Compounds And Methods For The Identification And Characterization Of Hdac Interacting Compounds

Cellzome AG January 20, 2012

CEL67592PC FLZ/NOT

Compounds and methods for the identification and characterization of HDAC interacting compounds The present invention relates to methods for the identification and characterization (e.g. selectivity profiling) of HDAC interacting compounds.

The approval of the drug vorinostat (Zolinza, Merck) by the FDA for the treatment of cutaneous T-cell lymphoma in October 2006 significantly increased the interest in developing inhibitors for the class of enzymes known as histone deacetylases (HDACs). The discovery of the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid; SAHA) resulted from efforts to improve first-generation hybrid polar compounds - originally derived from DMSO - that acted as differentiation inducers of transformed cells. Subsequently, it was discovered that SAHA potently inhibits several HDACs, for example HDAC1 , HDAC2, HDAC3 and HDAC6. Thus, SAHA represents a non-selective HDAC inhibitor and is sometimes referred to as a "pan-HDAC" inhibitor. The mechanisms underlying SAHA's anticancer activity are complex and not fully characterized. For example, the SAHA induces the accumulation of acetylated histones with an effect on gene expression. In addition, non-histone proteins are substrates for HDACs and the effects on these proteins could contribute to its anticancer activities (Marks and Breslow, 2007. Nature Biotechnology 25(l):84-90; Grant et al., 2007. Nature Reviews Drug Discovery 6, 21-22).

HDACs catalyse the removal of acetyl groups from lysine residues in histone amino termini, leading to chromatin condensation and changes in gene expression. In humans 18 HDACs have been identified and subdivided into four classes based on their homology to yeast HDACs, their subcellular localization and their enzymatic activities. The class I HDACs (1, 2, 3 and 8) are homologous to the yeast RPD3 protein, can generally be detected in the nucleus and show ubiquitous expression in human cell lines and tissues. Class II HDACs (4, 5, 6, 7, 9 and 10) are similar to the yeast Hda proteins and can shuttle between the nucleus and the cytoplasm. HDAC6 can deacetylate the cytoskeletal protein a- tubulin. The class III HDACs (SIRT1, 2, 3, 4, 5, 6 and 7) are homologues of the yeast protein Sir2 and require NAD+ for their activity. HDAC1 1 is the sole member of class IV HDACs (Bolden et al., 2006. Nat. Rev. Drug Discov. 5(9):769-784). Histone acetylation is a dynamic process controlled by HDACs and histone acetyltransferases (HATs) and the balance between these two processes together with other protein and DNA modifications such as DNA methylation regulates chromatin accessibility and gene expression. These reversible heritable changes in gene function occur without a change in the sequence of nuclear DNA and are therefore referred to as epigenetic gene regulation. HDACs play a key role in developmental processes, normal physiology and disease states. The ubiquitous expression and high homology between class I HDACs suggests functional redundancy among the HDACs in vivo. However, deletion of each member of the class I HDAC family leads to lethality, indicating a unique role of each HDAC in development (Haberland et al., 2009. Nat. Rev. Genet. 10(l):32-42).

Typically HDACs are present within multisubunit protein complexes together with other components that determine HDAC target gene specificity due to interactions with sequence-specific DNA-binding proteins (Cunliffe 2008. Curr. Opin. Genet. Dev. 18(5):404-410; Yang and Seto, 2008. Nat. Rev. Mol. Cell Biol. 9(3):206-218). Class I HDACs are found primarily in four distinct multiprotein complexes known as the Sin3, NuRD, CoREST and NCoR/SMRT complexes.

Mammalians have two Sin3 homologues, Sin3A and Sin3B. The Sin3A complex contains as components HDAC1, HDAC2, RbAp46 (retinoblastoma (RB) associated protein-46) and RbAp48. In addition, the complex contains RBPl (Rb-binding protein-1), SAP180 (which is similar to RBPl), SDS3, BRMS1 (breast cancer metastasis suppressor- 1, similar to SDS3), SAP30,SAP18 and ING1 (or ING2). Because some of the subunits have different isoforms, there are several distinct Sin3Acomplexes. The Sin3B complex shares some similarity to the Sin3Acomplex but might also contain distinct subunits (Yang and Seto, 2008. Nat. Rev. Mol. Cell Biol. 9(3):206-218).

The nucleosome remodeling and deacetylase (NuRD) complex contains a catalytic core of HDAC 1 , HDAC2, RbAp46 and RbAp48. In addition, this complex contains MTA (metastasis-associated) proteins, the nucleosome-remodelling ATPase Mi-2 (dermatomyositis-specific autoantigen), MBD2 (methyl CpG-binding domain-2) and p66. Through MBD2, this complex is recruited for DNA methylation-dependent gene silencing. Three mammalian MTA isoforms (MTA1, -2 and -3) have been identified that contain SANT domains, which are crucial for the integrity and deacetylase activity of NuRD. Mi-2 and p66 have a- and β-isoforms, enabling the formation of distinct complexes.

The CoREST complex contains the corepressor protein CoREST interacting with the transcriptional repressor REST, HDAC1 and HDAC2. This complex is also referred to as neuronal corepressor complex because the REST zinc finger protein to target sites in the promoters of neuronal genes. One subunit encodes the Lys-specific demethylase-1 (LSD1) protein which functions as a demethylase for K4-methylated histone H3. The NCoR/SNRT complex contains the SANT-domain-containing co-repressors SMRT, NCoR and SMRTER and is targeted to DNA by transcription factor such as CSL and nuclear receptors.

Several HDAC inhibitors are in preclinical development and clinical trials for the treatment of a wide variety of diseases including cancer, inflammatory, cardiac, and neurodegenerative diseases (Bolden et al., 2006. Nat. Rev. Drug Discov. 5(9):769-784; Haberland et al., 2009. Nat. Rev. Genet. 10(l):32-42). It is expected that the development of selective HDAC inhibitors targeting only one member of the HDAC family should lead to improved efficacy and drug safety compared to non-selective "pan-HDAC inhibitors" (Kalin et al., 2009. Curr. Opin. Chem. Biol. 13: 1-9; Balasubramanian et al., 2009. Cancer Lett. 280(2):21 1-21).

The majority of in vitro studies of HDAC activity are performed using in vitro biochemical assays. These assays are also used for the identification of HDAC inhibitors (Hauser et al., 2009. Curr. Top. Med. Chem. 9(3):227-234). For example, HDAC activity can be measured using purified recombinant enzyme in solution-based assays with acetylated peptide substrates (Blackwell et al., 2008. Life Sciences 82(21-22): 1050-1058).

Typically, these assays require the availability of purified or recombinant HDACs. However, not all HDACs can be produced will sufficient enzymatic activity to allow for inhibitor screening (Blackwell et al., 2008. Life Sciences 82(21-22): 1050-1058). In addition, some preparations of HDACs expressed in insect cells are contaminated with endogenous insect HDACs making the interpretation of assay results ambiguous. Another, although not in all instances necessary prerequisite for the identification of selective HDAC inhibitors is a method that allows to determine the target selectivity of these molecules. For example, it can be intended to provide molecules that bind to and inhibit a particular drug target but do not interact with a closely related target, inhibition of which could lead to unwanted side effects. Conventionally, large panels of individual enzyme assays are used to assess the inhibitory effect of a compound for HDACs (Khan et al., 2008. Biochem. J. 409(2):581-9; Blackwell et al., 2008. Life Sci. 82(21-22): 1050- 1058). More recently, chemical proteomics methods have been described using acitivity-based probes that irreversibly bind to HDACs (Salisbury and Cravatt, 2007. PNAS 104(4): 1 171- 1 176; Salisbury and Cravatt, 2008. J. Am. Chem. Soc. 130(7):2184-2194).

In view of the above, there is a need for providing effective tools and methods for the identification and selectivity profiling of HDAC interacting compounds.

In a first aspect, the present invention relates to a method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of a) providing an HDAC protein complex containing protein preparation derived from a cell endogenously expressing an HDAC, b) contacting the protein preparation with a given compound and with a ligand having the structure

(formula I) wherein R is H, C1-C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, thereby allowing the reversible binding of said ligand to said HDAC protein complex, and c) determining to what extent in step b) a binding between the ligand and the protein complex has occurred.

In a second aspect, the present invention relates to a method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of a) providing two aliquots of an HDAC protein complex containing protein preparation derived from a cell endogenously expressing an HDAC, b) contacting one aliquot with a ligand having the structure

wherein R is H, C1-C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, thereby allowing the reversible binding of said ligand to said HDAC protein complex, c) contacting the other aliquot with a given compound and with the ligand as defined in step b), thereby allowing the reversible binding of said ligand to said HDAC protein complex, and d) determining to what extent in steps b) and c) a binding between the ligand and the protein complex has occurred.

In a third aspect, the present invention relates to a method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of: a) providing two aliquots of a cell preparation each comprising at least one cell endogenously expressing an HDAC, b) incubating one aliquot with a given compound, c) harvesting the cells of each aliquot, lysing the cells of each aliquot in order to obtain protein preparations containing an HDAC protein complex, contacting the protein preparations with a ligand having the structure

wherein R is H, C1 -C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, thereby allowing the reversible binding of said ligand to said HDAC protein complex, and f) determining to what extent in step e) a binding between the ligand and the

protein complex has occurred.

In a fourth aspect, the present invention relates to a method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of a) providing at least two aliquots of an HDAC containing protein preparation derived from a cell endogenously expressing an HDAC, b) contacting one aliquot with a ligand as defined above thereby allowing the reversible binding of said ligand to HDAC, c) contacting the remaining aliquots with a ligand as defined above and with various concentrations of a compound thereby allowing the reversible binding of said ligand to HDAC, separating the HDAC from the the ligand, and e) determining by mass spectrometry or immunodetection the amount of HDAC eluted from the ligand, thereby determining to what extent in steps b) and c) a binding between the ligand and the protein has occurred. In the context of the present invention, it has been found that the ligand as defined above is especially suitable for binding to HDACs, which enables its use in screening assays for the identification of HDAC interacting compounds. Especially, as shown in example 2 with the help of said compound and method of the invention, it is possible to identify HDAC proteins (for example HDAC2, HDAC6, HDAC 8 and HDAC 10).

The methods according to the first three aspects of the invention are especially suitable for the identification of a histone deacetylase (HDAC) interacting compound because they rely on the use of HDAC complexes which should ensure that the HDAC is present as much as possible in its natural environment. The method according to the fourth aspect of the invention has been found especially suitable for the identification of a histone deacetylase (HDAC) interacting compound in that the binding characteristics of known HDAC inhibitors could be very well characterized using this method of the invention.

According to the present invention, the expression "HDAC" or "histone deacetylase" means enzymes that remove acetyl groups from histones or other substrate proteins. These enzymes are known in the art.

Examples of HDACs are:

Class I HDACs (HDAC1 , HDAC2, HDAC3, HDAC8);

class Ila HDAC (HDAC4, HDAC5, HDAC7, HDAC9);

class lib HDAC (HDAC6, HDAC 10);

class IV HDAC (HDAC 1 1).

Each of them can be used in the context of the present invention. Preferred examples are HDAC2, HDAC6, HDAC8, and HDA10.

According to the present invention, the expression "HDAC" relates to both human and other proteins of this family. The expression especially includes functionally active derivatives thereof, or functionally active fragments thereof, or a homologues thereof, or variants encoded by a nucleic acid that hybridizes to the nucleic acid encoding said protein under low stringency conditions. Preferably, these low stringency conditions include hybridization in a buffer comprising 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% BSA, 100 μg/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40°C, washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1-5 hours at 55°C, and washing in a buffer consisting of 2X SSC, 25 mM Tris-HCl (pH 7.4) 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60°C.

Moreover, according to the present invention, the expression "HDAC" includes mutant forms said HDACs.

In the aspects of the invention, first a protein preparation containing said HDAC (see the fourth aspect of the invention) or HDAC complex (see the first three aspects of the invention) is provided.

The methods of the present invention can be performed with any protein preparation as a starting material, as long as the respective HDAC or HDAC complex is present in the preparation and is derived from a cell endogenously expressing an HDAC. Examples include a liquid mixture of several proteins, a cell lysate, a partial cell lysate which contains not all proteins present in the original cell or a combination of several cell lysates. The term "protein preparation" also includes dissolved purified protein. In the context of the present invention, the term "endogenously" means that the respective cell expresses said HDAC without being transfected with an HDAC-encoding nucleic acid. This ensures that the HDAC is present, as much as possible, in its natural environment, especially is contained in a naturally occurring HDAC protein complex as discussed above. In another aspect of the invention, aliquots of a cell preparation are provided as the starting material. In the context of the present invention, the term "cell preparation" refers to any preparation containing at least one cell with the desired properties. Suitable cell preparations are described below. The presence of the HDACs in a protein preparation of interest can be detected on Western blots probed with antibodies that are specifically directed against said HDAC. Alternatively, also mass spectrometry (MS) could be used to detect the HDACs (see below). The presence of a given HDAC complex can be e.g. determined by determining whether, in a given protein preparation, HDAC is complexed to another known component of said complex. Corresponding methods are well known in the art and include co- immunoprecipitation or purification of HDAC under conditions allowing that the HDAC complex is not dissociated.

Cell lysates or partial cell lysates can be obtained by isolating cell organelles (e.g. nucleus, mitochondria, ribosomes, golgi etc.) first and then preparing protein preparations derived from these organelles. Methods for the isolation of cell organelles are known in the art (Chapter 4.2 Purification of Organelles from Mammalian Cells in "Current Protocols in Protein Science", Editors: John.E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471-14098-8).

In addition, protein preparations can be prepared by fractionation of cell extracts thereby enriching specific types of proteins such as cytoplasmic or membrane proteins (Chapter 4.3 Subcellular Fractionation of Tissue Culture Cells in "Current Protocols in Protein Science", Editors: John.E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471-14098-8).

Furthermore protein preparations from body fluids can be used (e.g. blood, cerebrospinal fluid, peritoneal fluid and urine). For example whole embryo lysates derived from defined development stages or adult stages of model organisms such as C. elegans can be used. In addition, whole organs such as heart dissected from mice can be the source of protein preparations. These organs can also be perfused in vitro in order to obtain a protein preparation. In a preferred embodiment of the methods of the invention, the provision of a protein preparation includes the steps of harvesting at least one cell containing the HDAC or the HDAC protein complex and lysing the cell.

Suitable cells for this purpose as well as for the cell preparations used as the starting material in one aspect of the present invention are those cells or tissues where the HDACs are endogenously expressed. In any given cell or tissue only a subset of the HDACs may be expressed. Therefore it may be necessary to generate multiple protein preparations from a variety of cell types and tissues to cover the HDAC family, especially for selectivity profiling of HDAC inhibitors. As established cell lines may not reflect the physiological expression pattern of HDACs, primary animal or human cells may be used, for example cells isolated from blood samples. Therefore, in a preferred embodiment, cells isolated from peripheral blood represent a suitable biological material. Procedures for the preparation and culture of human lymphocytes and lymphocyte subpopulations obtained from peripheral blood (PBLs) are widely known (W.E Biddison, Chapter 2.2 "Preparation and culture of human lymphocytes" in Current Protocols in Cell Biology, 1998, John Wiley & Sons, Inc.). For example, density gradient centrifugation is a method for the separation of lymphocytes from other blood cell populations (e.g. erythrocytes and granulocytes). Human lymphocyte subpopulations can be isolated via their specific cell surface receptors which can be recognized by monoclonal antibodies. The physical separation method involves coupling of these antibody reagents to magnetic beads which allow the enrichment of cells that are bound by these antibodies (positive selection).

As an alternative to primary human cells cultured cell lines (e.g. MOLT-4 cells, Jurkat, Ramos, HeLa or K-562 cells) can be used.

In a preferred embodiment, the cell is part of a cell culture system and methods for the harvest of a cell out of a cell culture system are known in the art (literature supra).

The choice of the cell will mainly depend on the expression of the HDACs, since it has to be ensured that the protein is principally present in the cell of choice. In order to determine whether a given cell is a suitable starting system for the methods of the invention, methods like Westernblot, PCR-based nucleic acids detection methods, Northernblots and DNA- microarray methods ("DNA chips") might be suitable in order to determine whether a given protein of interest is present in the cell.

The choice of the cell may also be influenced by the purpose of the study. If the in vivo efficacy for a given drug needs to be analyzed then cells or tissues may be selected in which the desired therapeutic effect occurs (e.g. B-cells). By contrast, for the elucidation of protein targets mediating unwanted side effects the cell or tissue may be analysed in which the side effect is observed (e.g. cardiomyocytes, vascular smooth muscle or epithelium cells).

Furthermore, it is envisaged within the present invention that the cell containing the HDACs or the HDAC complex may be obtained from an organism, e.g. by biopsy. Corresponding methods are known in the art. For example, a biopsy is a diagnostic procedure used to obtain a small amount of tissue, which can then be examined microscopically or with biochemical methods. Biopsies are important to diagnose, classify and stage a disease, but also to evaluate and monitor drug treatment. It is encompassed within the present invention that by the harvest of the at least one cell, the lysis is performed simultaneously. However, it is equally preferred that the cell is first harvested and then separately lysed.

Methods for the lysis of cells are known in the art (Karwa and Mitra: Sample preparation for the extraction, isolation, and purification of Nuclei Acids; chapter 8 in "Sample Preparation Techniques in Analytical Chemistry", Wiley 2003, Editor: Somenath Mitra, print ISBN: 0471328456; online ISBN: 0471457817). Lysis of different cell types and tissues can be achieved by homogenizers (e.g. Potter-homogenizer), ultrasonic desintegrators, enzymatic lysis, detergents (e.g. NP-40, Triton® X-100, CHAPS, SDS), osmotic shock, repeated freezing and thawing, or a combination of these methods.

According to the methods of the invention, the protein preparation containing the HDAC or the HDAC protein complex is contacted with a ligand as defined above thereby allowing the reversible binding of the HDAC complex or the HDAC to the ligand.

According the present invention, a ligand having the structure

(formula I) is used, wherein R is H, C1-C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, and wherein the ligand is immobilized on a solid support via the NHR-group.

Within the meaning of the present invention the terms are used as follows:

"Alkyl" means a straight-chain or branched saturated hydrocarbon chain. Each hydrogen of an alkyl carbon may be replaced by a substituent.

"Ci-6 alkyl" means an alkyl chain having 1 - 6 carbon atoms, e.g. if present at the end of a molecule: C alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert- butyl, n-pentyl, n-hexyl, or e.g. -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-, when two moieties of a molecule are linked by the alkyl group. Each hydrogen of a Ci-6 alkyl carbon may be replaced by a substituent.

"Cycloalkyl" means a cyclic alkyl chain having preferably 3 - 7 or 3 -5 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of a cycloalkyl carbon may be replaced by a substituent.

"Aryl" means an aromatic ring system.

"Solid support" relates to every undissolved support being able to immobilize a small molecule ligand on its surface. The solid support may be selected from the group consisting of agarose, modified agarose, sepharose® beads (e.g. NHS-activated sepharose®), latex, cellulose, and ferro- or ferrimagnetic particles.

In a preferred embodiment of the invention, the ligand has the structure

In a preferred embodiment, the ligand may further be labelled.

By "labeled" is meant that the respective substance is either directly or indirectly labeled with a molecule which provides a detection signal, e.g. radioisotope, fluorescent tag, chemiluminescent tag, a peptide or specific binding molecules. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin. The label can directly or indirectly provide a detectable signal. The tag can also be a peptide which can be used, for example, in an enzyme fragment complementation assay (e.g. beta- galactosidase enzyme fragment complementation; Zaman et al., 2006. Assay Drug Dev. Technol. 4(4):41 1-420). The labeled compounds would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for identifying kinase interacting compounds by inhibition of binding of the labeled compound, for example in kinase assays that contain such labeled compounds.

Radioisotopes are commonly used in biological applications for the detection of a variety of biomolecules and have proven to be useful in binding assays. Several examples of probes have been designed to incorporate H (also written as T for tritium) because it can replace hydrogen in a probe without altering its structure (Fenteany et al., 1995. Science 268:726-731). An "isotopically" or "radio-labeled" compound is a compound of the invention where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to 2H (also written D for Deuterium), UC, 13C, 14C, 13N, 15N, 150, 170, 180, 18F, 35S, 36C1, 82Br, 75Br, 76Br, 77Br, 123I, ,24I, 125I and 13 ,I. Guidance for the selection and methods for the attachment of fluorescent tags (e.g. fluorescein, rhodamine, dansyl, NBD (nitrobenz-2-oxa-l,3-diazole), BODIPY (dipyrromethene boron difluoride), and cyanine (Cy)-dyes) to small molecule ligands are generally known in the art (Vedvik et al., 2004. Assay Drug Dev. Technol. 2(2): 193-203; Zhang et al., 2005. Analytical Biochemistry 343(l):76-83). The application of fluorescent probes (fluorophores) in assays for high throughput screening (HTS) of protein kinases was described (Zaman et al., 2003. Comb. Chem. High Throughput Screen 6(4): 313-320). The change of the fluorescent properties after binding of the fluorescent probe to the target kinase can be determined by measuring for example fluorescence polarization (Kashem et al., 2007. J. Biomol. Screening 12(l):70-83), fluorescence resonance energy transfer (FRET; Zhang et al., 2005. Analytical Biochemistry 343(l):76-83) or fluorescence lifetime (Moger et al., 2006. J. Biomol. Screening 11(7): 765-772). In addition, the ALPHAScreen technology can be used where the excitation of a donor bead at 680 nm produces singlet oxygen which can diffuse to an acceptor bead undergoing a chemi luminescent reaction (Glickman et al., 2002. J. Biomol. Screen. 7(1):3-10).

The ligand may be coupled to the solid support either covalently or non-covalently. Non- covalent binding includes binding via biotin affinity ligands binding to steptavidin matrices. Preferably, the ligand is covalently coupled to the solid support. The person skilled in the art will understand that this means that the NHR-group may be modified accordingly if the ligand is covalently bound to the solid support.

Methods for immobilizing compounds on solid supports are known in the art and further exemplified in Example 1.

Methods for immobilizing compounds on solid supports are known in the art. In general, before the coupling, the matrixes can contain active groups such as NHS, Carbodiimide etc. to enable the coupling reaction with the ligand. The ligand can be coupled to the solid support by direct coupling (e.g. using functional groups such as amino-, sulfhydryl-, carboxyl-, hydroxyl-, aldehyde-, and ketone groups) and by indirect coupling (e.g. via biotin, biotin being covalently attached to the ligand and non-covalent binding of biotin to streptavidin which is bound directly to the solid support). The linkage to the solid support material may involve cleavable and non-cleavable linkers. The cleavage may be achieved by enzymatic cleavage or treatment with suitable chemical methods.

In the present invention, the term "allowing the reversible binding" means that reversibly a complex is formed between the HDAC complex or the HDAC and the ligand. Conditions allowing the reversible binding of molecules to proteins or protein complexes are known in the art. The skilled person will know which conditions can be applied in order to enable the formation of said complex. In general, the term "allowing the reversible binding" includes all conditions under which such binding is possible. This includes the possibility of having the solid support on an immobilized phase and pouring the lysate onto it. In another preferred embodiment, it is also included that the solid support is in a particulate form and mixed with the cell lysate. Such conditions are known to the person skilled in the art.

In the context of the present invention, compounds are identified which interfere with the binding between the ligand and an HDAC or an HDAC protein complex present in a cell or protein preparation. In the context of non-covalent binding, the binding between the ligand and the HDAC or the HDAC complex is, e.g., via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.

In a preferred embodiment, the steps of the formation of said complex are performed under essentially physiological conditions. The physical state of proteins within cells is described in Petty, 1998 (Howard R. Petty, Chapter 1, Unit 1.5 in: Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada (eds.) Current Protocols in Cell Biology Copyright © 2003 John Wiley & Sons, Inc. All rights reserved. DPI: 10.1002/0471 143030.cb0101 s00Online Posting Date: May, 2001Print Publication Date: October, 1998).

The contacting under essentially physiological conditions has the advantage that the interactions between the ligand, the cell preparation (e. g.. the HDAC involved) and optionally the compound reflect as much as possible the natural conditions. "Essentially physiological conditions" are inter alia those conditions which are present in the original, unprocessed sample material. They include the physiological protein concentration, pH, salt concentration, buffer capacity and post-translational modifications of the proteins involved. The term "essentially physiological conditions" does not require conditions identical to those in the original living organism, wherefrom the sample is derived, but essentially cell-like conditions or conditions close to cellular conditions. The person skilled in the art will, of course, realize that certain constraints may arise due to the experimental set-up which will eventually lead to less cell-like conditions. For example, the eventually necessary disruption of cell walls or cell membranes when taking and processing a sample from a living organism may require conditions which are not identical to the physiological conditions found in the organism. Suitable variations of physiological conditions for practicing the methods of the invention will be apparent to those skilled in the art and are encompassed by the term "essentially physiological conditions" as used herein. In summary, it is to be understood that the term "essentially physiological conditions" relates to conditions close to physiological conditions, as e. g. found in natural cells, but does not necessarily require that these conditions are identical.

For example, "essentially physiological conditions" may comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-37°C, and 0.001-10 mM divalent cation (e.g. Mg++, Ca++,); more preferably about 150 m NaCl or KC1, pH7.2 to 7.6, 5 mM divalent cation and often include 0.01-1.0 percent non-specific protein (e.g. BSA). A non-ionic detergent (Tween®, NP-40, Triton®-X100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (volume/volume). For general guidance, the following buffered aequous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HC1, pH5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents.

Preferably, "essentially physiological conditions" mean a pH of from 6.5 to 7.5, preferably from 7.0 to 7.5, and / or a buffer concentration of from 10 to 50 mM, preferably from 25 to 50 mM, and / or a concentration of monovalent salts (e.g. Na or ) of from 120 to 170 mM, preferably 150 mM. Divalent salts (e.g. Mg or Ca) may further be present at a concentration of from 1 to 5 mM, preferably 1 to 2 mM, wherein more preferably the buffer is selected from the group consisting of Tris-HCl or HEPES. The skilled person will appreciate that between the individual steps of the methods of the invention, washing steps may be necessary. Such washing is part of the knowledge of the person skilled in the art. The washing serves to remove non-bound components of the cell lysate from the solid support. Nonspecific (e.g. simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentrations in the wash buffer.

According to the methods of the invention, the read-out system is whether a binding between the HDAC complex and the ligand (first three aspects of the invention) or whether a binding HDAC and the ligand (fourth aspect of the invention) has occurred.

Methods for determining whether a binding between a ligand and a protein or protein complex has occurred are known in the art. These methods include in situ methods where the binding is assessed without separating the protein or protein complex from the ligand. For example, anti-HDAC antibodies can be used in combination with the ALPHAScreen technology can be used where the excitation of a donor bead at 680 nm produces singlet oxygen which can diffuse to an acceptor bead undergoing a chemiluminescent reaction (Glickman et al., 2002. J. Biomol. Screen. 7(1):3-10).

In a preferred embodiment of the first three aspects according to the invention as well as according to the fourth aspect of the invention, the binding between the ligand and the HDAC complex or protein is determined by separating bound HDAC or HDAC complex from the ligand and subsequent determination of the HDAC or the HDAC complex. This subsequent determination of the HDAC or HDAC complex may either be the detection of the HDAC or the HDAC complex in the eluate or the determination of their amount.

In general, the fact that, in the methods of the invention, a binding between the HDAC ligand and the HDAC complex or the HDAC as occurred preferably indicates that the compound does not completely inhibit the binding. On the other hand, if no binding takes place in the presence of the compound, the compound is presumably a strong interactor with the HDAC, which is indicative for its therapeutic potential. In case that the amount is determined, the less HDAC or HDAC complex can be detected in the eluate, the stronger the respective compound interacts with the HDAC, which is indicative for its therapeutic potential.

Accordingly, in a preferred embodiment of the first aspect of the invention, the extent of binding determined in step c) is indicative for the identification of a histone deacetylase (HDAC) interacting compound.

In another preferred embodiment, the extent of binding determined in step c) is compared to the extent of binding when steps a) to c) are performed in the absence of any compound. It is alternatively preferred that the extent of binding determined in step c) is compared to the extent of binding when steps a) to c) are performed in the presence of a different concentration of the given compound. In another preferred embodiment, a reduced extent of binding determined in step c) compared to the extent of binding determined in the absence of any compound, or equally preferred, compared to the extent of binding determined in the presence of the given compound at a lower concentration indicates that histone deacetylase (HDAC) is a target of the compound.

In yet another preferred embodiment, method steps a) to c) are performed in parallel with different given compounds, preferably with at least two different compounds, more preferably with at least 5, 10, 100, 1000, 5000 or 10000 different compounds, or with any other number of compounds in between. Preferably, steps a) to c) are performed with different given compounds in the context of a high throughput screening assay.

In the context of the present invention, the concentrations of the given compounds may either be similar or different to each other and may depend on the precise experimental set up of interest. Moreover, in the context of the present invention, the concentration(s) of the given compound generally refer to any concentration of interest, including, but not limited to, concentrations calculated by either weight per volume, volume per volume, or molarity.

In a preferred embodiment of the second, third or fourth aspect of the present invention, the method is performed by providing in step a) at least one further aliquot in addition (for example, a third, fourth, fifth or sixth aliquot etc.), and contacting and/or incubating this at least one further aliquot with the given compound, wherein the given compound has a different concentration than the concentration of the compound used in the previous aliquot. Alternatively preferred is that the methods of the invention are performed by providing in step a) in addition at least one further aliquot (for example, a third, fourth, fifth, or sixth aliquot etc.), and contacting and/or incubating this at least one further aliquot with a given compound, wherein the given compound is different from the compound used in the previous aliquot.

Consequently, in a preferred embodiment of the methods of the invention, a reduced binding observed for the aliquot incubated with the compound in comparison to the aliquot not incubated with the compound indicates that HDAC is a target of the compound In an alternatively preferred embodiment of the methods of the invention, a reduced binding observed for the aliquot incubated with the compound at a higher concentration in comparison to the aliquot incubated with the compound at a lower concentration indicates that HDAC is a target of the compound.

According to invention, separating means every action which destroys the interactions between the ligand and the HDAC or the HDAC protein complex. This includes in a preferred embodiment the elution of the HDAC or the HDAC protein complex from the ligand. The elution can be achieved by using non-specific reagents (ionic strength, pH value, detergents).

Such non-specific methods for destroying the interaction are principally known in the art and depend on the nature of the ligand enzyme interaction. Principally, change of ionic strength, the pH value, the temperature or incubation with detergents are suitable methods to dissociate the target enzymes from the immobilized compound. The application of an elution buffer can dissociate binding partners by extremes of pH value (high or low pH; e.g. lowering pH by using 0.1 M citrate, pH2-3), change of ionic strength (e.g. high salt concentration using Nal, KI, MgCl2, or KC1), polarity reducing agents which disrupt hydrophobic interactions (e.g. dioxane or ethylene glycol), or denaturing agents (chaotropic salts or detergents such as Sodium-docedyl-sulfate, SDS; Review: Subramanian A., 2002, Immunoaffinty chromatography).

In some cases, the solid support has preferably to be separated from the released material. The individual methods for this depend on the nature of the solid support and are known in the art. If the support material is contained within a column the released material can be collected as column flowthrough. In case the support material is mixed with the lysate components (so called batch procedure) an additional separation step such as gentle centrifugation may be necessary and the released material is collected as supernatant. Alternatively magnetic beads can be used as solid support so that the beads can be eliminated from the sample by using a magnetic device.

Methods for the detection of proteins (and, therefore, also for HDAC or a HDAC protein complex) or for the determination of their amounts are known in the art and include physico-chemical methods such as protein sequencing (e.g. Edmann degradation), analysis by mass spectrometry methods or immunodetection methods employing antibodies directed against the HDAC. According to a preferred embodiment of the methods according to the first, second and third aspect of the invention, the extent of the binding is determined by separating the HDAC protein complex from the ligand and subsequent detection of a component of the separated HDAC protein complex or subsequent determination of the amount of a component of the separated HDAC protein complex.

This preferred embodiment is especially advantageous, because it ensures that only HDAC protein complex is detected and not also free HDAC which has been bound to the ligand. In this context, the component may be any component known to be part of the respective HDAC complex which binding to the ligand is to be assessed. The detection of a component of the HDAC protein complex is especially suitable for identifying an HDAC interacting compound according to the methods of the invention. Consequently, in a preferred embodiment, the term "component of an HDAC protein complex" denotes a component of said complex which is not an HDAC protein.

Throughout the invention, if an antibody is used in order to detect a respective protein (e.g. HDAC or a component of the HDAC complex), a specific antibody may be used (Wu and Olson, 2002. J. Clin. Invest. 109(10): 1327-1333). As indicated above, such antibodies are known in the art. Furthermore, the skilled person is aware of methods for producing the same.

Preferably, mass spectrometry or immunodetection methods are used in the context of the methods of the invention. The identification of proteins with mass spectrometric analysis (mass spectrometry) is known in the art (Shevchenko et al., 1996, Analytical Chemistry 68: 850-858; Mann et al., 2001, Analysis of proteins and proteomes by mass spectrometry, Annual Review of Biochemistry 70, 437-473) and is further illustrated in the example section. Preferably, the mass spectrometry analysis is performed in a quantitative manner, for example by stable isotope labelling, to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides metabolically, by chemical means, enzymatically, or provided by spiked synthetic peptide standards (Bantscheff et al., 2007; Anal. Bioanal. Chem. 389(4): 1017-1031).

Preferably, the mass spectrometry analysis is performed in a quantitative manner, for example by using iTRAQ® technology (isobaric tags for relative and absolute quantification) or cICAT® (cleavable isotope-coded affinity tags) (Wu et al., 2006. J. Proteome Res. 5, 651-658). Alternatively, tandem mass tag (TMT®) reagents can be used (commercially available from, e.g., Thermo Scientific). The tandem mass tag (TMT®) reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides in up to six different biological samples enabling simultaneous identification and quantitation of peptides.

Accordingly, in a preferred embodiment of the methods of the invention, the detection and/or the determination of binding between the ligand and the given compound is performed by quantitative mass spectrometry.

According to a further preferred embodiment of the present invention, the characterization by mass spectrometry (MS) is performed by the identification of proteotypic peptides of the HDAC. The idea is that the HDAC or the component of the HDAC complex is digested with proteases and the resulting peptides are determined by MS. As a result, peptide frequencies for peptides from the same source protein differ by a great degree, the most frequently observed peptides that "typically" contribute to the identification of this protein being termed "proteotypic peptide". Therefore, a proteotypic peptide as used in the present invention is an experimentally well observable peptide that uniquely identifies a specific protein or protein isoform.

According to a preferred embodiment, the characterization is performed by comparing the proteotypic peptides obtained in the course of practicing the methods of the invention with known proteotypic peptides. Since, when using fragments prepared by protease digestion for the identification of a protein in MS, usually the same proteotypic peptides are observed for a given HDAC or component of the HDAC complex, it is possible to compare the proteotypic peptides obtained for a given sample with the proteotypic peptides already known for HDACs or the component and thereby identifying the HDAC or the component being present in the sample.

Suitable immunodetection methods include but are not limited to Western blots, ELISA assays, sandwich ELISA assays and antibody arrays or a combination thereof. The establishment of such assays is known in the art (Chapter 1 1 , Immunology, pages 1 1-1 to 1 1 -30 in: Short Protocols in Molecular Biology. Fourth Edition, Edited by F.M. Ausubel et al., Wiley, New York, 1999).

These assays can not only be configured in a way to detect and quantify a HDAC interacting protein of interest, but also to analyse posttranslational modification patterns of HDAC or of other components of the HDAC protein complex such as phosphorylation or ubiquitin modification.

As detailed above, the identification methods of the invention involve the use of compounds which are tested for their ability to be a HDAC interacting compound.

Principally, according to the present invention, such a compound can be every molecule which is able to interact with the HDAC, for example by inhibiting its binding to the Iigand. Preferably, the compound has an effect on the HDAC, e.g. a stimulatory or inhibitory effect.

Preferably, said compound is selected from the group consisting of synthetic or naturally occurring chemical compounds or organic synthetic drugs, more preferably small molecule organic drugs or natural small molecule compounds. Natural small molecules, also referred to as natural products, are chemical compounds produced by a living organism found in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and development (Cragg et al., 1997. J. Nat. Prod. 69, 52-60). Preferably, the compound has a molecular weight of less than 1000 Da. Preferably, said compound is identified starting from a library containing such compounds. Then, in the course of the present invention, such a library is screened.

Such small molecules are preferably not proteins or nucleic acids. Preferably, small molecules exhibit a molecular weight of less than 1000 Da, more preferred less than 750 Da, most preferred less than 500 Da.

A "library" according to the present invention relates to a (mostly large) collection of (numerous) different chemical entities that are provided in a sorted manner that enables both a fast functional analysis (screening) of the different individual entities, and at the same time provide for a rapid identification of the individual entities that form the library. Examples are collections of tubes or wells or spots on surfaces that contain chemical compounds that can be added into reactions with one or more defined potentially interacting partners in a high-throughput fashion. After the identification of a desired "positive" interaction of both partners, the respective compound can be rapidly identified due to the library construction. Libraries of synthetic and natural origins can either be purchased or designed by the skilled artisan.

Examples of the construction of libraries are provided in, for example, Breinbauer R, Manger M, Scheck M, Waldmann H. Natural product guided compound library development. Curr. Med. Chem. 2002; 9(23):2129-2145, wherein natural products are described that are biologically validated starting points for the design of combinatorial libraries, as they have a proven record of biological relevance. This special role of natural products in medicinal chemistry and chemical biology can be interpreted in the light of new insights about the domain architecture of proteins gained by structural biology and bioinformatics. In order to fulfill the specific requirements of the individual binding pocket within a domain family it may be necessary to optimise the natural product structure by chemical variation. Solid-phase chemistry is said to become an efficient tool for this optimisation process, and recent advances in this field are highlighted in this review article. The current drug discovery processes in many pharmaceutical companies require large and growing collections of high quality lead structures for use in high throughput screening assays. Collections of small molecules with diverse structures and "drug-like" properties have, in the past, been acquired by several means: by archive of previous internal lead optimisation efforts, by purchase from compound vendors, and by union of separate collections following company mergers. Although high throughput/combinatorial chemistry is described as being an important component in the process of new lead generation, the selection of library designs for synthesis and the subsequent design of library members has evolved to a new level of challenge and importance. The potential benefits of screening multiple small molecule compound library designs against multiple biological targets offers substantial opportunity to discover new lead structures.

In a preferred embodiment of the methods of the invention, the HDAC or HDAC protein complex containing protein preparation is first incubated with the compound and then with the ligand. However, the simultaneous incubation is equally preferred (competitive binding assay).

In case that the incubation with the compound is first, the HDAC or HDAC protein complex containing protein preparation is preferably first incubated with the compound for 10 to 60 minutes, more preferred 30 to 45 minutes at a temperature of 4°C to 37°C, more preferred 4°C to 25°C, most preferred 4°C. Preferably compounds are used at concentrations ranging from 1 nM to 100 μΜ, preferably from 10 nM to 10 μΜ. The second step, contacting with the ligand, is preferably performed for 10 to 60 minutes at 4°C. In case of simultaneous incubation, the HDAC or HDAC protein complex containing protein preparation is preferably simultaneously incubated with the compound and the ligand for 30 to 120 minutes, more preferred 60 to 120 minutes at a temperature of 4°C to 37°C, more preferred 4°C to 25°C, most preferred 4°C. Preferably compounds are used at concentrations ranging from 1 nM to 100 μΜ, preferably from 10 nM to 10 μΜ.

Furthermore, the methods of the invention may be performed with several protein preparations in order to test different compounds. This embodiment is especially interesting in the context of medium or high throughput screenings.

Preferably, the identification methods of the invention are performed as a medium or high throughput screening.

The interaction compound identified according to the present invention may be further characterized by determining whether it has an effect on the HDAC, for example on its HDAC activity (Khan et al., 2008. Biochem. J. 409(2):581 -9; Blackwell et al., 2008. Life Sci. 82(21-22): 1050-1058).

The compounds identified according to the present invention may further be optimized (lead optimisation). This subsequent optimisation of such compounds is often accelerated because of the structure-activity relationship (SAR) information encoded in these lead generation libraries. Lead optimisation is often facilitated due to the ready applicability of high-throughput chemistry (HTC) methods for follow-up synthesis. An example for lead optimization of HDAC inhibitors was reported (Remiszewski et al., 2003. J. Med. Chem. 46(21):4609-4624).

The invention further relates to a method for the preparation of a pharmaceutical composition comprising the steps of a) identifying a HDAC interacting compound as described above, and b) formulating the interacting compound to a pharmaceutical composition.

Methods for the formulation of identified compounds are known in the art. Furthermore, it is known in the art how to administer such pharmaceutical compositions.

The obtained pharmaceutical composition can be used for the prevention or treatment of diseases where the respective HDAC plays a role, e.g. for the prevention or treatment of cancer (Bolden et al., 2006. Nat. Rev. Drug Discov. 5(9):769-784). For example, HDAC inhibitors may be useful for the treatment of inflammatory diseases, cancer or neurodegenerative diseases. Furthermore, the invention relates to a compound having the structure

wherein R is H, C1-C6 alkyl or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl.

All definitions and embodiments disclosed above with respect to the ligand also apply to the compound of the invention. a preferred embodiment, said compound of the invention has the structure

The invention further related to the use of the compound according of the invention for the capture and screening of HDAC, preferably of HDAC6, in protein complexes. Preferably, the compound is immobilized on a solid support via the NHR-group.

All definitions and embodiments disclosed above with respect to the method of the invention also apply to said use of the invention.

The invention is further described by the following figures and examples, which are intended to illustrate, but not to limit the present invention. In case where in the following examples the term "affinity matrix" is used, this term refers to the immobilized ligand as defined in the present application. Short description of the figures

Figure 1: Synthesis of the compound of the invention. Steps: i) K2C03, DMF, 80°C, ii) NaOHaq 4N, MeOH, iii) H2NOTHP, l -ethyl-3-(3-dimethylaminopropyl) carbodiimide, HOBT, N-methyl morpholine, DMF, iv4N HC1 in Dioxane. Methods used in the synthesis of the immobilization compound are described in example 1.

Figure 2: Structure of the preferred compound of the invention. Figure 3: Amino acid sequence of human HDAC6 (ΓΡΙ0000571 1.4). Peptides identified by mass spectrometry are underlined (K562; experiment X012865).

Figure 4: The percentage (%) of competition of proteins by 200 μΜ bufexamac dissolved in DMSO is shown. Values have been normalized to 100 using the DMSO control sample as reference. The numbers above the bars indicate the number of quantified peptide spectra for each protein.

Examples Example 1: Preparation of the affinity matrix

This example describes the synthesis of compounds (Figure 1) and methods for their immobilization on a solid support yielding the affinity matrix used in the following examples for the capturing of HDACs from cell lysates.

All reagents and Bufexamac (Sigma B0760) were obtained from Sigma-Aldrich. Synthesis of methyl 2-(4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)acetate Methyl 2-(4-hydroxyphenyl)acetate (2.03 mmol, leq, 337mg), tert-butyl (2- bromoethyl)carbamate (2.03 mmol, leq, 453mg) and potassium carbonate (300mg) were stirred in DMF (5ml) at 80°C for 12 hours. The reaction was cooled and partitioned between ethyl acetate (20ml) and 1M aq sodium hydroxide (20ml). The phases were separated and the organic phase washed with water (10ml) and brine (10ml), dried

(MgS04), filtered and concentrated under reduced pressure to yield the title compound as a pale yellow oil. LCMS (method A) Rt 2.61 min, M+Na+ 332

Synthesis of 2-(4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)acetic acid Methyl 2-(4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)acetate (405mg) was dissolved in methanol (10ml) and 4M aq sodium hydroxide was added (3ml). The reaction was stirred at room temperature for four hours. The reaction was then acidified to pH 5 with 2M hydrochloric acid and concentrated under reduced pressure. The residue was triturated with methanol (3 x 5ml) and the combined organic extracts concentrated under reduced pressure to give the title compound as a white solid. LCMS (method A) Rt 2.29 min, M+Na+ 318 Synthesis of 2-(4-(2-aminoethoxy)phenyl)-N-hydroxyacetamide

2-(4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)acetic acid (52mg), l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (45 mg, 1.5eq), HOBT (18mg, 1.1 eq), 0-(tetrahydro- 2H-pyran-2-yl)hydroxylamine (19mg, 1.1 eq) and N-methylmorpholine (20μΐ8, 1.5eq) were stirred for 12 hours in DMF (1ml). The reaction was diluted with ethyl acetate (5ml) and washed with 2M hydrochloric acid (5ml) and brine (5ml). The organic layer was dried (MgS04), filtered and concentrated under reduced pressure to give the protected product. LCMS (method A) Rt 2.39 min, M+H+ 417

The crude residue was dissolved in 4M hydrogen chloride in dioxan (2ml). The resultant precipitate was collected by filtration and washed with ethyl acetate (2 x 2ml) then dried in vacuo to give the title compound as a white solid (15mg)

1NMR (DMSO-d6, 400MHz): δ= 10.54 (br s, 1H), 8.15 (br s, 3H), 7.20 (d, 2H), 6.92 (d, 2H) 4.14 (t, 2H), 3.57 (t, 2H), 3.20 (d, 2H).

LCMS conditions

Columns: Phenomenex Gemini-C18, 3.0 x 30 mm

Flow rate: 1.2 ml/mn

Temperature: 40°C

Wavelength: 254nm The mass spectrometry data were gathered in positive or negative mode, scanning for masses between 150 and 700amu, using a fragmentor set up to 100V for methods A

Solvents:

A= Water with 0.1% formic acid

B= Acetonitrile with 0.1 % formic acid

C= Water with 0.1% ammonia

D= (95% : 5%, acetonitrile : water) with 0.1% ammonia Gradient conditions

Table 1: Method A: Short Column Analytical, Low pH, Positive ion

Table 2: Abbreviations

Immobilization of compounds on beads (affinity matrix)

NHS-activated Sepharose® 4 Fast Flow (Amersham Biosciences, 17-0906-01) was equilibrated with anhydrous DMSO (Dimethylsulfoxid, Fluka, 41648, H20 <= 0.005%). 1 ml of settled beads was placed in a 15 ml Falcon tube, compound stock solution (usually 100 mM in DMF or DMSO) was added (final concentration 0.2-2 μπιοΐ/ηιΐ beads) as well as 15 μΐ of triethylamine (Sigma, T-0886, 99% pure). Beads were incubated at room temperature in darkness on an end-over-end shaker (Roto Shake Genie, Scientific Industries Inc.) for 16 - 20 hours. Coupling efficiency is determined by HPLC. Non- reacted NHS-groups were blocked by incubation with aminoethanol at room temperature on the end-over-end shaker over night. Beads were washed with 10 ml of DMSO and were stored in isopropanol at -20°C. These beads were used as the affinity matrix in the following examples. Control beads (no compound immobilized) were generated by blocking the NHS-groups by incubation with aminoethanol as described above.

Example 2: Bead experiment using an immobilized compound and K-562 cell lysate

This example demonstrates the use of an immobilized compound (structure shown in Figure 2) for the capturing and identification of proteins such as HDACs from cell lysate in a competition binding assay. To the first aliquot of cell lysate 200 μΜ bufexamac (Trommer et al., 2003. J. Pharm. Pharmacol. 55(10): 1379-88) was added and allowed to bind to proteins in the lysate. Then the affinity matrix with the immobilized compound (Example 1) was added to capture proteins that were not interacting with the previously added bufexamac. Beads were separated from the lysate and bead bound proteins were eluted in SDS sample buffer and subsequently separated by SDS-Polyacrylamide gel electrophoresis. Suitable gel bands were cut out and subjected to in-gel proteolytic digestion with trypsin. The second lysate aliquot was processed identically, however no bufexamac was added (DMSO solvent control). Peptides originating from samples 1 and 2 were labeled with iTRAQ® reagents (iTRAQ® 1 15 and iTRAQ® 1 16) and the combined samples were analyzed with a nano-flow liquid chromatography system coupled online to a tandem mass spectrometer (LC-MS/MS) experiment followed by iTRAQ® reporter ion quantification in the MS/MS spectra (Ross et al., 2004. Mol. Cell. Proteomics 3(12): 1 154- 1 169). Further experimental protocols can be found in WO2006/ 134056 and a previous publication (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044).

The identified proteins are shown in Table 4 and Figure 4 including the percent competition values for the sample to which 200 μΜ bufexamac was added. In total 4 different HDACs were identified. For illustration, the identified peptides for HDAC6 are shown in Figure 3. Sequence identifiers are defined by the International Protein Index (IPI) (Kersey et al., 2004. Proteomics 4(7): 1985-1988). 1. Cell culture

In this example K-562 cell lysate was used (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044). K-562 cells (American Type Culture Collection-No. CCL-243) were either obtained from an external supplier (CIL SA, Mons, Belgium) or grown in one litre Spinner flasks (Integra Biosciences, #182101) in suspension in RPMI 1640 medium (Invitrogen, #21875-034) supplemented with 10% Fetal Bovine Serum (Invitrogen, #10270-106). Cells were harvested by centrifugation, washed once with 1 x PBS buffer (Invitrogen, #14190-094) and cell pellets were frozen in liquid nitrogen and subsequently stored at -80°C.

2. Preparation of cell lysates

Cells were homogenized in a Potter S homogenizer in lysis buffer: 50 mM Tris-HCl, 0.8% NP40, 5% glycerol, 150 mM NaCl, 1.5 mM MgCl2, 25 mM NaF, 1 mM sodium vanadate, 1 mM DTT, pH 7.5. One complete EDTA-free tablet (protease inhibitor cocktail, Roche Diagnostics, 1 873 580) per 25 ml buffer was added. The material was dounced 20 times using a mechanized POTTER S, transferred to 50 ml falcon tubes, incubated for 30 minutes rotating at 4° C and spun down for 10 minutes at 20,000 x g at 4°C (10,000 rpm in Sorvall SLA600, precooled). The supernatant was transferred to an ultracentrifuge (UZ)- polycarbonate tube (Beckmann, 355654) and spun for 1 hour at 145.000 x g at 4°C (40.000 rpm in ΤΪ50.2, precooled). The supernatant was transferred again to a fresh 50 ml falcon tube, the protein concentration was determined by a Bradford assay (BioRad) and samples containing 50 mg of protein per aliquot were prepared. The samples were immediately used for experiments or frozen in liquid nitrogen and stored frozen at -80°C.

3. Capturing of proteins from cell lysate

Sepharose®-beads with the immobilized compound (35 μΐ beads per pull-down experiment) were equilibrated in lysis buffer and incubated with a cell lysate sample containing 5 mg of protein on an end-over-end shaker (Roto Shake Genie, Scientific Industries Inc.) for 2 hours at 4°C. Beads were collected, transferred to Mobicol-columns (MoBiTech 10055) and washed with 10 ml lysis buffer containing 0.4% NP40 detergent, followed by 5 ml lysis buffer containing 0.2 % detergent. To elute bound proteins, 60 μΐ 2x SDS sample buffer was added to the column. The column was incubated for 30 minutes at 50°C and the eluate was transferred to a siliconized microfuge tube by centrifugation. Proteins were then alkylated with 108 mM iodoacetamid. Proteins were then separated by SDS-Polyacrylamide electrophoresis (SDS-PAGE). 4. Protein Identification by Mass Spectrometry

4.1 Protein digestion prior to mass spectrometric analysis

Gel-separated proteins were digested in-gel essentially following a previously described procedure (Shevchenko et al., 1996, Anal. Chem. 68:850-858). Briefly, gel-separated proteins were excised from the gel using a clean scalpel, destained twice using 100 μΐ 5mM triethyl ammonium bicarbonate buffer (TEAB; Sigma T7408) and 40% ethanol in water and dehydrated with absolute ethanol. Proteins were subsequently digested in-gel with porcine trypsin (Promega) at a protease concentration of 10 ng/μΐ in 5mM TEAB. Digestion was allowed to proceed for 4 hours at 37°C and the reaction was subsequently stopped using 5 μΐ 5% formic acid.

4.2 Sample preparation prior to analysis by mass spectrometry

Gel plugs were extracted twice with 20 μΐ 1% formic acid and three times with increasing concentrations of acetonitrile. Extracts were subsequently pooled with acidified digest supernatants and dried in a vacuum centrifuge.

4.3 iTRAQ® labeling of peptide extracts

The peptide extracts of samples treated with 200 μΜ of free compound 6 (bufexamac) and the solvent control (0.5% DMSO) were treated with different variants of the isobaric tagging reagent (iTRAQ® Reagents Multiplex Kit, part number 4352135, Applied

Biosystems, Foster City, CA, USA). The iTRAQ® reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides on amino groups in up to four different biological samples enabling simultaneous identification and quantitation of peptides. The iTRAQ® reagents were used according to instructions provided by the manufacturer. The samples were resuspended in 10 μΐ 50 mM TEAB solution, pH 8.5 and 10 μΐ ethanol were added. The iTRAQ® reagent was dissolved in 120 μΐ ethanol and 10 μΐ of reagent solution were added to the sample. The labeling reaction was performed at room temperature for one hour on a horizontal shaker and stopped by adding 5 μΐ of 100 mM TEAB and 100 mM glycine in water. The two labeled sampled were then combined, dried in a vacuum centrifuge and resuspended in 10 μΐ of 0.1% formic acid in water.

4.4 Mass spectrometric data acquisition

Peptide samples were injected into a nano LC system (CapLC, Waters or nano-LC 1D+, Eksigent) which was directly coupled either to a quadrupole TOF (QTOF Ultima, QTOF Micro, Waters), ion trap (LTQ) or Orbitrap mass spectrometer. Peptides were separated on the LC system using a gradient of aqueous and organic solvents (see below). Solvent A was 0.1% formic acid and solvent B was 70% acetonitrile in 0.1% formic acid.

Table 3: Peptides elution off the LC system

4.5 Protein identification and quantitation

The peptide mass and fragmentation data generated in the LC-MS/MS experiments were used to query a protein data base consisting of an in-house curated version of the International Protein Index (IPI) protein sequence database combined with a decoy version of this database (Elias and Gygi, 2007. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods 4, 207-214). Proteins were identified by correlating the measured peptide mass and fragmentation data with data computed from the entries in the database using the software tool Mascot (Perkins et al., 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-3567). Search criteria varied depending on which mass spectrometer was used for the analysis. Protein acceptance thresholds were adjusted to achieve a false discovery rate of below 1% as suggested by hit rates on the decoy data base (Elias and Gygi, 2007. Target-decoysearch strategy for increased confidence in large-scale protein identifications by mass spectro- metry. Nature Methods 4, 207-214). Relative protein quantitation was performed using peak areas of iTRAQ® reporter ion signals essentially as described in an earlier publication (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044). Table 4: Identified proteins from mixed 562 cell lysate

Table 5: Pre aration of 5x-DP buffer

The 5x-DP buffer was filtered through a 0.22 μηι filter and stored in 40 ml-aliquots at -80°C. Stock solutions were obtained from the following suppliers: 1.0 M Tris/HCl pH 7.5 (Sigma, T-2663), 87% Glycerol (Merck, catalogue number 04091.2500); 1.0 M MgCl2 (Sigma, M-1028); 5.0 M NaCl (Sigma, S-5150).

Cellzome AG January 20, 2012

CEL67592PC FLZ/NOT

Claims

A method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of a) providing an HDAC protein complex containing protein preparation derived from a cell endogenously expressing an HDAC, b) contacting the protein preparation with a given compound and with a ligand having the structure

wherein R is H, C1 -C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, thereby allowing the reversible binding of said ligand to said HDAC protein complex, and c) determining to what extent in step b) a binding between the ligand and the protein complex has occurred.

2. The method of claim 1 , wherein the extent of binding determined in step c) is indicative for the identification of a histone deacetylase (HDAC) interacting compound. 2

2/098006 PCT/EP2012/000259

The method of claims 1 or 2, wherein the extent of binding determined in step c) is compared to the extent of binding when steps a) to c) are performed in the absence of any compound, or to the extent of binding when steps a) to c) are performed in the presence of the given compound, wherein the given compound has a different concentration.

The method of any of claims 1 to 3, wherein a reduced extent of binding determined in step c) compared to the extent of binding determined in the absence of any compound, or to the extent of binding determined in the presence of the given compound at a lower concentration indicates that histone deacetylase (HDAC) is a target of the compound.

The method of any of claims 1 to 4, wherein steps a) to c) are performed in parallel with different given compounds, preferably with at least two different compounds.

A method for the identification of a histone deacetylase (HDAC) interacting compound, comprising the steps of a) providing two aliquots of an HDAC protein complex containing protein preparation derived from a cell endogenously expressing an HDAC, b) contacting one aliquot with a ligand having the structure

wherein R is H, C1-C6 alkyl, or cycloalkyl and X is a C1 -C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, 3

WO 2012/098006 PCT/EP2012/000259

thereby allowing the reversible binding of said ligand to said HDAC protein complex, c) contacting the other aliquot with a given compound and with the ligand as defined in step b), thereby allowing the reversible binding of said ligand to said HDAC protein complex, and d) determining to what extent in steps b) and c) a binding between the ligand and the protein complex has occurred.

7. A method for the identification of a histone deacetylase (HDAC) interacting

compound, comprising the steps of: a) providing two aliquots of a cell preparation each comprising at least one cell endogenously expressing an HDAC, b) incubating one aliquot with a given compound, c) harvesting the cells of each aliquot, d) lysing the cells of each aliquot in order to obtain protein preparations

containing an HDAC protein complex, e) contacting the protein preparations with a ligand having the structure 4

WO 2012/098006 PCT/EP2012/000259

wherein R is H, C1-C6 alkyl, or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl, wherein the ligand is immobilized on a solid support via the NHR-group, thereby allowing the reversible binding of said ligand to said HDAC protein complex, and f) determining to what extent in step e) a binding between the ligand and the

protein complex has occurred.

8. The method of any of claims 1 to 7, wherein the ligand has the structure

9. The method of any of claims 1 to 8, wherein the HDAC is selected from the group consisting of HDAC2, HDAC6, HDAC 8, and HDAC10, preferably HDAC6. 10. The method of any of claims 6 to 9, wherein in step a) at least one further aliquot is provided in addition which is contacted and/or incubated with the given compound, wherein the given compound has a different concentration than the concentration of compound used in the previous aliquot.

1 1. The method of any of claims 6 to 9, wherein in step a) at least one further aliquot is provided in addition which is contacted and/or incubated with a given compound, wherein the given compound is different from the compound used in the previous aliquot.

12. The method of any of claims 6 to 9, wherein a reduced binding observed for the aliquot incubated with the compound in comparison to the aliquot not incubated with the compound indicates that HDAC is a target of the compound. 5

WO 2012/098006 PCT/EP2012/000259

13. The method of any of claims 6 to 9, wherein a reduced binding observed for the aliquot incubated with the compound at a higher concentration in comparison to the aliquot incubated with the compound at a lower concentration indicates that HDAC is a target of the compound.

14. The method of any of claims 1 to 13, wherein the extent of the binding is determined by separating the HDAC protein complex from the ligand and subsequent detection of a component of the separated HDAC protein complex or subsequent determination of the amount of a component of the separated HDAC protein complex.

15. The method of claim 14, wherein said detection or said determination is performed by mass spectrometry or immunodetection methods, preferably with an antibody directed against the component.

16. The method of claim 15, wherein the detection and/or the determination of binding between the ligand and the given compound is performed by quantitative mass spectrometry.

17. The method of any of claims 1 to 16, wherein said given compound is selected from the group consisting of synthetic compounds, or organic synthetic drugs, more preferably small molecule organic drugs, and natural small molecule compounds, even more preferable wherein the compound has a molecular weight of less than 1000 Da.

18. The method of any of claims 1 to 17, wherein the given compound is an HDAC inhibitor.

19. The method of any of claims 1 to 18, wherein the provision of a protein preparation includes the steps of harvesting at least one cell endogenously expressing an HDAC and lysing the cell.

20. The method of any of claims 1 to 19, wherein the binding of the ligand to the HDAC protein complex steps is performed under essentially physiological conditions. 21. The method of any of claims 1 to 20, performed as a high-throughput-method. 22. A compound having the structure

wherein R is H, C1-C6 alkyl or cycloalkyl and X is a C1-C6 alkyl, cycloalkyl or an aryl.

23. The compound of claim 22 having the structure

The use of the compound according to any of claims 22 and 23 for the capture and screening of HDAC, preferably of HDAC6, in protein complexes. 25. The use of any of claim 24, wherein the compound is immobilized on a solid support via the NHR-group.

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