WO2013030541A1

WO2013030541A1 - Histone deacetylase

Info

Publication number: WO2013030541A1
Application number: PCT/GB2012/052055
Authority: WO
Inventors: Peter James WATSON; John Walter Richard SCHWABE
Original assignee: University Of Leicester
Priority date: 2011-08-26
Filing date: 2012-08-22
Publication date: 2013-03-07
Also published as: GB201114806D0; GB2493982A

Abstract

The invention provides novel biological targets associated with histone deacetylases, and inhibitors and pharmaceutical compositions, medicaments and methods of treatment, for use in preventing, ameliorating or treating disease characterised by inappropriate histone deacetylation, including cancer, dementia or muscular dystrophy.

Description

HISTONE DEACETYLASE

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. The level of lysine acetylation is controlled through the opposing actions of histone acetyl transferases (HATs) and histone deacetylases (HDACs). Although chromatin is the best understood substrate for these enzymes, lysine acetylation is emerging as a general regulatory mechanism in a diverse array of cellular processes.

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. There are four classes of HDACs in mammalian cells. 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, on the other hand, 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.

In recent years, 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. In contrast, 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. 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.

As discussed above, HDACs are emerging as useful drug targets, especially for treating cancer, dementia and muscular dystrophy. Thus, it is an aim of embodiments of the present invention to gain a better understanding of the sites and mechanisms of interaction between HDAC3 and DAD within either SMRT or NCoR with a view to developing novel medicaments and therapies, which would result in a tighter control of the deacetylation of lysine residues in the tails of histone proteins. Since 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.

The inventors focused their attention on the class I HDACs (i.e. HDACi, 2 and 3), and in particular on HDAC3. The enzymatic activity of most class I HDACs (except HDAC8) requires recruitment to co-repressor complexes. As described in the Examples, the inventors are the first to report the detailed structure of an HDAC orepressor complex, i.e. HDAC3 with the deacetylase-activation-domain (DAD) from the SMRT co-repressor. This novel structure not only reveals the specificity and mechanism through which SMRT- DAD recruits and activates HDAC3, but also identifies a highly surprising key component of the complex that has the potential to regulate assembly of HDACs with their

corepressors. Accordingly, 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.

Thus, in a first aspect of the invention, there is provided a cancer treatment, dementia treatment or muscular dystrophy treatment composition comprising a therapeutically effective amount of an agent capable of:-

(a) inhibiting binding or interaction between a conserved motif represented by SEQ ID No:6 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, and its corresponding co-repressor protein;

(b) inhibiting binding or interaction between a class I HDAC and a conserved motif represented by SEQ ID No:i6 of its corresponding co-repressor protein, or a functional fragment or variant thereof;

(c) 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,

and optionally a pharmaceutically acceptable vehicle. The term "cancer treatment composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of cancer in a subject. The term "dementia treatment composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of dementia in a subject. Finally, the term "muscular dystrophy treatment composition" can mean a pharmaceutical

formulation used in the therapeutic amelioration, prevention or treatment of muscular dystrophy in a subject.

Surprisingly, 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.

Secondly, as discussed in Example 3, there is an essential inositol tetraphosphate molecule, Ins(i,4,5,6)P₄ (or "IP4"), acting as an 'intermolecular glue' between the two proteins, HDAC3 and DAD. The inventors believe that assembly of the complex formed between HDAC3 and DAD is dependent on the presence of the Ins(i,4,5,6)P₄. Although the inventors do not wish to be bound by theory, they postulate that Ins(i,4,5,6)P₄ can act as a regulator, and that this could explain why inositol phosphates and their kinases have been found to act as transcriptional regulators. 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.

In a second aspect, there is provided a composition of the first aspect, for use in therapy.

In a third aspect, there is provided the composition of the first aspect, for use in the treatment, prevention or amelioration of cancer, dementia treatment or muscular dystrophy.

In a fourth aspect, there is provided a process for making the composition according to the first aspect, the process comprising contacting a therapeutically effective amount of an agent capable of:-

(c) 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

with a pharmaceutically acceptable vehicle.

In a fifth aspect, there is provided an agent capable of:-

(c) 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,

for use in the treatment, prevention or amelioration of cancer, dementia or muscular dystrophy.

In a sixth aspect, there is provided use of an agent capable of:-

(e) inhibiting synthesis of inositol tetraphosphate or its release from intracellular stores and/or increasing degradation of inositol tetraphosphate,

for inhibiting histone deacetylation.

In a seventh aspect, there is provided a method of treating, preventing or ameliorating cancer, dementia or muscular dystrophy in a subject, the method comprising

administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of:- (a) inhibiting binding or interaction between a conserved motif represented by SEQ ID

No:6 of a class I histone deacetylase (HDAC), or a functional fragment or variant thereof, and its corresponding co-repressor protein;

(d) inhibiting synthesis of inositol tetraphosphate or its release from intracellular stores and/ or increasing degradation of inositol tetraphosphate,

to treat, prevent or ameliorate of cancer, dementia or muscular dystrophy in the subject. In an eighth aspect, there is provided a method of inhibiting histone deacetylation in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of:-

to inhibit histone deacetylation in the subject.

Preferably, the 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. The DNA and protein sequences of HDACi, 2 and 3 are available on freely accessible databases. The Accession Numbers for HDACi, 2 and 3 (DNA and amino acid) are: HDACi protein NP_004955.2, DNA

NM_004904.2; HDAC2 protein - NP_ooi5i8.3, DNA - ΝΜ_οοΐ527·3; and HDAC3 protein - NP_003874.2, DNA - ΝΜ_0038883·3·

It will be appreciated that HDACi, 2 and 3 are known to bind to a range of different corepressors in order to form the corresponding functional enzyme complex. For example, 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. The DAD both recruits and activates HDAC3. Therefore, the co-repressor protein may be selected from the group of co-repressors including: SMRT; NC0R1; NuRD; Sin3A;

C0REST1-3; MTA1-3; Sntip; MIER; and RERE.

The DNA and protein sequences of each of these corepressor proteins are available on freely accessible databases. The Accession Numbers (DNA and amino acid) are: SMRT protein NP_oo6303 , DNA NM_oo63i2.s; NCoRi protein NP_oo6302.2, DNA

NM_oo63ii.3; Sin3a protein NP_ooii38830.i, DNA NM_ooii45358.i; CoRESTi protein NP_05597i.i, DNA NM_oi5i56.2; C0REST2 protein NP_7758s8.2, ΝΜ_173587·3;

C0REST3 protein NP_ooii29695.i, NM_ooii36223.i; MTAi protein NP_oo468o.2, DNA ΝΜ_004689·3; MTA2 protein NP_004730.2, DNA NM_004739-3; MTA3 protein

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

NM_ooii78747.i. Thus, 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)P₄ are underlined, and key residues that mediate interaction with SMRT- DAD are in bold.

The amino acid sequence of a region of the human HDAC3 (i.e. HSHDAC3) is provided herein as SEQ ID No:i, as follows:

MAKWAYFYDPDVGNFHYGAGHPMKPHRIALTHSLVLHYGLYKKMIVFKPY-X(2oi)- SLGCDRLGCFN-X(₂₂)-GGGGYTVRNVARCW-X(_l6)-YSEYFEYFAPD

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:

TRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPH-X(2oo)- SLSGDRLGCFN-X(₂₂)-GGGGYTIRNVARCW-X(i6)-YNDYFEYFGPD

SEQ ID No: 2

The amino acid sequence of a region of human HDAC2 (i.e. HsHDAC2) is provided herein as SEQ ID No:3, as follows:

GKKKVCYYYDGDIGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEr RPH-X(2oo)- SLSGDRLGCFN-X(₂₂)-GGGGYTIRNVARCW-X(i6)-YNDYFEYFGPD SEQ ID No:3

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:

DKRRVAYFYDADVGNYAYGAGHPMKPHRIRMAHSLIMNYGLYKKMEIYRAK-X(2oo)- SLSGDRLGCFN- x₍₂₂)-GGGGYTMRNVARTW-X(_l6)-YNEYYEYYGPD

SEQ ID No:4

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

SEQ ID No:5

Thus, 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.

The amino acid consensus sequence of SEQ ID No's: 1-5 is provided herein as SEQ ID No:6, as follows:

NXHAYXXXGXXXKXH-X(₂₃₅/₂₃6)-R-X(34)-R-X(55)-YXXY-

SEQ ID No: 6

Figure 6B is a sequence alignment of the SANT domains from known co-reperssor protein for the various class I HDACs. In the following sequences, SEQ ID No: 7-16, key amino acid residues that mediate interaction with the Ins(i,4,5,6)P₄ are underlined, and key residues that mediate interaction with HDAC3 are in bold.

The amino acid sequence of the SANT domain of human SMRT (i.e. HsSMRT) is provided herein as SEQ ID No: 7, as follows:

MWSEQEKETFREKFMQHPKNFGLIAS-FLERKTVAECVLYYYLTKKNENYK

SEQ ID No: 7

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:

EWTVEDKVLFEOAFSFHGKTFHRIOO-MLPDKSIASLVKFYYSWKKTRTKT

SEQ ID No: 9

The amino acid sequence of the SANT domain of human C0REST2 (i.e. HsCoREST2) is provided herein as SEQ ID No: 10, as follows:

EWTVEDKVLFEQAFGFHGKCFQRIQQ-MLPDKLIPSLVKYYYSWKKTRSRT

SEQ ID No:lO

The amino acid sequence of the SANT domain of human C0REST3 (i.e. HSC0REST3) is provided herein as SEQ ID No: 11, as follows:

EWTVEDKVLFEQAFSFHGKSFHRIQQ-MLPDKTIASLVKYYYSWKKTRSRT

SEQ ID No:ii

The amino acid sequence of the SANT domain of human MTAi (i.e. HsMTAi) is provided herein as SEQ ID No: 12, as follows:

EWSASEANLFEEALEKYGKDFTDIQQDFLPWKSLTSIIEYYYMWKTTDRYV

SEQ ID No:l2

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:

EWSASEASLFEEALEKYGKDFNDIRQDFLPWKSLTSIIEYYYMWKTTDRYV

SEQ ID No: 14

The amino acid sequence of the SANT domain of yeast Sntip (i.e. ScSntip) is provided herein as SEQ ID No: 15, as follows:

I DHEHSLFLEGYLIHPKKFGKISHYMGGLRSPEECVLHYYRTKKTVNYK

SEQ ID No: 15

Thus, 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.

The amino acid consensus sequence of SEQ ID No's: 6-15 is provided herein as SEQ ID No: 16, as follows:

KXFXXXXXXXXXXXXhXXXhXXYYXXKK

SEQ ID No:i6 wherein h is Valine, Leucine or Isoleucine.

As discussed above in feature (a), 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. Thus, 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. It should be appreciated that 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.

Preferably, however, 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.

Furthermore, as discussed above in feature (b), 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;

Val463; Leu403; Ile403; Val407; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No:i6.

Preferably, 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.

As discussed above in feature (c), 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.

Furthermore, 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. Preferably, the inositol phosphate molecule comprises inositol tetraphosphate.

As discussed above in feature (d), 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. Thus, 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).

The Accession numbers for these two enzymes are: IPMK protein NP_6894i6.i, DNA NM_152230.4; PTEN protein ΝΡ_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. For example, a gene silencing technique could be used, such as RNAi, siRNA and/or shRNA molecules having sequences which would prevent expression of the enzyme. Such sequences could be determined based on the sequences of the target enzyme. Alternatively, a number of PTEN and IPMK inhibitors are known (see Zu et al., 2011, Eur. Journal Pharm. 650, 298-302; Mayr et al., 2005, J. Biol. Chem., 280, 14, 13229-13240), all of which are incorporated herein by reference, and could also be used for the treatment of cancer, dementia or muscular dystrophy. Such 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. In a further aspect, there is provided use of a class I histone deacetylase (HDAC) and/or an HDAC co-repressor protein as a biomarker for diagnosing a subject suffering from a disease characterised by inappropriate histone deacetylation, or a predisposition thereto, 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; Ile403; Val407; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No:i6. In another aspect, there is provided 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, the method 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; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No: 16, wherein presence of a mutation suggests that the subject is suffering from a disease characterized by inappropriate histone

deacetylation, or has a predisposition thereto, or provides a negative prognosis of the subject's condition. The skilled person will appreciate that inappropriate histone deacetylation in an unhealthy test subject involves abnormal deacetylation when compared to that in a healthy individual. Thus, 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.

Based on the detailed structure of the HDAC:corepressor complexes shown in the Figures and described in the Examples, 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.

A number of different agents may be used according to the invention. For example, 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.

The term "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.

Additionally, either one or both terminals of such peptides may be protected by 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.

According to another embodiment of the invention, 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).

Design of peptide inhibitors, based on the sequence of the natural protein partners has been successfully used previously. In the case of BCL6, peptides based on the BCOR

protein bind BCL6 and blocks SMRT from interacting at the same site and in doing so blocks BCL6-mediated transcriptional repression and kills lymphoma cells. Likewise, the design of a synthetic, cell-permeable, stabilised peptide that targets the protein-protein interface in the NOTCH transactivation complex has been successfully used in leukaemic cells in culture. Modifications/optimisation of the peptide sequence could be made to increase the affinity so that it is tighter than the wild-type, which is naturally a relatively weak and short-lived interaction.

It will be appreciated that such 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.

As described in Example 9, and with reference to Figures 10 and 11, 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:

Val4⁶3-Ala-Glu-Cys-Val-Leu-Tyr-Tyr-Tyr-Leu-Thr-Lys-Lys475-NH₂

[SEQ ID No: 17] Stapled peptide:

Val⁴⁶³-Ala-X-Cys-Val-Leu-X-Tyr-Tyr-Leu-Thr-Lys-Lys⁴⁷⁵-NH₂

[SEQ ID No: 18]

The results of these inhibition studies are shown in Figures 10 and 11. Surprisingly, both the native and stapled peptide exhibited a dose-dependent decrease in HDAC activity, and hence inhibition of the HDAC3-SMRT-DAD complex. The inventors were surprised to observe that the stapled peptide was able to inhibit the complex to a greater extent (8%) than the native peptide at the highest concentration.

Accordingly, 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.

In another embodiment, 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. Preferably, 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. Preferably 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. from a monoclonal antibody generated in a non-human hybridoma) with a C region (and ideally FRs from the V region) sequences from human antibodies. The resulting 'engineered' antibodies are less immunogenic in humans than the non-human antibodies from which they were derived and so are better suited for clinical use. 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. Typically, 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. Accordingly, 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

tetraphosphate.

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.

In 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 :-

(i) 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

(b) a first protein comprising a class I HDAC with a second protein comprising a conserved motif represented by SEQ ID No: 16 of the corresponding co-repressor protein, or a functional fragment or variant thereof; or

(c) an inositol phosphate molecule and a class I histone deacetylase (HDAC) and/or its corresponding co-repressor protein; and

(ii) detecting binding between the first and second proteins, wherein an alteration in binding as compared to a control is an indicator that the agent modulates the interaction of a class I histone deacetylase (HDAC), its corresponding co-repressor protein and/or an inositol phosphate molecule.

In a tenth aspect, there is provided a method for identifying an agent that modulates histone deacetylation, the method comprising the steps of :-

(i) contacting, in the presence of a test agent, either: -

(ii) detecting binding between the first and second proteins, wherein an alteration in binding as compared to a control is an indicator that the agent modulates histone deacetylation. In an eleventh aspect, there is provided a method for identifying a candidate agent, for use in the treatment, prevention or amelioration of cancer, dementia or muscular dystrophy, the method comprising the steps of :-

(i) 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

(b) a first protein comprising a class I HDAC with a second protein comprising a conserved motif represented by SEQ ID No:i6 of the corresponding co-repressor protein, or a functional fragment or variant thereof; or

(ii) detecting binding between the first and second proteins, wherein an alteration in binding as compared to a control is an indicator that the agent is a candidate for the treatment, prevention of amelioration of cancer, dementia or muscular dystrophy.

In the sections below, 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. Conversely, 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).

For example, in one embodiment, 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. In another embodiment, the association of HDAC and corepressor and inositol tetraphosphate in the presence of one or more test agents is assayed. This can be accomplished using, for example, a fluorescence resonance energy transfer system (FRET) comprising a donor fluorophore on one moiety (e.g., HDAC) and an 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. When association is reduced or prevented by a test agent, the FRET signal decreases indicating that the test agent inhibits interaction of HDAC and its corepressor. These assays are illustrative and not limiting. Using the teaching provided herein, numerous binding and/or HDAC orepressor interaction assays will be available to the skilled technician.

In certain embodiments, cells, tissues, and/or animals are provided that are transfected with a construct which encodes a mutant form of the HDAC or the corepressor. In other embodiments, 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.

It will be appreciated that 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. Alternatively, 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. Thus, for example, 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. It will be appreciated that the 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). In a preferred embodiment, 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. For example, 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).

It will be appreciated that the amount of the agent that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of

administration, the physiochemical properties of the modulator and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. 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. Generally, 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). Alternatively, the agent may require administration twice or more times during a day. As an example, 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. Alternatively, 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.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific

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. For example, 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.

In one embodiment, 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. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, 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. 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. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

However, 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. Suitable examples of 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). For parenteral administration, 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.

The agents and 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.

It will be appreciated that the invention extends to any 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. Preferably, 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. In order 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.

Having made the alignment, there are many different ways of calculating 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.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et ah, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et ah, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments:

ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino

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. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)*ioo.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, 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. By 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. Alternatively, 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. Due to the degeneracy of the genetic code, it is clear that any 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. For example 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.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:- Figure l relates to purification and crystallisation of the HDAC3/DAD complex. Figure lA is an SDS-PAGE gel, Figure lB is a gel filtration chromatogram showing purification of HDAC3:SMRT-DAD complex, and 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 μηι);

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 σ 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. 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)P₄ 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)P₄ binding in the basic pocket at the

HDAC3: SMRT-DAD interface. Figure 5E shows detailed interactions of Ins(i,4,5,6)P₄ 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)P₄ 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)P₄ 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)P₄. Figure 7A shows the SMRT-DAD (grey cartoon) and the Ins(i,4,5,6)P₄ 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

Ins(i,4,5,6)P₄ will reorient/stabilise pseudo helix Hi and loops Li and L6 (orange) leading to enhanced substrate binding. Figure 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)P₄ 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. Figure 8A shows surface representation of various HDACs coloured by crystallographic temperature factors (blue to red = low to high). Note that in the absence of inhibitor the HDAC8 surface around the active site is relatively disordered, yet the active site is accessible. Note also that in comparison to the inhibitor bound HDAC2 the HDAC3 is less mobile (bluer) especially in proximity to the SMRT-DAD and Ins(i,4,5,6)P₄; Figure 9 relates to pathways for the synthesis of Ins(i,4,5,6)P₄. Synthesis pathway for Ins(i,4,5,6)P₄ from PtdIns(3,4,5)P₃. Note that in yeast the Arg82 kinase acts on

Ins(i,4,5)P₃ to generate Ins(i,4,5,6)P₄. In mammals, this step may go through an

Ins(i,3,4,5,6)P₅ intermediate requiring both IPMK and PTEN;

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; and

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

Materials & Methods

Protein expression, purification and crystallisation

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). To transfect 250 ml of cells at lxio⁶ cell/ml final density, 0.25 mg DNA total was diluted in 25 ml of PBS (Sigma), vortexed briefly, 1 ml of 0.5 mg/ml PEI added, vortexed briefly, incubated for 20 min at room temperature then added to the cells. For larger volumes multiple flasks were used. Cells were harvested 48 h post transfection and lysed by sonication in buffer

containing 50 mM Tris pH 7.5, 100 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, Roche complete protease inhibitor (buffer A), the insoluble material was removed by centrifugation. 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

Superdex S200 column (GE healthcare) in buffer containing 25 mM Tris/HCl pH 7.5, 50 mM potassium acetate, 0.5 mM TCEP. The purified complex was concentrated to 7.5 mg/ml for crystallisation trials. Crystals were grown by sitting drop vapour diffusion at 4°C using o.i M HEPES pH 7.5, 0.2 M NaCl, 10 % v/v propan-2-ol. Crystals were cubic in nature, grew to a final dimension of 15 X 15 X 15 fm and belong to the space group C 2 2 2_t. Comparison of a fresh protein sample with protein from within the crystallisation drops (after 3 months) by SDS-PAGE showed the presence of a truncated form of HDAC3 (see Figure 2). Analysis by LC-MS/MS showed that this HDAC3 was truncated at the C-terminus to residue Q376.

Structure determination

Crystals were flash frozen in mother liquor containing 40% glycerol as a cryoprotectant. Diffraction data were collected on a single crystal in two 45⁰ 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). Initial model building was performed with ARP/wARP (http: / /www.embl-hamburg.de/ARP/), which was able to automatically build 95% of the HDAC3 protein chain, and two helices from the DAD. The additional HDAC3 and DAD sequences were fitted following multiple rounds of refinement and building using REFMAC and Coot (Collaborative Computational Project, Number 4, Acta Crystallogr D Biol Crystallogr 50, 760-763 (1994); Emsley et al., Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010)). Fo-Fc density consistent with the ligand, zinc/potassium ions and acetate/glycerol molecules observed during the refinement/ model building process was fitted and refined as they became apparent. 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. HDAC Activity assays

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. In order to standardise the assay 800 μg total protein was bound to 20 μΐ 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

Example l - Overall architecture of the complex

Since 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). During crystallisation, 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). Furthermore, the truncated HDAC3-SMRT-DAD complex retains deacetylase activity (see Figure 2). Small crystals (maximum dimension 15 μηι - see Figure 1) diffracted to 2A and the structure of the complex was solved by molecular replacement using the structure of HDAC8 (Dowling et al., Biochemistry 47, 13554-13563 (2008)). Model building and refinement yielded an excellent map to 2.1A resolution with clear density for both the HDAC3 and the SMRT-DAD (see Figure 3A).

Overall, the 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).

Example 2 - Structural rearrangement of the SMRT-DAD

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)). On forming a complex with HDAC3, 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). Along with a further extended region, this DAD-specific motif buries a surface of i,i78A². The remaining three helix bundle resembles a canonical SANT domain and buries a further i,i6oA² 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.

It has previously been suggested that the assembly of the SMRT-DAD with HDAC3 is dependent upon the TRiC chaperone complex since recombinant proteins expressed in bacteria will not interact until they have been incubated with rabbit reticulocyte lysate. After this incubation, a complex between SMRT-DAD and HDAC3 is formed and the TRiC chaperone complex remains partially associated. The apparent requirement for the chaperone could be explained by the need for the DAD to undergo a significant structural rearrangement to form a functional complex with HDAC3. Table 1 provides a summary of the crystal structure elucidated. Table 1 - Data collection and refinement statistics

Crystal 1

Data collection

Space group C 2 2 21

Cell dimensions

a, b, c (A) 86.5, 118.6, 190.71

a, b, c (A) 90, 90, 90

Resolution (A) 28.93-2.06 (2.17-

2.06)

-^sym Or -/Emerge 9-8 (32.8)

I/al 7.9 (2.7)

Completeness (%) 85.9 (68.9)

Redundancy 2.8 (2.1)

Refinement

Resolution (A) 95-35-2.06

No. reflections 49237

No. atoms

Protein 7095

Ligand/ion 94

Water 353

B-factors

Protein 26.57

Inositol 1,4,5,6 23.86

tetraphosphate

Zn ions, K ions and 23-76

acetate

Glycerols 33-40

Water 32-43

R.m.s deviations

Bond lengths (A) 0.010

Bond angles (°) 1.26 *Highest resolution shell is shown in parenthesis. Example 3 - An essential role for Insii.4. i.6^')P |

At the earliest stages of refinement, 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. During further refinement, 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)P₄ (see Figure 5B).

The Ins(i,4,5,6)P₄ 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)P₄ (Lys449, Tyr470, Tyr47i, Lys474 & Lys 475). HDAC3 contributes a further five residues (Hisi7, Gly2i, Lys25, Arg26s & Arg30i) (see Figure 5E).

It is surprising that the Ins(i,4,5,6)P₄ is sufficiently tightly bound to the complex that it is retained through the entire purification process. The inventors were also very surprised that the binding is highly specific for Ins(i,4,5,6)P₄ since the electron density shows that the ligand is uniquely Ins(i,4,5,6)P₄ rather than a mixture of inositol phosphates.

Example 4 - Mutually inter-dependent assembly

A careful examination of the structure of the complex suggests that Ins(i,4,5,6)P₄ binding is a requirement for the interaction between SMRT and HDAC3 and that it appears to act as a "molecular glue" that can cement the complex together. If the Ins(i,4,5,6)P₄ ligand were not present, then the many basic residues on either side of the binding cleft would likely prevent interaction through charge repulsion. Indeed, at the base of the cleft, the ζ atoms of SMRT Lys449 SMRT, and HDAC3 Lys25 are just 4.4A from each other.

Consequently, the assembly of the 3-way SMRT:HDAC3:Ins(i,4,5,6)P₄ complex appears to be mutually interdependent, such that both the SMRT-DAD and the Ins(i,4,5,6)P₄ are required for activation of the HDAC3 enzymatic functionality. These conclusions are supported by a mutagenesis study of the SMRT-DAD in which the inventors looked at the effect of mutations on both the interaction with, and the deacetylase activity of, HDAC3. Of the nineteen mutations tested, all of those that failed to activate HDAC3 also abolished or significantly impaired interaction (Codina et al., Proc Natl Acad Sci USA 102, 6009-6014 (2005)).

Importantly, mutations of three of the SMRT- DAD residues that mediate interaction with the Ins(i,4,5,6)P₄, i.e. Lys449, Tyr470 and Tyr47i, resulted in a total failure to activate the HDAC3. The requirement for Ins(i,4,5,6)P₄ for complex formation also provides a potential alternative explanation for the inability of recombinant proteins expressed in bacteria to interact, since bacteria probably do not contain sufficient Ins(i,4,5,6)P₄ to support complex formation.

Example , - Inositol phosphates and other HDACs

Having discovered that Ins(i,4,5,6)P₄ plays a key role in HDAC3 activation, the inventors asked whether inositol phosphates might contribute to the assembly and activation of other HDAC complexes. Examination of the amino acid sequences of other class I HDACs (1, 2 and 8) shows that the key residues that mediate interaction with the Ins(i,4,5,6)P₄ and the SANT domain from SMRT are conserved in both HDACi and 2 (which are known to have increased activity when assembled into their respective corepressor complexes), but not in HDAC8 which is intrinsically fully active (see Figure 6A). The inventors then examined the established corepressor partners for HDACi and 2 and asked if they contain a SANT domain capable of contributing to Ins(i,4,5,6)P4 binding. Remarkably, 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)P₄ 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'.

Since SMRT/NCoR, MTA(i-3) and CoREST(i-3) all share a related SANT domain, it seems likely that the specificity for the particular HDAC is conferred by the region amino- terminal to the SANT domain, i.e. the DAD specific motif in SMRT/NCoR and the ELM2 domains in the MTA and CoREST proteins.

Example 6 - Mechanism of activation of HDAC3

Although 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. With the HDAC3: SMRT-DAD structure in hand, the inventors sought to understand how the DAD and/or the Ins(i,4,5,6)P₄ results in activation of the enzyme. In the current structure, 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. Furthermore, a methionine sidechain (from an adjacent DAD in the crystal lattice) is bound in the active site tunnel mimicking a lysine residue.

The binding surfaces for the DAD and the Ins(i,4,5,6)P₄ 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)P₄ bind to HDAC3 and that these facilitate substrate access to the active site resulting in enhanced enzyme activity.

In particular, pseudo helix Hi along with loops Li and L6 participate in the interface between HDAC3 and the DAD/Ins(i,4,5,6)P₄. 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)P₄ 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)P₄ this loop is likely to be relatively mobile.

It is interesting to compare the HDAC3 structure with that of HDAC8, which does not require activation by complex formation. Figure 7D shows that HDAC8 differs significantly in the region where HDAC3 interacts with the SMRT-DAD and Ins(i,4,5,6)P₄. In HDAC8 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).

Example 7 - Does Insd,4,5,6)P₄ regulate HDACs?

The surprising finding that Ins(i,4,5,6)P₄ is essential for the assembly of class 1 HDAC repression complexes raises the question as to whether the Ins(i,4,5,6)P₄ is a signalling molecule with a role in regulating complex assembly, or whether it is simply an essential structural cofactor required for the assembly. At first sight, the fact that the Ins(i,4,5,6)P₄ remained bound to the complex through several purification steps would seem to suggest that the Ins(i,4,5,6)P₄ may be an irreversibly bound structural cofactor. However, in order to retain an intact complex during purification it was necessary to use low ionic strength buffers. In the nuclear environment, at physiological ionic strength, the Ins(i,4,5,6)P₄ is likely to be able to dissociate fairly readily and therefore has the potential to act as a regulator of complex assembly. With reference to Figure 9, 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)P₃ to Ins(i,4,5,6)P₄ 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)P₄ 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)P₃ to form Ins(i,3,4,s)P₄ and not Ins(i,4,5,6)P₄.

Consequently, in mammals, Ins(i,4,5,6)P₄ is most likely formed through phosphatase action converting Ins(i,3,4,5,6)P₅ to Ins(i,4,5,6)P₄, 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. Although not wishing to be bound by theory, the inventors postulate that loss of HDAC complex function might be one of the routes through which PTEN mutations contribute to oncogenesis.

It is important to note that in these various studies, Arg82 and Ins(i,4,5,6)P₄ have been found to have roles in both transcriptional repression and activation. At first sight it would seem that this is partly inconsistent with a requirement for Ins(i,4,5,6)P₄ in the assembly of a corepressor complex. However, genome-wide ChIP assays have shown that HDACs are also associated with active genes. It seems therefore that HDACs are required for both transcriptional repression and activation. It is also possible that Ins(i,4,5,6)P₄ is involved in the assembly of other transcriptional regulatory complexes. Example 8 - Monoclonal Antibody production based on SEP ID No: 6

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:

Step l - Immunisation and test bleeds

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

The best responding mice were chosen and given additional booster immunizations before their spleen cells were harvested for hybridoma production. To produce the hybridoma, lymphoid cells isolated from the spleen were fused with myeloma cells. Hybridomas were selected for by the use of selective medium, such as HAT

(hypoxanthine/ aminopterin/thymine) medium, as only the hybridomas would grow in this media.

Step 3 - Screening for positive supernatants

Supernatants from the cultured hybridomas were screened for secretion of the desired antibody. This was usually by ELISA due to the large numbers involved. Positive cell lines were selected and carried forward into the next stage.

Step 4 - Subcloning

Daughter cell lines were produced from the selected positive cell lines by subcloning, such that each daughter cell line was grown from a single parental cell. This ensured long term stability of the clones and that the cells were monoclonal. Step - Antibody production and purification

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.

Step 6 - Epitope mapping

Once antibodies have been produced and purified, epitope mapping was required to identify those antibodies which were immunospecific to SEQ ID No:6. Epitope mapping is where the binding sites (epitope) of an antibody on their target (antigen) are indentified. 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.

Example Q - HDAC3-SMRT-DAD Complex Peptide Interference

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.

Native peptide:

Val4⁶3-Ala-Glu-Cys-Val-Leu-Tyr-Tyr-Tyr-Leu-Thr-Lys-Lys475-NH₂

[SEQ ID No: 17]

Stapled peptide:

[SEQ ID No: 18]

These two peptides were prepared using solid-phase peptide synthesis methods, as described below and in Young- Woo Kim, Tom N Grossmann, Gregory L Verdine, (2011) Synthesis of all- hydrocarbon stapled a-helical peptides by ring-closing olefin metathesis. Nature Protocols, 6, 761-771.

Solid-Phase Peptide Synthesis General Experimental Procedures

Rink Resin swelling and deprotection

A 12 mL plastic filtration tube with polyethylene frit was charged with Rink MBHA resin (300 mg, 0.21 mmol, 0.7 mmol/g) and DMF (7 mL). The tube was sealed and shaken for 0.5 h. The resin was then filtered and taken up in freshly prepared 20% piperidine in DMF solution (7 mL), shaken for 30 min, filtered, retreated with 20% piperidine/DMF solution (7 mL) and shaken for 30 min. The resin was washed by successive agitations for 1 min and filtered from DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL). A positive Kaiser colour test indicated qualitatively the presence of free amine.

Amino Acid couplings

The resin was first swollen in DMF (7 mL) for 15 min. Meanwhile, a solution of N-

(Fmoc)amino acid (Fmoc-Xaa-OH, 3 equiv.), HBTU (3 equiv.) and DIEA (6 equiv.) in DMF (7 mL) was prepared in a small sample vial, stirred for 10 min and then added to the resin. The reaction mixture was shaken for 1 h at room temperature. The resin was then filtered and respectively washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL). A negative Kaiser test response indicated completion of the reaction. The resin was then dried in vacuo.

Peptide Cleavage

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%

piperidine/DMF solution (7 mL) and shaken for 15 min. The resin was then filtered and washed by shaking for 1 min with DMF (3 x 7 mL), MeOH (3 x 7 mL) and DCM (3 x 7 mL). A positive Kaiser colour test indicated qualitatively the presence of free amine. The peptide was then cleaved from the resin by shaking in TFA/H₂0/TES (7 mL, 95/2.5/2.5, v/v/v) for 2 h. The resin was filtered, washed with TFA (7 mL) and the combined filtrate and washings were concentrated in vacuo. The resulting residue was dissolved in a minimum volume of TFA (~i mL), transferred to a centrifuge tube and precipitated by the addition of ice-cold diethyl ether (40 mL). The peptide was then centrifuged and the diethyl ether was carefully decanted from the tube. The treatment of the precipitated peptide with cold diethyl ether wash was repeated twice. The resulting white solid was dissolved in water (10 mL) and freeze-dried to give a white foam that was purified by preparatory RP-HPLC, using the specified conditions. Native DAD Peptide (l)

Val4 -Ala-Glu-Cys-Val-Leu-Tyr-Tyr-Tyr-Leu-Thr-Lys-Lys475-NH₂

Peptide (1) [SEQ ID No 117] was prepared as described above to give the desired

peptide TFA salt (123 mg) of 50% 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 TFA salt (8 mg, 15%), as a white foam.

The purified product was analyzed by analytical RP-HPLC (UV 260) using a linear gradient of 5-100% B over 30 min (T_R 14.1) and 5-50% B over 30 min (T_R 22.5) and revealed to be of >98% purity. HRMS Calcd. for C8oH₁₂₅N_l80 [M+H]⁺ 1689.9216, found 1689.9167 (Δ = -2.9 ppm)

Hydrocarbon Stapled DAD Peptide (2)

A is- X- C y ϊ^■ 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

TFA salt (5 mg, 9%), as a white foam. The purified product was analyzed by analytical RP-HPLC (UV 260) using a linear gradient of 5-100% B over 30 min (T_R 17.5) and 5- 50% B over 30 min (T_R 28.4) and revealed to be of >98% purity. HRMS Calcd. for

C8oHi₃iNi₈Oi₉ [M+H]⁺ 1647.9838, found 1647.9861 (Δ = 1.4 ppm)

Once prepared the native peptide and the stapled peptide were then tested for their inhibitory activity against the HDAC3-SMRT-DAD complex and thus their ability to inhibit HDAC activity. Methods

The HDAC3-SMRT-DAD complex was expressed and purified as described in (Watson et. al. 2012, Nature, Jan 9;48ι(738ι):335-4θ). 0.5 mM HDAC3-SMRT-DAD complex was incubated for ihr 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).

Results

The results of these inhibition studies are shown in Figures 10 and 11. Both the native and stapled peptide show a dose-dependent decrease in deacetylase activity, and hence inhibition of the complex. The stapled peptide was able to inhibit the complex to a greater extent (8%) than the native peptide at the highest concentration.

Conclusions

The inventors have determined the first structure of a histone deacetylase in complex with its activating corepressor protein. On binding to HDAC3, 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)P₄, bridges the interface between the carboxy terminal SANT domain of the SMRT-DAD and HDAC3. This Ins(i,4,5,6)P₄ molecule acts as an 'intermolecular glue' contributing to the stabilisation, and hence activation, of the HDAC enzyme. The conservation of key residues in the SANT domains of MTA(i-3) and CoREST(i-3) suggest that the 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.

These findings show that Ins(i,4,5,6)P₄ plays a key role in corepressor assembly and activation of class I HDACs, from yeast to man. The finding that Ins(i,4,5,6)P₄ is essential for the corepressor HDAC assembly presents novel opportunities for therapeutic

intervention that could complement 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)P₄ binding site itself, as well as those which target the enzymes responsible for Ins(i,4,5,6)P₄ 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.

Finally, as described in 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. Thus, these two peptides represent useful agents according to the invention, and can be used in therapeutic compositions, uses and methods described herein.

Claims

1. A cancer treatment, dementia treatment or muscular dystrophy treatment composition comprising a therapeutically effective amount of an agent capable of:-

and optionally a pharmaceutically acceptable vehicle.

2. A composition according to claim l, wherein the agent is capable of inhibiting binding or interaction between HDACi, HDAC2 or HDAC3, or a functional fragment or variant thereof, and its corresponding co-repressor protein.

3. A composition according to either claim 1 or 2, wherein the agent is capable of inhibiting binding or interaction between HDAC3, or a functional fragment or variant thereof, and its corresponding co-repressor protein.

4. A composition according to any preceding claim, wherein the co-repressor protein is selected from the group of co-repressors including: SMRT; NC0R1; NuRD; Sin3A;

C0REST1-3; MTA1-3; Sntip; MIER; and RERE.

5. A composition according to any preceding claim, wherein the co-repressor protein with which HDAC3 forms a complex is SMRT, and preferably a DAD domain thereof, optionally, wherein HDACi and HDAC2 are found together in the Sin3A, NuRD and CoREST complexes.

6. A composition according to any preceding claim, wherein the agent is 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.

7. A composition according to any preceding claim, wherein the agent is 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.

8. A composition according to any preceding claim, wherein the agent is 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; Tyri7; Gly2i; Lys25; His27; Arg26s; Arg30i; Tyr328 and Tyr 331 of SEQ ID No:6.

9. A composition according to any preceding claim, wherein the agent is 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.

10. A composition according to any preceding claim, wherein the agent is 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; Val403; Leu403; Ile463; Val407; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No: 16.

11. A composition according to any preceding claim, wherein the agent is 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; Val463; Leu403; Ile463; Val407; Leu407; Ile407 of SEQ ID No:i6.

12. A composition according to any preceding claim, wherein the agent is 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; Arg205 and Arg30i of SEQ ID No:6.

13. A composition according to any preceding claim, wherein the agent is 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:i6.

14. A composition according to any preceding claim, wherein the inositol phosphate molecule comprises inositol diphosphate, inositol triphosphate, inositol tetraphosphate, inositol pentaphosphate or inositol heptaphosphate.

15. A composition according to any preceding claim, wherein the inositol phosphate molecule comprises inositol tetraphosphate.

16. A composition according to any preceding claim, wherein the agent is 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).

17. A composition according to claim 16, wherein the agent comprises an RNAi, siRNA and/or shRNA molecule having a sequence which would prevent expression of the enzyme.

18. A composition according to any preceding claim, wherein the agent comprises an PTEN and IPMK inhibitor selected from the group consisting of Ellagic Acid, Gossypol, ECG, EGCG, ATA, Hypericin, bpV(phen) or 3-PT-PIP3.

19. A composition according to any preceding claim, wherein the agent is capable of binding specifically to HDAC, its corepressor and/or the inositol phosphate molecule in order to prevent the formation of the functional complex.

20. A composition according to any preceding claim, wherein the agent comprises a competitive polypeptide or a peptide-like molecule, or a derivative or analogue thereof; an antibody or a fragment or derivative thereof; an aptamer; 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.

21. A composition according to any preceding claim, wherein the agent comprises 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.

22. A composition according to any preceding claim, wherein the agent comprises 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.

23. A composition according to any one of claims 1-22, for use in therapy.

24. A composition according to any one of claims 1-22, for use in the treatment, prevention or amelioration of cancer, dementia or muscular dystrophy.

25. A process for making the composition according to any one of claims 1-22, the process comprising contacting a therapeutically effective amount of an agent capable of:-

(a) inhibiting binding or interaction between a conserved motif represented by SEQ ID

(b) inhibiting binding or interaction between a class I HDAC and a conserved motif represented by SEQ ID No: 16 of its corresponding co-repressor protein, or a functional fragment or variant thereof;

(c) 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

with a pharmaceutically acceptable vehicle.

26. An agent capable of:-

(d) inhibiting synthesis of inositol tetraphosphate or its release from intracellular

stores and/or increasing degradation of inositol tetraphosphate, for use in the treatment, prevention or amelioration of cancer, dementia or muscular dystrophy.

27. Use of an agent capable of:-

stores and/or increasing degradation of inositol tetraphosphate,

for inhibiting histone deacetylation.

28. 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 :-

(i) contacting, in the presence of a test agent, either: -

29. A method for identifying an agent that modulates histone deacetylation, the method comprising the steps of :- (i) contacting, in the presence of a test agent, either: -

(ii) detecting binding between the first and second proteins, wherein an alteration in binding as compared to a control is an indicator that the agent modulates histone deacetylation.

30. A method for identifying a candidate agent, for use in the treatment, prevention or amelioration of cancer, dementia or muscular dystrophy, the method comprising the steps of :-

(i) contacting, in the presence of a test agent, either: -

31. A method according to any one of claims 28-30, wherein 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 is 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.

32. A method according to any one of claims 28-31, wherein 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 is 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.

33. A method according to any one of claims 28-32, wherein the methods is carried out ex vivo, optionally the contacting is conducted in a substantially cell-free system.

34. Use of a class I histone deacetylase (HDAC) and/or an HDAC co-repressor protein as a biomarker for diagnosing a subject suffering from a disease characterised by inappropriate histone deacetylation, or a predisposition thereto, 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; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No: 16.

35. 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, the method 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; Val463; Leu403; Ile463; Val407; Leu407; Ile467; Tyr470; Tyr47i; Lys474 and Lys475 of SEQ ID No:i6, wherein presence of a mutation suggests that the subject is suffering from a disease characterized by inappropriate histone deacetylation, or has a predisposition thereto, or provides a negative prognosis of the subject's condition.

36. A use according to claim 34, or a method according to claim 35, wherein the disease characterized by inappropriate histone deacetylation is cancer, a developmental disease, dementia or muscular dystrophy.