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WO2013030541A1 - Histone désacétylase - Google Patents

Histone désacétylase Download PDF

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Publication number
WO2013030541A1
WO2013030541A1 PCT/GB2012/052055 GB2012052055W WO2013030541A1 WO 2013030541 A1 WO2013030541 A1 WO 2013030541A1 GB 2012052055 W GB2012052055 W GB 2012052055W WO 2013030541 A1 WO2013030541 A1 WO 2013030541A1
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hdac
seq
protein
class
binding
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Peter James WATSON
John Walter Richard SCHWABE
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University of Leicester
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2878—Muscular dystrophy

Definitions

  • the present invention relates to histone deacetylases, and in particular to the role played by histone deacetylases in regulating gene expression and their impact on a range of diseases characterised by inappropriate histone deacetylation, including developmental diseases, cancer, dementia and muscular dystrophy.
  • the invention provides novel biological targets associated with histone deacetylases, and pharmaceutical compositions, medicaments and methods of treatment, for use in preventing, ameliorating or treating disease characterised by inappropriate histone deacetylation.
  • the acetylation of lysine residues in the tails of histone proteins plays an important role in the regulation of gene expression in eukaryotic cells.
  • HATs histone acetyl transferases
  • HDACs histone deacetylases
  • HDACs catalyse the removal of acetyl groups from lysine residues in the tails of histone proteins, and this results in an increased positive charge on the histone. This increased positive charge strengthens the electrostatic attraction between the positively charged histones and the negatively charged DNA, resulting in chromatin condensation, which renders the DNA less accessible for transcription.
  • Class I HDACs are Zn-dependent enzymes, and include HDACs l, 2, 3 and 8. Of these, only HDAC8 is found as a functional enzyme in isolation.
  • HDACs 1, 2 and 3 require association with large multi-subunit co-repressor complexes for full activity. These co-repressor complexes bring about the repression of gene expression when recruited to repressive transcription factors, but also contribute to the 'resetting' of chromatin after rounds of transcriptional activation.
  • HDACs have become important targets for the treatment of a number of cancers. Cancer cell lines treated with HDAC inhibitors undergo terminal differentiation, growth arrest and/or apoptosis. Several HDAC inhibitors are at various stages in clinical trials and two drugs, Vorinostat and Romidepsin, have been approved for the treatment of cutaneous T-cell lymphomas. In addition, since HDACs are so ubiquitous, HDAC inhibitors are useful in treating many other diseases, including developmental diseases, dementia (Fischer et al., 2007, Nature, 447178-82) and muscular dystrophy (Minetti et al., Nat. Med., 2006, 12, 1147-50). HDACs l and 2 are found in three repression complexes: NuRD, CoREST and Sin3A.
  • HDAC3 appears to be uniquely recruited to the SMRT/NCoR complex where it interacts with a conserved deacetylase-activation-domain (DAD) within SMRT or NCoR.
  • DAD deacetylase-activation-domain
  • the DAD both recruits and activates HDAC3.
  • Recruitment of HDAC3 to the DAD is essential for repression by certain nuclear receptors and for the maintenance of normal circadian physiology. It has been proposed that the assembly of the HDAC3 and SMRT- DAD requires a chaperone complex, since they do not form a complex when these proteins are expressed in bacteria.
  • the DAD contains an extended SANT-like domain with an amino-terminal DAD-specific motif. Deletion of this motif results in both loss of binding and failure to activate HDAC3.
  • the structure of the isolated DAD from SMRT has been reported, and this revealed that part of the DAD-specific motif forms an extra helix that is folded against the three helices of the SANT domain to form a four-helix bundle.
  • the amino-terminal portion of the DAD- specific motif is unstructured in solution.
  • HDACs are emerging as useful drug targets, especially for treating cancer, dementia and muscular dystrophy.
  • acetylation of lysines in the histones is known to play an important role in the regulation of gene expression in eukaryotic cells, it is believed that the medicaments can be used for treating cancer, as well as dementia and muscular dystrophy.
  • HDACi class I HDACs
  • HDAC8 class I HDACs
  • DAD deacetylase-activation-domain
  • the structure of the complex can be used as a means to design molecules (or agents), which are capable of blocking the formation of the
  • HDAC:corepressor complex and thereby inhibit histone deacetylation, and prepare pharmaceutical compositions comprising such agents for use in the effective treatment of cancer, dementia and muscular dystrophy.
  • a cancer treatment, dementia treatment or muscular dystrophy treatment composition comprising a therapeutically effective amount of an agent capable of:-
  • HDAC histone deacetylase
  • HDAC histone deacetylase
  • cancer treatment composition can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of cancer in a subject.
  • distalia treatment composition can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of dementia in a subject.
  • muscle dystrophy treatment composition can mean a pharmaceutical
  • Example 2 the structure elucidated by the inventors, as described in Example l, reveals two remarkable features. Firstly, as described in Example 2, the SMRT-DAD protein undergoes a large structural rearrangement on forming the complex with HDAC3.
  • Example 3 there is an essential inositol tetraphosphate molecule, Ins(i,4,5,6)P 4 (or “IP4"), acting as an 'intermolecular glue' between the two proteins, HDAC3 and DAD.
  • IP4 inositol tetraphosphate molecule
  • HDAC3 This mechanism for the activation of HDAC3 appears to be conserved in other class I HDACs, including HDACi and 2, from yeast to man, and opens up significant novel therapeutic opportunities. Since HDACs are so ubiquitous, the composition and agents described herein are useful in treating many diseases, including cancer, developmental diseases, dementia and muscular dystrophy.
  • composition of the first aspect for use in therapy.
  • composition of the first aspect for use in the treatment, prevention or amelioration of cancer, dementia treatment or muscular dystrophy.
  • a process for making the composition according to the first aspect comprising contacting a therapeutically effective amount of an agent capable of:-
  • HDAC histone deacetylase
  • an agent capable of:-
  • HDAC histone deacetylase
  • inositol phosphate molecule inhibiting binding or interaction between an inositol phosphate molecule and either: (i) a class I histone deacetylase (HDAC), or (ii) its corresponding co- repressor protein; or (d) inhibiting synthesis of inositol tetraphosphate or its release from intracellular stores and/or increasing degradation of inositol tetraphosphate,
  • HDAC histone deacetylase
  • HDAC histone deacetylase
  • HDAC histone deacetylase
  • a seventh aspect there is provided a method of treating, preventing or ameliorating cancer, dementia or muscular dystrophy in a subject, the method comprising
  • HDAC histone deacetylase
  • HDAC histone deacetylase
  • a method of inhibiting histone deacetylation in a subject comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of:-
  • HDAC histone deacetylase
  • HDAC histone deacetylase
  • compositions and agents of the invention may be used for treating cancer.
  • the agent may be capable of inhibiting binding or interaction between any of the class I HDACs, for example HDACi, HDAC2 or HDAC3, or a functional fragment or variant thereof, and its corresponding co-repressor protein.
  • HDACi, 2 and 3 are available on freely accessible databases.
  • the Accession Numbers for HDACi, 2 and 3 are: HDACi protein NP_004955.2, DNA
  • HDACi, 2 and 3 are known to bind to a range of different corepressors in order to form the corresponding functional enzyme complex.
  • HDACs 1 and 2 are found in three repression complexes: NuRD, CoREST and Sin3A, whereas HDAC3 appears to be uniquely recruited to the SMRT/NCoR complex where it interacts with a conserved deacetylase-activation-domain (DAD) within SMRT or NCoR.
  • DAD deacetylase-activation-domain
  • the co-repressor protein may be selected from the group of co-repressors including: SMRT; NC0R1; NuRD; Sin3A;
  • DNA and protein sequences of each of these corepressor proteins are available on freely accessible databases.
  • the Accession Numbers are: SMRT protein NP_oo6303 , DNA NM_oo63i2.s; NCoRi protein NP_oo6302.2, DNA
  • NP_o65795.i DNA NM_020744.2; MIERi protein NP_o65999.2, NM_020948.3; RERE protein NP_ooi036i46.i, NM_ooi04268i.i; Sntip protein NP_009902.2, DNA
  • the co-repressor protein with which HDAC3 may form a complex may be SMRT, and preferably a DAD domain thereof.
  • HDACi and HDAC2 may be found together in the Sin3A, NuRD and CoREST complexes.
  • Figure 6A and 6B shows two sequence alignments demonstrating the clear conservation of certain key residues, which suggest that class I HDACs, from yeast to man, require inositol tetraphosphates for their assembly and activation.
  • Figure 6A shows an alignment of class I HDACs from H. sapiens and S. cerevisiae. In the following sequences, SEQ ID No: 1-6, key amino acid residues that mediate interaction with the Ins(i,4,5,6)P 4 are underlined, and key residues that mediate interaction with SMRT- DAD are in bold.
  • HSHDAC3 The amino acid sequence of a region of the human HDAC3 (i.e. HSHDAC3) is provided herein as SEQ ID No:i, as follows:
  • SEQ ID No:i The amino acid sequence of a region of human HDACi (i.e. HsHDACi) is provided herein as SEQ ID No:2, as follows:
  • HsHDAC2 human HDAC2
  • SEQ ID No:4 The amino acid sequence of a region of a class I S. cerevisiae HDAC (i.e. ScRPD3p) is provided herein as SEQ ID No:4, as follows:
  • HsHDAC8 The amino acid sequence of a region of human HDAC8 (i.e. HsHDAC8) is provided herein as SEQ ID No:5, as follows: QSLVPVYIYSPEYVSMCDSLAK ⁇ IPKRASMVHSLIEAYALHKQMRrVKPK-X(2oo)- TIAGDPMCSFN-X( 22 )-GGGGYNLANTARCW-X(i6)-DHEFFTAYGPD
  • the agent may be capable of inhibiting binding or interaction between any one of SEQ ID No: 1-5 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, and its corresponding co-repressor protein.
  • HDAC histone deacetylase
  • amino acid consensus sequence of SEQ ID No's: 1-5 is provided herein as SEQ ID No:6, as follows:
  • Figure 6B is a sequence alignment of the SANT domains from known co-reperssor protein for the various class I HDACs.
  • SEQ ID No: 7-16 key amino acid residues that mediate interaction with the Ins(i,4,5,6)P 4 are underlined, and key residues that mediate interaction with HDAC3 are in bold.
  • SANT domain of human SMRT i.e. HsSMRT
  • SEQ ID No: 7 amino acid sequence of the SANT domain of human SMRT (i.e. HsSMRT) is provided herein as SEQ ID No: 7, as follows:
  • amino acid sequence of the SANT domain of human NC0R1 i.e. HsNCoRi
  • SEQ ID No: 8 The amino acid sequence of the SANT domain of human NC0R1 (i.e. HsNCoRi) is provided herein as SEQ ID No: 8, as follows: VWTDHEKEIFKDKFIQHPKNFGLIAS-YLERKSVPDCVLYYYLTKKNENYK
  • SEQ ID No: 8 The amino acid sequence of the SANT domain of human CoRESTi (i.e. HsCoRESTi) is provided herein as SEQ ID No:9, as follows:
  • amino acid sequence of the SANT domain of human C0REST2 i.e. HsCoREST2
  • SEQ ID No: 10 amino acid sequence of human C0REST2 (i.e. HsCoREST2) is provided herein as SEQ ID No: 10, as follows:
  • amino acid sequence of the SANT domain of human C0REST3 i.e. HSC0REST3
  • SEQ ID No: 11 amino acid sequence of human C0REST3 (i.e. HSC0REST3) is provided herein as SEQ ID No: 11, as follows:
  • amino acid sequence of the SANT domain of human MTAi i.e. HsMTAi
  • SEQ ID No: 12 amino acid sequence of human MTAi (i.e. HsMTAi)
  • amino acid sequence of the SANT domain of human MTA2 i.e. HsMTA2
  • SEQ ID No: 13 The amino acid sequence of the SANT domain of human MTA2 (i.e. HsMTA2) is provided herein as SEQ ID No: 13, as follows: EWSASEAMLFEEALEKYGKDFNDIRQDFLPWKSIASIVQFYYMWKTTDRYI
  • SEQ ID No:i3 The amino acid sequence of the SANT domain of human MTA3 (i.e. HSMTA3) is provided herein as SEQ ID No: 14, as follows:
  • SANT domain of yeast Sntip i.e. ScSntip
  • SEQ ID No: 15 amino acid sequence of yeast Sntip (i.e. ScSntip) is provided herein as SEQ ID No: 15, as follows:
  • the agent may be capable of inhibiting binding or interaction between any one of SEQ ID No:7-i5 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, and its corresponding co-repressor protein.
  • HDAC histone deacetylase
  • amino acid consensus sequence of SEQ ID No's: 6-15 is provided herein as SEQ ID No: 16, as follows:
  • the agent may be adapted to inhibit binding or interaction between certain conserved amino acid residues in the HDAC and its corresponding co-repressor protein.
  • the agent may be capable of inhibiting interaction or binding between an HDAC co-repressor protein and one or more amino acid residues in the HDAC selected from the group of residues consisting of: Asms; Hisi7; Tyrl7; Gly2l; Lys25; His27; Arg26s; Arg30l; Tyr328 and Tyr 331 of SEQ ID No:6.
  • the numbering used herein when referring to SEQ ID No: 6 is that of the amino acid sequence of HDAC3, i.e. H17 in HDAC3 is Y24 in HDACi, and so on.
  • the agent may be capable of inhibiting interaction or binding between an HDAC co-repressor protein and one or more amino acid residues in the HDAC selected from the group of residues consisting of: Asms; His27; Tyr328 and Tyr 331 of SEQ ID No:6.
  • the agent may be adapted to inhibit binding or interaction between the HDAC and certain conserved amino acid residues in its corresponding co-repressor protein.
  • the agent may therefore be capable of inhibiting interaction or binding between an HDAC and one or more amino acid residues in the co- repressor protein selected from the group of residues consisting of: Lys449; Phe45i;
  • the agent may be capable of inhibiting interaction or binding between an HDAC and one or more amino acid residues in the co-repressor protein selected from the group of residues consisting of: Phe45i; Val403; Leu403; Ile403; Val407; Leu407; Ile407 of SEQ ID No:i6.
  • the agent may also be adapted to inhibit binding or interaction between an inositol phosphate molecule and either: (i) a class I histone deacetylase (HDAC), or (ii) its corresponding co-repressor protein. Therefore, the agent may be capable of inhibiting interaction or binding between an inositol phosphate molecule and one or more amino acid residues in the HDAC selected from the group of residues consisting of: Hisi7; Tyri7; Gly2i; Lys25; Arg26s and Arg30i of SEQ ID No:6.
  • HDAC histone deacetylase
  • the agent may be capable of inhibiting interaction or binding between an inositol phosphate molecule and one or more amino acid residues in the corepressor selected from the group of residues consisting of: Lys449; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No:l6.
  • the inositol phosphate molecule may comprise inositol diphosphate, inositol triphosphate, inositol tetraphosphate, inositol pentaphosphate or inositol heptaphosphate.
  • the inositol phosphate molecule comprises inositol tetraphosphate.
  • the agent may be capable of inhibiting synthesis of inositol tetraphosphate or its release from intracellular stores and/or increasing degradation of inositol tetraphosphate.
  • Figure 9 shows the metabolic pathway of the synthesis of Ins(i,4,5,6)P4.
  • the agent may be capable of repressing any of the enzymes that are involved in the synthesis of inositol tetraphosphate, for example inositol polyphosphate multikinase (IPMK) and/or phosphatase and tensin homologue (PTEN).
  • IPMK inositol polyphosphate multikinase
  • PTEN phosphatase and tensin homologue
  • IPMK protein NP_6894i6.i DNA NM_152230.4
  • PTEN protein ⁇ _000305 ⁇ 3, DNA NM_000314 The skilled person would readily appreciate how the activity of such an enzyme may be repressed based on their sequences.
  • a gene silencing technique could be used, such as RNAi, siRNA and/or shRNA molecules having sequences which would prevent expression of the enzyme.
  • sequences could be determined based on the sequences of the target enzyme.
  • PTEN and IPMK inhibitors are known (see Zu et al., 2011, Eur. Journal Pharm. 650, 298-302; Mayr et al., 2005, J. Biol.
  • inhibitors may include Ellagic Acid, Gossypol, ECG, EGCG, ATA or Hypericin (see page 13232 of J. Biol. Chem., 280, 14, 13229-13240), bpV(phen), 3-PT-PIP3.
  • HDAC histone deacetylase
  • HDAC co-repressor protein comprises a mutation at one or more amino acid residues selected from the group of residues consisting of: Asms; Hisi7; Tyri7; Gly2i; Lys25; His27; Arg26s; Arg30i; Tyr328 and Tyr 331 of SEQ ID No: 6, and wherein the HDAC co-repressor protein comprises a mutation at one or more amino acid residues selected from the group of residues consisting of: Lys449; Phe45i; Val403; Leu403; Ile403; Val407; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No:i6.
  • a method for diagnosing a subject suffering from a disease characterized by inappropriate histone deacetylation, or a predisposition thereto, or for providing a prognosis of the subject's condition comprising screening, in a bodily sample obtained from a test subject, for the presence of a mutation in a class I histone deacetylase (HDAC) and/or an HDAC co-repressor protein, wherein the HDAC comprises a mutation at one or more amino acid residues selected from the group of residues consisting of: Asms; Hisi7; Tyri7; Gly2i; Lys25; His27; Arg26s; Arg30i; Tyr328 and Tyr 331 of SEQ ID No:6, and wherein the HDAC co-repressor protein comprises a mutation at one or more amino acid residues selected from the group of residues consisting of: Lys449; Phe45i; Val403; Leu403; Ile463; Val407; Leu40
  • deacetylation or has a predisposition thereto, or provides a negative prognosis of the subject's condition.
  • inappropriate histone deacetylation in an unhealthy test subject involves abnormal deacetylation when compared to that in a healthy individual.
  • inappropriate histone deacetylation may involve increased or decreased levels compared to the corresponding level in a healthy subject.
  • the disease characterized by inappropriate histone deacetylation may be cancer, a developmental disease, dementia or muscular dystrophy.
  • the sample may be any bodily sample, for example a biopsy, or a bodily fluid, such as lymph, interstitial fluid, urine or blood.
  • the skilled person would readily appreciate how a suitable agent could be prepared, which would be capable of locating itself inside the binding pocket that is formed between the HDAC and its corepressor, thereby blocking the location of the inositol tetraphosphate molecule, and achieving the desired level of inhibition.
  • the agent is preferably capable of binding specifically to HDAC, its corepressor and/or the inositol phosphate molecule in order to prevent the formation of the functional complex.
  • the agent may comprise a competitive polypeptide or a peptide-like molecule, or a derivative or analogue thereof; an antibody or a fragment or derivative thereof; an aptamer (nucleic acid or peptide); a peptide-binding partner; or a small molecule that binds specifically to the HDAC, its corepressor and/or the inositol phosphate molecule to prevent formation of the complex.
  • the agent may comprise a molecule which mimics the structure of inositol phosphate (preferably inositol tetraphosphate), such that it can position at least a portion of itself inside the binding pocket, while still preventing inositol tetraphosphate from binding or interacting with HDAC and/or its corepressor, thereby preventing formation of the functional complex.
  • the agent may comprise a small molecule having a molecule weight of less than loooDa.
  • derivative or analogue thereof can mean a polypeptide within which amino acids residues are replaced by residues (whether natural amino acids, non-natural amino acids or amino acid mimics) with similar side chains or peptide backbone properties.
  • N- and C- terminal protecting groups for example groups with similar properties to acetyl or amide groups. It will be appreciated that the amino acid sequenced may be varied, truncated or modified once the final polypeptide is formed or during the development of the peptide.
  • short peptides may be use to inhibit interaction or binding between HDAC, its corepressor and/or inositol tetraphosphate, to prevent the complex forming.
  • These peptides may be isolated from libraries of peptides by identifying which members of the library are able to bind to the peptide of SEQ ID No: l- 16. Suitable libraries may be generated using phage display techniques (e.g. as disclosed in Smith & Petrenko (1997) Chem Rev 97 P391-410).
  • inhibitory peptides, peptide mimics or small molecules will exploit the inventor's knowledge of the interaction between HDAC, its coreperssor and inositol tetraphosphate, and be based upon the sequences, as described herein, that have been identified as being important to that interaction.
  • the agent may bind tightly to the HDAC protein, preferably those amino acids shown to be important in its binding with either its corepressor and/or inositol tetraphosphate.
  • Example 9 the inventors synthesised and tested the ability of certain peptides to inhibit the HDAC3-SMRT-DAD complex formation, and thus inhibit HDAC activity.
  • Two test peptides were prepared, which were based on residues 463-475 of SMRT, and these were referred to as a native peptide and a stapled peptide.
  • the stapled peptide contains an intermolecular "staple" between successive turns of the a-helix which induces a-helical conformation in the peptide.
  • Native peptide Native peptide:
  • Val4 6 3-Ala-Glu-Cys-Val-Leu-Tyr-Tyr-Tyr-Leu-Thr-Lys-Lys475-NH 2
  • a suitable agent according to the invention may comprise a peptide which comprises an amino acid sequence substantially as set out in either SEQ ID No: 17 or 18, or a functional fragment or variant thereof.
  • the peptide may comprise an intermolecular "staple" between successive turns of the a-helix which induces a-helical conformation in the peptide.
  • antibodies, and fragments and derivatives thereof represent preferred agents for use according to the invention.
  • Antibodies according to the invention may be produced as polyclonal sera by injecting antigen into animals.
  • Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen (e.g. a fragment of the HDAC, its corepressor and/or the inositol tetraphosphate molecule using techniques known to the art.
  • Polyclonal antibodies for use in treating human subjects, may be raised against any of SEQ ID No:i-i6, or a fragment of variant thereof.
  • SEQ ID No.6 and/or SEQ ID No: 16 may be used as an antigen.
  • Example 8 provides details as to how antibodies specific for SEQ ID No.6 can be generated.
  • the antibody may be monoclonal. Conventional hybridoma techniques may be used to raise the antibodies.
  • the antigen used to generate monoclonal antibodies according to the present invention may be the same as would be used to generate polyclonal sera.
  • the antigen comprises, or is the peptide of SEQ ID No:i-i6, preferably SEQ ID No:6 or 16, or a fragment or variant thereof.
  • Preferred antibodies, and functional derivatives thereof, according to the invention may comprise the variable regions (i.e. complementarity determining regions), which exhibit immunospecificity for HDAC, its corepressor and/or inositol tetraphosphate, preferably the binding pocket formed therebetween.
  • a derivative of the antibody may comprise at least 75% sequence identity, more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity. It will be appreciated that most sequence variation may occur in the framework regions (FRs) whereas the sequence of the CDRs of the antibodies, and functional derivatives thereof, according to the first aspect of the invention should be most conserved.
  • the antibody may be humanised, by splicing V region sequences (e.g.
  • Aptamers represent another preferred agent for use according to the invention. Aptamers are nucleic acid or peptide molecules that assume a specific, sequence-dependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand.
  • aptamers may comprise either single- or double-stranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA).
  • Peptide aptamers consist of a short variable peptide domain, attached at both ends to a protein scaffold. Aptamers may be used to bind both nucleic acid and non-nucleic acid targets.
  • the binding pocket is formed when HDAC3/DAD bind to each other and in the presence of the IP4 (as the IP4 bridges the charge's). Thus, blocking the formation of the pocket is preferred.
  • the aptamer may recognise the "half-binding pocket" on either the HDAC molecule or its corepressor. Accordingly aptamers may be generated that recognise the binding pocket formed between the HDAC, its corepressor and/or inositol
  • Suitable aptamers may be selected from random sequence pools, from which specific aptamers may be identified which bind to the selected target molecules (e.g. a peptide of SEQ ID No. 1-16) with high affinity.
  • Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.
  • Preferred methodologies for producing aptamers include those disclosed in WO 2004/042083.
  • a ninth aspect there is provided a method for identifying an agent that modulates the interaction of a class I histone deacetylase (HDAC), its corresponding co-repressor protein and/or an inositol phosphate molecule, the method comprising the steps of :-
  • HDAC histone deacetylase
  • a first protein comprising a conserved motif represented by SEQ ID No:6 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, with a second protein comprising the corresponding co-repressor protein; or
  • HDAC histone deacetylase
  • a method for identifying an agent that modulates histone deacetylation comprising the steps of :-
  • a first protein comprising a conserved motif represented by SEQ ID No:6 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, with a second protein comprising the corresponding co-repressor protein; or
  • HDAC histone deacetylase
  • test agent contacting, in the presence of a test agent, either: - (a) a first protein comprising a conserved motif represented by SEQ ID No:6 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, with a second protein comprising the corresponding co-repressor protein; or
  • HDAC histone deacetylase
  • the HDAC orepressor interaction is used as an example as to how an agent may be developed, though it will be appreciated that similar methods may be used to develop agents that are capable of inhibiting any of the other interactions (i.e. the interaction between the HDAC and inositol tetraphosphate, or the interaction between the corepressor and inositol tetraphosphate) described herein.
  • a decrease in binding of the first protein to the second protein in the presence of the test agent as compared to a negative control may be an indicator that the test agent reduces interaction of a class I histone deacetylase (HDAC), its corresponding co-repressor protein and/or an inositol phosphate molecule, or reduces histone deacetylation.
  • HDAC histone deacetylase
  • an increase in binding of the first protein to the second protein in the presence of the test agent as compared to a negative control may be an indicator that the test agent increases interaction of a class I histone deacetylase (HDAC), its corresponding co-repressor protein and/ or an inositol phosphate molecule, or increases histone deacetylation.
  • Any of the methods described herein may be carried out ex vivo.
  • the contacting may be in a substantially cell-free system.
  • Any of the method may comprise screening an agent that shows a positive indication for the same activity in a cell-based system and/or in vivo in a non-human mammal.
  • the HDAC orepressor recognition sequence can be used as the basis for screens aimed at identifying small molecules that specifically disrupt HDAC orepressor interaction, e.g. by targeting this region of HDAC. Accordingly, in certain embodiments, screening systems are contemplated that screen for the ability of test agents to bind the specific residues of HDAC and its corepressor. Methods of screening for agents that bind HDAC:corepressor:inositol tetraphosphate are readily available to the skilled technician (Colas 2008).
  • the HDAC or its corepressor is immobilized and probed with test agents. Detection of the test agent (e.g., via a label attached to the test agent) indicates that the agent binds to the target moiety and is a good candidate modulator of HDAC: corepressor interaction.
  • the association of HDAC and corepressor and inositol tetraphosphate in the presence of one or more test agents is assayed.
  • FRET fluorescence resonance energy transfer system
  • HDAC donor fluorophore on one moiety
  • acceptor fluorophore on the corepressor molecule or inositol phosphate molecule The donor and acceptor quench each other when brought into proximity by the interaction of HDAC and corepressor.
  • the FRET signal decreases indicating that the test agent inhibits interaction of HDAC and its corepressor.
  • cells, tissues, and/or animals are provided that are transfected with a construct which encodes a mutant form of the HDAC or the corepressor.
  • cells, tissues, and/or animals in which HDAC or its corepressor is "knocked out" are provided. It will be appreciated that one or both of these constructs may be used in screens for suitable agents of the invention for inhibiting any of the HDAC orepressor interactions.
  • agents according to the invention may be used in a medicament which may be used in a monotherapy (i.e. use of only an agent which inhibits binding between HDAC and its corepressor), for treating, ameliorating, or preventing cancer, dementia or muscular dystrophy.
  • agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing cancer, dementia or muscular dystrophy.
  • the agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.
  • Medicaments comprising agents according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.
  • Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
  • Agents according to the invention may also be incorporated within a slow- or delayed- release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site, e.g. a tumour. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • agents and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment.
  • the medicament may be injected at least adjacent a tumour. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
  • the amount of the agent that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the cancer, dementia or muscular dystrophy. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • a daily dose of between o.o ⁇ g/kg of body weight and soomg/kg of body weight of the agent according to the invention may be used for treating, ameliorating, or preventing cancer, dementia or muscular dystrophy, depending upon which agent is used. More preferably, the daily dose is between o.oimg/kg of body weight and 400mg/kg of body weight, more preferably between o.img/kg and 200mg/kg body weight, and most preferably between approximately lmg/kg and loomg/kg body weight.
  • the agent may be administered before, during or after onset of cancer, dementia or muscular dystrophy.
  • Daily doses may be given as a single administration (e.g. a single daily injection).
  • the agent may require administration twice or more times during a day.
  • agents may be administered as two (or more depending upon the severity of the cancer being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4- hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.
  • formulations comprising the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).
  • a "subject” may be a vertebrate, mammal, or domestic animal. Hence, agents,
  • compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.
  • a “therapeutically effective amount” of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the cancer, dementia or muscular dystrophy, or produce the desired effect, such as inhibiting histone deacetylation, and increasing gene expression.
  • the therapeutically effective amount of agent used may be from about o.oi mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is preferred that the amount of agent is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent e.g.
  • the peptide or antibody may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo- regulators.
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the agents used according to the invention can also be administered orally either in liquid or solid composition form.
  • Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • the terms "substantially the amino acid/nucleotide/peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No: 1-6 (i.e. HDAC) or its encoding nucleotide, or 40% identity with the polypeptide identified as SEQ ID No:7-i6 (i.e. the HDAC corepressor protein) or its encoding nucleotide, and so on.
  • amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences.
  • an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith- Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to any sequences referred to herein or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/ sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/o.i% SDS at approximately 20-65°C.
  • a substantially similar polypeptide may differ by at least l, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-18.
  • nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.
  • Figure lA is an SDS-PAGE gel
  • Figure lB is a gel filtration chromatogram showing purification of HDAC3:SMRT-DAD complex
  • Figure lC is an image of a HDAC3:SMRT-DAD crystal mounted in a loop at the Diamond Synchrotron beamline I24 (Box represents 25.9 * 25.9 ⁇ );
  • Figure 2 relates to carboxy-terminal truncated HDAC3 remains associated with SMRT- DAD and is catalytically active.
  • Figure 2A is an SDS-PAGE gel showing the HDAC3:SMRT- DAD complex fresh and after 3 months in crystallisation trials.
  • Figure 2B shows the results of HDAC activity assays showing HDAC activity of the DADA alone, HDAC3:SMRT-DAD complex and the truncated complex;
  • Figure 3 relates to the overall structure of the HDAC3/DAD complex and structural rearrangement of the SMRT DAD.
  • Figure 3A shows electron density (2F0-FC) contoured at 1 ⁇ around the hydrophobic core of the DAD (green sticks) and the interface with HDAC3 (grey sticks).
  • Figure 3B shows the interaction of the SMRT-DAD (green ribbon) with the HDAC3 (grey surface). Side chains in the DAD that mediate interaction with the HDAC3 are shown in stick form.
  • Figure 3C shows the structure of the DAD domain in solution compared with that bound to HDAC3 (helices are individually coloured to facilitate comparison);
  • Figure 4 relates to comparison of loop L5 in class I HDAC structures.
  • Figure 4A shows an overlay of HDAC3 (grey), HDAC2 (blue) 34 and HDAC8 (green) 32 highlighting the tyrosine insertion in loop L5 of HDAC3. Residues and loops are labelled with respect to HDAC3.
  • Figure 4B shows a structural alignment between HDACs 3, 2 and 8, loop L5 is indicated by a box and the tyrosine is highlighted in grey;
  • Figure 5 relates to D-myo-inositol-i,4,5,6-tetrakisphosphate binding to the
  • HDAC3:SMRT-DAD complex HDAC3:SMRT-DAD complex.
  • Figure 5A shows a striking feature in the difference electron density map (Fo-Fc at 3 o) observed following molecular replacement.
  • Figure 5B shows electron density corresponding to the Ins(i,4,5,6)P 4 ligand following refinement (2F0-FC at 2.25 o).
  • Figure 5C shows electrostatic surface representation of the HDAC3: SMRT-DAD complex.
  • a strikingly basic pocket is located at the HDAC3:SMRT-DAD interface. The interface is indicated by a dashed green line.
  • the active site pocket of HDAC3 is indicated by a yellow cross.
  • Figure 5D shows Ins(i,4,5,6)P 4 binding in the basic pocket at the
  • HDAC3 SMRT-DAD interface.
  • Figure 5E shows detailed interactions of Ins(i,4,5,6)P 4 with HDAC3 (blue) and SMRT DAD (grey);
  • Figure 6 relates to conservation of key residues suggesting that class I HDACs, from yeast to man, require inositol phosphates for assembly and activation.
  • Figure 6A are alignments of class I HDACs from H. sapiens and S. cerevisiae. Key residues that mediate interaction with the Ins(i,4,5,6)P 4 and SMRT-DAD are highlighted in blue and red respectively. Other conserved residues are highlighted grey.
  • Figure 6B is a sequence alignment of SANT domains from known interaction partners for class I HDACs. Key residues that mediate interaction with the Ins(i,4,5,6)P 4 and HDAC3 are highlighted in blue and red respectively. Other conserved residues are highlighted grey. Residues highlighted by a green arrow impair HDAC3 recruitment and activation when mutated to alanine;
  • Figure 7 relates to a mechanism of activation of HDAC3 by binding SMRT-DAD and Ins(i,4,5,6)P 4 .
  • Figure 7A shows the SMRT-DAD (grey cartoon) and the Ins(i,4,5,6)P 4 bind adjacent to the HDAC3 (charged surface representation) active site. Acetate and a methionine (lysine mimic) are located in the active site.
  • Figure 7B shows details of the HDAC3 active site. A bound acetate (cyan) and crystal packing methionine (salmon) mimic the reaction products. Zinc (grey sphere) ligands are shown in yellow. Tyr298 and Hisi34 (magenta) form hydrogen bonds with the product acetate. Leu266, Phei44, Phe200 and Hisi35 form the walls of the active site tunnel. Binding of SMRT-DAD (grey) and
  • FIG. 7C shows pseudo helix Hi and loops Li and L6 are shown in blue on the surface of HDAC3. These regions are influenced / stabilised by SMRT-DAD and Ins(i,4,5,6)P 4 binding.
  • Figure 7D shows a comparison of the structures of HDAC3 and HDAC8. Regions of significant difference are coloured blue (HDAC3) and red (HDAC8);
  • Figure 8 relates to insights into mechanism of activation of HDAC3 by SMRT-DAD from comparison with HDACs 2 and 8.
  • this step may go through an
  • Figure 10 shows HDAC activity inhibition using a Native Peptide: Showing the mean and standard error of the mean of three replicates for each peptide concentration. HDAC activity is expressed as a percentage HDAC activity with no peptide;
  • Figure 11 shows HDAC activity inhibition using a Stapled Peptide: Showing the mean and standard error of the mean of three replicates for each peptide concentration. HDAC activity is expressed as a percentage HDAC activity with no peptide. Examples
  • the DAD domain (SMRT 389 - 480) and full length HDAC3 were cloned into pcDNA3 vector (Invitrogen).
  • the DAD domain construct contained a N-terminal ioxHis-3xFLAG tag and a TEV protease cleavage site.
  • HEK293F cells (Invitrogen) were co-transfected with both constructs using 25 kDa branched Polyethylenimine (PEI) (Sigma).
  • the lysate was pre-cleared using Sepharose 4B (Sigma) and the complex was then bound to FLAG resin (Sigma), washed three times with buffer A, three times with buffer B (50 mM Tris pH 7.5, 300 mM potassium acetate, 5 % v/v glycerol) and three times with buffer C (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.5 mM TCEP).
  • the complex was eluted from the resin by overnight cleavage at 4°C with TEV protease in buffer C. The eluted protein was further purified by gel filtration on a
  • Crystals were flash frozen in mother liquor containing 40% glycerol as a cryoprotectant. Diffraction data were collected on a single crystal in two 45 0 wedges at the Diamond synchrotron microfocus beamline I24 and processed using XDS (Kabsch, W. XDS. Acta Crystallogr D Biol Crystallogr 66, 125-132 (2010). The structure was solved by molecular replacement using HDAC8 ((PDB code 3EW8) Dowling et al. Biochemistry 47, 13554- 13563 (2008)), as a search model in Phaser (McCoy, A. J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674 (2007).
  • the final model contains amino acids 2-370 chain A and 2-370 chain B of HDAC3, amino acids 408-476 chain C and 408- 475 chain D of the DAD.
  • the model also contains two inositol-1,4,5,6 tetraphosphate molecules, two zinc ions, four potassium ions, 2 acetate molecules, and 4 glycerol molecules.
  • the final model has 97.8% residues in the favoured region, 2.0% in the allowed region and 0.2% in the outlier region of the Ramachandran plot.
  • FLAG tagged HDAC3 and myc- tagged DAD were co-expressed in HEK 293 cells as described above.
  • Cells were lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 5 % v/v glycerol, 0.3 % v/v Triton X-100, Roche complete protease inhibitor.
  • the assay 800 ⁇ g total protein was bound to 20 ⁇ FLAG resin (Sigma) for 2 hrs at 4°C, then washed 4 times with lysis buffer.
  • HDAC activity was measured using the HDAC Assay Kit (Active Motif) and read on a Victor X5 plate reader (Perkin Elmer). Results
  • HDAC3 and SMRT-DAD do not form a complex when expressed in bacterial cells
  • full-length HDAC3 and FLAG-tagged SMRT-DAD (aa: 389-480) were expressed in suspension grown mammalian HEK293 cells.
  • the complex remained tightly associated during a three-step purification including size exclusion chromatography (see Figure 1).
  • the carboxy-terminal tail was proteolysed (see Figure 2).
  • Size exclusion chromatography confirmed that the tail is not required for complex stability (data not shown).
  • the truncated HDAC3-SMRT-DAD complex retains deacetylase activity (see Figure 2).
  • HDAC3 structure is similar to the previously determined class I HDAC structures of HDAC8 and HDAC2 (Somoza et al., Structure 12, 1325-1334 (2004), Bressi et al., Bioorg Med Chem Lett 20, 3142-3145 (2010)), and consists of an eight-stranded parallel beta-sheet surrounded by a number of alpha-helices.
  • the active site lies at the base of a tunnel leading from the surface of the protein.
  • a solvent-exposed tyrosine residue is located on the surface of the enzyme immediately adjacent to the active site tunnel. This tyrosine is unique to HDAC3 and it seems likely that this residue will interact with substrate and hence contribute to substrate specificity (see Figure 4).
  • the NMR structure of the isolated SMRT-DAD domain in solution shows that the four helices are folded together to form a single domain (Codina et al., Proc Natl Acad Sci USA 102, 6009-6014 (2005)).
  • the amino terminal helix of the DAD undergoes a major structural rearrangement such that it no longer forms part of the core structure, but lies along the surface of HDAC3 making extensive intermolecular interactions (see Figure 3B & 3C).
  • this DAD-specific motif buries a surface of i,i78A 2 .
  • the remaining three helix bundle resembles a canonical SANT domain and buries a further i,i6oA 2 at the interface with HDAC3.
  • This SANT domain interacts with HDAC3 in a region that is well-conserved between HDACsi-3 but rather divergent in HDAC8.
  • This region (residues 10-30 in HDAC3) is well-ordered but lacks a defined secondary structure, whereas the equivalent region in HDAC8 adopts a well-defined alpha-helix Hi.
  • the inventors subsequently refer to this region as pseudo- helix Hi in HDAC3.
  • the electron density difference map revealed a well- ordered small molecule bound at the interface between HDAC3 and the DAD (see Figure 5A).
  • the electron density was sufficiently well-defined that the small molecule could be readily identified as inositol tetraphosphate.
  • it could be unambiguously assigned as D-myo-inositol-i,4,5,6-tetrakisphosphate (based on the axial orientation of the hydroxyl group on carbon 2) and is hereafter termed Ins(i,4,5,6)P 4 (see Figure 5B).
  • the Ins(i,4,5,6)P 4 molecule is sandwiched between HDAC3 and the DAD making extensive contacts to both proteins. It sits in a highly basic pocket formed at the interface of the two molecules (see Figure 5C and 5D). Five side-chains from the DAD make key hydrogen bonds and salt bridges to the Ins(i,4,5,6)P 4 (Lys449, Tyr470, Tyr47i, Lys474 & Lys 475). HDAC3 contributes a further five residues (Hisi7, Gly2i, Lys25, Arg26s & Arg30i) (see Figure 5E).
  • MTA(i-3) and CoREST(i-3) contain SANT domains that are very similar to the SMRT-DAD, and the key Ins(i,4,5,6)P 4 binding residues are almost entirely conserved (see Figure 6B). This strongly suggests that these complexes also rely on an inositol phosphate to provide an 'intermolecular glue'.
  • HDAC3 has little or no activity by itself, it has been shown that its activity is greatly increased when in complex with the SMRT or NCoR corepressor proteins.
  • HDAC3: SMRT-DAD structure the inventors sought to understand how the DAD and/or the Ins(i,4,5,6)P 4 results in activation of the enzyme.
  • the active site of the HDAC3 resembles the product complex (see Figure 7 A and 7B).
  • An acetate molecule (present during purification) is bound at the active site, making hydrogen bonds to the catalytic zinc and side chains of Ty298 and Hisi34.
  • a methionine sidechain is bound in the active site tunnel mimicking a lysine residue.
  • the binding surfaces for the DAD and the Ins(i,4,5,6)P 4 are located to one side of the HDAC3 active site (see Figure 7A and 7B).
  • the inventors propose that changes in both conformation and dynamics occur when the DAD and Ins(i,4,5,6)P 4 bind to HDAC3 and that these facilitate substrate access to the active site resulting in enhanced enzyme activity.
  • pseudo helix Hi along with loops Li and L6 participate in the interface between HDAC3 and the DAD/Ins(i,4,5,6)P 4 . These regions are coloured blue on the
  • HDAC3 surface in Figure 7C It appears that the SMRT-SANT domain interacts with, and stabilises, pseudo helix Hi and loop Li. This region of protein contributes to one side of the active site tunnel. There is a key interaction between the Ins(i,4,5,6)P 4 and Arg26s in loop L6 (coloured orange in Figure 7B). This loop seems to be very important for access to the active site since Leu266 forms one wall of the active site tunnel and in the absence of the Ins(i,4,5,6)P 4 this loop is likely to be relatively mobile.
  • HDAC8 differs significantly in the region where HDAC3 interacts with the SMRT-DAD and Ins(i,4,5,6)P 4 .
  • the pseudo helix Hi has a regular stable helical structure, loop Li is two amino acids shorter and loop L6 contains a proline residue that partly orientates the loop away from the active site (see Figure 4D).
  • the inventors suggest that together these differences give the substrate better access to that active site of HDAC8 that would be possible in the uncomplexed HDAC3.
  • the pattern of crystallographic temperature factors for the various structures support this interpretation (see Figure 8).
  • Arg82 is a yeast protein that acts as a transcriptional regulator coordinating the expression of genes involved in arginine metabolism. It is required for the repression of arginine anabolic genes and the induction of catabolic genes. Arg82 is an inositol phosphate kinase that converts Ins(i,4,5)P 3 to Ins(i,4,5,6)P 4 and this activity is required for at least part of its role in transcriptional regulation (Science 287, 2026-2029 (2000)).
  • the kinase activity of Arg82 is required for chromatin remodelling activities in the cell and controls promoter accessibility of the PI105 gene. Arg82 mutations lead to changes in expression, both up- and down-regulation, of many genes in yeast consistent with a perturbation in the transcriptional machinery. Ins(i,4,5,6)P 4 is able to modulate the activity of ATP-dependent chromatin remodelling complexes and consequently stimulate nucleosome mobilisation. In mammals, the homologue of Arg82 is known as IPMK. However, in contrast to Arg82, IPMK has been reported to phosphorylate Ins(i,4,5)P 3 to form Ins(i,3,4,s)P 4 and not Ins(i,4,5,6)P 4 .
  • Ins(i,4,5,6)P 4 is most likely formed through phosphatase action converting Ins(i,3,4,5,6)P 5 to Ins(i,4,5,6)P 4 , as shown in Figure 9.
  • Two enzymes have been reported to possess such activity in mammalian cells. One of these is MINPPi, but this enzyme is restricted to the lumen of the endoplasmic reticulum and may therefore not be relevant in the nucleus.
  • the other enzyme, the well-known phosphatase and tumour suppressor gene PTEN is known to be active in the nucleus and to play a role in chromosome stability.
  • the inventors postulate that loss of HDAC complex function might be one of the routes through which PTEN mutations contribute to oncogenesis.
  • Mouse monoclonal antibodies are usually produced by the hybridoma method. They possess monovalent affinity and so bind to the same target (epitope) and as such are the best choice to produce an antibody specific to SEQ ID No:6. Described below are the steps required to produce and purify a monoclonal antibody against SEQ ID No:6:
  • mice were injected with recombinant purified HDAC protein with a suitable adjuvant (such as freund's complete adjuvant or incomplete freund's adjuvant).
  • adjuvants enhance the immune response thus increasing antibody production. Immunisations and test-bleeds would typically be carried out over a 5-week period. Test bleeds would be assayed for anti-HDAC antibodies by ELISA or western blotting, and antibody titers determined.
  • Step 2 Mice selection and hybridoma production
  • mice were chosen and given additional booster immunizations before their spleen cells were harvested for hybridoma production.
  • lymphoid cells isolated from the spleen were fused with myeloma cells.
  • Hybridomas were selected for by the use of selective medium, such as HAT
  • Step 3 Screening for positive supernatants
  • Antibodies were produced by either continued in vitro cell culture or by in vivo propagation as ascitic tumours.
  • the ascitic tumours were produced by injecting antibodies into the peritoneal cavity of a mouse, and a tumour formed that secreted antibody rich ascitic fluid.
  • Antibodies were purified from the cell culture medium or ascitic fluid by numerous methods. These included ion exchange chromatography, protein A/G affinity chromatography, and affinity chromatography.
  • Epitope mapping is where the binding sites (epitope) of an antibody on their target (antigen) are indentified.
  • epitope mapping There are various methods for epitope mapping, and these include: X-ray co-crystallography, Site- directed mutagenesis, Mutagenesis mapping, Hydrogen/Deuterium exchange mass spectrometry and docking.
  • the aim of this experiment was to test the ability of peptides based on residues 463-475 of SMRT to inhibit the HDAC3-SMRT-DAD complex and thus inhibit HDAC activity.
  • Two peptides were generated, a native peptide and a stapled peptide.
  • the stapled peptide contains an intermolecular "staple" between successive turns of the a-helix which induces a-helical conformation in the peptide.
  • Val4 6 3-Ala-Glu-Cys-Val-Leu-Tyr-Tyr-Tyr-Leu-Thr-Lys-Lys475-NH 2
  • the resin was first swollen in DCM in a plastic filtration tube with polyethylene frit, as described for Fmoc removal above, treated with a freshly prepared 20% piperidine / DMF solution (7 mL), shaken for 15 min, filtered, treated with a second portion of 20%
  • Peptide (1) [SEQ ID No 117] was prepared as described above to give the desired
  • A is- X- C y ⁇ ⁇ Vsi- Leu -X- T yr- Lys- ⁇ H -
  • Peptide (2) [SEQ ID No:i8] was prepared as described above and in reference 1 to give the desired hydrocarbon stapled peptide TFA salt (231 mg) of 22% crude purity as analyzed by analytical RP-HPLC (5-100% B, 30 min gradient). Purification was then carried out by preparatory RP-HPLC (5-100% B, 30 min gradient) to give the desired hydrocarbon stapled peptide TFA salt (231 mg) of 22% crude purity as analyzed by analytical RP-HPLC (5-100% B, 30 min gradient). Purification was then carried out by preparatory RP-HPLC (5-100% B, 30 min gradient) to give the desired
  • HDAC3-SMRT-DAD complex was expressed and purified as described in (Watson et. al. 2012, Nature, Jan 9;48 ⁇ (738 ⁇ ):335-4 ⁇ ).
  • 0.5 mM HDAC3-SMRT-DAD complex was incubated for Ran at room temperature with or without peptide as required, in buffer A (50 mM Tris pH 7.5, 50 mM potassium acetate), in a black 96 well plate (Corning).
  • HDAC activity was then measured using a fluorescence based HDAC activity assay. 100 mM final concentration BOC acetyl-lysine was added to each well and the plate was then incubated in the dark at 37°C for 120 min.
  • Quenching of the deacetylase activity and trypsin cleavage of the substrate was then performed by adding 50 ml of 2 mM Trichostatin A in 10 mg/ml trypsin solution (in 50 mM Tris pH 7.5, 100 mM NaCl). Fluorescence was measured after incubation at room temperature for 10 min, with an excitation wavelength of 360 nM and an emission wavelength of 470 nM on a Victor X5 plate reader (Perkinelmer).
  • the inventors have determined the first structure of a histone deacetylase in complex with its activating corepressor protein.
  • the SMRT-DAD corepressor undergoes a structural rearrangement such that the amino terminal region wraps over the surface of the deacetylase.
  • a highly unexpected small molecule, Ins(i,4,5,6)P 4 bridges the interface between the carboxy terminal SANT domain of the SMRT-DAD and HDAC3. This Ins(i,4,5,6)P 4 molecule acts as an 'intermolecular glue' contributing to the stabilisation, and hence activation, of the HDAC enzyme.
  • HDAC1/2 corepressor complexes will also require inositol phosphates at the intermolecular interface. Specificity for the particular HDAC is likely to depend not on the SANT domain, but on the region amino- terminal to the SANT domain, i.e. the DAD-specific motif in SMRT and ELM2 motifs in the MTA and CoREST proteins.
  • HDAC inhibitors which target the active site of the enzymes.
  • the inventors have demonstrated that it will be useful to develop molecules that target the Ins(i,4,5,6)P 4 binding site itself, as well as those which target the enzymes responsible for Ins(i,4,5,6)P 4 synthesis, i.e. enzymes which produce and remove the compound, as shown in Figure 9. Since HDACs are so ubiquitous, HDAC inhibitors, including the agents and compositions described herein, will be useful in treating many diseases, including developmental diseases, dementia, cancer and muscular dystrophy.
  • Example 9 the inventors have synthesised and tested two inhibitory peptides, SEQ ID No: 17 & 18, which show a dose-dependent decrease in deacetylase activity, and hence inhibition of the complex.
  • these two peptides represent useful agents according to the invention, and can be used in therapeutic compositions, uses and methods described herein.

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Abstract

L'invention concerne de nouvelles cibles biologiques associées aux histone désacétylases, et des inhibiteurs et des compositions pharmaceutiques, des médicaments et des procédés de traitement, destinés à une utilisation dans la prévention, l'amélioration ou le traitement de maladies, caractérisées par une désacétylation de l'histone inadéquate, incluant le cancer, la démence ou la dystrophie musculaire.
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