The present invention relates to immobilization compounds, immobilization products and preparations thereof as well as methods and uses for the identification of histone demethylase interacting compounds or for the purification or identification of histone demethylase proteins.

Epigenetic information refers to heritable changes in gene function that are stable between cell divisions but without changing the DNA sequence. Part of the epigenetic mechanism has been ascribed to modifications of histone proteins or DNA that affects the expression of specific genes. The post-translational modifications of histone tails such as the methylation of arginine and lysine amino acid residues are important for the storage of epigenetic information (Agger et al., 2008. Curr. Opin. Genet. Dev. 18(2): 159- 168).

The histone-modifying enzymes that catalyse the demethylation of lysine residues (lysine demethylases, KDMs) and arginine residues (arginine demethylases, RDMs) are of substantial interest from the perspective of drug discovery and medicinal chemistry. These enzymes play important roles in controlling gene regulation, and there is evidence that the enzymatic activities of several of these proteins have pathogenic roles in for example in diseases such as cancer (Kampranis and Tsichlis, 2009. Adv. Cancer Res. 102:103-169). Of particular interest are lysine demethylases of the Jumonji family which as a common feature share the catalytic Jumonji C (JmjC) protein domain. There are 27 different human JmjC domain proteins of which 15 have been shown to demethylate specific lysines in the histone 3 (H3) tail and one to demethylate methylated arginine. The catalytic JmjC domain is essential for the oxidative lysine demethylation reaction that requires Fe(II) and a- ketoglutarate (aKG) as cofactors (Agger et al., 2008. Curr. Opin. Genet. Dev. 18(2).T 59- For the development of drugs targeting histone demethylases it is important to have assays that allow the identification and characterization of small molecule inhibitors in terms of potency and selectivity across the histone demethylase family (Hamada et al, 2010. J. Med. Chem. 53(15):5629-5638).

Therefore another step for the identification of selective histone demethylase 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 panels of individual enzyme assays are used to assess the inhibitory effect of a compound for protein histone demethylases (Hamada et al, 2010. J. Med. Chem. 53(15):5629-5638).

The in vitro investigation of histone demethylase activity is typically performed using enzyme assays. For example, the recombinant histone demethylases GASC1, JMJD2a and JMJD2b were expressed using baculovirus vectors in insect cells and tested in demethylation assays (Cloos et al., 2006. Nature 442(7100):307-31 1). However, it can be advantageous to use endogenous histone demethylases as isolated from mammalian cells.

Several reports described histone demethylase inhibitors (Hamada et al., 2009. Bioorg Med Chem Lett. 19(10):2852-2855; Hamada et al, 2010. J. Med. Chem. 53(15):5629-5638; Rose et al., 2010. J. Med. Chem. 53(4): 1810-1818; WO2010043866A3).

In view of the above, there is a need for providing effective tools and methods for the identification and selectivity profiling of histone demethylase interacting compounds as well as for the purification of histone demethylases.

The present invention relates inter alia to an immobilization compound of formula (I) or a salt thereof, wherein

R ¹ , R ² are independently selected from the group consisting of H; Ci

n is 0, 1, or 2;

m is 0, 1, 2, 3, or 4;

R ³ is halogen; CH ₃ ; CF ₃ ; or OCH ₃ ;

R ⁴ is X ¹ -(CH ₂ ) _ol -X ² -(CH ₂ )o2-X ³ -(CH ₂ )o3-X ⁴ ;

X ¹ is *0; *N(R ⁵ ); *OC(0); *N(R ⁵ )C(0); *OS(0) ₂ ; or *N(R ⁵ )S(0) ₂ , wherein the asterisk indicates the attachment to the phenyl ring shown in formula (I);

ol, o2, o3 are independently selected from the group consisting of 0, 1, and 2;

X ² is a chemical bond; optionally substituted phenylene; optionally substituted 5 to 6 membered heteroarylene; or (CH ₂ CH ₂ 0)o4;

o4 is 1, 2, or 3;

X ³ is a chemical bond; O; N(R ^5a ); OC(O); C(0)0; N(R ^5a )C(0); C(0)N(R ^5a ); OS(0) ₂ ; N(R ^5a )S(0) ₂ ; S(0) ₂ 0; or S(0) ₂ N(R ^5a );

4 is N(R ^5b )H; COOH; or

R ⁵ ; R ^5a ; R ^5b are independently selected from the group consisting of H; and Ci _-6 alkyl. In case a variable or substituent can be selected from a group of different variants and such variable or substituent occurs more than once the respective variants can be the same or different. Within the meaning of the present invention the terms are used as follows:

"Alkyl" means a straight-chain or branched hydrocarbon chain. Each hydrogen of an alkyl carbon may be replaced by a substituent as further specified. "C alkyl" means an alkyl chain having 1 - 4 carbon atoms, e.g. if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or e.g. -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH(C ₂ H ₅ )-, -C(CH ₃ ) ₂ -, when two moieties of a molecule are linked by the alkyl group. Each hydrogen of a C _1-4 alkyl carbon may be replaced by a substituent as further specified herein.

"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-penty], n-hexyl, or e.g. -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, - CH(C ₂ H ₅ )-, -C(CH ₃ ) 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 as further specified herein.

"Halogen" means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

"Optionally substituted phenylene" means a bivalent (preferably 1 ,4- or 1,3-bivalent, more preferably 1 ,4-bivalent) phenyl group, which is unsubstituted or substituted with one or more substituents, which are the same or different and selected from the group consisting of halogen; CH ₃ ; CF ₃ ; and OCH ₃ . Preferably, an optionally substituted phenylene is unsubstituted.

"Optionally substituted 5 to 6 membered heteroarylene" means a bivalent aromatic heterocyclic ring with 5 or 6 ring atoms (preferably 6 membered, more preferably preferably 1 ,4- or 1 ,3-bivalent, even more preferably 1 ,4-bivalent), wherein at least one ring atom up to 4 ring atoms are represented by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(0) ₂ -), oxygen and nitrogen (including =N(0)-) and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom. An "optionally substituted 5 to 6 membered heteroarylene" is unsubstituted or substituted with one or more substituents, which are the same or different and selected from the group consisting of halogen; C¾; CF ₃ ; and OCH ₃ . Preferably, an optionally substituted 5 to 6 membered heteroarylene is unsubstituted. Examples for a 5 to 6 membered heteroaryls are furan, thiophene, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, isothiazoline, thiadiazole, pyridine, pyridazine, pyrazine, pyrimidine, tetrazole, triazole.

Preferred compounds of formula (I) are those compounds in which one or more of the residues contained therein have the meanings given below, with all combinations of preferred substituent definitions being a subject of the present invention. With respect to all preferred compounds of the formula (I) the present invention also includes all tautomeric and stereoisomeric forms and mixtures thereof in all ratios, and their pharmaceutically acceptable salts.

In preferred embodiments of the present invention, the substituents mentioned below independently have the following meaning. Hence, one or more of these substituents can have the preferred or more preferred meanings given below.

Preferably, R ¹ and R ² are the same. Preferably, R ¹ and R ² are H; or CH ₃ . Preferably, n is 1. Preferably, m is 0. Preferably, X ¹ is *OC(0); or O. Preferably, ol is 0. Preferably, o2 is 0; or 1. Preferably, X ³ is a chemical bond; or C(0)NH. Preferably, o3 is 0 or 2. Preferably, X ⁴ is NH ₂ . Preferably, X ⁴ is

Preferably, X ² is an optionally substituted phenylene; or optionally substituted 5 to 6 membered heteroarylene. More preferably, X ² is an optionally substituted phenylene.

Preferably, o2 is 0 and X ³ is a chemical bond. A preferred compound of formula (I) is a compound, wherein

m is 0;

X ¹ is *0; *OC(0) or *OS(0) ₂ wherein the asterisk indicates the attachment to the phenyl ring shown in formula (I);

X ² is an optionally substituted phenylene;

X ³ is a chemical bond; O; N(R ^5a )C(0); C(0)N(R ^5a ); N(R ^5a )S(0) ₂ or S(0) ₂ N(R ^5a ); and

X ⁴ is N(R ^5b )H. Compounds of formula (I) in which some or all of the above-mentioned groups have the preferred meanings are also an object of the present invention.

In case the immobilization compounds according to formula (I) contain one or more acidic or basic groups, the invention also comprises their corresponding salts. Thus, the immobilization compounds of the formula (I) which contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. Compounds of the formula (I) which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p- toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the immobilization compounds of the formula (I) simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts according to the formula (I) can be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. A preferred immobilization compound of formula (I) is

Further preferred compounds of the present invention are selected from the group consisting of

(R)-3-(4-((4-(aminomethyl)benzyl)oxy)phenyl)-2-(carboxyfo rmamido)propanoic acid;

(S)-3-(4-((4-(aminomethyl)benzyl)oxy)phenyl)-2-(carboxyfo rmamido)propanoic acid;

(R)-3-(4-((3-((2-aminoethyl)carbamoyl)benzyl)oxy)phenyl)- 2

(carboxyformamido)propanoic acid hydrochloride;

(R)-4-(3-methoxy-2-(2-methoxy-2-oxoacetamido)-3-oxopropyl )phenyl3-(piperazin-l- ylmethyljbenzoate;

(R)-2-(carboxyformamido)-3-(4-((3- iperazin-l-ylmethyl)benzoyl)oxy)phenyl)propanoic acid;

(R)-3-(4-((5-aminopentyl)oxy)phenyl)-2-(carboxyformamido) propanoic acid; (R)-3-(4-((5-aminopropyl)oxy)phenyl)-2-(carboxyformamido)pro panoic acid.

The immobilization compounds of the present invention can be prepared by methods well known in the art.

Exemplary routes for the preparation of compounds of the present invention are described below. It is clear to a practitioner in the art to combine or adjust such routes especially in combination with the introduction of activating or protective chemical groups.

General routes for the preparation of exemplary compounds according to the present invention are outlined in Schemes 1 to 3.

Scheme 1

R= Ci _-6 alkyl i) DCM, DIPEA, MeOCOCOCCl; iia) AcN, K ₂ C0 ₃ , tert-butyl 4- (bromomethyl)benzylcarbamate, b) NaOHaq; iii) HC1. Scheme 2

i) AcN, K ₂ C0 ₃ , tert-butyl (2-(3-(chloromethyl)benzamido)ethyl)carbamate; ii) 4M HC1 dioxane; iii) NaOHaq, H ₂ 0.

Scheme 3

i) DCM, tert-butyl (3-hydroxypropyl)carbamate, DIAD; ii) LiOHaq; iii) HC1 or TFA.

The invention further relates to a method for the preparation of an immobilization product, wherein at least one immobilization compound according to the invention is immobilized on a solid support. Such immobilization products obtainable according to the method of the invention are e.g. useful in the methods of the invention for the identification of histone demethylase interacting compounds or in diagnostic methods for the diagnosis of inflammatory diseases, proliferative diseases and metabolic diseases. According to the method of the invention, at least one immobilization compound of the invention is immobilized on a solid support. Throughout the invention, the term "solid support" relates to every undissolved support being able to immobilize a small molecule ligand on its surface.

According to the invention, the term "at least one immobilization compound" means either that at least one immobilization compound of the same type is immobilized on the solid support or that one or more different immobilization compounds (each of them either in singular or plural) may be immobilized on the solid support. Preferably, one or two different immobilization compounds are immobilized on the solid support, more preferably the preferred immobilization compounds of formula I)

is immobilized. 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 case that the solid support is a material comprising various entities, e.g. in case that the solid support comprises several beads or particles, it is envisaged within the present invention that, if different immobilization compounds are immobilized, on each single entity, e.g. each bead or particle, one or more different immobilization compounds are immobilized. Therefore, in case that two immobilization compounds are used, it is envisaged within the present invention that on each single entity one or two different immobilization compounds are immobilized. If no measures are taken that on one entity only one different immobilization compound is immobilized, it is very likely that on each entity all different immobilization compounds will be present.

The immobilization compound or compounds of the invention 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 immobilization compound or compounds are covalently coupled to the solid support.

Methods for immobilizing compounds on solid supports are known in the art and further exemplified in the Example section. Especially, the skilled person will understand that if the immobilization compound is covalently coupled to said solid support, the residues forming part of said coupling will have to be modified accordingly.

In general, before the coupling, the matrixes can contain active groups such as NHS, Carbodimide etc. to enable the coupling reaction with the immobilization compound. The immobilization compound 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 immobilization product of the invention 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.

Therefore, according to a preferred embodiment of the invention, the immobilization product results from a covalent direct or linker mediated attachment of the at least one immobilization compound of the invention to the solid support. This linker may be a CMO alkylene group, which is optionally interrupted or terminated by one or more atoms or functional groups selected from the group consisting of S, O, NH, C(0)0, OC(O), C(O), NHC(O), and C(0)NH and wherein the linker is optionally substituted with one or more substituents independently selected from the group consisting of halogen, OH, NH ₂ , C(0)H, C(0)NH ₂ , S0 ₃ H, N0 ₂ , and CN.

The term„Ci_io alkylene" means an alkylene chain having 1 - 10 carbon atoms, e.g. methylene, ethylene, n-propylene and the like, wherein each hydrogen of a carbon atom may be replaced by a substituent as indicated herein. The term "Ci _-6 alkylene" as used herein is defined accordingly.

The term "interrupted" means that the one or more atoms or functional groups are inserted between two carbon atoms of the alkylene chain or -when "terminated"- at the end of said chain.

The invention further relates to an immobilization product, obtainable by the method of the invention.

Furthermore, the present invention relates to an immobilization product, comprising the immobilization compound of the invention immobilized on a solid support, in particular wherein the solid support is selected from the group consisting of agarose, modified agarose, sepharose beads (e.g. NHS-activated sepharose), latex, cellulose, and ferro- or ferrimagnetic particles.

Therefore, the present invention relates inter alia to an immobilization product comprising a compound of formula (I)

or a salt thereof, wherein Rl, R2 are independently selected from the group consisting of H; Cl-6 alkyl; n is 0, 1 , or 2;

m is 0, 1, 2, 3, or 4;

R3 is halogen; CH3; CF3; or OCH3;

R4 is Xl-(CH2)ol-X2-(CH2)o2-X3-(CH2)o3-X4;

XI is *0; *N(R5); *OC(0); *N(R5)C(0); *OS(0)2; or *N(R5)S(0)2, wherein the asterisk indicates the attachment to the phenyl ring shown in formula (I);

ol, o2, o3 are independently selected from the group consisting of 0, 1, and 2;

X2 is a chemical bond; optionally substituted phenylene; optionally substituted 5 to

6 membered heteroarylene; or (CH2CH20)o4;

o4 is 1, 2, or 3;

X3 is a chemical bond; O; N(R5a); OC(O); C(0)0; N(R5a)C(0); C(0)N(R5a); OS(0)2; N(R5a)S(0)2; S(0)20; or S(0)2N(R5a);

COOH; or

R5; R5a; R5b are independently selected from the group consisting of H; and Cl-6 alkyl, wherein the compound of formula (I) is immobilized on a solid support.

In preferred embodiments, the immobilisation product of the invention comprising a compound of formula (I) is characterized as defined above.

Methods and strategies for choosing appropriate solid supports and for coupling compounds to said solid supports are known in the art (see e.g. Wong, Shan S. Chemistry of protein conjugation and cross-linking (1991), CRC Press, Inc. ISBN 0-8493-5886-8 Chapter 12: Conjugation of proteins to solid matrices, pages 295-318).

Therefore, an immobilization product which is obtainable by the method of the invention is or comprises an immobilization compound of the present invention immobilized on a solid support. This immobilization product will be referred to in the following as the immobilization product of the invention and is used in the methods of the present invention.

In a preferred embodiment, the immobilization compound or immobilization product of the invention may further be labeled.

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, dioxigenin and anti- dioxigenin antibodies. 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 histone demethylase interacting compounds by inhibition of binding of the labeled compound, for example in histone demethylase 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 ³ 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 ² H (also written D for Deuterium), ⁿ C, ¹³ C, ^,4 C, ¹³ N, ¹⁵ N, ¹⁵ 0, ^,7 0, ^,8 0, ¹⁸ F, ³⁵ S, ³⁶ C1, ⁸² Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and ¹³¹ 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 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 histone demethylase 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(1):76- 83) or fluorescence lifetime (Moger et al., 2006. J. Biomol. Screening 1 1(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).

One possible use of the immobilization products of the invention is in the context of the identification of compounds interacting with histone demethylases. Therefore, the present invention also relates to such methods and uses.

In a first aspect of the methods of the invention, the invention therefore relates to a method for the identification of a histone demethylase interacting compound, comprising the steps of a) providing a protein preparation containing at least one histone demethylase, b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of one or more different complexes between one of the histone demethylases and the immobilization product, c) incubating the one or more different complexes with a given compound, and d) determining whether the compound is able to separate the histone demethylase from the immobilization product. In a second aspect, the present invention relates into a method for the identification of a histone demethylase interacting compound, comprising the steps of a) providing a protein preparation containing at least one histone demethylase, b) contacting the protein preparation with the immobilization product of the invention and with a given compound under conditions allowing the formation of one or more different complexes between one of the histone demethylases and the immobilization product, and c) detecting the complex or the complexes formed in step b).

In a third aspect, the present invention relates to a method for the identification of a histone demethylase interacting compound, comprising the steps of: a) providing two aliquots of a protein preparation containing at least one histone demethylase, b) contacting one aliquot with the immobilization product of the invention under conditions allowing the formation of one or more different complexes between one of the histone demethylases and the immobilization product, c) contacting the other aliquot with the immobilization product of the invention and with a given compound under conditions allowing the formation of one or more different complexes between one of the histone demethylases and the immobilization product, and d) determining the amount of the complex or the complexes formed in steps b) and c).

In a fourth aspect, the invention relates to a method for the identification of a histone demethylase interacting compound, comprising the steps of: a) providing two aliquots of a cell preparation comprising each at least one cell containing at least one histone demethylase, b) incubating one aliquot with a given compound, c) harvesting the cells of each aliquot, d) lysing the cells in order to obtain protein preparations,

e) contacting the protein preparations with the immobilization product of the

invention under conditions allowing the formation of one or more different complexes between one of the histone demethylases and the immobilization product, and f determining the amount of the complex or the complexes formed in each

aliquot in step e).

In the context of the present invention, it has been found that the immobilization products of the present invention are suitable for the identification of compounds interacting with histone demethylases.

The immobilization products of the present invention bind several histone demethylases. For example, the following histone demethylases were identified in the biological examples (Tables 6 and 8): JMJD6, JMJD2A and JMJD2B. Consequently, in the context of the present invention, the term "at least one histone demethylase" means that at least one type of histone methylase (e.g. JMJD6, JMCD2A, JMJD2B, or HIFIAN) is present in the respective sample, e.g. the cell or the protein preparation.

Furthermore, the skilled person will appreciate that the respective sample will contain one or more histone demethylases of said type. Therefore, the person skilled in the art will understand that in the context of the present invention, with respect to a histone demethylase, the singular and the plural may be used interchangeably.

According to the present invention, the expression "histone demethylase" denotes a protein that contains a Jumonji C (JmjC) domain. Preferably the histone demethylase is an enzyme that can demethylate methylated lysine or arginine residues. Even more preferred the histone demethylase is an enzyme that can demethylate methylated lysine residues (Agger et al., 2008. Curr. Opin. Genet. Dev. 18(2): 159- 168).

Consequently, in the methods of the present invention, these immobilization products can be used to identify compounds binding to histone demethylases.

Several histone demethylases have been implicated in human cancers. For example, the GASC1/KDM4C protein which belongs to the JMJD2 family was initially identified as a gene amplified in cell lines from esophageal squamous cell carcinomas. Recent evidence suggests that GASC1 may be involved in a cascade of events that contributes to the maintenance of pluripotency and self renewal of stem cells (Kampranis and Tsichlis, 2009. Adv. Cancer Res. 102:103-169).

According to the present invention, the expression "histone demethylase" 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 ug/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 "histone demethylase" includes mutant forms said histone demethylases.

In some aspects of the invention, first a protein preparation containing said histone demethylase is provided. The methods of the present invention can be performed with any protein preparation as a starting material, as long as the respective histone demethylase is solubilized in the preparation. 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 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 preparation are described below.

The presence of the histone demethylases in a protein preparation of interest can be detected on Western blots probed with antibodies that are specifically directed against said histone demethylase. Alternatively, also mass spectrometry (MS) could be used to detect the histone demethylases (see below).

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 nuclear proteins (Dignam et al., 1983. Nucleic Acids Res. 1 1(5): 1475-89). Furthermore protein preparations from body fluids can be used (e.g. blood, cerebrospinal fluid, peritoneal fluid and urine).

Furthermore, the protein preparation may be a preparation containing the histone demethylase or the histone demethylases which has been recombinantely produced. Methods for the production of recombinant proteins in prokaryotic and eukaryotic cells are widely established (Chapter 5 Production of Recombinant Proteins in "Current Protocols in Protein Science", Editors: John. E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, 1995, ISBN: 0-471 -14098-8). 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 histone demethylase or the histone demethylases 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 e.g. those cells or tissues where the histone demethylases are expressed. In any given cell or tissue only a subset of the histone demethylases may be expressed. Therefore it may be necessary to generate multiple protein preparations from a variety of cell types and tissues to cover the histone demethylase family of proteins, especially for selectivity profiling of histone demethylase inhibitors. As established cell lines may not reflect the physiological expression pattern of histone demethylases, 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, HL-60 or HeLa 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 histone demethylases, 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. T-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).

Furthermore, it is envisaged within the present invention that the cell containing the histone demethylases or the histone demethylase 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 Nucleic 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 one or more histone demethylases is contacted with the immobilization product under conditions allowing the formation of a complex between the said histone demethylase and the immobilization product of the invention. In the present invention, the term "a complex between a histone demethylase and the immobilization product" denotes a complex where the immobilization product interacts with a histone demethylase , e.g. by covalent or, most preferred, by non-covalent binding. In the context of the present invention, compounds are identified which interfere with the formation of a complex between the immobilization product and a histone demethylase present in a cell or protein preparation. In case that only one histone demethylase is to be detected or present, the formation of one complex is observed and tested. In case that several histone demethylases are to be detected or present, the formation of several, different complexes is observed and tested.

The skilled person will know which conditions can be applied in order to enable the formation of said complex. In the context of the present invention, the term "under conditions allowing the formation of the complex" includes all conditions under which such formation, preferably 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 non-covalent binding, the binding between the immobilization product and the histone demethylase 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 sOOOnline 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 (i. e. the histone demethylase to be characterized) 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 KCl, 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 KCl, 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-Xl OO) 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 HCl, 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 K) 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 identification methods of the invention, the read-out system is either the detection or determination of a histone demethylase (first aspect of the invention), the detection of the complex between a histone demethylase and the immobilization product (second aspect of the invention), or the determination of the amount of the complex between a histone demethylase and the immobilization product (second, third and fourth aspect of the invention).

In the method according to the first aspect of the invention, the detection or determination of the amount of separated histone demethylase is preferably indicative for the fact that the compound is able to separate the histone demethylase from the immobilization product. This capacity indicates that the respective compound interacts, preferably binds to the histone demethylase, which is indicative for its therapeutic potential.

In one embodiment of the method according to the second aspect of the invention, the complex formed during the method of the invention is detected. The fact that such complex is formed preferably indicates that the compound does not completely inhibit the formation of the complex. On the other hand, if no complex is formed, the compound is presumably a strong interactor with the histone demethylase, which is indicative for its therapeutic potential.

According to the methods of the second, third and fourth aspect of the invention the amount of the complex formed during the method is determined. In general, the less complex in the presence of the respective compound is formed, the stronger the respective compound interacts with the histone demethylase, which is indicative for its therapeutic potential.

The detection of the complex formed according to the second aspect of the invention can be performed by using labeled antibodies directed against the histone demethylase and a suitable readout system.

According to a preferred embodiment of the second aspect of the invention, the complex between one histone demethylase and the immobilization product is detected by determining its amount.

In the course of the second, third and fourth aspect of the invention, it is preferred that the histone demethylase are separated from the immobilization product in order to determine the amount of said complex.

According to invention, separating means every action which destroys the interactions between the immobilization compound and the histone demethylase. This includes in a preferred embodiment the elution of the histone demethylase from the immobilization compound.

The elution can be achieved by using non-specific reagents as described in detail below (ionic strength, pH value, detergents). In addition, it can be tested whether a compound of interest can specifically elute the histone demethylase from the immobilization compound. Such histone demethylase interacting compounds are described further in the following sections.

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, MgCl ₂ , 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.

In step d) of the method according to the first aspect of the invention, it is determined if the histone demethylase has been separated from the immobilization product of the invention. This may include the detection of the histone demethylase or the determination of the amount of the histone demethylase.

Consequently, at least in preferred embodiments of all identification methods of the invention, methods for the detection of a separated histone demethylase or for the determination of their amount are used. Such methods 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 histone demethylase.

Throughout the invention, if an antibody is used in order to detect a histone demethylase or in order to determine its amount (e.g. via ELISA), the skilled person will understand that, if a specific histone demethylase is to be detected or if the amount of a histone demethylase is to be determined, a specific antibody may be used (Gray et al., 2006. J. Biol. Chem. 280(31):28507-28518). As indicated above, such antibodies are known in the art. Furthermore, the skilled person is aware of methods for producing the same.

Preferably, a histone demethylase is detected or the amount of a histone demethylase is determined by mass spectrometry or immunodetection methods. The identification of proteins with mass spectrometric analysis (mass spectrometry, MS) is known in the art (Shevchenko et al., 1996, Analytical Chemistry 68: 850-858; Mann et al., 2001. 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 labeling 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 stable isotope is introduced into proteins by metabolic labeling during cell growth and division, for example by the stable isotope labeling by amino acids in cell culture (SILAC) approach (Ong et al., 2002; Mol. Cell. Proteomics. l(5):376-386).

Preferably, the mass spectrometry analysis is performed in a quantitative manner, for example by using iTRAQ technology (isobaric tags for relative and absolute qualification) or cICAT (cleavable isotope-coded affinity tags) (Wu et al., 2006. J. Proteome Res. 5, 651- 658). 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 histone demethylase. The idea is that the histone demethylase 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 histone demethylase, it is possible to compare the proteotypic peptides obtained for a given sample with the proteotypic peptides already known for histone demethylases and thereby identifying the histone demethylase being present in the sample. As an alternative to mass spectrometry analysis, the eluted histone demethylase (including coeluted binding partners such as regulatory subunits), can be detected or its amount can be determined by using a specific antibody directed against the histone demethylase.

Furthermore, in another preferred embodiment, once the identity of the coeluted binding partner (e.g. regulatory subunit) has been established by mass spectrometry analysis, each binding partner can be detected with specific antibodies directed against this protein.

Suitable antibody-based assays 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 histone demethylase interacting protein of interest, for example a component of a histone demethylase protein complex (Gray et al., 2006. J. Biol. Chem. 280(31):28507-28518), but also to analyse posttranslational modification patterns such as phosphorylation or ubiquitin modification.

Furthermore, the identification methods of the invention involve the use of compounds which are tested for their ability to be a histone demethylase interacting compound.

Principally, according to the present invention, such a compound can be every molecule which is able to interact with the histone demethylase, eg. by inhibiting its binding to the immobilization product of the invention. Preferably, the compound has an effect on the histone demethylase, e.g. a stimulatory or inhibitory effect.

Preferably, the compound is an inhibitor of the enzyme activity of said histone demethylase, including, but not limited to, active site inhibitors which may also inhibit binding of the enzyme to the immobilized ligand, and/or allosteric inhibitors of unknown mode of action which may not necessarily prevent binding to the immobilized ligand.

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. 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 second and third aspect of the invention, the histone demethylase containing protein preparation is first incubated with the compound and then with the immobilization product. However, the simultaneous incubation of the compound and the immobilization product of the invention (coincubation) with the histone demethylase containing protein preparation is equally preferred (competitive binding assay).

In case that the incubation with the compound is first, the histone demethylase 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 1 mM, preferably from 1 nM to 100 μΜ, preferably from 1 nM to 10μΜ. The second step, contacting with the immobilized ligand, is preferably performed for 10 to 60 minutes at 4°C.

In case of simultaneous incubation, the histone demethylase is preferably simultaneously incubated with the compound and the immobilization product of the invention 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 1 mM, preferably from 1 nM to 100 μΜ, preferably from 1 nM to 10μΜ. Furthermore, steps a) to c) of the second aspect 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.

In a preferred embodiment of the method of the invention according to the third or fourth aspect, the amount of the complex formed in step c) is compared to the amount formed in step b)

In a preferred embodiment of the method of the invention according to the third or fourth aspect, a reduced amount of the complex formed in step c) in comparison to step b) indicates that a histone demethylase is a target of the compound. This results from the fact that in step c) of this method of the invention, the compound competes with the immobilized compound for the binding of the histone demethylase. If less histone demethylase is present in the aliquot incubated with the compound, this means preferably that the compound has competed with the inhibitor for the interaction with the enzyme and is, therefore, a direct target of the protein and vice versa.

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 histone demethylase, for example on its histone demethylase activity (Hamada et al, 2010. J. Med. Chem. 53(15):5629-5638). The compounds identified according to the present invention may further be optimized in terms of potency and selectivity. An example for lead optimization of histone demethylase inhibitors was reported (Hamada et al, 2010. J. Med. Chem. 53(15):5629-5638).

The invention further relates to a method for the preparation of a pharmaceutical composition comprising the steps of a) identifying a histone demethylase 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 histone demethylase plays a role, e.g. for the prevention or treatment of cancer (Kampranis and Tsichlis, 2009. Adv. Cancer Res. 102: 103-169). For example, histone demethylase inhibitors may be useful for the treatment of inflammatory diseases, cancer or metabolic diseases.

The invention further relates to a method for the purification of a histone demethylase, comprising the steps of a) providing a protein preparation containing said histone demethylase, b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of a complex between the histone demethylase and the immobilization product, and c) separating the histone demethylase from the immobilization product.

As mentioned above, it has been surprisingly found that the compound of the invention and therefore also the immobilization product of the invention is a ligand which recognizes the histone demethylases mentioned above. This enables efficient purification methods for said histone demethylases.

With respect to the histone demethylases, the protein preparation containing the histone demethylases, the conditions for contacting with the immobilization product of the invention, the immobilization product of the invention, the complex between the histone demethylases and the immobilization product of the invention, the separation of the histone demethylases from the immobilization product of the invention, and the detection of the histone demethylases or the determination of its amount, the embodiments as defined above for the identification methods of the invention also apply to the purification method of the invention.

In a preferred embodiment, the purification method of the invention further comprises after step c) the identification of proteins being capable of binding to said histone demethylases. This is especially interesting when the formation of the complex is performed under essentially physiological conditions, because it is then possible to preserve the natural condition of the enzyme which includes the existence of binding partners, enzyme subunits or post-translational modifications, which can then be identified with the help of mass spectrometry.

Consequently, in a preferred embodiment, the purification method of the invention further comprises after step c) the determination whether the histone demethylase is further posttranslationally modified, e.g. by ubiquitin modification.

The binding proteins or the posttranslational modifications can be determined as explained above for the detection of histone demethylases or the determination of the amount of histone demethylases. Preferably, said methods include mass spectrometry of immunodetection methods as described above.

The invention further relates to a method for determining the presence of one or more histone demethylases in a sample, comprising the steps of: a) providing a protein preparation expected to contain said one or more histone demethylases, b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of a complex between one of the histone demethylases and the immobilization product, and c) detecting whether one or more histone demethylases have formed a complex with the immobilization product. In a preferred embodiment of the invention, said detecting in step c) is performed by separating said one or more histone demethylases from the immobilization product and further identification of said one or more histone demethylases. Said identification may be performed by mass spectrometry or immunodetection methods as described above.

According to an especially preferred embodiment of this method of the invention, the histone demethylase contains at least one mutation.

With respect to said one or more histone demethylases, the protein preparation containing said histone demethylases, the conditions for contacting with the immobilization product of the invention, the immobilization product of the invention, the complex between said histone demethylase and the immobilization product of the invention, the separation of histone demethylases from the immobilization product of the invention, and the detection of histone demethylases or the determination of its amount, the embodiments as defined above for the identification methods of the invention also apply to the purification method of the invention.

The invention further relates to the use of the immobilization compound or the immobilization product of the invention for the identification of a histone demethylase interacting compound and for the purification of a histone demethylase. The embodiments as defined above also apply to the uses of the invention.

The invention further relates to a kit comprising the compound or the immobilization product of the invention. Such a kit is especially useful for performing the methods of the invention. Further components of the kit may be antibodies for the detection of histone demethylase proteins. Such antibodies and their use are known in the art and they are commercially available (Gray et al., 2006. J. Biol. Chem. 280(31):28507-28518). Furthermore, the kit may contain further auxiliary components like buffers, means for the detection of antibodies, and positive controls. Such components are known in the art.

In a further preferred embodiment of the methods or uses of the present invention, the affinity of the histone demethylase interacting compound for the histone demethylase is determined. This can be done by incubating different aliquots of the protein preparation or cell preparation with different amounts of the compound and subsequently correlating the amount of complexes with the concentration of the compound. Plotting the amount of complexes against the concentration of the compounds will in most cases result in a curve with sigmoidal shape, with which the IC ₅₀ value or the KD value of the compound for the histone demethylase can be determined according to standard methods and as described e.g. in Bantscheff et al, Nature Biotechnology 25: 1035-1044 (2007).

The invention is further illustrated by the following figures and examples, which are not considered as being limiting for the scope of protection conferred by the claims of the present application. In case where in the following examples the term "affinity matrix" is used, this term refers to an immobilization product as defined in the present application.

Brief description of the figures Figure 1 : Amino acid sequence of human HIF 1 AN (IPI00299906.4). Peptides identified by mass spectrometry are underlined (Experiment X01 1753; HeLa and Jurkat cells).

Figure 2: Amino acid sequence of human JMJD6 (IPI00604598.1). Peptides identified by mass spectrometry are underlined (Experiment X01 1753; HeLa and Jurkat cells).

Figure 3: Amino acid sequence of human JMJD2A (IPI00005666.1). Peptides identified by mass spectrometry are underlined (Experiment X012215; HL60 cells).

Figure 4: Amino acid sequence of human HIF1 AN (IPI00299906.4). Peptides identified by mass spectrometry are underlined (Experiment X012215; HL60).

Examples

Synthesis of compounds

The following examples describe the synthesis of compounds and methods for their immobilization on a solid support yielding the affinity matrix used in the biological examples for the capturing of histone demethylases from cell lysates. Table 1: Abbreviations

ACN Acetonitrile

aq Aqueous

br Broad

Boc Tert-Butoxycarbonyl

BuLi Butyllithium

d Doublet

DCM Dichloromethane

dd Doubledoublet

DIAD Diisopropyl azodicarboxylate

DIPEA Diisopropylethylamine

DME 1 ,2-Dimethoxyethane

DMF-DMA Ν,Ν-dimethylformamide dimethylacetal

EDC 1 -Ethyl-3 -(3 -dimethylaminopropyl)carbodiimide

EtOAc Ethyl acetate

EtOH Ethanol

eq equivalents

g grams

h hours

HC1 Hydrochloric acid

H ₂ 0 Water

H ₂ S Hydrogen sulfide

HOBt 1 -Hydroxybenzotriazole

HPLC High performance Layer Chromatography

LCMS Liquid chromatography mass spectroscopy m Multiplet

M Molar

MeOH Methanol

mg Milligrams

min Minutes mL Millilitres

Mmol Millimoles

mol% Molar percent

NMR Nuclear magnetic resonance

q Quartet

rt Room temperature

RT Retention time

s Singlet

sat. saturated

t Triplet

tert Tertiary

TFA Trifluoroacetic acid

THF Tetrahydrofuran

Analytical Methods

NMR spectra were obtained on a Brucker dpx400. LCMS Methods A-C were carried out on an Agilent 1 100 using a ZORBAX ^® SB-C18, 4.6 x 150 mm, 5microns, ZORBAX ^® SB- CI S, 4.6 x 75 mm, 3.5 micron or Gemini™ CI 8, 3 x 30 mm, 3 microns column. Column flow was 1.0 or 1.2 mL/min. and solvents used were water and acetonitrile (0.1% formic acid) with an injection volume of 3 or l Oul. Wavelengths were 254 and 210nm.

Method A

Column: ZORBAX ^® SB-C18, 4.6 x 150 mm, 5microns

Table 2

Time (min) Water Acetonitrile

0 95 5

1 1 5 95

13 5 95

13.01 95 5

14.00 STOP Method B

Column: ZORBAX ^® SB-C18, 4.6 x 75 mm, 3.5 microns

Table 3

Method C

Column: Gemini CI 8, 3 x 30 mm, 3 microns Flow rate: 1.2mL/min

Table 4

LC/MS Method D was conducted on an Acquity UPLC BEH CI 8 column (50 mm x 4.6 mm i.d. 2.7 μηι packing diameter) at 40 degrees centigrade, eluting with 0.05% v/v solution of formic Acid in Water (Solvent A) and 0.05% v/v solution of formic Acid in Acetonitrile (Solvent B) using the following elution gradient 0-l .Omin 5-95% B, 1.0-2.0min 95% B, 2.0 - 2.01min 95-5% B, 2.01 - 2.5min 5% B at a flow rate of 1.8ml/min. The UV detection was a summed signal from wavelength of 214nm to 254nm. The mass spectra were recorded on a Waters ZQ Mass Spectrometer using Positive Electrospray. Ionisation data was rounded to the nearest integer. Preparative HPLC was conducted on a Gemini 5u CI 8 column (150 x 21.2 mm) eluting with acetonitrile (Solvent A) and 0.1% TFA in water (Solvent B) using the following elution gradient at a flow rate of 20ml/min and injection volume of 4ml/injection:

The UV detection was from wavelength 214 nm to 254 nm. Intermediate 1

(R)-methyl 3-(4-hydroxyphenyl)-2-(2-methoxy-2-oxoacetamido)propanoate

A suspension of D-tyrosine methyl ester (5.0 g, 0.027mol) in DCM (50 mL) was treated with DIPEA (4.45 mL, 2.0 equivalents) followed by dropwise addition of methyl chloro(oxo)acetate (3.0 mL, 1.2 equivalents) and the resultant mixture stirred at rt for 24hours. The reaction was quenched by addition of H ₂ 0 and the organics collected washed with brine, dried (MgS04) and evaporated to dryness. Purification by FC (100% DCM to 50:50 DCM:EtOAc) gave an oil which solidified on standing (4.5g, 62%). Ή NMR (400 MHz, d ₆ -DMSO) δ 9.24 (s, IH), 9.22 (d, IH), 7.01 (d, 2H), 6.65 (d, 2H), 4.47 (m, IH), 3.77 (s, 3H), 3.63 (s, 3H), 2.98 (ddd, 2H); LCMS method A, (ES+) 382, RT = 0.69 min.

Intermediate 2

(R)-3-(4-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)o xy)phenyl)-2- (carboxyformamido)propanoic acid

A solution of Intermediate 1 (300mg, 1.06 mmol) in AcN (20 mL) was treated with tert- butyl 4-(bromomethyl)benzylcarbamate (384 mg, 1.2 equivalents) and K ₂ C0 ₃ (300 mg, 2.0 equivalents) and the mixture stirred at 50°C for 24 hours. The resultant suspension was filtered and evaporated to dryness. The resultant oil was redissolved in THF (1.0 mL) and treated with NaOHaq (1.0 mol, 1.0 mL) and stirred for 24 hours. The resultant solution was filtered and evaporated to dryness to give the desired product as a thick oil (380 mg, 75%). Ή NMR (400 MHz, de-DMSO) δ 7.99 (d, IH), 7.39 (d, 4H), 7.04 (d, 2H), 6.84 (d, 2H), 5.07 (s, 2H), 4.33 (td, IH), 3.86 (s, 2H), 2.97 (dd, 2H), 1.62 (s, 9H); LCMS method A, (ES+) 472, RT 0.75 min.

Intermediate 3

Tert-butyl (2-(3-(chloromethyl)benzamido)ethyl)carbamate

To an ice-cooled solution of 3-(chloromethyl)benzoyl chloride (375 μΐ, 2.6 mmol) and DIPEA (509 μΐ, 2.86 mmol) in DCM (25 mL) was added dropwise a solution of BOC- ethylenediamine (423 mg, 2.60 mmol) in DCM (5.0 mL) over 5 minutes. The solution was allowed to warm to room-temperature and stirring was continued overnight. The mixture was then partitioned between brine (50 mL) and DCM (20 mL), the organics were collected and passed through a hydrophobic frit, and the solvent removed in vacuo to yield the product as beige solid (715 mg, 88%). Ή NMR (400 MHz, d ₆ -DMSO) δ 8.52 (t, 1H), 7.89 (d, 1H), 7.80 (d, 1H), 7.62 - 7.55 (m, 1H), 7.47 (t, 1H), 6.92 (tlH), 4.81 (s, 2H), 3.30 (q, 2H), 3.1 1 (q, 2H), 1.38 (s, 9H). LCMS method A, (ES+) 313, RT = 1.00 min

Intermediate 4

(R)-3-(4-((3-((2-((tert-butoxycarbonyl)amino)ethyl)carbam oyl)benzyl)oxy)phenyl)-2- (carboxyformamido)propanoic acid

Intermediate 1 (200 mg, 0.71 mmol) was dissolved in DMF (10 mL) containing potassium carbonate (147 mg, 1.07 mmol) at room-temperature and stirred for 30 minutes. Tert-butyl (2-(3-(chloromethyl)benzamido)ethyl) carbamate (223 mg, 0.71 mmol) was then added portion wise, followed by a catalytic amount of KI, and the resultant mixture stirring overnight. The reaction mixture was then poured into water 50 ml) and extracted with DCM (2 x 30 mL) which was passed through a hydrophobic frit and evaporated in vacuo to yield an oily residue, LCMS method A, (ES+) 558 RT = 1.02 min

This residue was dissolved in 1M NaOH _(aq) and stirred at room-temperature overnight. The volume was reduced in vacuo and the product obtained by preparative HPLC chromatography, yielding a white crystalline solid (215mg 57 %). Ή NMR (400 MHz, d ₆ - DMSO) δ 9.39 (d, 1H), 8.05 (s, 1H), 8.02 (d, 1H), 7.66 (t, 1H), 7.57 (t, 1H), 7.33 (d, 2H), 7.20 (d2H), 4.67 - 4.55 (m, 1H), 3.32 (s, 2H), 3.16 (ddd, 2H), 2.41 - 2.25 (m, 2H), 1.39 (s, 9H). LCMS method A, (ES+) 530, RT = 0.89 min

Intermediate s

(R) -Tert-butyl (2-(4-(chloromethyl)benzamido)ethyl)carbamate

3-(Chloromethyl)benzoyl chloride (151 mg, 1.07 mmol) was added dropwise to an ice- cooled solution of Intermediate 1 (300 mg, 1.07 mmol) and DIPEA (200 μΐ, 1.12 mmol) in DCM (10 mL) with rapid stirring. The reaction mixture was allowed to reach room- temperature overnight. The mixture was then poured into brine (20 mL) and DCM (10 mL) and the organics separated, washed with water (20 mL) and filtered through a hydrophobic frit before the solvent was concentrated in vacuo. The resultant residue was purified by column chromatography using a gradient of 100% DCM to 50% EtOAc : DCM. The relevant fractions were combined and evacuated to leave the product as a white solid (422 mg, 91 %). Ή NMR (400 MHz, DMSO) δ 9.40 (d, 1H), 8.20 (s, 1H), 8.09 (d, 1H), 7.82 (d, 1H), 7.63 (t, 1H), 7.34 (d, 2H), 7.21 (d, 2H), 4.91 (s, 2H), 4.61 (td, 1H), 3.78 (s, 3H), 3.67 (s, 3H), 3.16 (ddd, 2H); LCMS method A, (ES+) 434, RT = 1.12 min.

Intermediate 6

R)-Tert-butyl4-(3-((4-(3-methoxy-2-(2-methoxy-2-oxoacetam ido)-3- oxopropyl)phenoxy)carbonyl)benzyl )piperazine-l -carboxylate

Intermediate 5 (400 mg, 0.92 mmol), tert-butyl piperazine-1 -carboxylate (172 mg, 0.92 mmol) and potassium carbonate (255 mg, 1.84 mmol) were combined in DMF (10 ml) at room-temperature, catalytic I was then added to the mixture which was heated to 80°C and stirred overnight. The mixture was then poured carefully into stirring brine, yielding the product as a white precipitate which was filtered, washed with water and dried in vacuo. (140 mg, 26 %). 1H NMR (400 MHz, d ₆ -DMSO) δ 9.39 (d, 1H), 8.05 (s, 1H), 8.02 (d, 1H), 7.66 (t, 1H), 7.57 (t, 1H), 7.33 (d2H), 7.20 (d2H), 4.67 - 4.54 (m, 1H), 3.78 (s, 3H), 3.66 (s, 3H), 3.59 (s, 2H), 3.32 (m, 4H), 3.16 (ddd, 2H), 2.41 - 2.26 (m, 4H), 1.39 (s, 9H). LCMS method A, (ES+) 584, RT = 1.20 min.

Intermediate 7

(R)-3-(4-((5-((tert-butoxycarbonyl)amino)pentyl)oxy)pheny l)-2- (carboxyformamido)propanoic acid

A solution of triphenylphosphine (934 mg, 3.56 mmol) and DIAD (720 mg, 3.56 mmol) in 30 mL of DCM was stirred at rt for 0.5 h. A solution of Intermediate 4 (500 mg, 1.78 mmol) and tert-butyl (5-hydroxypentyl)carbamate (723 mg, 3.56 mmol) in 10 mL of DCM was added at 0°C and the mixture was stirred at reflux for 20 h. The mixture was diluted with EtOAc (50 mL), washed with water (50 mL), sat. aq. Na ₂ C0 ₃ solution (50 mL) and brine (50mL). The organics were dried over Na ₂ S04, filtered and concentrated, then purified by column chromatography on silica gel, using a gradient of 1 :3 to 1 :1 EtOAc : petroleum ether, to give a yellow oil that was used without further purification (470mg, 1H NMR and LCMS analysis showed that sample contained significant triphenylphosphine oxide impurity). LCMS method D, (MNa+) 489, (MH+ -Boc) 367, RT = 1.57 min.

To a solution of this yellow oil (470mg, crude) in a mixture of THF:water (30 mL, 4: 1) was added LiOH.H20 (420 mg, 10 mmol), and the mixture was stirred at rt overnight. THF was removed under reduced pressure and the residue was dissolved in water (20 mL) and washed with EtOAc (3 x 20 mL). The aqueous layer was acidified with 2N. aqueous HC1 solution and extracted with EtOAc (3 x 30 mL). The organics were washed with brine (2 x 50 mL), dried over Na ₂ S04, filtered and concentrated to give the title compound as a yellow oil (125 mg). LCMS method D, (MNa+) 461 , (MH+ -Boc) 339, RT = 1.50 min. Intermediate 8

(R)-3-(4-((5-((tert-butoxycarbonyl)amino)propyl)oxy)phenyl)- 2- (carboxyformamido)propanoic acid

A solution of Intermediate 4 (500 mg, 1.78 mmol) and tert-butyl (3- hydroxypropyl)carbamate (343 mg, 1.96 mmol) in 15 mL of DCM was added slowly to a solution of triphenylphosphine (513 mg, 1.96 mmol) and DIAD 396 mg, 1.96mmol) in 30 mL of DCM at 0°C. The mixture was then stirred at reflux for 40 h. Another portion of triphenylphosphine (513 mg, 1.96 mmol) and DIAD 396 mg, 1.96mmol) in 5mL of DCM was added and the mixture was stirred at reflux for 5 h. The mixture was diluted with EtOAc (50 mL), washed with water (50 mL), sat. aq. Na ₂ C0 ₃ solution (2 x 50 mL) and brine (50mL). The organics were dried over Na ₂ S04, filtered and concentrated, then purified by column chromatography on silica gel, using a gradient of 1 :5 to 1 : 1 EtOAc : petroleum ether, to give a yellow oil that was used without further purification (830mg, 1H NMR and LCMS analysis showed that sample contained significant triphenylphosphine oxide impurity). LCMS method D, (MNa+) 461, (MH+ -Boc) 339, RT = 1.54 min.

To a solution of this yellow oil (830mg, crude) in a mixture of THF:water (30 mL, 4:1) was added LiOH.H20 (796 mg, 19 mmol), and the mixture was stirred at rt overnight. THF was removed under reduced pressure and the residue was dissolved in water (20 mL) and washed with EtOAc (3 x 20 mL) and acidified with 2N. aqueous HCl solution to pH 3-4. The aqueous solution was extracted with EtOAc (3 x 30 mL). The organics were washed with brine (2 x 50 mL), dried over Na ₂ S04, filtered and concentrated to give the title compound as a yellow oil (190 mg). LCMS method D, (MNa+) 433, (MH+ -Boc) 311, RT = 1.30 min. Example 1

(R)-3-(4-((4-(aminomethyl)benzyl)oxy)phenyl)-2-(c rboxyformamido)propanoic acid

4M HCl in dioxane (1.0 mL, 4.0 mmol) was added to a stirring solution of (R)-3-(4-((4- (((tert-butoxycarbonyl)amino)methyl)benzyl)oxy)phenyl)-2-(ca rboxyformamido)propanoic acid (200 mg, 0.42 mmol) in THF (5 mL). Stirring was continued overnight, yielding a white precipitate which was filtered, the filtrate was evaporated to dryness, purification by prep HPLC provided the desired product as a colourless solid (30mg, 20%). Ή NMR (400 MHz, d ₆ -DMSO) δ 7.99 (d, 1H), 7.39 (d, 4H), 7.04 (d, 2H), 6.84 (d, 2H), 5.07 (s, 2H), 4.33 (td, 1H), 3.95 (s, 2H), 2.97 (dd, 2H); LCMS method A, (ES+) 372, RT = 0.69 min.

Example 2

(S)-3-(4-((4-(aminomethyl)benzyl)oxy)phenyl)-2-(carboxyfo rmamido)propanoic acid

(S)-3-(4-((4-(aminomethyl)benzyl)oxy)phenyl)-2-(carboxyforma mido)propanoic acid was prepared following the procedure for the preparation of Example 1 using L-tyrosine methyl ester in the initial reaction. 1H NMR (400 MHz, d ₆ -DMSO) δ 7.99 (d, IH), 7.39 (d, 4H), 7.04 (d, 2H), 6.84 (d, 2H), 5.07 (s, 2H), 4.33 (td, IH), 3.95 (s, 2H), 2.97 (dd, 2H); LCMS method A, (ES+) 372, RT = 0.69 min.

Example 3

(R)-3-(4-((3-((2-aminoethyl)carbamoyl)benzyl)oxy)phenyl)-2- (carboxyformamido)propanoic acid hydrochloride

4M HCl in dioxane (1.0 mL, 4.0 mmol) was added to a stirring solution Intermediate 4 (200 mg, 0.37 mmol) in DCM (5.0 mL). Stirring was continued overnight, yielding a white precipitate which was collected by filtration, washed with cold DCM and dried (95mg, 52%). Ή NMR (400 MHz, d ₆ -DMSO) δ 8.87 (t, lH), 8.84 - 8.75 (m, 1H), 8.08 (s, 3H), 8.00 (s, 1H), 7.90 (t, 1H), 7.66 - 7.56 (m, 1H), 7.49 (t, 1H), 7.17 (d, 2H), 6.94 (d, 2H), 5.77 (s, 2H), 5.1 1 (s, 2H), 4.42 (td, 1H), 3.56 - 3.49 (m, H), 3.16 - 2.85 (m, 4H); LCMS method A, (ES+) 430 RT = 0.71 min.

Example 4

(R)-4-(3-methoxy-2-(2-methoxy-2-oxoacetamido)-3-oxopropyl)ph enyl3-(piperazin-l- ylmethyl)benzoate

4 M HCl in dioxane (1.0 ml) was added to a solution of Intermediate 6 (100 mg, 0.17 mmol) in DCM (3.0 ml) which was stirred overnight. The reaction mixture was then completely evacuated in vacuo, providing the desired product as a pinkish crystalline solid (32 mg, 32 %) . Ή NMR (400 MHz, d ₆ -DMSO) δ 8.24 (s, 1H), 8.18 (d, 1H), 7.88 - 7.79 (m, 1H), 7.67 (t, 1H), 7.33 - 7.23 (m, 2H), 7.15 (d, 2H), 4.59 (dd, 1H), 4.37 (s, 2H), 3.74 (s, 3H), 3.61 (t, 3H), 3.35 (s, 8H), 3.26 - 2.93 (m, 2H); LCMS method A, (ES+) 484, RT = 0.99 min Example 5

(R)-2-(carboxyformamido)-3-(4-((3-^iper∑in-]-ylmethyl)benz oyl)oxy)phenyl)propanoic acid

A solution of Example 4 (25 mg, 0.052mmol) in THF (0.5 mL) was treated with NaOHaq (0.25 mL) And the reaction stirred at room temperatre for 24 hours, the resultant mixture was evaporated to dryness and purified by prep HPLC to give the desired product (2.0 mg, 8%)

LCMS method A, (ES+) 456, RT = 0.66min

Example 6

(R)-3-(4-((5-aminopentyl)oxy)phenyl)-2-(carboxyformamido)pro panoic acid

.TFA To a solution of Intermediate 7 (125 mg, 0.29 mmol) in 20 mL of anhydrous DCM was added TFA (1 mL). The mixture was stirred at rt overnight. The mixture was concentrated and purified by preparative HPLC to give the title compound as a white solid (50 mg). LCMS method D, (MH+) 339, RT = 1.05 min.

Example 7

(R)-3-(4-((5-aminopropyl)oxy)phenyl)-2-(carboxyformamido)pro panoic acid

To a solution of Intermediate 8 (220 mg, 0.5 mmol) in 20 mL of THF was added concentrated HC1 (2 mL) and the solution was stirred at rt overnight. The solvent was evaporated and the residue was purified by preparative HPLC to give the title compound as a white solid (80 mg). LCMS method D, (MH+) 31 1, RT = 0.84 min.

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/ml 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.

Biological examples

Bead assay using immobilized compound 1 and a mix of HeLa and Jurkat cell lysates (Experiment X011753)

This example demonstrates the use of immobilized compound 1 (example 1) for the capturing and identification of histone demethylases from cell lysate in a competition binding assay. To the first aliquot of cell lysate 200 μΜ of the free compound 1 was added and allowed to bind to proteins in the lysate. Then the affinity matrix with the immobilized compound 1 was added to capture proteins that were not interacting with the previously added free compound. 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 free compound was added (DMSO solvent control). Peptides originating from samples 1 and 2 were labeled with iTRAQ reagents (iTRAQ 1 14 and iTRAQ 115) 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 previous publications (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044; Bantscheff et al., 201 1. Nat. Biotechnol. 29, 255-265).

The identified histone demethylases are shown in Table 6 including the percent competition values for the sample to which 200 μΜ free compound had been added. Two different histone demethylases were identified and competed by different degrees. For illustration, the identified peptides for HIF1AN and JMJD6 are shown in Figures 1 and 2. Sequence identifiers are defined by the International Protein Index (IPI) (Kersey et al., 2004. Proteomics 4(7): 1985-1988).

1. Cell culture

Jurkat cells (ATCC number TIB- 152) and HeLa cells (ATCC number CCL-2.2) 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) at a density between 0.2 x 10 ⁶ and 1.0 x 10 ⁶ cells/ml. 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 ly sates

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 MgCl ₂ , 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 histone demethylases from cell lysate

Sepharose-beads with the immobilized compound (100 μΐ beads per pull-down experiment) were equilibrated in lysis buffer and incubated with a cell lysate sample containing 50 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 triethylammonium 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 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 5: 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 spectrometry. 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 6: Identified histone demethylases with compound 1 from mixed HeLa and Jurkat cell lysates

Representative Protein Protein Quantified % Competition Sequence Name Domain Spectra

IPI00299906.4 HIF1AN JmjC 21 94.8

IPI00604598.1 JMJD6 JmjC 3 58.9

Table 7: Pre aration of 5x-DP buffer

The 5x-DP buffer was filtered through a 0.22 μπι 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 MgCl ₂ (Sigma, M-1028); 5.0 M NaCl (Sigma, S-5150).

Bead assay using immobilized compound 1 and HL60 nuclear extract (Experiment X012215)

This example demonstrates the use of immobilized compound 1 (example 1) for the capturing and identification of histone demethylases from cell lysate in (nuclear extract) a competition binding assay. To the first aliquot of cell lysate 200 μΜ of the free compound 1 was added and allowed to bind to proteins in the lysate. Then the affinity matrix with the immobilized compound 1 was added to capture proteins that were not interacting with the previously added free compound. 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 free compound was added (DMSO solvent control). Peptides originating from samples 1 and 2 were labeled with iTRAQ reagents (iTRAQ 1 15 and iTRAQ 117) 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 previous publications (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044; Bantscheff et al., 201 1. Nat. Biotechnol. 29, 255-265). The identified histone demethylases are shown in Table 8 including the percent competition values for the sample to which 200 μΜ free compound had been added. Three different histone demethylases were identified and competed by different degrees. For illustration, the identified peptides for JMJD2A and HIF1 AN are shown in Figures 3 and 4. Sequence identifiers are defined by the International Protein Index (IPI) (Kersey et al., 2004. Proteomics 4(7): 1985-1988).

Cell culture

Human HL-60 cells (DSMZ, Braunschweig, Germany; DSMZ number ACC3) 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) at a density between 0.2 x 10 ⁶ and 1.0 x 10 ⁶ cells/ml.

Preparation of cell lysates (nuclear extracts)

Cells were harvested by centrifugation for 6 minutes at 2,370 rpm (Sorvall R12BP, Newtown, CO, USA) and washed twice in Phosphate Buffer Saline (137 mM NaCl (Sigma S5150), 2.7 mM KC1 (Merck 1.04936), 8 mM Na ₂ HP0 ₄ (Sigma S7907), 1.46 mM KH ₂ PO ₄ (Sigma P5504)). Washed cells were centrifuged for 5 minutes (first wash) or 10 minutes (second wash) at 2,370 rpm (Heraeus centrifuge 75004375). The cell pellet was resuspended in two volumes of hypotonic buffer (10 mM TRIS-Cl, pH 7.4, 1.5 mM MgCl ₂ (Sigma M-1028), 10 mM KC1 , 25 mM NaF (Sigma S7920), 1 mM Na ₃ Vo ₄ (Sigma S6508), 1 mM DTT (Biomol 04010, Plymouth Meeting, PA, USA). The cells were allowed to swell for 10 minutes (swelling checked under microscope) and homogenized by 20 strokes in a homogenizer (VWR SCERSP885300-0015, Radnor, PA, USA) and the homogenate was centrifuged for 10 minutes at 3,300 rpm (Heraeus centrifuge). The supernatant was discarded and the pellet was resuspended in one volume of extraction buffer (50 mM TRIS-Cl, pH 7.4, 1.5 mM MgCl ₂ , 20 % glycerol (Merck Z835091), 420 mM NaCl (Sigma S5150), 25 mM NaF, 1 mM Na ₃ V0 ₄ , 1 mM DTT, 400 units/ml of DNAsel (Sigma D4527), and protease inhibitors (1 tablet for 25 ml; Roche, 13137200, Basel, Switzerland)) and the homogenate was incubated for 30 minutes with gentle mixing at 4°C. The homogenate was then diluted in dilution buffer (1.8 ml buffer per 1 ml supernatant; 50 mM TRIS-Cl, pH 7.4, 1.5 mM MgCl ₂ , 25 mM NaF, 1 mM Na ₃ V0 ₄ , 0.6 % Igepal CA-630 (Sigma, 13021), 1 mM DTT and protease inhibitors (1 tablet for 25 ml)). After 10 minutes incubation on ice, the lysate was centrifuged for one hour at 33,500 rpm in a ΤΪ50.2 rotor (Beckman Coulter LE90K, 392052, Brea, CA, USA) and the supernatant was frozen in liquid nitrogen and stored at -80°C. After thawing of the nuclear lysate the protein concentration was adjusted to 5 mg/ml. The final buffer composition was 50 mM TRIS pH 7.4, 5% Glycerol, 150 mM NaCl, 25 mM NaF, 1.5 mM MgCl ₂ , 0.4% Igepal CA- 630, 1 mM DTT and protease inhibitors (1 tablet for 25 ml lysate). The lysate was then submitted to ultracentrifugation at 33,500 rpm for 20 minutes in a Ti50.2 rotor.

The capturing of histone demethylases from cell lysate and the protein identification by mass spectrometry was performed as described in the previous example. Table 8: Identified histone demethylases with compound 1 from mixed HL60 cell lysate

Representative Protein Protein Quantified % Competition

Sequence Name Domain Spectra

IPI00005666.1 JMJD2A TUDOR, PHD, JmjN, JmjC 5 49.8

IPI00013205.6 JMJD2B TUDOR, PHD, JmjN, JmjC 13 69.9

IPI00299906.4 HIF1AN JmjC 5 86.1