Hmgb1 modulator binding domain

Info

Publication number
EP1592707A2
EP1592707A2 EP04710953A EP04710953A EP1592707A2 EP 1592707 A2 EP1592707 A2 EP 1592707A2 EP 04710953 A EP04710953 A EP 04710953A EP 04710953 A EP04710953 A EP 04710953A EP 1592707 A2 EP1592707 A2 EP 1592707A2
Authority
EP
European Patent Office
Prior art keywords
hmgbl
modulator
binding domain
carbon atoms
modulator binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04710953A
Other languages
German (de)
French (fr)
Inventor
Marco E. San Raffaele Scientific Inst. BIANCHI
Giovanna A. San Raffaele Scientific Inst. MUSCO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fondazione Telethon
Ospedale San Raffaele SRL
Original Assignee
Fondazione Telethon
Ospedale San Raffaele SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fondazione Telethon, Ospedale San Raffaele SRL filed Critical Fondazione Telethon
Publication of EP1592707A2 publication Critical patent/EP1592707A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention relates to an HMGBl modulator binding domain, an HMGBl modulator binding domain having a modulator which is bound to it, modulators of an HMGBl modulator binding domain and their uses in therapy.
  • HMGBl The non-histone nuclear protein HMGBl belongs to the B family of HMG proteins also known as the high mobility group. HMGBl was originally identified as a ubiquitously expressed, abundant nuclear protein. When bound to double stranded DNA, it induces structure distortion, allowing the formation of nucleoprotein complexes where several DNA-binding proteins can contact each other while bound to their respective DNA cognate sites (Muller et al., 2001).
  • HMGBl HMGBl mediates endotoxin lethality, acute lung injury, arthritis induction, activation of macrophages, smooth muscle cell chemotaxis, and epithelial cell barrier dysfunction. It is released as a late mediator during inflammation and participates in the pathogenesis of systemic inflammation after the early mediator response has resolved (Andersson et al., 2002).
  • WOOO/47104 describes the use of a pharmaceutical composition comprising an antagonist or agonist HMG1 (now designated HMGBl) for treating conditions characterised by activation of the inflammatory cytokine cascade, including sceptic shock.
  • HMG1 now designated HMGBl
  • HMGBl occupies a critical role as a proinflammatory mediator passively released by necrotic but not apoptotic cells.
  • International Patent Application No. PCT/IT03/00265 demonstrates that HMGBl released by necrotic cells can be the initial trigger for inflammatory responses such as inflammatory cell activation, and that released HMGBl itself can activate inflammatory cells. It further demonstrates that HMGBl acts as a chemoattractant and proliferation factor for stem cells.
  • HMGBl has been identified as one of the ligands binding to the RAGE receptor (Receptor for advanced glycation endproducts) (Hori et al., 1995).
  • RAGE is a multiligand receptor of the immunoglobulin superfamily, is expressed in many cell types, including endothelial cells, mononuclear phagocytes, and neurons (Brett et al., 1993; Neeper et al., 1992) and is implicated in several different pathological processes, such as diabetes, amyloidoses and atherosclerosis (Scmidt et al., 1999).
  • HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage.
  • SMC smooth muscle cells
  • HMGBl can be used to upregulate or downregulate an antigen mediated immune response.
  • a mediator in the inflammatory cytokine cascade as a chemoattractant and proliferation factor for stem cells, a regulator of antigen mediated immune responses, and a factor in promoting atherosclerosis and restenosis after vascular damage there is a pressing need for the development of effective modulators of HMGBl function.
  • the present invention addresses this problem.
  • an HMGBl modulator binding domain having the structure substantially as represented by the space filling balls in Figure 4 or Figure 11.
  • an HMGBl modulator binding domain comprising: at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45; or a homologue or mutant thereof.
  • the domain comprises all of the amino acid residues 11, 12, 13, 17, 20, 23, 25 and 27 and in helix 1 and/or all of the amino acid residues 43, 44 and 45 in helix 2. In another embodiment the domain comprises all of the aforementioned amino acid residues in helix 1 and helix 2.
  • the domain comprises all of the amino acid residues 17, 20, 23, 25 and 26 in helix 1 and/or residue 42 in helix 2. In another embodiment the domain comprises all of the aforementioned amino acid residues in helix 1 and 2. In one embodiment the HMGBl modulator binding domain is defined by a mutation or substitution or derivatisation in or of any one or more of the amino acid residues defined above.
  • the HMGBl modulator binding further comprises a modulator bound to the modulator binding domain or a portion thereof.
  • an HMGBl modulator binding domain wherein the modulator comprises a pentacyclic triterpene moiety as represented by general formula II (see later).
  • the modulator is a compound of formula III:
  • each R 1 is independently CH 3 , H, OH, CH 2 OY ! , CH 2 O-X-OH, OEfcO-X-OY 1 , CH2O-X-Y 2 , CH 2 O-X-Y 3 .
  • R 2 or R 3 is R 1 , OY 1 , O-X-OH, O-X-OY 1 , O-X-Y 2 , Y 3 , NHY 1 , Y ⁇ , Y 3 , NH-X-OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY ! -X-OH, NY ⁇ X-Y 2 , NY ⁇ X-Y 3 , NY'-X-OY 1 , monosaccharide, disaccharide, oligosaccharide or polysaccharide or a derivative thereof, provided that one of R or R is H or that together they form an oxo group;
  • R 4 is H, R 1 , provided that at least one R 4 is H or together they form an oxo group;
  • R 5 is H, OH, OY 1 OR Y 3 ;
  • Y 1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
  • Y 2 is NH 2 , NHY 1 OR NY 2 ;
  • Y 3 is -(O(CH 2 ) m ) procurR 5 or -(O(CH 2m ) justifyY 2 ;
  • n 1-4;
  • n 1-50;
  • X is -OC(CH 2 ) p CO-
  • P is 1-22;
  • the modulator is glycyrrhizic acid (glycyrrhizin), glycyrrhetinic acid, ursolic acid or carbenoxolone or a salt or a mimetic thereof.
  • a method for screening for a modulator capable of binding to an HMGBl modulator binding domain of the present invention comprising contacting the HMGBl modulator binding domain with a test agent, and determining if said agent binds to said HMGBl modulator binding domain.
  • the modulator identified by any of the above methods is capable of spatially fitting into an HMGBl modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4.
  • the modulator identified by any of the above methods is capable of interacting with at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl : 11, 12, 13, 17, 20, 23, 25 and 27; and or at least one or more of the following amino acid residues of helix 2 of HMGBl: 43, 44 and 45.
  • the domain also comprises residue 91.
  • a pharmaceutical composition comprising a modulator identified above by any of the above methods of the present invention and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant or any combination thereof.
  • a method of modulating the activity of HMGBl comprising administering a modulator of the present invention as defined above.
  • a method of preventing and/or treating a disorder associated with HMGBl comprising administering a modulator according to the present invention, or a pharmaceutical composition of the present invention, wherein said modulator or pharmaceutical composition is capable of modulating HMGBl to cause a beneficial and/or therapeutic effect.
  • the method is for the treatment of a condition associated with activation of the inflammatory cytokine cascade.
  • the method is for downregulating an immune specific response.
  • the method is for the treatment of an inflammatory or autoimmune disease, allergy or transplant rejection.
  • the method is for inhibiting stem cell migration and/or proliferation.
  • the method is for inhibiting epithelial cell migration and/or proliferation.
  • the method is for the treatment of vascular diseases, in particular atherosclerosis and/or restenosis that occur during angioplasty and/or angiography.
  • the method is for the blocking, retarding or reducing connective tissue regeneration.
  • the modulators are released by catheters, surgical instruments or stents for angioplasty.
  • the modulator is glycyrrhizic acid or a salt or mimetic thereof.
  • the HMGBl is acetylated.
  • a method for predicting, simulating or modelling the molecular characteristics and/or molecular interactions of an HMGBl modulator binding domain comprising the use of a computer model, said computer model comprising using or depicting the structure substantially as presented by the space filling balls in Figure 4 or Figure 11 and/or the structural coordinates of a modulator binding domain as provided by at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45, to provide an image of said HMGBl modulator binding domain, and optionally to display said image.
  • the method comprises the use of a computer model to provide an image of said modulator, and optionally to display said image.
  • the method comprises providing an image of said modulator in association with said HMGBl modulator binding domain, and optionally displaying said image.
  • said modulator is prepared and optionally formulated as a pharmaceutical composition.
  • a computer readable medium having stored thereon parameters capable of displaying a representation of a 3D model comprising the HMGBl modulator binding domain.
  • said model is built for all or part of the HMGB 1 modulator binding domain as defined by the present invention
  • a computer controlled method for designing a modulator capable of binding to HMGBl comprising:
  • a machine- readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a representation of an HMGBl modulator binding domain of a homologue thereof.
  • a computer comprising the above storage medium of the present invention.
  • HMG-1 As reported in WOOO/47104, HMGBl (indicated there as HMG-1) is a 25 kDa chromosomal nucleoprotein belonging to the burgeoning high mobility group (HMG) of non-histone chromatin-associated proteins.
  • HMG proteins recognise unique DNA structures and have been implicated in diverse cellular functions, including determination of nucleosome structure and stability, as well as in transcription and/or replication.
  • the HMG proteins were first characterised by Johns and Goodwin as chromatin components with a high electrophoretic mobility in polyacrylamide gels (see in The HMG Chromosomal Proteins, E.W. Johns, Academic Press, London, 1982).
  • HMG proteins are highly conserved across species, ubiquitously distributed and highly abundant, and are extractable from chromatin in 0.35 M NaCI and are soluble in 5% perchloric or trichloroacetic acid.
  • HMGB proteins are thought to bend DNA and facilitate binding of various transcription factors to their cognate sequences, including for instance, progesterone receptor, estrogen receptor, HOX proteins, and Octl, Oct2 and Oct6.
  • HMGBl a large, highly diverse group of proteins including several transcription factors and other DNA-interacting proteins, contain one or more regions similar to HMGBl, and this feature has come to be known as the HMG box or HMGB domain.
  • cDNAs coding for HMGBl have been cloned from human, rat, mouse, mole rat, trout, hamster, pig and calf cells, and HMGBl is believed to be abundant in all vertebrate cell nuclei. The protein is highly conserved with interspecies sequence identities in the 80% range.
  • HMGBl In chromatin, HMGBl binds to linker DNA between nucleosomes and to a variety of non-B-DNA structures such as palindromes, cruciforms and stem-loop structures, as well as cisplatin-modified DNA. DNA binding by HMGBl is generally believed to be sequence insensitive. HMGBl is most frequently prepared from washed nuclei or chromatin, but the protein has also been detected in the cytoplasm. (Reviewed in Landsman and Bustin, BioEssays 1993; Baxevanis and Landsman, 1995).
  • HMGBl has a tripartite structure, composed by two homologous DNA- binding domains, the HMG-boxes entitled box A and box B, and a C-terminal domain of aspartic and glutamic acids (reviewed in Bustin, 1999, Bianchi and Beltrame, 2000, and Thomas and Travers, 2001).
  • the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) has been expressed in Escherichia coli its structure has been determined by 2D 1H-NMR spectroscopy (Weir et al., 1993).
  • the structure of box B has also been determined by Read (Nucleic Acids Res 1993 Jul 25;21(15):3427-36).
  • the structure of the A-domain has been determined using heteronuclear three- and four-dimensional NMR spectroscopy (Hardman et al., Biochemistry 1995).
  • the A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D (Jones et al., 1994).
  • HMGBl In most cells, HMGBl is located in the nucleus, where it acts as an architectural protein that facilitates nucleoprotein assembly. It binds to the minor groove of DNA, inducing a local distortion of the double helix. Its lack of sequence-specificity is offset by recruitment via protein-protein interaction by different types of nuclear factors (including the Hox and Pou proteins, the steroid hormone receptors, p53, TBP, some viral proteins and the RAG1 recombinase). HMGBl can bind to nucleosomes (Falciola et al., 1997; Nightingale et al., 1996) but in vivo its association with chromatin is very dynamic.
  • HMGBl Surprisingly, beyond its intranuclear function, HMGBl also has a pivotal function outside of the cell (reviewed by M ⁇ ller et al., 2001b). Wang et al. (1999a) identified HMGBl as a late mediator of endotoxin lethality in mice, and showed that macrophages and myeloid cells stimulated by LPS, TNF or IL-1 secrete HMGBl as a delayed response. HMGBl can then act as a cytokine, eliciting several different responses in cells that are equipped with receptors to it. For example, HMGBl recruits inflammatory cells and promotes the secretion of TNF.
  • HMGBl In addition to monocytes, developing neurons and a few other cell types also secrete HMGBl in response to specific stimuli (reviewed by M ⁇ ller et al., 2001b). However, most cells are not able to secrete HMGBl in an active manner.
  • HMGBl protein In myeloid cells, secretion does not involve HMGBl protein newly made in the cytoplasm, but proceeds through the depletion of nuclear stores. The secretion of a nuclear protein poses daunting challenges. We recently showed that activation of myeloid cells results in the redistribution of HMGBl from the nucleus to secretory lysosomes (Gardella et al., 2002). HMGBl does not traverse the endoplasmic reticulum and the Golgi apparatus, consistent with the absence of a leader peptide in the protein.
  • IL-l ⁇ interleukin-l ⁇
  • HMGBl lysophosphatidylcholine
  • Myeloid cells acetylate HMGBl in response to activation: in promyelocytic cells, binding of LPS or inflammatory cytokines to their surface receptors promotes the activation of the MAP kinase pathway that impinges on ERK.
  • HMGBl is acetylated and moves from the nucleus to the cytoplasm, where it is concentrated in secretory lysosomes and can be secreted in response to a second signal, LPC.
  • myeloid cells have a signaling pathway that allows them to regulate HMGBl acetylation is response to inflammatory stimuli, switching a chromatin protein into a cytokine.
  • Methods of modulating HMGBl activity according to the present invention include methods of modulating acetylated HMGBl activity.
  • HMGBl is claimed in International as a chemoattractant and proliferation factor for stem cells and a method to induce stem cell migration and/or proliferation in cell culture or in vivo comprising the step of exposing such cells to effective amounts of HMGBl protein or functional parts thereof is provided. It was demonstrated that HMGBl released by necrotic cells could be the initial trigger for inflammatory responses and that released HMGBl itself could activate inflammatory cells. HMGBl diffusion can take place in a matter of seconds or minutes and is thus a very early event in inflammation.
  • HMGBl has been identified as one of the ligands binding to the RAGE receptor (Receptor for advanced glycation endproducts) (Hori et al., 1995).
  • WO 02/074337 showed that HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage.
  • SMC smooth muscle cells
  • the present invention also relates to variants, derivatives and fragments of HMGBl and HMGBl modulator binding domain according to the invention.
  • the variant sequences etc. are at least as biologically active as the sequences presented herein.
  • HMGBl includes actetylated HMGBl.
  • biologically active refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.
  • variants, derivative and fragments comprise one or both of the HMG boxes.
  • protein includes single-chain polypeptide molecules as well as multiple- polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.
  • polypeptide includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
  • amino acid sequences for use in the invention are not limited to the particular sequences or fragments thereof or sequences obtained from a particular protein but also include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • the present invention covers variants, homologues or derivatives of the amino acid sequences for use in the present invention, as well as variants, homologues or derivatives of the nucleotide sequence coding for the amino acid sequences used in the present invention.
  • HMGBl modulator binding domain means the region of HMGBl which is responsible for modulator binding.
  • HMGBl modulator binding domain also includes a homologue of the modulator binding domain or a portion thereof.
  • the HMGBl modulator binding domain of the present invention may be contained within box A and/or box B.
  • HMGBl modulator binding domain of the present invention is contained within box A of HMGB 1.
  • portion thereof means the structural co-ordinates corresponding to a sufficient number of amino acid residues of HMGBl modulator binding domain (or homologues thereof) that are capable of interacting with an agent capable of binding to the of HMGBl modulator binding domain.
  • the HMGBl modulator binding domain may be defined by its association with the modulator.
  • the HMGBl modulator binding domain of the present invention is as substantially as presented by the space filling balls in Figure 4 or Figure 11.
  • an HMGBl modulator binding domain comprising: at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45; or a homologue or mutant thereof.
  • an HMGBl modulator binding domain which further comprises a modulator bound to the modulator binding domain or a portion thereof.
  • the HMGBl modulator binding domain may be associated with a modulator.
  • the modulator may be any compound which is capable of interacting stably and specifically with the HMGBl modulator binding domain.
  • the modulator may, for example, be an inhibitor of the HMGB 1 modulator binding domain.
  • the HMGBl modulator binding domain of the present invention substantially as presented by the space filling balls in Figure 4 or Figure 11 has the structural co-ordinates corresponding to at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45 which may be used for the identification of a modulator capable of binding to the HMGB 1 modulator binding domain.
  • structural co-ordinates refer to a set of values that define the position of one or more amino acid residues with reference to a system of axes.
  • the term refers to a data set that defines the three dimensional structure of a molecule or molecules (e.g. Cartesian coordinates, temperature factors, and occupancies).
  • Structural coordinates can be slightly modified and still render nearly identical three dimensional structures.
  • Structural coordinates that render three dimensional structures in particular a three dimensional structure of an SGC domain) that deviate from one another by a root-mean-square deviation of less than 5 A, 4 A, 3 A, 2 A, or 1.5 A may be viewed by a person of ordinary skill in the art as very similar.
  • the solution structures of HMGBl box A and B have been determined. (Biochemistry 1995 Dec 26;34(51):16596-607 and EMBO J. 1993 Apr; 12 (4): 1311-9).
  • an HMGBl modulator binding domain which is defined by a mutation or substitution or derivatisation in any one or more amino acids.
  • the term "mutant” refers to any organism that has undergone mutation or that carries a mutant gene that is expressed in the phenotype of that organism.
  • a mutation may arise due to a substitution of one nucleotide for another or from a deletion of a nucleotide or an insertion of a nucleotide relative to a referenced wild type sequence.
  • SNPs single nucleotide polymorphisms
  • SNPs may occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other SNPs may occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other SNPs may have no phenotypic effects.
  • mutant refers to HMGBl modulator binding domain comprising any one or more changes in the sequence and of the amino acid residues.
  • the mutated amino acid residue(s) is/are located in N-terminal helix 1 of HMGB land/or selected from amino acid residues of helix 2 of HMGBl wherein the amino acid changes in the HMGBl modulator binding domain may be selected from amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and or selected from amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45.
  • mutant is not limited to the above mutations which are reflected in amino acid substitutions of the key amino acid residues in the HMGBl modulator binding domain but may also include and is not limited to other deletions or insertions of nucleotides in the wild type sequence which may result in changes in the amino acid residues in the deduced amino acid sequence of the HMGBl modulator binding domain.
  • mutant also includes uncharacterised mutants.
  • the term "homologue” refers to an HMGBl modulator binding domain or a portion thereof which may have deletions, insertions or substitutions of amino acid residues as long as the binding specificity of the HMGBl modulator binding domain is retained.
  • deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the binding specificity of the HMGBl modulator binding domain is retained.
  • a conservative substitution which may produce a silent change which may result in a functionally equivalent HMGBl modulator binding domain.
  • variant or derivative in relation to the amino acid sequences HMGBl or HMGBl modulator binding domain includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has HMGBl activity and/or HMGBl modulator binding activity, preferably having at least the same activity as human HMGB 1.
  • HMGBl or HMGBl modulator binding domain of the present invention may be modified for use in the present invention. Typically, modifications are made that maintain the activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the HMGBl modulator binding activity and/or HMGBl activity. Amino acid substitutions may include the use of non-naturally occurring analogues.
  • Proteins for use in the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins for use in the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein of interest.
  • Proteins for use in the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention.
  • nucleotide sequence refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof) which comprise the nucleotide sequences encoding HMGBl or an HMGBl modulator binding domain of the present invention.
  • Polynucleotides for use in the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides for use in the invention.
  • variant in relation to the nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a polypeptide having the capability of binding HMGBl modulators.
  • Nucleotide homology comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.
  • Polynucleotides for use in the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%), preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • Preferred polynucleotides for use in the invention will comprise regions homologous to the HMG box, preferably at least 80 or 90% and more preferably at least 95% homologous to the HMG box.
  • the term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide for use in the invention is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening.
  • background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Nol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • both strands of the duplex are encompassed by the present invention.
  • the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.
  • Polynucleotides which are not 100% homologous to the sequences used in the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells e.g. rat, mouse, bovine and primate cells
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the human HMGBl sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the protein or nucleotide sequences for use in the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
  • Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as a DNA polynucleotides and probes for use in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides for use in the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
  • HMGBl or an HMGBl modulating domain according to the present invention may be produced by bacterial cells (Bianchi 1991, Lee et al. 1998) by yeasts (Mistry et al. 1997), or by purification from cell cultures or from mammalian tissues.
  • Vectors/polynucleotides for use in the invention may introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adeno viruses, direct injection of nucleic acids and biolistic transformation.
  • retroviruses such as retroviruses, herpes simplex viruses and adeno viruses
  • Host cells comprising polynucleotides of the invention may be used to express proteins for use in the invention.
  • Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention.
  • Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • Proteins for use in the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
  • ⁇ -helix means a helical or spiral configuration of a polypeptide chain in which successive turns of the helix are held together by hydrogen bonds between the amide (peptide) links, the carbonyl group of any given residue being hydrogen-bonded to the imino group of the third residue behind it in the chain. This is the case for all of the carbonyl and amide groups of the peptide bonds of the main chain.
  • the ⁇ -helix has 3, 6 residues per turn and the translation or pitch along the helical axis is 1.5 A per residue and 5.4A per turn.
  • the helix may be left- or right-handed, the latter being much more common.
  • the ⁇ -helix is one of the two basic elements of the secondary structure adopted by the polypeptide chain within the hydrophobic core of a globular protein. The other basic element is the ⁇ strand.
  • key residues refers to one or more amino acid residues in a HMGBl modulator binding domain, capable of moderating modulator binding.
  • the residues may be any one of the key residues within the HMGBl modulator binding domain as described herein or mutants thereof or they may be residues with homology to the residues or mutants thereof.
  • the key amino acid residues of the HMGBl modulator binding domain may be any one or more of the amino acid residues selected from the group consisting of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25 and 27; and/or any one or more of the following amino acid residues of helix 2 of HMGBl : 43, 44 and 45; or a homologue or mutant thereof.
  • the invention relates to a method of screening for an agent capable of binding to a HMGBl modulator domain of the present invention the method comprising contacting the HMGBl modulator domain with an agent and determining if said agent binds to said HMGBl modulator domain.
  • agent includes, but is not limited to, a compound, such as a test compound, which may be obtainable from or produced by any suitable source, whether natural or not.
  • the agent may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds.
  • the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic test compound, a semi- synthetic test compound, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised test compound, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant test compound, a natural or a non-natural test compound, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
  • modulator refers to an agent capable of binding to the HMGBl modulator binding domain, preferably to one or more key residues in the HMGBl modulator binding domain.
  • a modulator may increase or decrease HMGBl, or change its characteristics, or functional or immunological properties. It may be an inhibitor that decreases the biological or immunological activity of the protein.
  • Modulators include but are not limited to peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies, carbohydrates, nucleosides or nucleotides or parts thereof, and small organic or inorganic molecules.
  • a modulator may be an endogenous physiological compound, or it may be a natural or synthetic compound.
  • a modulator of the present invention agent may also be capable of displaying one or more other beneficial functional properties.
  • the modulator may be an antagonist or agonist of HMGBl.
  • the agent is an antagonist of HMGBl.
  • the agent selectively downregulates an immune specific response and or may be used for the treatment of a condition associated with activation of the inflammatory cytokine cascade and/or may be used for the treatment of an inflammatory or autoimmune disease, allergy or transplant rejection.
  • the agent may be used for inhibiting stem cell migration and/cell proliferation and/or inhibiting epithelial cell migration and/or proliferation or may be used for the treatment of vascular diseases.
  • the modulator has at least about a 25, 50, 75, 100 fold selectivity to the desired target, preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 250 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least about a 350 fold selectivity to the desired target.
  • the modulator or agent can be an amino acid sequence or a chemical derivative thereof.
  • the substance may even be an organic compound or other chemical.
  • the agent may even be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence.
  • the agent may even be an antibody.
  • an HMGBl modulator wherein the modulator comprises a saccharide moiety.
  • saccharide we include any of a group of water-soluble carbohydrates of relatively low molecular weight and typically having a sweet taste.
  • the simple sugars are called monosaccharides.
  • More complex sugars comprise generally between two and ten monosaccharides linked together: disaccharides contain two, trisaccharides contain three and so on.
  • the modulator contains a disaccharide residue.
  • the modulator also comprises a hydrocarbon ring system.
  • hydrocarbon we mean that the ring is generally composed of hydrogen and carbon, but may include other atoms which do not substantially affect the properties of the ring system, for example the ring system may include one or more heteroatoms.
  • the hydrocarbon ring system is preferably substituted, for example with hydrogen, an alkyl, oxo, COOH or ester group.
  • the ring system comprises one or more rings, which may or may not be aromatic - a combination of aromatic and non-aromatic rings may be employed.
  • the ring system may comprises two, three, four, or preferably five rings.
  • X and Y each represent a hydrogen atom or form together an oxo group
  • Z represents a monosacchande; disaccharide, oligosaccharide or polysaccharide or a derivative thereof;
  • R' is a hydrogen atom, an optionally substituted alkyl or optionally substituted alkenyl group; or a salt thereof.
  • R' is an optionally substituted C ⁇ - 6 alkyl group.
  • R' is an optionally substituted C ⁇ - 6 alkenyl group. More preferably R' is a hydrogen atom.
  • X and Y form together to form an oxo group
  • Z is a disaccharide
  • an HMGBl modulator wherein the modulator comprises a triterpene moiety or a derivative or analog thereof.
  • Triterpenes [C 3 oI- 8 ] are abundunt in nature, particularly in resins and may occur as either esters or glycosides (often called saponins - molecules made up of sugars linked to steroids or triterpenes - due to their ability to make aqueous solutions appear foamy).
  • esters or glycosides often called saponins - molecules made up of sugars linked to steroids or triterpenes - due to their ability to make aqueous solutions appear foamy.
  • pentacyclic moeities are useful in the present invention, and in particular triterpenic sapnonins and their derivatives and analogues.
  • the moduator may or may not include the suga side chain.
  • the modulator of the present invention is glycyrrhizic acid, glycyrrhetinic acid or a salt or a mimetic thereof.
  • the modulator of the present invention is glycyrrhizic acid ammonium salt.
  • Glycyrrhizic acid is degraded by the intestinal flora when ingested per os, and its concentration in plasma is essentially zero.
  • a solution of glycyrrhizic acid (0.2%)) and glutatathione has been used parenterally in Japan under the name of Stronger Neo-Mynophagen as antiviral agent (Yoh et al., Dig. Dis. Sci. 47:1775-81, 2002).
  • We have found that glycyrrhizic acid binds to HMGBl and inhibits its extracellular activity, presumably by abolishing the interactions of HMGBl with its receptor(s).
  • Glycyrrhizic acid (Glycyrrhizin) consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. The active part of the molecule is believed to be the triterpenoid part (without the glucuronic acid).
  • Pentacyclic triterpenes which may be used in the present invention also include glycyrrhetinic acid, betulin, betulinin acid, ursolic acid, oleanolic acid, betulin mono- and di-succinate or glutarate, carbenoxolone, as well as polyethylene glycol derivatives and salts thereof.
  • pentacyclic triterpenes having the following general formula II:
  • the present invention may employ compounds of the following formula III:
  • each R 1 is independently CH 3 , H, OH, C ⁇ OY 1 , CH 2 O-X-OH, CH 2 O-X-OY ! , CH 2 O-X-Y 2 , CH 2 O-X-Y 3 .
  • R 2 or R 3 is R 1 , OY 1 , O-X-OH, O-X-OY 1 , O-X-Y 2 , Y 3 , NHY ⁇ NY ⁇ , Y 3 , NH-X-OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY ⁇ X-OH, NY ⁇ X-Y 2 , NY ⁇ X-Y 3 , NY ⁇ X-OY 1 , monosaccharide, disaccharide, oligosaccharide or polysaccharide or a derivative thereof, provided that one of R or R is H or that together they form an oxo group;
  • R 4 is H, R 1 , provided that at least one R 4 is H or together they form an oxo group;
  • R 5 is H, OH, O ⁇ OR Y 3 ;
  • Y 1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
  • Y 2 is NH 2 , NHY 1 OR NY ! 2 ;
  • Y 3 is -(O(CH 2 ) m ) n R 5 or -(O(CH 2m ) n Y 2 ;
  • n 1-4;
  • n 1-50;
  • X is -OC(CH 2 ) p CO-
  • P is 1-22;
  • R 1 or R 2 or R 3 is CH 3 , H, COOY 1 , COOY 1 , CO 2 (CH 2 )COOY 1 or OH.
  • R 4 is H or together form an oxo group. Hydrocarbyl
  • the term "hydrocarbyl group” means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the modulator or agent may contain halo groups.
  • halo means fluoro, chloro, bromo or iodo.
  • the modulator or agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, which may be unbranched or branched-chain.
  • the modulator or agent may be in the form of, and/or may be administered as, a pharmaceutically acceptable salt such as an acid addition salt or a base salt, or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt such as an acid addition salt or a base salt, or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
  • Suitable base salts are formed from bases which form non-toxic salts and examples are the sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine salts.
  • the modulator or agent of the present invention may exist in polymorphic form.
  • the modulator of the present invention may contain one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. Where a modulator contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur.
  • the present invention includes the individual stereoisomers of the modulator and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
  • Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the modulator or agent or a suitable salt or derivative thereof.
  • An individual enantiomer of a modulator or agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
  • the present invention also includes all suitable isotopic variations of the modulator or agent or a pharmaceutically acceptable salt thereof.
  • An isotopic variation of a modulator or agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the modulator or agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as H, H, C, C, 15 N, 17 O, 18 0, 31 P, 32 P, 35 S, 18 F and 36 C1, respectively.
  • isotopic variations of the modulator or agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the test compounds of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagent.
  • the modulator of the present invention may be derived from a prodrug.
  • prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be admimstered (such as orally or parenterally) and thereafter metabolised in the body to form the modulator of the present invention which are pharmacologically active.
  • pro-moiety it will be further appreciated that certain moieties known as “pro-moieties”, for example as described in “Design of Prodrugs” by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the modulators or agents. Such prodrugs are also included within the scope of the invention.
  • the modulator or agent of the present invention may be an antibody.
  • HMGBl modulator binding domain of the present invention or derivatives or variants thereof, or cells expressing the same can be used to produce antibodies immunospecific for such polypeptides.
  • immunospecific means that the antibodies have substantially greater affinity for the HMGBl modulator binding domain of the present invention than for other related polypeptides.
  • polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing an HMGBl epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
  • Monoclonal antibodies directed against epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
  • Antibodies both monoclonal and polyclonal, which are directed epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti- idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the agent against which protection is desired.
  • anti-idiotype antibodies are known in the art. These anti- idiotype antibodies may also be useful in therapy.
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
  • agonist means any modualtor, which is capable of binding to an HMGBl modulator binding domain and which is capable of increasing a proportion of HMGBl that is in an active form, resulting in an increased biological response.
  • the term includes partial agonists and inverse agonists.
  • Antagonist means any agent that reduces the action of another agent, such as an agonist.
  • the antagonist may act at the same receptor as the agonist.
  • the antagonistic action may result from a combination of the substance being antagonised (chemical antagonism) or the production of an opposite effect through a different receptor (functional antagonism or physiological antagonism) or as a consequence of competition for the binding site of an intermediate that links receptor activation to the effect observed (indirect antagonism).
  • modulating means inducing an increase or a decrease in the activity of the HMGBl through binding of an agent to an HMGBl modulator binding domain.
  • mimetic relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a known agent. That is, the mimetic is a functional equivalent of a known agent.
  • derivative or "derivatised” as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
  • the agent will be prepared by recombinant DNA techniques and/or chemical synthesis techniques.
  • the modulator or agent of the present invention may be prepared by chemical synthesis techniques.
  • the modulator or agent of the present invention or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesise the modulator or agent in whole or in part.
  • peptides can be synthesised by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY).
  • the composition of the . synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • Direct synthesis of the modulator or agent or variants, homologues, derivatives, fragments or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al., 1995) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • Agents and modulators which are identified with the HMGBl modulator binding domain structure of the present invention can be screened in assays such as are well known in the art. Screening can be, for example in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity. The biological assay, may also be an assay for ligand binding activity of a compound that selectively binds to the LBD compared to other nuclear receptors.
  • HTS high capacity-high throughput screening
  • a method for screening of a modulator capable of binding to an HMGBl modulator binding domain of the present invnetion comprising contacting the HMGBl modulator binding domain with an agent, and determining if said agent binds to said HMGBl modulator binding domain
  • the present invention provides a method of screening for an agent capable of interacting with a key amino acid residue of the HMGBl modulator binding domain.
  • Another preferred aspect of the invention provides a process comprising the steps of:
  • a further preferred aspect of the invention provides a process comprising the steps of:
  • Yet another preferred aspect of the invention provides a process comprising the steps of:
  • the information from the structure of the present invention is useful in the design of potential modulators capable of interacting with the HMGBl modulator binding domain and/or capable of modulating the activity of HMGBl modulator binding domain, and the models of the present invention are useful to examine the effect such a modulator is likely to have on the structure and/or function of the
  • the present invention relates to a modulator identified using such screening methods.
  • the modulator is capable of spatially fitting into an HMGBl modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4 or Figure 11.
  • the modulator is capable of interacting with at least some of the following amino acid residues of the N-terminal helix 1 ofHMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45.
  • the present invention also relates to a pharmaceutical composition comprising a modulator identified by any of the screening methods of the present invention.
  • a method of preventing and/or treating a disorder associated with HMGBl comprising administering a modulator of the present invention, or a pharmaceutical composition comprising a modulator of the present invention, wherein said modulator or pharmaceutical composition is capable of modulating HMGBl to cause a beneficial and/or therapeutic effect.
  • a modulator may be administered for treating conditions (diseases) mediated by the inflammatory cytokine cascade, such as sepsis.
  • Sepsis is an often fatal clinical syndrome that develops after infection or injury. Sepsis is the most frequent cause of mortality in hospitalised patients.
  • Experimental models of gram negative sepsis based on administration of bacterial endotoxin (lipopolysaccharide, LPS) have led to an improved understanding of the pathogenic mechanisms of lethal sepsis and conditions related to sepsis by virtue of the activation of a common underlying inflammatory cytokine cascade.
  • This cascade of host- response mediators includes TNF, IL-1, PAF and other macrophage-derived factors that have been widely studied as acute, early mediators of eventual lethality in severe endotoxemia (Zhang and Tracey, In The Cytokine Handbook, 3rd ed. Ed. Thompson (Academic Press Limited, USA). 515-547, 1998).
  • Systemic Inflammatory Response Syndrome which includes:
  • ARDS Acute pancreatitis Adult respiratory distress syndrome
  • Cardiovascular Disease which includes Cardiac stun syndrome
  • Obstetrics/Gynecology including:
  • Inflammatory Disease/Autoimmunity which includes:
  • Malignancy which includes:
  • Transplants including:
  • Dermatologic which includes:
  • Neurologic which includes:
  • Migraine headache Renal which includes:
  • Toxicity which includes: OKT3 therapy
  • Metabolic/ldiopathic which includes:
  • HMGBl is also an earlier marker of inflammation in the case of large scale necrosis.
  • HMGBl may be released by the necrotic cells.
  • the present invention may be used in the treatment of the first stages of inflammation in the case of large scale necrosis, such as occurs in intestinal infarction, acute pancreatitis and extensive trauma.
  • a modulator or pharmaceutical composition of the present invention may be administered so as to downregulate an immune specific response and or treat an inflammatory or autoimmune disease, allergy or transplant rejection.
  • an inhibitor HMGBl is used in a method of modulating an immune response and/or treating an inflammatory or autoimmune disease, allergy or transplant rejection.
  • pathogens in addition to triggering non-specific mechanisms, pathogens, e.g. during an infection, also trigger the antigen-specific adaptive immune response.
  • the adaptive immune response to infection involves both the T and B cell mediated compartments of the immune system.
  • APCs antigen presenting cells
  • APC function is also required for maintenance of the adaptive immune response.
  • APCs constitute a complex of cells capable of internalising an antigen, processing it and expressing epitopes thereof in association with class I and class II
  • the group mainly comprises dendritic cells, activated macrophages, microglial cells of the central nervous system and B lymphocytes.
  • the dendritic cells are particularly specialised in antigen presentation and constitute a population with distinctive characteristics and are widely distributed in tissues.
  • the DCs are involved in the activation of the immune response, which takes place by stimulation of the T lymphocytes in the course of various pathologies such as infections, autoimmune diseases and transplant rejection. Activation or maturation of DCs is a necessary process for "priming" the T cells and initiating the immune response.
  • DCs stimulated with cells in the initial apoptotic state, or with their culture medium are not activated (Gallucci, et al. 1999, Sauter et al. 2000, Ignatius et al. 2000 and Rovere et al., 1998).
  • the DCs are not activated by necrotic polymorphonuclear (PMN) leukocytes.
  • PMN necrotic polymorphonuclear
  • HMGBl is capable of activating the maturation of APCs.
  • activating we include inducing maturation of APCs.
  • an antagonist capable of minding to an HMGBl modulator binding domain which is capable of preventing or reducing the activation of an APC.
  • an antagonist of HMGBl is added to a population of APCs in conditions in which maturation is capable of occurring, fewer APCs proceed to maturity than in the absence of the HMGBl antagonist.
  • a modulator of the present invention may be admimstered for treating a vascular disease, comprising administering an effective amount of a modulator of the invention.
  • the vascular disease may arise due to diagnostic and/or surgical techniques, for example involving the use of diagnostic or surgical instruments, such as catheters, surgical instruments or stents.
  • the diagnostic or surgical technique involves angioplasty and/or angiography.
  • the present invention is used to treat atherosclerosis and/or restenosis that occur during angioplasty and/or angiography.
  • the treatment may include blocking, retarding or reducing connective tissue regeneration.
  • the modulators are released by catheters, surgical instruments or stents for angioplasty.
  • the modulators may however be used before, during or after invasive diagnostic and/or surgical techniques.
  • the insertion of instruments, such as catheters, into vessels may damage the endothelial lining and lead to inflammation and/or stenosis of the vessel.
  • the present invention can be used in the treatment and/or prevention of such a condition.
  • HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage.
  • Vascular smooth muscles cells (SMC) are the most predominant cells of the larger blood vessels. When the endothelium is damaged, either after mechanical or inflammatory injuries, SMC switch to a synthetic phenotype and undergo cell division and migration. The migration of SMC cells from the tunica media to the tunical intima plays an important role in the pathophysiology of many vascular disorders, such as atherosclerosis and restenosis after coronary angioplasty.
  • HMGBl is a strong chemoattractant and induces SMC cell shape changes, and cytoskeleton reorganisation. These events are inhibited by addition of an ant-RAGE antibody and by pertussis toxin, underlying that both RAGE and Gi/o protein might be involved in the pathway. Furthermore, the evidence that HMGBl promotes the translocation of phosphorylated ERK 1 and 2 proteins into the nucleus, indicates the involvement of the MAP kinase pathway.
  • HMGBl is released by damage or necrosis of a variety of cell types, including endothelial cells. Furthermore, it was shown that HMGBl fragments may be more efficacious than the entire full-length molecule. Consequently, every kind of molecule able to block the interaction between HMGBl and a receptor for HMGBl, such as the RAGE receptor, can efficiently be used for the production of pharmacological preparation in order to avoid, retard or inhibit atherosclerosis and restenosis after vascular epithelium damage even due to angioplasty.
  • APCs as described above may be cultured in a suitable culture medium such as DMEM or other defined media, optionally in the presence of fetal calf serum.
  • HMGBl may be admimstered to APCs and by introducing nucleic acid constructs/viral vectors encoding the protein into cells under conditions that allow for expression of the polypeptide in the APC.
  • nucleic acid constructs encoding antisense constructs may be introduced into the APCs and by transfection, viral infection or viral transduction.
  • Antigen presenting cells include macrophages, dentritic cells, B cells and virtually any other cell type capable of expressing an MHC molecule.
  • Macrophages are phagocytic cells of the monocytic lineage residing within tissues and are particularly well equipped for effective antigen presentation. They generally express MHC class II molecules and along with their phagocytic properties are extremely efficient at engulfing macromolecular or particulate material, digesting it, processing it with an extensive lysosomal system to antigenic peptide form, and expressing it on the cell surface for recognition by T lymphocytes.
  • Dendritic cells so named for their highly branched morphology, are found in many organs throughout the body, are bone marrow-derived and usually express high levels of MHC class II antigen. Dendritic cells are actively motile and can recirculate between the bloodstream and tissues. In this way, they are considered the most important APCs. Langerhans cells are an example of dendritic cells that are located in the skin.
  • B lymphocytes while not actively phagocytic, are class II-positive and possess cell surface antigen-specific receptors, immunoglobulin, or antibody molecules. Due to their potential for high affinity antigen binding, B cells are uniquely endowed with the capacity to concentrate low concentrations of antigen on their surface, endocytose it, process it and present it in the context of antigenic peptide in association with MHC antigen on their surface. In this manner, B cells become extremely effective APCs.
  • Immature dendritic cells for use in the present invention can be obtained from haematopoietic precursors or from stem cells, for example from PBMC cells, by suitable treatment with cytokines such as GM-CSF, IL-4 and flt3-L.
  • cytokines such as GM-CSF, IL-4 and flt3-L.
  • the activation or maturation of antigen-presenting cells can be effected starting from a culture of immature or inactive cells, by adding HMGBl protein and possibly other co-adjuvants such as cytokines to the culture medium.
  • the antigen-presenting cells can be used for the activation of T lymphocytes in response to particular antigens; the lymphocytes thus activated can then be administered to a subject to stimulate their immune response to the said antigens.
  • the indicators of activation can vary according to the cell type under consideration. With regard to macrophages, microglia and B lymphocytes, for example, it is a functional activation with increase in membrane expression of MHC molecules and co-stimulatory molecules following contact with other adjuvants, as described in (27, 28).
  • those cells that display increased expression of markers characteristic of the "maturation phenotype", such as the CD83 and CD86 surface molecules, or reduced expression of markers characteristic of the immature phenotype, such as CD115, CD14, CD68 and CD32, are regarded as activated or mature.
  • An ex vivo method for the activation of T lymphocytes may comprise the following steps: a) bringing a preparation of inactive APCs into contact with HMGBl, or with its biologically active fragments, so as to induce their activation; b) bringing the activated APCs into contact with a particular antigen; c) exposing the T lymphocytes to the APCs that have been activated and exposed to the antigen.
  • dendritic cells are used as APCs.
  • Steps a) - c) indicated above can be executed in a different order.
  • the antigen can be added to a culture of immature or inactive APCs before the HMGBl protein or its fragments.
  • the APCs or DCs can be transfected with a vector for the expression of a particular antigen or of a polypeptide derived from it, or alternatively a vector for the expression of a specific MHC molecule.
  • Antigens associated with microorganisms, viruses, tumours or autoimmune diseases can be used for the activation of lymphocytes according to the method described.
  • tumour antigens in addition to the proteins or their fragments isolated from tumour tissues or cells, it is possible to use whole cells that have been killed by apoptosis or necrosis. It is also possible to use antigens associated with viruses or retroviruses, especially HIV, or with intracellular pathogens, such as mycobacteria or plasmodia.
  • a pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. It preferably includes a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • “Therapeutically effective amount” refers to the amount of the therapeutic agent which is effective to achieve its intended purpose. While individual patient needs may vary, determination of optimal ranges for effective amounts of HMGBl modulator is within the skill of the art.
  • the dosage regimen for treating a condition with the compounds and/or compositions of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient,, the severity of the dysfunction, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound used, whether a drug delivery system is used, and whether the compound is administered as part of a drug combination and can be adjusted by one skilled in the art.
  • the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth herein.
  • Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
  • the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • each conjugate may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the amount of nucleic acid administered may typically be in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg.
  • Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE-dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the invention further provides a method of preventing and/or treating a disorder associated with HMGBl, the method comprising administering to a mammal a modulator according to the present invention.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient and severity of the condition.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • compositions (or component parts thereof) of the present invention may be administered orally.
  • the compositions (or component parts thereof) of the present invention may be administered by direct injection.
  • the compositions (or component parts thereof) of the present invention may be administered topically.
  • the compositions (or component parts thereof) of the present invention may be administered by inhalation.
  • the compositions (or component parts thereof) of the present invention may also be administered by one or more of: parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration means, and are formulated for such administration.
  • the pharmaceutical composition of the present invention may be administered in accordance with a regimen of 1 to 10 times per day, such as once or twice per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • administered also includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
  • the pharmaceutical composition of the present invention may be administered by one or more of the following routes: oral administration, injection (such as direct injection), topical, inhalation, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration or transdermal administration.
  • One aspect of the present invention is related to a model.
  • the HMGBl modulator binding domain structure substantially as presented by the space filling balls in Figure 4 or Figure 11 of the present invention can be used to generate a model such as a 3D structural model (or a representation thereof) comprising a HMGBl modulator binding domain or portion thereof.
  • the structure may be used to generate a computer model for the structure.
  • the model comprising the HMGBl modulator binding domain comprises the structural coordinates of a modulator binding domain corresponding to at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26and 27; and/or at least some of the following amino acid residues of helix 2 ofHMGBl: 42, 43, 44 and 45.
  • the model structure may comprise the amino acid residues of the HMGBl modulator binding domain, or a portion of the HMGBl modulator binding domain or a homologue thereof useful in the modelling and design of test compounds capable of binding to the HMGBl modulator binding domain.
  • modeling includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models.
  • the term “modelling” includes conventional numeric-based molecular dynamic and energy minimisation models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure- based constraint models.
  • the HMGB 1 modulator binding domain structure substantially as presented by the space filling balls in Figure 4 or a portion thereof may be applied to a model screening system.
  • model screening system may be a solid 3D screening system or a computational screening system. Using this model, modulators can be modelled that fit spatially and preferentially into the HMGBl modulator binding domain.
  • the modulators are positioned in the HMGBl modulator binding domain through computational docking. In another prefened aspect, the modulators are positioned in the HMGBl modulator binding domain through manual docking.
  • the term "fits spatially” means that the three-dimensional structure of a modulator is accommodated geometrically in a cavity or pocket of a HMGBl modulator binding domain.
  • modelling is performed using a computer and may be further optimised using known methods. This is called modelling optimisation. Overlays and super positioning with a three dimensional model of the HMGBl modulator binding domain, and/or a portion thereof, can also be used for modelling optimisation.
  • Alignment and/or modelling can be used as a guide for the placement of mutations on the HMGBl modulator binding domain surface to characterise the nature of the site in the context of a cell.
  • the present invention also relates to a method of screening for a modulator capable of binding to the HMGBl modulator binding domain of the present invention.
  • the method may employ a solid 3D screening system or a computational screening system. Using these systems, agents may be screened to find those which interact spatially and preferentially with the HMGBl modulator binding domain, through either computational or manual docking.
  • HMGBl modulator binding domain can be accomplished by utilising the atomic structural coordinates that define an HMGBl modulator binding domain structure according to the present invention, or a part thereof.
  • Structure-based modulator design identification methods are powerful techniques that can involve searches of computer data bases containing a variety of potential modulators and chemical functional groups. (See Kuntz et al., 1994, Ace. Chem. Res. 27:117; Guida, 1994, Current Opinion in Struc. Biol. 4: 777; and Colman, 1994, Current Opinion in Struc. Biol. 4: 868, for reviews of structure-based drug design and identification; and Kuntz et al 1982, J. Mol. Biol.
  • Modulators of an HMGBl modulator binding domain may be identified by docking the computer representation of compounds from a data base of molecules.
  • Data bases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall).
  • Computer programs such as CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions.
  • the computer programs may comprise the following steps:
  • a method for identifying potential modulators according to the invention.
  • the method utilises the structural coordinates of an HMGBl modulator binding domain three-dimensional structure, or binding site thereof.
  • the method comprises the steps of (a) generating a computer representation of an HMGBl modulator binding domain structure, and docking a computer representation of a compound from a computer data base with a computer representation of the HMGBl modulator binding domain to form a complex; (b) determining a conformation of the complex with a favourable geometric fit or favourable complementary interactions; and (c) identifying compounds that best fit the HMGBl modulator binding domain as potential modulators of HMGBl modulator binding domain function.
  • the initial HMGBl modulator binding domain structure may or may not have compounds bound to it.
  • a favourable geometric fit occurs when the surface areas of a compound in a compound-HMGBl modulator binding domain complex is in close proximity with the surface area of the HMGBl modulator binding domain without forming unfavourable interactions.
  • a favourable complementary interaction occurs where a compound in a compound-HMGBl modulator binding domain complex interacts by hydrophobic, aromatic, ionic, or hydrogen donating and accepting forces, with the HMGBl modulator binding domain without forming unfavourable interactions. Unfavourable interactions may be steric hindrance between atoms in the compound and atoms in the HMGBl modulator binding domain.
  • “Docking” refers to a process of placing a compound in close proximity with an active site of a polypeptide (i.e. an HMGBl modulator binding domain), or a process of finding low energy conformations of a compound/polypeptide complex (i.e. compound/ HMGB 1 modulator binding domain complex) .
  • Examples of other computer programs that may be used for structure-based modulator design are CAVEAT (Bartlett et al., 1989, in "Chemical and Biological Problems in Molecular Recognition", Roberts, S.M. Ley, S.N.; Campbell, ⁇ .M. eds; Royal Society of Chemistry: Cambridge, pp 182-196); FLOG (Miller et al., 1994, J. Comp.
  • potential modulators are identified utilising an HMGBl modulator binding domain structure with or without compounds bound to it.
  • the method comprises the steps of (a) modifying a computer representation of an HMGBl modulator binding domain having one or more compounds bound to it, where the computer representations of the compound or compounds and HMGBl modulator binding domain are defined by atomic structural coordinates; (b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and (c) identifying the compounds that best fit the
  • HMGBl modulator binding domain as potential modulators.
  • a computer representation may be modified by deleting or adding a chemical group or groups.
  • Computer representations of the chemical groups can be selected from a computer database.
  • modulators can be modified within the computer representation of an HMGBl modulator binding domain. This technique is described in detail in Molecular Simulations User Manual, 1995 in LUDI.
  • the computer representation of a modulator may be modified by deleting a chemical group or groups, or by adding a chemical group or groups. After each modification to a compound, the atoms of the modified compound and binding site can be shifted in conformation and the distance between the modulator and the binding site atoms may be scored on the basis of geometric fit and favourable complementary interactions between the molecules. Compounds with favourable scores are potential modulators.
  • Compounds designed by modulator building or modulator searching computer programs may be screened to identify potential modulators.
  • Examples of such computer programs include programs in the Molecular Simulations Package (Catalyst), ISIS/HOST, ISIS/BASE, and ISIS/DRAW (Molecular Designs Limited), and UNITY (Tripos Associates).
  • a building program may be used to replace computer representations of chemical groups in a compound complexed with an HMGBl modulator binding domain with groups from a computer database.
  • a searching program may be used to search computer representations of compounds from a computer database that have similar three dimensional structures and similar chemical groups as a compound that binds to an HMGBl modulator binding domain.
  • the programs may be operated on the structure of the HMGBl modulator binding domain structure.
  • a typical program may comprise the following steps:
  • mapping chemical features of a compound such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites;
  • a method of identifying potential modulators of an HMGBl modulator binding domain is provided using the three dimensional conformation of the HMGBl modulator binding domain in various modulator construction or modulator searching computer programs on compounds complexed with the HMGBl modulator binding domain.
  • the method comprises the steps of (a) generating a computer representation of one or more compounds complexed with an HMGBl modulator binding domain; (b) (i) searching a data base for a compound with a similar geometric structure or similar chemical groups to the generated compounds using a computer program that searches computer representations of compounds from a database that have similar three dimensional structures and similar chemical groups, or (ii) replacing portions of the compounds complexed with the HMGBl modulator binding domain with similar chemical structures (i.e. nearly identical shape and volume) from a database using a compound construction computer program that replaces computer representations of chemical groups with groups from a computer database, where the representations of the compounds are defined by structural coordinates.
  • HMGBl modulator binding domain identified using the above-described methods may be prepared using methods described in standard reference sources utilised by those skilled in the art.
  • organic compounds may be prepared by organic synthetic methods described in references such as March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill.
  • the invention contemplates all optical isomers and racemic forms of the modulators of the invention.
  • Figure 1 shows the structure of glycyrrhizic acid ammonium salt.
  • Figure 2 shows the heat effects of subsequent injections of glycyrrhizic acid into a solution of HMGB 1 Box A.
  • Figure 3 shows the average chemical shift changes of the IHN and 15N nuclei of HMGBl box A induced upon binding of glycyrrhizic acid as a function of residue number.
  • Figure 4 shows the residues ofHMGBl which interact with glycyrrhizic acid.
  • the C ⁇ backbone is depicted as a ribbon; interacting residues in space-filling representation as represented by the space filling balls.
  • Figure 5 shows the migratory response of Bovine Artery Endothelial Cells (BAEC) in the presence and absence of glycyrrhizic acid.
  • BAEC Bovine Artery Endothelial Cells
  • Figure 6 shows the proliferation of BAEC in the presence and absence of glycyrrhizic acid.
  • Figure 8 shows the amino acid sequence ofHMGBl with box A underlined and box B indicated in bold.
  • Figure 9A shows 15N-HSQC spectrum ofHMGBl Box A, 0.3 mM, 10 mM phosphate buffer, 150 mM NaCI, pH 7.
  • Figure 9B shows 15N-HSQC spectrum ofHMGBl Box Axarbenoxolone (1:5).
  • Figure 11 shows the residues of HMGBl which interact with carbenoxolone.
  • the C ⁇ backbone is depicted as a ribbon; interacting residues in space-filling representation as space filling balls.
  • Figure 12 shows the averaged chemical shift displacement of residue R23 vs. carbenoxolone concentration (Cbnx) (raw data and curve fitting).
  • Figure 13 shows the averaged chemical shift displacement of residue R23 vs. glycerrhetic acid (GLY) concentration (raw data and curve fitting).
  • Figure 14 shows the effect of glycyrrhetic acid on BAEC migration.
  • Figure 15 shows the effect of carbenoxolone or glycyrrhizin on BAEC migration.
  • Figure 16A shows the structure of glycyrrhizin
  • Figure 16B shows the structure of carbenoxolone
  • Figure 16C shows the structure of ursolic acid
  • Figure 16D shows the structure of a sodium salt of gycyrrhetic acid.
  • Glycyrrhizic acid binds to HMGBl boxA with a dissociation constant ofl3+/-7 ⁇ M.
  • the thermodynamics of the interaction of the single HMG boxes A and B with glycyrrhizic acid were analyzed using isothermal titration calorimetry. In the temperature interval used (10-27°C), HMGBl has been reported to be completely folded (Ramstein J., Locker D., Bianchi M.E. and Leng M. Eur. J. Biochem. 260: 692- 700, 1999).
  • glycyrrhizic acid was titrated into a solution of box A or box B, respectively.
  • Figure 2 shows the heat effects of subsequent injections of GA into a solution of box A. Box B was similar.
  • the equilibrium constant K B was determined as 7.5 +/-4 10 4 .
  • Example 2 Mapping on HMGBl boxA of the binding site for glycyrrhizic acid Quantification of chemical shift changes in a protein upon ligand binding is a sensitive method for measuring the strength of such interactions and for defining the protein's interaction surface.
  • HMGBl boxA concentration upon titration with was 0.2 mM, at pH 5.0, 150 mM NaCI.
  • the ligand binding is in the fast chemical exchange limit; although the affinity is low ( - d in the 10 "5 M range) the binding is specific, as it delineates a well defined area on the surface ofHMGBl boxA ( Figure 4; C ⁇ backbone as a ribbon; interacting residues in space-filling representation) .
  • Endothelial cells respond to extracellular HMGBl by reorganizing their cytoskeleton (not shown) and migrating.
  • the migratory response of BAEC was assayed with modified Boyden chambers for 4 hours at 37°C, as described by Degryse et al. (J. Cell Biol. 152:1197-2006, 2001) for Rat Smooth Muscle cells (RSMC).
  • RSMC Rat Smooth Muscle cells
  • BAEC cells respond maximally to 1 ng/ml HMGBl. Addition of 30 ⁇ M glycyrrhizic acid to the medium abolishes the migratory response completely (Fig. 5).
  • HMGBl -induced migratory response of, vascular smooth muscle cells and fibroblasts is similarly inhibited (not shown).
  • BAEC respond to HMGBl by proliferating for at least 2 days in the absence of serum.
  • Glycyrrhizic acid (30 ⁇ M) abolished completely the effect ofHMGBl (Fig. 6).
  • FCS fetal calf serum
  • Carbenoxolone is able to bind HMGBl with a high Kd.
  • Figure 9A and B show that upon addition of carbenoxolone, several peaks change their position indicating that carbenoxolone intereacts with Box A.
  • the spectra were acquired on a Bruker 500 MHz spectromter at 20°C.
  • Figure 10 shows the average chemical shift changes of 1H and 15N nuclei ofHMGBl Box A induced upon binding of carbenoxolone shown as a function of residue number.
  • the largest chemical shift changes are clustered around helix 1 (17, 20, 23, 25 and 26) and around residue 42.
  • Residue 91 is actually the NH 2 of residue Q20.
  • the surface of interaction is very similar to the one with glycyrrhizic acid.
  • Figure 12 shows averaged chemical shift difference of the residue R23 as a function of carbenoxolone concentration. Assuming a simple binary reaction between Box A and carbenoxolone, analysis by nonlinear curve fitting yields values of Kd ⁇ 0.08 mM.
  • Figure 13 shows averaged chemical shift difference of the residue R23 as a function of glycerrhetic acid concentration. Assuming a simple binary reaction between Box A and glycerrhetic acid, analysis by nonlinear curve fitting yields values of Kd ⁇ 1.3 mM
  • Figs. 14 and 15 show the inhibition of BAEC migration by glycerrhetic acid (glycyrrhetinic acid) and carbenoxolone, respectively.
  • Glycerrhetic acid is very insoluble, and may be forming miscelles, and may be the more active of the three inhibitors used in these Examples.
  • glycyrrhizin and the like are specific for HMGBl, since they do not inhibit the migration of BAEC induced by fMLP (formyl-Methionine-Leucine- Proline), a chemoattractant that mimics bacterial protecins (Degryse et al).

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Abstract

An HMGB 1 modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4 or Figure 11.

Description

HMGBl Modulator Binding Domain
Field of the invention
The present invention relates to an HMGBl modulator binding domain, an HMGBl modulator binding domain having a modulator which is bound to it, modulators of an HMGBl modulator binding domain and their uses in therapy.
Background of the invention
The non-histone nuclear protein HMGBl belongs to the B family of HMG proteins also known as the high mobility group. HMGBl was originally identified as a ubiquitously expressed, abundant nuclear protein. When bound to double stranded DNA, it induces structure distortion, allowing the formation of nucleoprotein complexes where several DNA-binding proteins can contact each other while bound to their respective DNA cognate sites (Muller et al., 2001).
Recently, additional roles of HMGBl outside the cell nucleus have come into focus. Stimulated macrophages actively secrete HMGBl to promote inflammation and in turn, stimulate production of multiple, proinflammatory cytokines. HMGBl mediates endotoxin lethality, acute lung injury, arthritis induction, activation of macrophages, smooth muscle cell chemotaxis, and epithelial cell barrier dysfunction. It is released as a late mediator during inflammation and participates in the pathogenesis of systemic inflammation after the early mediator response has resolved (Andersson et al., 2002).
WOOO/47104 describes the use of a pharmaceutical composition comprising an antagonist or agonist HMG1 (now designated HMGBl) for treating conditions characterised by activation of the inflammatory cytokine cascade, including sceptic shock.
HMGBl occupies a critical role as a proinflammatory mediator passively released by necrotic but not apoptotic cells. International Patent Application No. PCT/IT03/00265 demonstrates that HMGBl released by necrotic cells can be the initial trigger for inflammatory responses such as inflammatory cell activation, and that released HMGBl itself can activate inflammatory cells. It further demonstrates that HMGBl acts as a chemoattractant and proliferation factor for stem cells. Thus, a succession of events can be envisaged whereby necrosis and primary HMGBl release leads to inflammatory cell activation and the secretion of cytokines, which in turn leads to further activation and recruitment of inflammatory cells which then secrete HMGBl as a late mediator of inflammation.
HMGBl has been identified as one of the ligands binding to the RAGE receptor (Receptor for advanced glycation endproducts) (Hori et al., 1995). RAGE is a multiligand receptor of the immunoglobulin superfamily, is expressed in many cell types, including endothelial cells, mononuclear phagocytes, and neurons (Brett et al., 1993; Neeper et al., 1992) and is implicated in several different pathological processes, such as diabetes, amyloidoses and atherosclerosis (Scmidt et al., 1999). Interaction of HMGBl and RAGE induces neurite outgrowth and the block of tumour growth and metastasis is observed preventing the interactions between HMGBl and RAGE. Inhibition of this interaction has also been shown to suppress activation of mitogen- activated protein (MAP) kinases and the expression of matrix metalloproteinases. WO 02/074337 shows that HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage.
Our co-pending International Patent Application No. PCT/IB2003/005718 describes how surprisingly we found that the form of protein HMGBl which mediates the late phases of inflammation is an acetylated form and provides for methods for treating conditions associated with the inflammatory cytokine cascade.
Moreover, our co-pending International Patent Application Publication No. WO03/026691 describes how we have found that HMGBl can be used to upregulate or downregulate an antigen mediated immune response. Given the numerous diseases associated with HMGBl and its role as, among others, a mediator in the inflammatory cytokine cascade, as a chemoattractant and proliferation factor for stem cells, a regulator of antigen mediated immune responses, and a factor in promoting atherosclerosis and restenosis after vascular damage there is a pressing need for the development of effective modulators of HMGBl function. The present invention addresses this problem.
Statements of the Invention
According to one aspect of the invention there is provided an HMGBl modulator binding domain having the structure substantially as represented by the space filling balls in Figure 4 or Figure 11.
According to another aspect of the present invention there is provided an HMGBl modulator binding domain comprising: at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45; or a homologue or mutant thereof.
In one embodiment the domain comprises all of the amino acid residues 11, 12, 13, 17, 20, 23, 25 and 27 and in helix 1 and/or all of the amino acid residues 43, 44 and 45 in helix 2. In another embodiment the domain comprises all of the aforementioned amino acid residues in helix 1 and helix 2.
In another embodiment the domain comprises all of the amino acid residues 17, 20, 23, 25 and 26 in helix 1 and/or residue 42 in helix 2. In another embodiment the domain comprises all of the aforementioned amino acid residues in helix 1 and 2. In one embodiment the HMGBl modulator binding domain is defined by a mutation or substitution or derivatisation in or of any one or more of the amino acid residues defined above.
In another embodiment the HMGBl modulator binding further comprises a modulator bound to the modulator binding domain or a portion thereof.
According to another aspect of the present invention there is provided an HMGBl modulator binding domain wherein the modulator comprises a pentacyclic triterpene moiety as represented by general formula II (see later).
More preferably the modulator is a compound of formula III:
wherein each R1 is independently CH3, H, OH, CH2OY!, CH2O-X-OH, OEfcO-X-OY1, CH2O-X-Y2, CH2O-X-Y3. CH-2NHY1, CH2NY12, CH2Y3, CH2NH-X-OH, CH2NH-X- Y2, CH2NH-X-Y3, CH2NH-X-OY1, CH2OC(O)-OY1, COY3, COY2, CHO, CH=N(CH2)m(O(CH2)ra)„R5 or CH=N(CH2)m(O(CH2)m)„Y2;
R2 or R3 is R1, OY1, O-X-OH, O-X-OY1, O-X-Y2, Y3, NHY1, Y^, Y3, NH-X-OH, NH-X-Y2, NH-X-Y3, NH-X-OY1, NY!-X-OH, NY^X-Y2, NY^X-Y3, NY'-X-OY1, monosaccharide, disaccharide, oligosaccharide or polysaccharide or a derivative thereof, provided that one of R or R is H or that together they form an oxo group;
R4 is H, R1, provided that at least one R4 is H or together they form an oxo group;
R5 is H, OH, OY1 OR Y3;
Y1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
Y2 is NH2, NHY1 OR NY 2;
Y3 is -(O(CH2)m)„R5 or -(O(CH2m)„Y2;
m is 1-4;
n is 1-50;
X is -OC(CH2)pCO-; and
P is 1-22;
or a salt, derivative or analogue thereof.
In one embodiment, the modulator is glycyrrhizic acid (glycyrrhizin), glycyrrhetinic acid, ursolic acid or carbenoxolone or a salt or a mimetic thereof. According to another aspect of the present invention there is provided a method for screening for a modulator capable of binding to an HMGBl modulator binding domain of the present invention, the method comprising contacting the HMGBl modulator binding domain with a test agent, and determining if said agent binds to said HMGBl modulator binding domain.
According to another aspect of the present invention there is provided a process comprising the steps of: (a) performing the method for screening of a modulator described above;
(b) identifying one or more modulators capable of binding to an HMGBl modulator binding domain; and
(c) preparing a quantity of those one or more modulators.
According to another aspect of the present invention there is provided a process comprising the steps of:
(a) performing the method for screening of a modulator described above;
(b) identifying one or more modulators capable of binding to an HMGBl modulator binding domain; and (c) preparing a pharmaceutical composition comprising those one or more modulators.
According to another aspect of the present invention there is provided a process comprising the steps of:
(a) performing the method for screening of a modulator described above; (b) identifying one or more modulators capable of binding to an HMGBl modulator binding domain;
(c) modifying those one or more modulators;
(d) performing the method for screening of a modulator described above; and
(e) optionally preparing a pharmaceutical composition comprising those one or more modified modulators. According to another aspect of the present invention there is provided a modulator identifiable by any of the above methods.
In one embodiment the modulator identified by any of the above methods is capable of spatially fitting into an HMGBl modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4.
In another embodiment the modulator identified by any of the above methods is capable of interacting with at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl : 11, 12, 13, 17, 20, 23, 25 and 27; and or at least one or more of the following amino acid residues of helix 2 of HMGBl: 43, 44 and 45.
In another embodiment the domain also comprises residue 91.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising a modulator identified above by any of the above methods of the present invention and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant or any combination thereof.
According to another aspect of the present invention there is provided a method of modulating the activity of HMGBl comprising administering a modulator of the present invention as defined above.
According to another aspect of the present invention there is provided a method of preventing and/or treating a disorder associated with HMGBl comprising administering a modulator according to the present invention, or a pharmaceutical composition of the present invention, wherein said modulator or pharmaceutical composition is capable of modulating HMGBl to cause a beneficial and/or therapeutic effect. In one embodiment the method is for the treatment of a condition associated with activation of the inflammatory cytokine cascade.
In another embodiment the method is for downregulating an immune specific response.
In a further embodiment the method is for the treatment of an inflammatory or autoimmune disease, allergy or transplant rejection.
In another embodiment the method is for inhibiting stem cell migration and/or proliferation.
In one embodiment the method is for inhibiting epithelial cell migration and/or proliferation.
In another embodiment the method is for the treatment of vascular diseases, in particular atherosclerosis and/or restenosis that occur during angioplasty and/or angiography.
In one embodiment the method is for the blocking, retarding or reducing connective tissue regeneration.
Preferably the modulators are released by catheters, surgical instruments or stents for angioplasty.
Preferably the modulator is glycyrrhizic acid or a salt or mimetic thereof.
In one embodiment the HMGBl is acetylated.
According to another aspect of the present invention there is provided a method for predicting, simulating or modelling the molecular characteristics and/or molecular interactions of an HMGBl modulator binding domain comprising the use of a computer model, said computer model comprising using or depicting the structure substantially as presented by the space filling balls in Figure 4 or Figure 11 and/or the structural coordinates of a modulator binding domain as provided by at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45, to provide an image of said HMGBl modulator binding domain, and optionally to display said image. Preferably the method comprises the use of a computer model to provide an image of said modulator, and optionally to display said image. Preferably the method comprises providing an image of said modulator in association with said HMGBl modulator binding domain, and optionally displaying said image.
In one embodiment said modulator is prepared and optionally formulated as a pharmaceutical composition.
According to another aspect of the present invention there is provided a computer readable medium having stored thereon parameters capable of displaying a representation of a 3D model comprising the HMGBl modulator binding domain.
In one embodiment said model is built for all or part of the HMGB 1 modulator binding domain as defined by the present invention
According to another aspect of the present invention there is provided a computer controlled method for designing a modulator capable of binding to HMGBl comprising:
(i) providing a model of the structure of the HMGBl modulator binding domain; (ii) analysing said model to design a ligand which binds to the HMGBl modular binding domain; and optionally (iii) determining the effect of said modulator on HMGB 1. According to another aspect of the present invention there is provided a machine- readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a representation of an HMGBl modulator binding domain of a homologue thereof.
According to another aspect of the present invention there is provided a computer comprising the above storage medium of the present invention.
According to another aspect of the present invention there is provided the use of a computer according to the present invention in an industrial context, such as identifying putative modulators.
Detailed description of the Invention
Narious preferred features and embodiments of the present invention will now be described by way of non-limiting example.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DΝA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.
HMGBl
As reported in WOOO/47104, HMGBl (indicated there as HMG-1) is a 25 kDa chromosomal nucleoprotein belonging to the burgeoning high mobility group (HMG) of non-histone chromatin-associated proteins. As a group, the HMG proteins recognise unique DNA structures and have been implicated in diverse cellular functions, including determination of nucleosome structure and stability, as well as in transcription and/or replication. The HMG proteins were first characterised by Johns and Goodwin as chromatin components with a high electrophoretic mobility in polyacrylamide gels (see in The HMG Chromosomal Proteins, E.W. Johns, Academic Press, London, 1982). Higher eukaryotes exhibit three families of HMG proteins; the HMGA family (previously HMG-I/Y), the HMGB family (previously HMG1, HMG2 and HMG4), and the HMGN family (previously HMG-14/-17). The families are distinguishable by size and DNA-binding properties. HMG proteins are highly conserved across species, ubiquitously distributed and highly abundant, and are extractable from chromatin in 0.35 M NaCI and are soluble in 5% perchloric or trichloroacetic acid. Generally, HMGB proteins are thought to bend DNA and facilitate binding of various transcription factors to their cognate sequences, including for instance, progesterone receptor, estrogen receptor, HOX proteins, and Octl, Oct2 and Oct6. Recently, it has become apparent that a large, highly diverse group of proteins including several transcription factors and other DNA-interacting proteins, contain one or more regions similar to HMGBl, and this feature has come to be known as the HMG box or HMGB domain. cDNAs coding for HMGBl have been cloned from human, rat, mouse, mole rat, trout, hamster, pig and calf cells, and HMGBl is believed to be abundant in all vertebrate cell nuclei. The protein is highly conserved with interspecies sequence identities in the 80% range. In chromatin, HMGBl binds to linker DNA between nucleosomes and to a variety of non-B-DNA structures such as palindromes, cruciforms and stem-loop structures, as well as cisplatin-modified DNA. DNA binding by HMGBl is generally believed to be sequence insensitive. HMGBl is most frequently prepared from washed nuclei or chromatin, but the protein has also been detected in the cytoplasm. (Reviewed in Landsman and Bustin, BioEssays 1993; Baxevanis and Landsman, 1995).
In more detail, HMGBl has a tripartite structure, composed by two homologous DNA- binding domains, the HMG-boxes entitled box A and box B, and a C-terminal domain of aspartic and glutamic acids (reviewed in Bustin, 1999, Bianchi and Beltrame, 2000, and Thomas and Travers, 2001). The HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) has been expressed in Escherichia coli its structure has been determined by 2D 1H-NMR spectroscopy (Weir et al., 1993). The structure of box B has also been determined by Read (Nucleic Acids Res 1993 Jul 25;21(15):3427-36). The structure of the A-domain has been determined using heteronuclear three- and four-dimensional NMR spectroscopy (Hardman et al., Biochemistry 1995). The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D (Jones et al., 1994). There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain.
There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues.
In most cells, HMGBl is located in the nucleus, where it acts as an architectural protein that facilitates nucleoprotein assembly. It binds to the minor groove of DNA, inducing a local distortion of the double helix. Its lack of sequence-specificity is offset by recruitment via protein-protein interaction by different types of nuclear factors (including the Hox and Pou proteins, the steroid hormone receptors, p53, TBP, some viral proteins and the RAG1 recombinase). HMGBl can bind to nucleosomes (Falciola et al., 1997; Nightingale et al., 1996) but in vivo its association with chromatin is very dynamic. Photobleaching experiments indicated that the average residence time of HMGBl molecules on chromatin is less than 2 seconds (Scaffidi et al., 2002). The phenotype of Hmgbl knockout mice confirmed the functional importance of HMGBl as regulator of transcription: they die shortly after birth due and show a defect in the transcriptional control exerted by the glucocorticoid receptor (Calogero et al., 1999).
Surprisingly, beyond its intranuclear function, HMGBl also has a pivotal function outside of the cell (reviewed by Mϋller et al., 2001b). Wang et al. (1999a) identified HMGBl as a late mediator of endotoxin lethality in mice, and showed that macrophages and myeloid cells stimulated by LPS, TNF or IL-1 secrete HMGBl as a delayed response. HMGBl can then act as a cytokine, eliciting several different responses in cells that are equipped with receptors to it. For example, HMGBl recruits inflammatory cells and promotes the secretion of TNF. In addition to monocytes, developing neurons and a few other cell types also secrete HMGBl in response to specific stimuli (reviewed by Mϋller et al., 2001b). However, most cells are not able to secrete HMGBl in an active manner.
In myeloid cells, secretion does not involve HMGBl protein newly made in the cytoplasm, but proceeds through the depletion of nuclear stores. The secretion of a nuclear protein poses formidable challenges. We recently showed that activation of myeloid cells results in the redistribution of HMGBl from the nucleus to secretory lysosomes (Gardella et al., 2002). HMGBl does not traverse the endoplasmic reticulum and the Golgi apparatus, consistent with the absence of a leader peptide in the protein. The early mediator of inflammation interleukin (IL)-lβ is also secreted by myeloid cells through a non-classical pathway involving exocytosis of secretory lysosomes (Andrei et al., 1999). However, in keeping with the roles of IL-lβ and HMGBl as early and late inflammatory factors, the discharge of the secretory vesicles that contain these 2 proteins responds at different times to different stimuli: IL-lβ secretion is induced earlier by ATP, autocrinally released by myeloid cells soon after activation; HMGBl secretion is triggered by lysophosphatidylcholine (LPC), a lipid generated later in the inflammation site (Gardella et al., 2002).
In our co-pending International Patent Application No. PCT/IB2033/005718 we found that in activated myeloid cells HMGBl is extensively modified by acetylation, and that the two major clusters of acetylated lysines belong to 2 independent nuclear localization signals. We also proved that HMGBl has non-classical nuclear export signals (NESs). Thus, in most cells HMGBl shuttles continually from the nucleus to the cytoplasm, but the equilibrium is almost completely shifted towards a nuclear accumulation. Treatment of cells with deacetylase inhibitors causes HMGBl acetylation, that shuts off its import into the nucleus but leaves export unaffected - the protein is then relocated to the cytoplasm. Myeloid cells acetylate HMGBl in response to activation: in promyelocytic cells, binding of LPS or inflammatory cytokines to their surface receptors promotes the activation of the MAP kinase pathway that impinges on ERK. As a result, HMGBl is acetylated and moves from the nucleus to the cytoplasm, where it is concentrated in secretory lysosomes and can be secreted in response to a second signal, LPC. Thus, myeloid cells have a signaling pathway that allows them to regulate HMGBl acetylation is response to inflammatory stimuli, switching a chromatin protein into a cytokine. Methods of modulating HMGBl activity according to the present invention include methods of modulating acetylated HMGBl activity.
HMGBl is claimed in International as a chemoattractant and proliferation factor for stem cells and a method to induce stem cell migration and/or proliferation in cell culture or in vivo comprising the step of exposing such cells to effective amounts of HMGBl protein or functional parts thereof is provided. It was demonstrated that HMGBl released by necrotic cells could be the initial trigger for inflammatory responses and that released HMGBl itself could activate inflammatory cells. HMGBl diffusion can take place in a matter of seconds or minutes and is thus a very early event in inflammation.
Little is known about the signalling mechanisms by which HMGBl activates cells to respond. HMGBl has been identified as one of the ligands binding to the RAGE receptor (Receptor for advanced glycation endproducts) (Hori et al., 1995). WO 02/074337 showed that HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage.
The present invention also relates to variants, derivatives and fragments of HMGBl and HMGBl modulator binding domain according to the invention. Preferably, the variant sequences etc. are at least as biologically active as the sequences presented herein.
The term "HMGBl" includes actetylated HMGBl.
As used herein "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.
Preferably such variants, derivative and fragments comprise one or both of the HMG boxes.
The term "protein" includes single-chain polypeptide molecules as well as multiple- polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. The term "polypeptide" includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
It will be understood that amino acid sequences for use in the invention are not limited to the particular sequences or fragments thereof or sequences obtained from a particular protein but also include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
Thus, the present invention covers variants, homologues or derivatives of the amino acid sequences for use in the present invention, as well as variants, homologues or derivatives of the nucleotide sequence coding for the amino acid sequences used in the present invention.
HMGBl modulator binding domain
As used herein, the term "HMGBl modulator binding domain" means the region of HMGBl which is responsible for modulator binding. The term "HMGBl modulator binding domain" also includes a homologue of the modulator binding domain or a portion thereof.
The HMGBl modulator binding domain of the present invention may be contained within box A and/or box B.
Preferably the HMGBl modulator binding domain of the present invention is contained within box A of HMGB 1.
As used herein, the term "portion thereof means the structural co-ordinates corresponding to a sufficient number of amino acid residues of HMGBl modulator binding domain (or homologues thereof) that are capable of interacting with an agent capable of binding to the of HMGBl modulator binding domain.
The HMGBl modulator binding domain may be defined by its association with the modulator.
In a highly preferred embodiment, the HMGBl modulator binding domain of the present invention is as substantially as presented by the space filling balls in Figure 4 or Figure 11.
According to one aspect of the present invention, there is provided an HMGBl modulator binding domain comprising: at least one or more of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least one or more of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45; or a homologue or mutant thereof.
According to one aspect of the present invention, there is provided an HMGBl modulator binding domain according to the present invention which further comprises a modulator bound to the modulator binding domain or a portion thereof. In other words the HMGBl modulator binding domain may be associated with a modulator. The modulator may be any compound which is capable of interacting stably and specifically with the HMGBl modulator binding domain. The modulator may, for example, be an inhibitor of the HMGB 1 modulator binding domain.
Structural Coordinates
In a highly preferred embodiment, the HMGBl modulator binding domain of the present invention substantially as presented by the space filling balls in Figure 4 or Figure 11 has the structural co-ordinates corresponding to at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45 which may be used for the identification of a modulator capable of binding to the HMGB 1 modulator binding domain.
As used herein, the term "structural co-ordinates" refer to a set of values that define the position of one or more amino acid residues with reference to a system of axes. The term refers to a data set that defines the three dimensional structure of a molecule or molecules (e.g. Cartesian coordinates, temperature factors, and occupancies). Structural coordinates can be slightly modified and still render nearly identical three dimensional structures. Structural coordinates that render three dimensional structures (in particular a three dimensional structure of an SGC domain) that deviate from one another by a root-mean-square deviation of less than 5 A, 4 A, 3 A, 2 A, or 1.5 A may be viewed by a person of ordinary skill in the art as very similar. The solution structures of HMGBl box A and B have been determined. (Biochemistry 1995 Dec 26;34(51):16596-607 and EMBO J. 1993 Apr; 12 (4): 1311-9).
Mutant
According to one aspect of the present invention there is provided an HMGBl modulator binding domain according to the invention which is defined by a mutation or substitution or derivatisation in any one or more amino acids. As used herein, the term "mutant" refers to any organism that has undergone mutation or that carries a mutant gene that is expressed in the phenotype of that organism. A mutation may arise due to a substitution of one nucleotide for another or from a deletion of a nucleotide or an insertion of a nucleotide relative to a referenced wild type sequence. These single nucleotide variations are sometimes referred to as single nucleotide polymorphisms (SNPs). Some SNPs may occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other SNPs may occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other SNPs may have no phenotypic effects.
As used herein, the term "mutant" refers to HMGBl modulator binding domain comprising any one or more changes in the sequence and of the amino acid residues. Preferably the mutated amino acid residue(s) is/are located in N-terminal helix 1 of HMGB land/or selected from amino acid residues of helix 2 of HMGBl wherein the amino acid changes in the HMGBl modulator binding domain may be selected from amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and or selected from amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45.
The term "mutant" is not limited to the above mutations which are reflected in amino acid substitutions of the key amino acid residues in the HMGBl modulator binding domain but may also include and is not limited to other deletions or insertions of nucleotides in the wild type sequence which may result in changes in the amino acid residues in the deduced amino acid sequence of the HMGBl modulator binding domain. The term "mutant" also includes uncharacterised mutants.
Homologues
As used herein, the term "homologue" refers to an HMGBl modulator binding domain or a portion thereof which may have deletions, insertions or substitutions of amino acid residues as long as the binding specificity of the HMGBl modulator binding domain is retained. In this regard, deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the binding specificity of the HMGBl modulator binding domain is retained. Here, a conservative substitution which may produce a silent change which may result in a functionally equivalent HMGBl modulator binding domain.
HMGBl Variants and Derivatives
The terms "variant" or "derivative" in relation to the amino acid sequences HMGBl or HMGBl modulator binding domain includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has HMGBl activity and/or HMGBl modulator binding activity, preferably having at least the same activity as human HMGB 1.
The HMGBl or HMGBl modulator binding domain of the present invention may be modified for use in the present invention. Typically, modifications are made that maintain the activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the HMGBl modulator binding activity and/or HMGBl activity. Amino acid substitutions may include the use of non-naturally occurring analogues.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Proteins for use in the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins for use in the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein of interest.
Proteins for use in the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention. Nucleotide Sequences
As used herein, the term "nucleotide sequence" refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof) which comprise the nucleotide sequences encoding HMGBl or an HMGBl modulator binding domain of the present invention.
It will be understood by a skilled person that numerous different polynucleotides can encode the same protein as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the proteins for use in the invention are to be expressed.
Polynucleotides for use in the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides for use in the invention.
The terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a polypeptide having the capability of binding HMGBl modulators. Preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequences presented herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.
The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.
Polynucleotides for use in the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%), preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred polynucleotides for use in the invention will comprise regions homologous to the HMG box, preferably at least 80 or 90% and more preferably at least 95% homologous to the HMG box.
The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide for use in the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P.
Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Nol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.
Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC (lxSSC = 0.15 M ΝaCl, 0.015 M Νa3 Citrate pH 7.0)).
Where the polynucleotide for use in the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.
Polynucleotides which are not 100% homologous to the sequences used in the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the human HMGBl sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the protein or nucleotide sequences for use in the invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as a DNA polynucleotides and probes for use in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
h general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector
Nucleotide vectors
Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides for use in the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
In preferred embodiments HMGBl or an HMGBl modulating domain according to the present invention may be produced by bacterial cells (Bianchi 1991, Lee et al. 1998) by yeasts (Mistry et al. 1997), or by purification from cell cultures or from mammalian tissues.
Vectors/polynucleotides for use in the invention may introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adeno viruses, direct injection of nucleic acids and biolistic transformation.
Protein Expression and Purification
Host cells comprising polynucleotides of the invention may be used to express proteins for use in the invention. Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention. Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
Proteins for use in the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.
Alpha helix (α-Helix)
As used herein, the term "α-helix" means a helical or spiral configuration of a polypeptide chain in which successive turns of the helix are held together by hydrogen bonds between the amide (peptide) links, the carbonyl group of any given residue being hydrogen-bonded to the imino group of the third residue behind it in the chain. This is the case for all of the carbonyl and amide groups of the peptide bonds of the main chain. Typically, the α-helix has 3, 6 residues per turn and the translation or pitch along the helical axis is 1.5 A per residue and 5.4A per turn. The helix may be left- or right-handed, the latter being much more common. The α-helix is one of the two basic elements of the secondary structure adopted by the polypeptide chain within the hydrophobic core of a globular protein. The other basic element is the β strand.
Key residues
As used herein the term "key residues" refers to one or more amino acid residues in a HMGBl modulator binding domain, capable of moderating modulator binding. The residues may be any one of the key residues within the HMGBl modulator binding domain as described herein or mutants thereof or they may be residues with homology to the residues or mutants thereof. The key amino acid residues of the HMGBl modulator binding domain may be any one or more of the amino acid residues selected from the group consisting of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25 and 27; and/or any one or more of the following amino acid residues of helix 2 of HMGBl : 43, 44 and 45; or a homologue or mutant thereof.
Agent
In one aspect, the invention relates to a method of screening for an agent capable of binding to a HMGBl modulator domain of the present invention the method comprising contacting the HMGBl modulator domain with an agent and determining if said agent binds to said HMGBl modulator domain.
As used herein, the term "agent" includes, but is not limited to, a compound, such as a test compound, which may be obtainable from or produced by any suitable source, whether natural or not. The agent may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds. By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic test compound, a semi- synthetic test compound, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised test compound, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant test compound, a natural or a non-natural test compound, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
Modulator
As used herein the term "modulator" refers to an agent capable of binding to the HMGBl modulator binding domain, preferably to one or more key residues in the HMGBl modulator binding domain. A modulator may increase or decrease HMGBl, or change its characteristics, or functional or immunological properties. It may be an inhibitor that decreases the biological or immunological activity of the protein.
Modulators include but are not limited to peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies, carbohydrates, nucleosides or nucleotides or parts thereof, and small organic or inorganic molecules. A modulator may be an endogenous physiological compound, or it may be a natural or synthetic compound.
A modulator of the present invention agent may also be capable of displaying one or more other beneficial functional properties.
The modulator may be an antagonist or agonist of HMGBl. Preferably the agent is an antagonist of HMGBl. Preferably the agent selectively downregulates an immune specific response and or may be used for the treatment of a condition associated with activation of the inflammatory cytokine cascade and/or may be used for the treatment of an inflammatory or autoimmune disease, allergy or transplant rejection. Preferably the agent may be used for inhibiting stem cell migration and/cell proliferation and/or inhibiting epithelial cell migration and/or proliferation or may be used for the treatment of vascular diseases. For some applications, preferably the modulator has at least about a 25, 50, 75, 100 fold selectivity to the desired target, preferably at least about a 150 fold selectivity to the desired target, preferably at least about a 200 fold selectivity to the desired target, preferably at least about a 250 fold selectivity to the desired target, preferably at least about a 300 fold selectivity to the desired target, preferably at least about a 350 fold selectivity to the desired target.
The modulator or agent can be an amino acid sequence or a chemical derivative thereof. The substance may even be an organic compound or other chemical. The agent may even be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence. The agent may even be an antibody.
According one aspect of the present invention there is provided an HMGBl modulator wherein the modulator comprises a saccharide moiety. By "saccharide" we include any of a group of water-soluble carbohydrates of relatively low molecular weight and typically having a sweet taste. The simple sugars are called monosaccharides. More complex sugars comprise generally between two and ten monosaccharides linked together: disaccharides contain two, trisaccharides contain three and so on. Preferably the modulator contains a disaccharide residue.
In one embodiment the modulator also comprises a hydrocarbon ring system. By hydrocarbon we mean that the ring is generally composed of hydrogen and carbon, but may include other atoms which do not substantially affect the properties of the ring system, for example the ring system may include one or more heteroatoms. The hydrocarbon ring system is preferably substituted, for example with hydrogen, an alkyl, oxo, COOH or ester group. The ring system comprises one or more rings, which may or may not be aromatic - a combination of aromatic and non-aromatic rings may be employed. The ring system may comprises two, three, four, or preferably five rings.
In a preferred embodiment the modulator is a compound of formula I:
wherein
X and Y each represent a hydrogen atom or form together an oxo group;
Z represents a monosacchande; disaccharide, oligosaccharide or polysaccharide or a derivative thereof; and
R' is a hydrogen atom, an optionally substituted alkyl or optionally substituted alkenyl group; or a salt thereof.
Preferably R' is an optionally substituted Cι-6 alkyl group. Preferably R' is an optionally substituted Cι-6 alkenyl group. More preferably R' is a hydrogen atom.
Preferably X and Y form together to form an oxo group
Preferably Z is a disaccharide.
In one preferred aspect of the present invention there is provided an HMGBl modulator wherein the modulator comprises a triterpene moiety or a derivative or analog thereof. The terpenes have long been associated with the term Essential Oils comprising resins, steroids and rubber. In fact, they are hydrocarbons that usually contain one or more C=C double bonds, while the terpenoids are oxygen-containing analogues of the terpenes. They are thoroughly distributed in the plant kingdom, especially in those plants that have abundant chlorophyll. Among these are compounds which fall in the general class of terpenes, compounds made up of 5- carbon units, often called isoprene units, put together in a regular pattern. Triterpenes [C3oI- 8] are abundunt in nature, particularly in resins and may occur as either esters or glycosides (often called saponins - molecules made up of sugars linked to steroids or triterpenes - due to their ability to make aqueous solutions appear foamy). We have found that pentacyclic moeities are useful in the present invention, and in particular triterpenic sapnonins and their derivatives and analogues. We have also found that the moduator may or may not include the suga side chain.
In one embodiment, the modulator of the present invention is glycyrrhizic acid, glycyrrhetinic acid or a salt or a mimetic thereof. For example, the modulator of the present invention is glycyrrhizic acid ammonium salt.
Glycyrrhizic acid is degraded by the intestinal flora when ingested per os, and its concentration in plasma is essentially zero. However, a solution of glycyrrhizic acid (0.2%)) and glutatathione has been used parenterally in Japan under the name of Stronger Neo-Mynophagen as antiviral agent (Yoh et al., Dig. Dis. Sci. 47:1775-81, 2002). We have found that glycyrrhizic acid binds to HMGBl and inhibits its extracellular activity, presumably by abolishing the interactions of HMGBl with its receptor(s).
Glycyrrhizic acid (Glycyrrhizin) consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. The active part of the molecule is believed to be the triterpenoid part (without the glucuronic acid).
Pentacyclic triterpenes which may be used in the present invention also include glycyrrhetinic acid, betulin, betulinin acid, ursolic acid, oleanolic acid, betulin mono- and di-succinate or glutarate, carbenoxolone, as well as polyethylene glycol derivatives and salts thereof. Thus the present invention may preferably employ pentacyclic triterpenes having the following general formula II:
More particularly, the present invention may employ compounds of the following formula III:
wherein each R1 is independently CH3, H, OH, C^OY1, CH2O-X-OH, CH2O-X-OY!, CH2O-X-Y2, CH2O-X-Y3. CH2-NHY1, CH2NY1 2, CH2Y3, CH2NH-X-OH, CH2NH-X- Y2, CH2NH-X-Y3, CH2NH-X-OY1, CH2OC(O)-OY1, CH-aO-X-OY1, CO2Y\ COY3, COY2, CHO, CH=N(CH2)m(O(CH2)m)nR5 or CH=N(CH2)m(O(CH2)m)nY2;
R2 or R3 is R1, OY1, O-X-OH, O-X-OY1, O-X-Y2, Y3, NHY^NY^, Y3, NH-X-OH, NH-X-Y2, NH-X-Y3, NH-X-OY1, NY^X-OH, NY^X-Y2, NY^X-Y3, NY^X-OY1, monosaccharide, disaccharide, oligosaccharide or polysaccharide or a derivative thereof, provided that one of R or R is H or that together they form an oxo group;
R4 is H, R1, provided that at least one R4 is H or together they form an oxo group;
R5 is H, OH, O^ OR Y3;
Y1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
Y2 is NH2, NHY1 OR NY! 2;
Y3 is -(O(CH2)m)nR5 or -(O(CH2m)nY2;
m is 1-4;
n is 1-50;
X is -OC(CH2)pCO-; and
P is 1-22;
or a salt, derivative or analogue thereof.
Preferably R1 or R2 or R3 is CH3, H, COOY1, COOY1, CO2(CH2)COOY1 or OH. Preferably R4 is H or together form an oxo group. Hydrocarbyl
If the modulator or agent is an organic compound then for some applications that organic compound will typically comprise one or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
The modulator or agent may contain halo groups. Here, "halo" means fluoro, chloro, bromo or iodo.
The modulator or agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, which may be unbranched or branched-chain.
Pharmaceutically acceptable salt
The modulator or agent may be in the form of, and/or may be administered as, a pharmaceutically acceptable salt such as an acid addition salt or a base salt, or a solvate thereof, including a hydrate thereof. For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.
Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. Suitable acid addition salts are formed from acids which form non-toxic salts and examples are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and examples are the sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine salts.
Polymorphic form(s)/asymmetric carbon(s)
The modulator or agent of the present invention may exist in polymorphic form.
The modulator of the present invention may contain one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. Where a modulator contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the modulator and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the modulator or agent or a suitable salt or derivative thereof. An individual enantiomer of a modulator or agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
Isotopic Variations The present invention also includes all suitable isotopic variations of the modulator or agent or a pharmaceutically acceptable salt thereof. An isotopic variation of a modulator or agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the modulator or agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as H, H, C, C, 15N, 17O, 180, 31P, 32P, 35S, 18F and 36C1, respectively. Certain isotopic variations of the modulator or agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the test compounds of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagent.
Pro-Drug
It will be appreciated by those skilled in the art that the modulator of the present invention may be derived from a prodrug. Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be admimstered (such as orally or parenterally) and thereafter metabolised in the body to form the modulator of the present invention which are pharmacologically active.
Pro-moiety It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the modulators or agents. Such prodrugs are also included within the scope of the invention.
Antibodies
In one embodiment of the present invention, the modulator or agent of the present invention may be an antibody.
The HMGBl modulator binding domain of the present invention or derivatives or variants thereof, or cells expressing the same can be used to produce antibodies immunospecific for such polypeptides. The term "immunospecific" means that the antibodies have substantially greater affinity for the HMGBl modulator binding domain of the present invention than for other related polypeptides.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing an HMGBl epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.
Monoclonal antibodies directed against epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.
Antibodies, both monoclonal and polyclonal, which are directed epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti- idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the agent against which protection is desired.
Techniques for raising anti-idiotype antibodies are known in the art. These anti- idiotype antibodies may also be useful in therapy.
For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
Agonist
As used herein, the term "agonist" means any modualtor, which is capable of binding to an HMGBl modulator binding domain and which is capable of increasing a proportion of HMGBl that is in an active form, resulting in an increased biological response. The term includes partial agonists and inverse agonists.
Antagonist As used herein, the term "antagonist" means any agent that reduces the action of another agent, such as an agonist. The antagonist may act at the same receptor as the agonist. The antagonistic action may result from a combination of the substance being antagonised (chemical antagonism) or the production of an opposite effect through a different receptor (functional antagonism or physiological antagonism) or as a consequence of competition for the binding site of an intermediate that links receptor activation to the effect observed (indirect antagonism).
Modulating
The term "modulating" means inducing an increase or a decrease in the activity of the HMGBl through binding of an agent to an HMGBl modulator binding domain.
Mimetic
As used herein, the term "mimetic" relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a known agent. That is, the mimetic is a functional equivalent of a known agent.
Derivative
The term "derivative" or "derivatised" as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
Typically the agent will be prepared by recombinant DNA techniques and/or chemical synthesis techniques.
Once an agent capable of interacting with a key amino acid residue in the HMGBl modulator binding domain has been identified, further steps may be carried out either to select and/or to modify compounds and/or to modify existing compounds, to modulate the interaction with the key amino acid residues in the HMGBl modulator binding domain.
Chemical Synthesis Methods
The modulator or agent of the present invention may be prepared by chemical synthesis techniques.
The modulator or agent of the present invention or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesise the modulator or agent in whole or in part. For example, peptides can be synthesised by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the . synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
Direct synthesis of the modulator or agent or variants, homologues, derivatives, fragments or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al., 1995) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
Biological Screens
Agents and modulators which are identified with the HMGBl modulator binding domain structure of the present invention can be screened in assays such as are well known in the art. Screening can be, for example in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity. The biological assay, may also be an assay for ligand binding activity of a compound that selectively binds to the LBD compared to other nuclear receptors.
According to one aspect of the present invention there is provided a method for screening of a modulator capable of binding to an HMGBl modulator binding domain of the present invnetion, the method comprising contacting the HMGBl modulator binding domain with an agent, and determining if said agent binds to said HMGBl modulator binding domain
In one embodiment, the present invention provides a method of screening for an agent capable of interacting with a key amino acid residue of the HMGBl modulator binding domain.
Another preferred aspect of the invention provides a process comprising the steps of:
(a) performing the method of screening for a modulator as described above;
(b) identifying one or more modulators capable of binding to an HMGBl modulator binding domain; and (c) preparing a quantity of said one or more modulators.
A further preferred aspect of the invention provides a process comprising the steps of:
(a) performing the method of screening for a modulators as described above;
(b) identifying one or more ligands capable of binding to an HMGBl modulator binding domain; and
(c) preparing a pharmaceutical composition comprising said one or more modulators.
Yet another preferred aspect of the invention provides a process comprising the steps of:
(a) performing the method of screening for a modulator as described above; (b) identifying one or more modulators capable of binding to an HMGBl modulator binding domain;
(c) modifying said one or more modulators capable of binding to an HMGBl modulator binding domain; (d) performing said method of screening for a modulator as described above;
(e) optionally preparing a pharmaceutical composition comprising said one or more modulators.
Thus, the information from the structure of the present invention is useful in the design of potential modulators capable of interacting with the HMGBl modulator binding domain and/or capable of modulating the activity of HMGBl modulator binding domain, and the models of the present invention are useful to examine the effect such a modulator is likely to have on the structure and/or function of the
HMGBl modulator binding domain.
In one aspect the present invention relates to a modulator identified using such screening methods.
Preferably the modulator is capable of spatially fitting into an HMGBl modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4 or Figure 11.
More preferably the modulator is capable of interacting with at least some of the following amino acid residues of the N-terminal helix 1 ofHMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45.
The present invention also relates to a pharmaceutical composition comprising a modulator identified by any of the screening methods of the present invention. Disorders
According to one aspect of the present invention there is provided a method of preventing and/or treating a disorder associated with HMGBl comprising administering a modulator of the present invention, or a pharmaceutical composition comprising a modulator of the present invention, wherein said modulator or pharmaceutical composition is capable of modulating HMGBl to cause a beneficial and/or therapeutic effect.
Inflammatory cytokine cascade
In one embodiment, a modulator may be administered for treating conditions (diseases) mediated by the inflammatory cytokine cascade, such as sepsis.
Sepsis is an often fatal clinical syndrome that develops after infection or injury. Sepsis is the most frequent cause of mortality in hospitalised patients. Experimental models of gram negative sepsis based on administration of bacterial endotoxin (lipopolysaccharide, LPS) have led to an improved understanding of the pathogenic mechanisms of lethal sepsis and conditions related to sepsis by virtue of the activation of a common underlying inflammatory cytokine cascade. This cascade of host- response mediators includes TNF, IL-1, PAF and other macrophage-derived factors that have been widely studied as acute, early mediators of eventual lethality in severe endotoxemia (Zhang and Tracey, In The Cytokine Handbook, 3rd ed. Ed. Thompson (Academic Press Limited, USA). 515-547, 1998).
Unfortunately, therapeutic approaches based on inhibiting these individual "early" mediators of endotoxemia have met with only limited success in large prospective clinical trials against sepsis in human patients. It is possible to infer from these disappointing results that later-appearing factors in the host response might critically determine pathogenesis and/or lethality in sepsis and related disorders. Accordingly, there is a need to discover such putative "late" mediators necessary and/or sufficient for part or all of the extensive multisystem pathogenesis, or for the lethality, of severe endotoxemnia, particularly as endotoxemia is representative of clinical sepsis and related clinical disorders.
Diseases and conditions mediated by the inflammatory cytokine cascade are numerous. Such conditions include the following grouped in disease categories:
Systemic Inflammatory Response Syndrome, which includes:
Sepsis syndrome
Gram positive sepsis Gram negative sepsis
Culture negative sepsis
Fungal sepsis
Neutropenic fever
Urosepsis Meningococcemia
Trauma hemorrhage
Hums
Ionizing radiation exposure
Acute pancreatitis Adult respiratory distress syndrome (ARDS)
Reperfusion Injury, which includes
Post-pump syndrome
Ischemia-reperfusion injury
Cardiovascular Disease, which includes Cardiac stun syndrome
Myocardial infarction
Congestive heart failure
Infectious Disease, which includes
HIN infection/HIN neuropathy Meningitis
Hepatitis
Septic arthritis Peritonitis
Pneumonia Epiglottitis
E. coli 0157:H7
Hemolytic uremic syndromc/thrombolytic thrombocytopcnic purpura Malaria
Dengue hemorrhagic fever
Leishmaniasis
Leprosy
Toxic shock syndrome Streptococcal myositis
Gas gangrene
Mycobacterium tuberculosis
Mycobaclerium aviun intracellulare
Pyneumocystis carinii pneumonia Pelvic inflammatory disease
Orchitis/epidydimitis
Legionella
Lyme disease
Influenza A Epstein-Barr Virus
Viral associated hemiaphagocytic syndrome
Viral encephalitis/aseptic meningitis
Obstetrics/Gynecology, including:
Premature labor Miscarriage
Infertility
Inflammatory Disease/Autoimmunity, which includes:
Rheumatoid arthritis/seronegative arthropathies
Osteoarthritis Inflationary bowel disease
Systemic lupus erythematosis
Iridoeyelitis/uveitistoptic neuritis Idiopathic pulmonary fibrosis
Systemic vasculitis/Wegener's gramilomatosis
Sarcoidosis
Orchitis/vasectomy reversal procedures Allergic/Atopic Diseases, which includes:
Asthma
Allergic rhinitis
Eczema
Allergic contact dermatitis Allergic conjunctivitis
Hypersensitivity pneumonitis
Malignancy, which includes:
ALL
AML CML
CLL
Hodgkin's disease, non-Hodgkin's lymphoma
Kaposi's sarcoma
Colorectal carcinoma Nasopharyngeal carcinoma
Malignant histiocytosis
Paraneoplastic syndrome/hypercalcemia of malignancy
Transplants, including:
Organ transplant rejection Graft-versus-host disease
Cachexia
Congenital, which includes:
Cystic fibrosis
Familial hematophagocytic lymphohistiocytosis Sickle cell anemia
Dermatologic, which includes:
Psoriasis Alopecia
Neurologic, which includes:
Multiple sclerosis
Migraine headache Renal, which includes:
Nephrotic syndrome
Hemodialysis
Uremia
Toxicity, which includes: OKT3 therapy
Anti-CD3 therapy
Cytokine therapy
Chemotherapy
Radiation therapy Chronic salicylate intoxication
Metabolic/ldiopathic, which includes:
Wilson's disease
Hemachromatosis
Alpha- 1 antitrypsin deficiency Diabetes
Hashimoto's thyroiditis
Osteoporosis
Hypothalamic-pituitary-adrenal axis evaluation
Primary biliary cirrhosis
HMGBl is also an earlier marker of inflammation in the case of large scale necrosis.
In particular, HMGBl may be released by the necrotic cells. Thus, the present invention may be used in the treatment of the first stages of inflammation in the case of large scale necrosis, such as occurs in intestinal infarction, acute pancreatitis and extensive trauma.
Immune Response In one embodiment, a modulator or pharmaceutical composition of the present invention may be administered so as to downregulate an immune specific response and or treat an inflammatory or autoimmune disease, allergy or transplant rejection.
In a preferred embodiment of the present invention an inhibitor HMGBl is used in a method of modulating an immune response and/or treating an inflammatory or autoimmune disease, allergy or transplant rejection.
As described in our co-pending International Patent Application Publication No. WO03/026691, in addition to triggering non-specific mechanisms, pathogens, e.g. during an infection, also trigger the antigen-specific adaptive immune response. The adaptive immune response to infection involves both the T and B cell mediated compartments of the immune system. During the so-called induction phase during antigen presenting cells (APCs) are involved in the initiation of the adaptive immune response. APC function is also required for maintenance of the adaptive immune response.
In more detail, APCs constitute a complex of cells capable of internalising an antigen, processing it and expressing epitopes thereof in association with class I and class II
MHC molecules. In general it can be said that the common characteristic of the cells of the group of APCs used medically is the expression of MHC molecules of class II as well as class I on the cell surface. The group mainly comprises dendritic cells, activated macrophages, microglial cells of the central nervous system and B lymphocytes. Among these, the dendritic cells (DCs) are particularly specialised in antigen presentation and constitute a population with distinctive characteristics and are widely distributed in tissues. The DCs are involved in the activation of the immune response, which takes place by stimulation of the T lymphocytes in the course of various pathologies such as infections, autoimmune diseases and transplant rejection. Activation or maturation of DCs is a necessary process for "priming" the T cells and initiating the immune response. In autoimmune diseases and in transplant rejection, i.e. in the absence of pathogenic agents, induction of maturation of the dendritic cells takes place by means of endogenous molecules possessing immunostimulatory activity in vivo. It is known that cells that are dying contain and release molecules that are able to amplify the immune response (Gallucci, et al. 1999, Sauter et al. 2000, Ignatius et al. 2000, Shi et al. 2000, Basu, et al. 2000, Larsson et al., 2001). These molecules, normally segregated inside living cells, remain thus in the apoptotic process while they are released during cell death.
Cellular constituents released in the culture medium after cell death are able to provoke maturation of the DCs (Gallucci, et al. 1999, Sauter et al. 2000). On the other hand, DCs stimulated with cells in the initial apoptotic state, or with their culture medium, are not activated (Gallucci, et al. 1999, Sauter et al. 2000, Ignatius et al. 2000 and Rovere et al., 1998). Similarly, the DCs are not activated by necrotic polymorphonuclear (PMN) leukocytes.
In our co-pending International patent application publcation No. WO03/026691 we have found that HMGBl is capable of activating the maturation of APCs. By "activating" we include inducing maturation of APCs. Thus, in one embodiment of the present invention there is provided an antagonist capable of minding to an HMGBl modulator binding domain which is capable of preventing or reducing the activation of an APC. Thus, for example, when an antagonist of HMGBl is added to a population of APCs in conditions in which maturation is capable of occurring, fewer APCs proceed to maturity than in the absence of the HMGBl antagonist.
Vascular Diseases
In one embodiment, a modulator of the present invention may be admimstered for treating a vascular disease, comprising administering an effective amount of a modulator of the invention. The vascular disease may arise due to diagnostic and/or surgical techniques, for example involving the use of diagnostic or surgical instruments, such as catheters, surgical instruments or stents. In one embodiment the diagnostic or surgical technique involves angioplasty and/or angiography.
Preferably the present invention is used to treat atherosclerosis and/or restenosis that occur during angioplasty and/or angiography.
In one embodiment, the treatment may include blocking, retarding or reducing connective tissue regeneration.
In a one embodiment, the modulators are released by catheters, surgical instruments or stents for angioplasty.
The modulators may however be used before, during or after invasive diagnostic and/or surgical techniques. The insertion of instruments, such as catheters, into vessels may damage the endothelial lining and lead to inflammation and/or stenosis of the vessel. The present invention can be used in the treatment and/or prevention of such a condition.
WO 02/074337 showed that HMGBl has a potent biological effect on smooth muscle cells (SMC), one of the cell types where RAGE is expressed on the surface and demonstrated the role of HMGBl in promoting atherosclerosis and restenosis after vascular damage. Vascular smooth muscles cells (SMC) are the most predominant cells of the larger blood vessels. When the endothelium is damaged, either after mechanical or inflammatory injuries, SMC switch to a synthetic phenotype and undergo cell division and migration. The migration of SMC cells from the tunica media to the tunical intima plays an important role in the pathophysiology of many vascular disorders, such as atherosclerosis and restenosis after coronary angioplasty.
After vessel wall injury, the release of several growth factors and or chemoattractants either by circulating monocytes, macrophages and platelets, or by damaged endothelial cells can induce SMC cells switch to the synthetic phenotypes. WO 02/074337 demonstrated that HMGBl is a strong chemoattractant and induces SMC cell shape changes, and cytoskeleton reorganisation. These events are inhibited by addition of an ant-RAGE antibody and by pertussis toxin, underlying that both RAGE and Gi/o protein might be involved in the pathway. Furthermore, the evidence that HMGBl promotes the translocation of phosphorylated ERK 1 and 2 proteins into the nucleus, indicates the involvement of the MAP kinase pathway. It was also demonstrated that HMGBl is released by damage or necrosis of a variety of cell types, including endothelial cells. Furthermore, it was shown that HMGBl fragments may be more efficacious than the entire full-length molecule. Consequently, every kind of molecule able to block the interaction between HMGBl and a receptor for HMGBl, such as the RAGE receptor, can efficiently be used for the production of pharmacological preparation in order to avoid, retard or inhibit atherosclerosis and restenosis after vascular epithelium damage even due to angioplasty.
Introduction of nucleic acid sequences into APCs
APCs as described above may be cultured in a suitable culture medium such as DMEM or other defined media, optionally in the presence of fetal calf serum.
HMGBl may be admimstered to APCs and by introducing nucleic acid constructs/viral vectors encoding the protein into cells under conditions that allow for expression of the polypeptide in the APC. Similarly, nucleic acid constructs encoding antisense constructs may be introduced into the APCs and by transfection, viral infection or viral transduction.
APCs
Antigen presenting cells (APCs) include macrophages, dentritic cells, B cells and virtually any other cell type capable of expressing an MHC molecule.
Macrophages are phagocytic cells of the monocytic lineage residing within tissues and are particularly well equipped for effective antigen presentation. They generally express MHC class II molecules and along with their phagocytic properties are extremely efficient at engulfing macromolecular or particulate material, digesting it, processing it with an extensive lysosomal system to antigenic peptide form, and expressing it on the cell surface for recognition by T lymphocytes.
Dendritic cells, so named for their highly branched morphology, are found in many organs throughout the body, are bone marrow-derived and usually express high levels of MHC class II antigen. Dendritic cells are actively motile and can recirculate between the bloodstream and tissues. In this way, they are considered the most important APCs. Langerhans cells are an example of dendritic cells that are located in the skin.
B lymphocytes, while not actively phagocytic, are class II-positive and possess cell surface antigen-specific receptors, immunoglobulin, or antibody molecules. Due to their potential for high affinity antigen binding, B cells are uniquely endowed with the capacity to concentrate low concentrations of antigen on their surface, endocytose it, process it and present it in the context of antigenic peptide in association with MHC antigen on their surface. In this manner, B cells become extremely effective APCs.
Methods
Immature dendritic cells for use in the present invention can be obtained from haematopoietic precursors or from stem cells, for example from PBMC cells, by suitable treatment with cytokines such as GM-CSF, IL-4 and flt3-L.
The activation or maturation of antigen-presenting cells can be effected starting from a culture of immature or inactive cells, by adding HMGBl protein and possibly other co-adjuvants such as cytokines to the culture medium.
Once led to maturation or activated, the antigen-presenting cells, especially the DCs, can be used for the activation of T lymphocytes in response to particular antigens; the lymphocytes thus activated can then be administered to a subject to stimulate their immune response to the said antigens. The indicators of activation can vary according to the cell type under consideration. With regard to macrophages, microglia and B lymphocytes, for example, it is a functional activation with increase in membrane expression of MHC molecules and co-stimulatory molecules following contact with other adjuvants, as described in (27, 28).
In the case of the dendritic cells, those cells that display increased expression of markers characteristic of the "maturation phenotype", such as the CD83 and CD86 surface molecules, or reduced expression of markers characteristic of the immature phenotype, such as CD115, CD14, CD68 and CD32, are regarded as activated or mature.
An ex vivo method for the activation of T lymphocytes may comprise the following steps: a) bringing a preparation of inactive APCs into contact with HMGBl, or with its biologically active fragments, so as to induce their activation; b) bringing the activated APCs into contact with a particular antigen; c) exposing the T lymphocytes to the APCs that have been activated and exposed to the antigen.
According to a preferred embodiment, dendritic cells are used as APCs.
Steps a) - c) indicated above can be executed in a different order. For example, the antigen can be added to a culture of immature or inactive APCs before the HMGBl protein or its fragments. In addition, the APCs or DCs can be transfected with a vector for the expression of a particular antigen or of a polypeptide derived from it, or alternatively a vector for the expression of a specific MHC molecule. Antigens associated with microorganisms, viruses, tumours or autoimmune diseases can be used for the activation of lymphocytes according to the method described. As tumour antigens, in addition to the proteins or their fragments isolated from tumour tissues or cells, it is possible to use whole cells that have been killed by apoptosis or necrosis. It is also possible to use antigens associated with viruses or retroviruses, especially HIV, or with intracellular pathogens, such as mycobacteria or plasmodia.
Pharmaceutical compositions
A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. It preferably includes a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
"Therapeutically effective amount" refers to the amount of the therapeutic agent which is effective to achieve its intended purpose. While individual patient needs may vary, determination of optimal ranges for effective amounts of HMGBl modulator is within the skill of the art. Generally the dosage regimen for treating a condition with the compounds and/or compositions of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient,, the severity of the dysfunction, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound used, whether a drug delivery system is used, and whether the compound is administered as part of a drug combination and can be adjusted by one skilled in the art. Thus, the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth herein.
Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Typically, each conjugate may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
When the polynucleotides/vectors are administered as a naked nucleic acid, the amount of nucleic acid administered may typically be in the range of from 1 μg to 10 mg, preferably from 100 μg to 1 mg.
Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition.
The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.
Administration
The invention further provides a method of preventing and/or treating a disorder associated with HMGBl, the method comprising administering to a mammal a modulator according to the present invention.
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient and severity of the condition. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
The compositions (or component parts thereof) of the present invention may be administered orally. In addition or in the alternative the compositions (or component parts thereof) of the present invention may be administered by direct injection. In addition or in the alternative the compositions (or component parts thereof) of the present invention may be administered topically. In addition or in the alternative the compositions (or component parts thereof) of the present invention may be administered by inhalation. In addition or in the alternative the compositions (or component parts thereof) of the present invention may also be administered by one or more of: parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration means, and are formulated for such administration.
By way of further example, the pharmaceutical composition of the present invention may be administered in accordance with a regimen of 1 to 10 times per day, such as once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
The term "administered" also includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
Hence, the pharmaceutical composition of the present invention may be administered by one or more of the following routes: oral administration, injection (such as direct injection), topical, inhalation, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration or transdermal administration.
Model
One aspect of the present invention is related to a model.
The HMGBl modulator binding domain structure substantially as presented by the space filling balls in Figure 4 or Figure 11 of the present invention can be used to generate a model such as a 3D structural model (or a representation thereof) comprising a HMGBl modulator binding domain or portion thereof. Alternatively, the structure may be used to generate a computer model for the structure.
Preferably the model comprising the HMGBl modulator binding domain comprises the structural coordinates of a modulator binding domain corresponding to at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26and 27; and/or at least some of the following amino acid residues of helix 2 ofHMGBl: 42, 43, 44 and 45.
Thus, for example, the model structure may comprise the amino acid residues of the HMGBl modulator binding domain, or a portion of the HMGBl modulator binding domain or a homologue thereof useful in the modelling and design of test compounds capable of binding to the HMGBl modulator binding domain.
As used herein, the term "modelling" includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models. The term "modelling" includes conventional numeric-based molecular dynamic and energy minimisation models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure- based constraint models.
In another aspect of the present invention, the HMGB 1 modulator binding domain structure substantially as presented by the space filling balls in Figure 4 or a portion thereof may be applied to a model screening system.
As used herein, the term "model screening system" may be a solid 3D screening system or a computational screening system. Using this model, modulators can be modelled that fit spatially and preferentially into the HMGBl modulator binding domain.
In one preferred aspect, the modulators are positioned in the HMGBl modulator binding domain through computational docking. In another prefened aspect, the modulators are positioned in the HMGBl modulator binding domain through manual docking.
As used herein, the term "fits spatially" means that the three-dimensional structure of a modulator is accommodated geometrically in a cavity or pocket of a HMGBl modulator binding domain.
Preferably, modelling is performed using a computer and may be further optimised using known methods. This is called modelling optimisation. Overlays and super positioning with a three dimensional model of the HMGBl modulator binding domain, and/or a portion thereof, can also be used for modelling optimisation.
Alignment and/or modelling can be used as a guide for the placement of mutations on the HMGBl modulator binding domain surface to characterise the nature of the site in the context of a cell.
The present invention also relates to a method of screening for a modulator capable of binding to the HMGBl modulator binding domain of the present invention.
The method may employ a solid 3D screening system or a computational screening system. Using these systems, agents may be screened to find those which interact spatially and preferentially with the HMGBl modulator binding domain, through either computational or manual docking.
The rational design and identification of modulators of HMGBl modulator binding domain can be accomplished by utilising the atomic structural coordinates that define an HMGBl modulator binding domain structure according to the present invention, or a part thereof. Structure-based modulator design identification methods are powerful techniques that can involve searches of computer data bases containing a variety of potential modulators and chemical functional groups. (See Kuntz et al., 1994, Ace. Chem. Res. 27:117; Guida, 1994, Current Opinion in Struc. Biol. 4: 777; and Colman, 1994, Current Opinion in Struc. Biol. 4: 868, for reviews of structure-based drug design and identification; and Kuntz et al 1982, J. Mol. Biol. 162:269; Kuntz et al., 1994, Ace. Chem. Res. 27: 117; Meng et al., 1992, J. Compt. Chem. 13: 505; Bohm, 1994, J. Comp. Aided Molec. Design 8: 623 for methods of structure-based modulator design).
Modulators of an HMGBl modulator binding domain may be identified by docking the computer representation of compounds from a data base of molecules. Data bases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall). Computer programs such as CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions.
The computer programs may comprise the following steps:
(a) docking a computer representation of a structure of a compound into a computer representation of an HMGBl modulator binding domain defined in accordance with the invention using the computer program, or by interactively moving the representation of the compound into the representation of the binding site;
(b) characterising the geometry and the complementary interactions formed between the atoms of the binding site and the compound; optionally
(c) searching libraries for molecular fragments which can fit into the empty space between the compound and binding site and can be linked to the compound; and
(d) linking the fragments found in (c) to the compound and evaluating the new modified compound. Methods are also provided for identifying a potential modulator of an HMGBl modulator binding domain function by docking a computer representation of a compound with a computer representation of a structure of an HMGBl modulator binding domain according to the present invention.
In an embodiment of the invention, a method is provided for identifying potential modulators according to the invention. The method utilises the structural coordinates of an HMGBl modulator binding domain three-dimensional structure, or binding site thereof. The method comprises the steps of (a) generating a computer representation of an HMGBl modulator binding domain structure, and docking a computer representation of a compound from a computer data base with a computer representation of the HMGBl modulator binding domain to form a complex; (b) determining a conformation of the complex with a favourable geometric fit or favourable complementary interactions; and (c) identifying compounds that best fit the HMGBl modulator binding domain as potential modulators of HMGBl modulator binding domain function. The initial HMGBl modulator binding domain structure may or may not have compounds bound to it. A favourable geometric fit occurs when the surface areas of a compound in a compound-HMGBl modulator binding domain complex is in close proximity with the surface area of the HMGBl modulator binding domain without forming unfavourable interactions. A favourable complementary interaction occurs where a compound in a compound-HMGBl modulator binding domain complex interacts by hydrophobic, aromatic, ionic, or hydrogen donating and accepting forces, with the HMGBl modulator binding domain without forming unfavourable interactions. Unfavourable interactions may be steric hindrance between atoms in the compound and atoms in the HMGBl modulator binding domain.
"Docking" refers to a process of placing a compound in close proximity with an active site of a polypeptide (i.e. an HMGBl modulator binding domain), or a process of finding low energy conformations of a compound/polypeptide complex (i.e. compound/ HMGB 1 modulator binding domain complex) . Examples of other computer programs that may be used for structure-based modulator design are CAVEAT (Bartlett et al., 1989, in "Chemical and Biological Problems in Molecular Recognition", Roberts, S.M. Ley, S.N.; Campbell, Ν.M. eds; Royal Society of Chemistry: Cambridge, pp 182-196); FLOG (Miller et al., 1994, J. Comp. Aided Molec. Design 8:153); PRO Modulator (Clark et al., 1995 J. Comp. Aided Molec. Design 9:13); MCSS (Miranker and Karplus, 1991, Proteins: Structure, Function, and Genetics 8:195); and, GRID (Goodford, 1985, J. Med. Chem. 28:849).
In another embodiment, potential modulators are identified utilising an HMGBl modulator binding domain structure with or without compounds bound to it. The method comprises the steps of (a) modifying a computer representation of an HMGBl modulator binding domain having one or more compounds bound to it, where the computer representations of the compound or compounds and HMGBl modulator binding domain are defined by atomic structural coordinates; (b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and (c) identifying the compounds that best fit the
HMGBl modulator binding domain as potential modulators. A computer representation may be modified by deleting or adding a chemical group or groups.
Computer representations of the chemical groups can be selected from a computer database.
Another way of identifying potential modulators is to modify an existing modulator (e.g., glycyrrhizic acid) in a polypeptide binding site. The computer representation of modulators can be modified within the computer representation of an HMGBl modulator binding domain. This technique is described in detail in Molecular Simulations User Manual, 1995 in LUDI. The computer representation of a modulator may be modified by deleting a chemical group or groups, or by adding a chemical group or groups. After each modification to a compound, the atoms of the modified compound and binding site can be shifted in conformation and the distance between the modulator and the binding site atoms may be scored on the basis of geometric fit and favourable complementary interactions between the molecules. Compounds with favourable scores are potential modulators. Compounds designed by modulator building or modulator searching computer programs may be screened to identify potential modulators. Examples of such computer programs include programs in the Molecular Simulations Package (Catalyst), ISIS/HOST, ISIS/BASE, and ISIS/DRAW (Molecular Designs Limited), and UNITY (Tripos Associates). A building program may be used to replace computer representations of chemical groups in a compound complexed with an HMGBl modulator binding domain with groups from a computer database. A searching program may be used to search computer representations of compounds from a computer database that have similar three dimensional structures and similar chemical groups as a compound that binds to an HMGBl modulator binding domain. The programs may be operated on the structure of the HMGBl modulator binding domain structure.
A typical program may comprise the following steps:
(a) mapping chemical features of a compound such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites;
(b) adding geometric constraints to selected mapped features; (c) searching data bases with the model generated in (b).
In an embodiment of the invention a method of identifying potential modulators of an HMGBl modulator binding domain is provided using the three dimensional conformation of the HMGBl modulator binding domain in various modulator construction or modulator searching computer programs on compounds complexed with the HMGBl modulator binding domain. The method comprises the steps of (a) generating a computer representation of one or more compounds complexed with an HMGBl modulator binding domain; (b) (i) searching a data base for a compound with a similar geometric structure or similar chemical groups to the generated compounds using a computer program that searches computer representations of compounds from a database that have similar three dimensional structures and similar chemical groups, or (ii) replacing portions of the compounds complexed with the HMGBl modulator binding domain with similar chemical structures (i.e. nearly identical shape and volume) from a database using a compound construction computer program that replaces computer representations of chemical groups with groups from a computer database, where the representations of the compounds are defined by structural coordinates.
Potential modulators of HMGBl modulator binding domain identified using the above-described methods may be prepared using methods described in standard reference sources utilised by those skilled in the art. For example, organic compounds may be prepared by organic synthetic methods described in references such as March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill.
The invention contemplates all optical isomers and racemic forms of the modulators of the invention.
Description of the Figures
The present invention will now be described further with reference to the following non-limiting Examples and Figures in which:
Figure 1 shows the structure of glycyrrhizic acid ammonium salt.
Figure 2 shows the heat effects of subsequent injections of glycyrrhizic acid into a solution of HMGB 1 Box A.
Figure 3 shows the average chemical shift changes of the IHN and 15N nuclei of HMGBl box A induced upon binding of glycyrrhizic acid as a function of residue number.
Figure 4 shows the residues ofHMGBl which interact with glycyrrhizic acid. The Cα backbone is depicted as a ribbon; interacting residues in space-filling representation as represented by the space filling balls.
Figure 5 shows the migratory response of Bovine Artery Endothelial Cells (BAEC) in the presence and absence of glycyrrhizic acid.
Figure 6 shows the proliferation of BAEC in the presence and absence of glycyrrhizic acid.
Figure 7A shows the 1H-15N HSQC spectrum of Box B of HMGBl (10 mM phosphate, 15 mM NaCI, pH 5.5, T=293K, [BoxB]=0.6mM).
Figure 7B represents the 1H-15N HSQC spectrum of Box B of HMGBl (10 mM phosphate, 15 mM NaCI, pH 5.5T=293K, [BoxB]=0.6mM) in presence of glycyrrhizic acid (ratio 1 :4 Box B: glycyrrhizic acid).
Figure 8 shows the amino acid sequence ofHMGBl with box A underlined and box B indicated in bold.
Figure 9A shows 15N-HSQC spectrum ofHMGBl Box A, 0.3 mM, 10 mM phosphate buffer, 150 mM NaCI, pH 7.
Figure 9B shows 15N-HSQC spectrum ofHMGBl Box Axarbenoxolone (1:5).
Figure 10 shows chemical shift changes for HMGBl Box Axarbenoxolone (pH = 7; final ratio 1:5).
Figure 11 shows the residues of HMGBl which interact with carbenoxolone. The Cα backbone is depicted as a ribbon; interacting residues in space-filling representation as space filling balls.
Figure 12 shows the averaged chemical shift displacement of residue R23 vs. carbenoxolone concentration (Cbnx) (raw data and curve fitting). Figure 13 shows the averaged chemical shift displacement of residue R23 vs. glycerrhetic acid (GLY) concentration (raw data and curve fitting).
Figure 14 shows the effect of glycyrrhetic acid on BAEC migration.
Figure 15 shows the effect of carbenoxolone or glycyrrhizin on BAEC migration.
Figure 16A shows the structure of glycyrrhizin; Figure 16B shows the structure of carbenoxolone, Figure 16C shows the structure of ursolic acid and Figure 16D shows the structure of a sodium salt of gycyrrhetic acid.
Examples
Example 1 - Binding of glycyrrhizic acid to HMGBl boxA
Glycyrrhizic acid binds to HMGBl boxA with a dissociation constant ofl3+/-7μM. The thermodynamics of the interaction of the single HMG boxes A and B with glycyrrhizic acid were analyzed using isothermal titration calorimetry. In the temperature interval used (10-27°C), HMGBl has been reported to be completely folded (Ramstein J., Locker D., Bianchi M.E. and Leng M. Eur. J. Biochem. 260: 692- 700, 1999). In the experiments performed, glycyrrhizic acid was titrated into a solution of box A or box B, respectively. Figure 2 shows the heat effects of subsequent injections of GA into a solution of box A. Box B was similar.
The equilibrium constant KB was determined as 7.5 +/-4 104.
Example 2 - Mapping on HMGBl boxA of the binding site for glycyrrhizic acid Quantification of chemical shift changes in a protein upon ligand binding is a sensitive method for measuring the strength of such interactions and for defining the protein's interaction surface. Here we show by solution heteronuclear NMR spectroscopy that glycyrrhizic acid binds directly to HMGBl boxA. HMGBl boxA concentration upon titration with was 0.2 mM, at pH 5.0, 150 mM NaCI.
In Figure 3 the average chemical shift changes of the IHN and 15N nuclei ofHMGBl boxA induced upon binding of glycyrrhizic acid (GA) are shown as a function of residue number. The largest changes in chemical shifts are clustered on the N-terminal helix 1 around residues 11, 12, 13, 17, 20, 23, 25, 27 and around residues 43, 44, 45 in helix 2.
The ligand binding is in the fast chemical exchange limit; although the affinity is low ( -d in the 10"5 M range) the binding is specific, as it delineates a well defined area on the surface ofHMGBl boxA (Figure 4; Cα backbone as a ribbon; interacting residues in space-filling representation) .
Example 4 - The migratory response of Bovine Artery Endothelial Cells (BAEC)
Endothelial cells respond to extracellular HMGBl by reorganizing their cytoskeleton (not shown) and migrating. The migratory response of BAEC was assayed with modified Boyden chambers for 4 hours at 37°C, as described by Degryse et al. (J. Cell Biol. 152:1197-2006, 2001) for Rat Smooth Muscle cells (RSMC). BAEC cells respond maximally to 1 ng/ml HMGBl. Addition of 30 μM glycyrrhizic acid to the medium abolishes the migratory response completely (Fig. 5).
The HMGBl -induced migratory response of, vascular smooth muscle cells and fibroblasts is similarly inhibited (not shown).
Example 5 - Inhibition of HMGBl-induced proliferation by glycyrrhizic acid
BAEC respond to HMGBl by proliferating for at least 2 days in the absence of serum. Glycyrrhizic acid (30 μM) abolished completely the effect ofHMGBl (Fig. 6). Per se, glycyrrhizic acid does not inhibit the proliferation of BAEC in the presence of 10% fetal calf serum (FCS), nor does it reduce their viability when added to medium without serum.
The proliferation of vascular smooth muscle cells in response to HMGBl is similarly inhibited (not shown).
Example 6 - Binding of glycyrrhizic acid to HMGBl box B
Figure 7A represents the 1H-15N HSQC spectrum of Box B of HMGBl (10 mM phosphate, 15 mM NaCI, pH 5.5, T=293K, [BoxB]=0.6mM). Figure 7B represents the 1H-15N HSQC spectrum of Box B ofHMGBl (10 mM phosphate, 15 mM NaCI, pH 5.5T=293K, [BoxB]=0.6mM) in presence of glycyrrhizic acid (ratio 1:4 Box B: glycyrrhizic acid). Upon addition of glycyrrhizic acid, several peaks change their position in the spectrum. At substocbiometric concentration of BoxB:glycyrrhizic acid two sets of resonances of Box B are observed, one corresponding to unligated Box B, the other to the complex. This indicates that glycyrrhizic acid interacts with Box B, and that the complex is in slow chemical exchange in the NMR time scale.
Example 7 - Binding of Carbenoxolone to HMGBl box A
Carbenoxolone is able to bind HMGBl with a high Kd. Carbenoxolone binds to the first helix of Box A and to a set of amino acids similar to those involved in binding glycyrrhizic acid (Fig. 10). From the nmr data it can be deduced that the shift of Q20 (the NH2 residue on amino acid 20) is important and the modulator may bind to the oxygen (=O covalent binidng) in the terpene.
In more detail, Figure 9A and B show that upon addition of carbenoxolone, several peaks change their position indicating that carbenoxolone intereacts with Box A. The spectra were acquired on a Bruker 500 MHz spectromter at 20°C.
Figure 10 shows the average chemical shift changes of 1H and 15N nuclei ofHMGBl Box A induced upon binding of carbenoxolone shown as a function of residue number. The largest chemical shift changes are clustered around helix 1 (17, 20, 23, 25 and 26) and around residue 42. Residue 91 is actually the NH2 of residue Q20. The surface of interaction is very similar to the one with glycyrrhizic acid.
Figure 12 shows averaged chemical shift difference of the residue R23 as a function of carbenoxolone concentration. Assuming a simple binary reaction between Box A and carbenoxolone, analysis by nonlinear curve fitting yields values of Kd ~ 0.08 mM.
Figure 13 shows averaged chemical shift difference of the residue R23 as a function of glycerrhetic acid concentration. Assuming a simple binary reaction between Box A and glycerrhetic acid, analysis by nonlinear curve fitting yields values of Kd ~ 1.3 mM
Example 8 - Migratory Response of BAEC
Figs. 14 and 15 show the inhibition of BAEC migration by glycerrhetic acid (glycyrrhetinic acid) and carbenoxolone, respectively. Glycerrhetic acid is very insoluble, and may be forming miscelles, and may be the more active of the three inhibitors used in these Examples.
We have also found that glycyrrhizin and the like are specific for HMGBl, since they do not inhibit the migration of BAEC induced by fMLP (formyl-Methionine-Leucine- Proline), a chemoattractant that mimics bacterial protecins (Degryse et al).
Narious modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or biology or related fields are intended to be covered by the present invention. All publications mentioned in the above specification are herein incorporated by reference.
References - (herein incorporated by reference)
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Scmidt et al., 1999 Circ. Res., 84: 489-497
Landsman and Bustin, 1993 BioEssays 15:539-546 Baxevanis and Landsman, 1995 Nucleic Acids Research 23:514-523
Bustin, M. 1999, Mol. Cell Biol. 19, 5237-46
Bianchi, M. E., and Beltrame, M. 2000. EMBO Rep. 1, 109-114
Thomas, J. O., and Travers, A. A.2001, Trends Biochem. Sci. 26, 161-11
Weir et al., 1993 EMBO J Apr;12(4):1311-9 Read et al, 1993 Nucleic Acids Res. Jul 25;21(15):3427-36
Hardman et al., 1995 Biochemistry Dec 26;34(51): 16596-607.
Jones et al., 1994 Structure 2, 609-627
Falciola, L. et al., 1997 J. Cell Biol. 137, 19-26.
Nightingale, K. et al., 1996 EMBO J. 15, 548-561. Scaffidi, P., et al., 2002 Nature 418, 191-195.
Calogero, et al., 1999 Nature Genet. 22, 276-280
Muller, et al.,. 2001b EMBO J. 20, 4337-4340.
Wang et al. 1999a Science 285, 248-51
Gardella, et al., 2002 EMBO Rep. 3. Andrei, C, et al., 1999 Mol. Biol. Cell 10, 1463-75
Hori et al., 1995, J. Biol. Chem., 270:25752-25761
Bianchi 1991, Gene 104: 271-275
Lee et al. 1998, Gene 225: 97-105 Mistry et al. 1997, Biotechniques 22: 718-729
Yoh et al., 2002, Dig. Dis. Sci. 47:1775-81
Berge et al, J. Pharm. Sci., 1977, 66, 1-19
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Gallucci, et al., 1999 Nat Med. 5, 1249-1255
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Ignatius et al., 2000 J. Virol. 74, 11329-11338
Shi et al., 2000 Proc. Natl. Acad. Sci. U.S.A. 97, 14590-14595
Basu, S., et al., 2000 Int. Immunol. 12, 1539-1546
Larsson, M., et al., 2001 Trends Immunol. 3, 141-148
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Kuntz et al, 1994, Ace. Chem. Res. 27:117 Guida, 1994, Current Opinion in Struc. Biol. 4: 777
Colman, 1994, Current Opinion in Struc. Biol. 4: 868
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Kuntz et al., 1994, Ace. Chem. Res. 27: 117
Meng et al., 1992, J. Compt. Chem. 13: 505 Bohm, 1994, J. Comp. Aided Molec. Design 8: 623
Miller et al., 1994, J. Comp. Aided Molec. Design 8:153
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Degryse B et al, 2001, J. Cell Biol. 152:1197-2006

Claims

1. An HMGBl modulator binding domain having the structure substantially as presented by the spacing filling balls in Figure 4 or Figure 11.
2. An HMGBl modulator binding domain comprising: at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43,
44 and 45; or a homologue or mutant thereof.
3. An HMGBl modulator binding domain defined by a mutation or substitution or derivatisation in or of any one or more of the amino acid residues as defined in claim 2.
4. An HMGBl modulator binding domain according to any one of the preceding claims which further comprises a modulator bound to the modulator binding domain or a portion thereof.
5. An HMGBl modulator binding domain according to any preceding claim wherein the modulator comprises a triterpene moiety.
6. An HMGBl modulator binding domain according to claim 5 wherein the modulator also comprises a monosaccharide, disaccharide, oligosacchari.de or polysaccharide or a derivative thereof.
7. An HMGBl modulator binding domain according to claim 5 or 6 wherein the modulator is a compound of formula III :
wherein
each R1 is independently CH3, H, OH, CH2OY1, CH2O-X-OH, CH2O-X-OY1, CH2O- X-Y2, CH2O-X-Y3. CH2NHY1, CH2NY12, CH2Y3, CH2NH-X-OH, CH2NH-X-Y2, CH2NH-X-Y3, CHzNH-X-OY1, CH2OC(O)-OY1, CH2O-X-OY1, CO2Y!, COY3, COY2, CHO, CH=N(CH2)m(O(CH2)m)nR5 or CH=N(CH2)m(O(CH2)m)„Y2;
R2 or R3 is R1, OY1, O-X-OH, O-X-OY1, O-X-Y2, Y3, NHY1 > NY12, Y3, NH-X-OH, NH-X-Y2, NH-X-Y3, NH-X-OY1, NY^X-OH, NY^X-Y2, NY!-X-Y3, NY'-X-OY1, monosaccharide, disaccharide, ohgosaccharide or polysaccharide or a derivative
9 thereof, provided that one of R or R is H or that together they form an oxo group;
R4 is H, R1, provided that at least one R4 is H or together they form an oxo group;
R5 is H, OH, OY1 OR Y3;
Y1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
Y2 is NH2, NHY1 OR NY! 2;
Y3 is -(O(CH2)ra)„R5 or -(O(CH2m)„Y2;
m is 1-4;
n is 1-50;
X is -OC(CH2)pCO-; and
P is 1-22;
or a salt, derivative or analogue thereof.
8. A modulator binding domain according to claim 8 wherein the modulator is glycyrrhizic acid, glycyrrhetinic acid, ursolic acid, carbenoxolone or a salt or a mimetic thereof.
9. A method for screening of a modulator capable of binding to an HMGBl modulator binding domain as defined in any preceding claim, the method comprising contacting the HMGBl modulator binding domain with an agent, and determining if said agent binds to said HMGBl modulator binding domain.
10. A process comprising the steps of: performing the method according to claim 9; identifying one or more modulators capable of binding to an HMGBl modulator binding domain; and preparing a quantity of those one or more modulators.
11. A process comprising the steps of: performing the method according to claim 9; identifying one or more modulators capable of binding to an HMGBl modulator binding domain; and preparing a pharmaceutical composition comprising those one or more modulators.
12. A process comprising the steps of: performing the method according to claim 9; identifying one or more modulators capable of binding to an HMGBl modulator binding domain; modifying those one or more modulator; performing said method according to claim 9; and optionally preparing a pharmaceutical composition comprising those one or more modified modulators.
13. A modulator identifiable by the method of any one of claims 9 to 12.
14. A modulator according to claim 13 wherein the modulator is capable of spatially fitting into an HMGBl modulator binding domain having the structure substantially as presented by the space filling balls in Figure 4 or Figure 11.
15. A modulator according to claim 13 wherein the modulator is capable of interacting with at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45.
16. A pharmaceutical composition comprising a modulator according to any one of claims 13 to 15 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant or any combination thereof.
17. A method of modulating the activity of HMGBl comprising administering a modulator according to any one of claims 13 to 15, or a compound comprising a triterpene moiety.
18. The method according to claim 17 wherein the modulator also comprises a monosaccharide, disaccharide, ohgosaccharide or polysaccharide or a derivative thereof.
19. The method according to claim 18 wherein the compound is a compound of formula III:
wherein
each R1 is independently CH3, H, OH, CH2O- X-Y2, CH2O-X-Y3. CHzNHY1, CH2NY1 2, CH2Y3, CH2NH-X-OH, CH2NH-X-Y2, CH2NH-X-Y3, CH2NH-X-OY1, CH2OC(O)-OY1, CHzO-X-OY1, CO2Y1, COY3, COY2, CHO, CH=N(CH2)m(O(CH2)m)nR5 or CH=N(CH2)m(O(CH2)m)nY2;
R2 or R3 is R1, OY1, O-X-OH, O-X-OY1, O-X-Y2, Y3, NHY^NY^, Y3, NH-X-OH, NH-X-Y2, NH-X-Y3, NH-X-OY1, NY^X-OH, NY^X-Y2, NY -X-Y3, NY^X-OY1, monosaccharide, disaccharide, ohgosaccharide or polysaccharide or a derivative thereof, provided that one of R or R is H or that together they form an oxo group; R4 is H, R1, provided that at least one R4 is H or together they form an oxo group;
R5 is H, OH, OY1 ORY3;
Y1 is H, alkyl of 1-30 carbon atoms, preferably 1-6 carbon atoms, more preferably methyl, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkenyl of 2-30 carbon atoms, preferably 2-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms or phenoxyalkyl of 7-30 carbon atoms, a metal ion, such as a Na, K, Mg or Ca ion;
Y2 is NH2, NHY1 OR NY^;
Y3 is -(O(CH2)m)nR5 or-(O(CH2m)„Y2;
m is 1-4;
n is 1-50;
X is -OC(CH2)pCO-; and
P is 1-22;
or a salt, derivative or analogue thereof.
20. A method of preventing and/or treating a disorder associated with HMGBl comprising administering a modulator according to any one of claims 13 to 15, or a compound as defined in any one of claims 17 to 19, or a pharmaceutical according to claim 16, wherein said modulator or pharmaceutical is capable of modulating HMGBl to cause a beneficial and/or therapeutic effect.
21. A method according to claim 20 for the treatment of a condition associated with activation of the inflammatory cytokine cascade.
22. A method according to claim 20 for downregulating an immune specific response.
23. A method according to claim 20 for the treatment of an inflammatory or autoimmune disease, allergy or transplant rejection.
24. A method according to claim 23 for the treatment of the first stages of inflammation relating to a necrosis, such as an intestinal infarction, acute pancreatitis or trauma.
25. A method according to claim 20 for inhibiting stem cell migration and/or proliferation.
26. A method according to claim 20 for inhibiting epithelial cell migration and/or proliferation.
27. A method according to claim 20 for the treatment of vascular diseases.
28. A method according to claim 27 wherein the vascular disease relates to diagnostic and/or surgical techniques involving the insertion of catheters into vessels.
29. A method according to claim 28 wherein the diagnostic and/or surgical technique is angioplasty and/or angiography.
30. A method according to claim 27 where the vascular diseases comprise atherosclerosis and/or restenosis that occur during angioplasty and/or angiography.
31. A method according to any one of claims 27 to 30 for the treatment of blocking, retarding or reducing connective tissue regeneration.
32. A method according to any one of claims 27 to 31 wherein said modulators are released by catheters, surgical instruments or stents for angioplasty and/or angiography.
33. A method according to any one of claims 20 to 32 wherein the modulator is glycyrrhizic acid or a salt or mimetic thereof.
34. A method according to any one of claims 20 to 33 wherein the HMGBl is acetylated.
35. A method for predicting, simulating or modelling the molecular characteristics and/or molecular interactions of an HMGBl modulator binding domain comprising the use of a computer model, said computer model comprising using or depicting the structure substantially as presented by the space filling balls in Figure 4 or
Figure 11 and/or the structural coordinates of a modulator binding domain as provided by at least some of the following amino acid residues of the N-terminal helix 1 of HMGBl: 11, 12, 13, 17, 20, 23, 25, 26 and 27; and/or at least some of the following amino acid residues of helix 2 of HMGBl: 42, 43, 44 and 45, to provide an image of said HMGBl modulator binding domain, and optionally to display said image.
36. A method according to claim 35 wherein said method further comprises the use of a computer model to provide an image of a modulator, and optionally to display said image.
37. A method according to claim 36 wherein said method further comprises providing an image of said modulator in association with said HMGBl modulator binding domain, and optionally displaying said image.
38. A method according to claim 37 wherein said modulator is prepared and optionally formulated as a pharmaceutical composition.
39. A computer readable medium having stored thereon parameters capable of displaying a representation of a 3D model comprising the HMGBl modulator binding domain.
40. A computer readable medium according to claim 39 wherein said model is built for all or part of the HMGBl modulator binding domain as defined in any one of claims 1, 2, or 5.
41. A computer controlled method for designing a modulator capable of binding to HMGBl comprising: providing a model of the structure of the HMGBl modulator binding domain; analysing said model to design a ligand which binds to the HMGBl modular binding domain; and optionally determining the effect of said modulator on HMGB 1.
42. A machine-readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a representation of an HMGBl modulator binding domain of a homologue thereof.
43. A computer comprising a storage medium according to claim 42.
44. The use of a computer according to claim 43 in an industrial context, such as identifying putative modulators.
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