WO2012150042A1 - Composés et procédés pour identifier des molécules ayant une interaction avec les histones déméthylases et pour purifier des protéines d'histone déméthylase - Google Patents

Composés et procédés pour identifier des molécules ayant une interaction avec les histones déméthylases et pour purifier des protéines d'histone déméthylase Download PDF

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Publication number
WO2012150042A1
WO2012150042A1 PCT/EP2012/001926 EP2012001926W WO2012150042A1 WO 2012150042 A1 WO2012150042 A1 WO 2012150042A1 EP 2012001926 W EP2012001926 W EP 2012001926W WO 2012150042 A1 WO2012150042 A1 WO 2012150042A1
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compound
histone demethylase
histone
immobilization product
immobilization
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PCT/EP2012/001926
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English (en)
Inventor
Jay Freeman
John Harrison
Ulrich Kruse
Robert John SHEPHERD
David Matthew Wilson
Michael David Woodrow
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Cellzome Ag
Glaxo Group Limited
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Publication of WO2012150042A1 publication Critical patent/WO2012150042A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • JmjC domain proteins 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-
  • 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).
  • 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).
  • histone demethylase activity is typically performed using enzyme assays.
  • 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).
  • 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;
  • n 0, 1, or 2;
  • n 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 );
  • R 5 ; R 5a ; R 5b are independently selected from the group consisting of H; and Ci -6 alkyl.
  • 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.
  • 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.
  • 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.
  • 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 .
  • an optionally substituted phenylene is unsubstituted.
  • 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; C3 ⁇ 4; CF 3 ; and OCH 3 .
  • 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.
  • the present invention also includes all tautomeric and stereoisomeric forms and mixtures thereof in all ratios, and their pharmaceutically acceptable salts.
  • 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.
  • R 1 and R 2 are the same.
  • R 1 and R 2 are H; or CH 3 .
  • n is 1.
  • m is 0.
  • X 1 is *OC(0); or O.
  • ol is 0.
  • o2 is 0; or 1.
  • X 3 is a chemical bond; or C(0)NH.
  • o3 is 0 or 2.
  • X 4 is NH 2 .
  • X 4 is
  • X 2 is an optionally substituted phenylene; or optionally substituted 5 to 6 membered heteroarylene. More preferably, X 2 is an optionally substituted phenylene.
  • o2 is 0 and X 3 is a chemical bond.
  • a preferred compound of formula (I) is a compound, wherein
  • n 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.
  • the invention also comprises their corresponding salts.
  • 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.
  • acids 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.
  • 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.
  • the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).
  • 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
  • the immobilization compounds of the present invention can be prepared by methods well known in the art.
  • 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.
  • 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.
  • at least one immobilization compound of the invention is immobilized on a solid support.
  • solid support relates to every undissolved support being able to immobilize a small molecule ligand on its surface.
  • 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.
  • one or two different immobilization compounds are immobilized on the solid support, more preferably the preferred immobilization compounds of formula I)
  • 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.
  • 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.
  • the immobilization compound or compounds are covalently coupled to the solid support.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention relates inter alia to an immobilization product comprising a compound of formula (I)
  • Rl, R2 are independently selected from the group consisting of H; Cl-6 alkyl; n is 0, 1 , or 2;
  • n 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
  • 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);
  • 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.
  • the immobilisation product of the invention comprising a compound of formula (I) is characterized as defined above.
  • 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.
  • the immobilization compound or immobilization product of the invention may further be labeled.
  • label 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.
  • 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.
  • fluorescent tags e.g. fluorescein, rhodamine, dansyl, NBD (nitrobenz-2-oxa-l,3-diazole), BODIPY (dipyrromethene boron difluoride), and cyanine (Cy)-dyes
  • fluorescent tags e.g. fluorescein, rhodamine, dansyl, NBD (nitrobenz-2-oxa-l,3-diazole), BODIPY (dipyrromethene boron difluoride), and cyanine (Cy)-dyes
  • fluorescent probes fluorophores
  • HTS high throughput screening
  • the ALPHAScreen technology can be used where the excitation of a donor bead at 680 nm produces singlet oxygen which can diffuse to an acceptor bead undergoing a chemi luminescent reaction (Glickman et al., 2002. J. Biomol. Screen. 7(1):3-10).
  • the present invention also relates to such methods and uses.
  • 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.
  • 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).
  • 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).
  • the invention in a fourth aspect, 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,
  • 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.
  • 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.
  • histone demethylases e.g. JMJD6, JMCD2A, JMJD2B, or HIFIAN
  • histone demethylase denotes a protein that contains a Jumonji C (JmjC) domain.
  • 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).
  • these immobilization products can be used to identify compounds binding to histone demethylases.
  • 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).
  • 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.
  • 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.
  • histone demethylase includes mutant forms said histone demethylases.
  • 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.
  • aliquots of a cell preparation are provided as the starting material.
  • 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.
  • MS mass spectrometry
  • 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).
  • cell organelles e.g. nucleus, mitochondria, ribosomes, golgi etc.
  • 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).
  • protein preparations from body fluids can be used (e.g. blood, cerebrospinal fluid, peritoneal fluid and urine).
  • 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).
  • 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.
  • lymphocytes and lymphocyte subpopulations obtained 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.).
  • 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).
  • 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.
  • 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).
  • the cell containing the histone demethylases or the histone demethylase may be obtained from an organism, e.g. by biopsy.
  • an organism e.g. by biopsy.
  • 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.
  • the lysis is performed simultaneously.
  • the cell is first harvested and then separately lysed.
  • 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.
  • homogenizers e.g. Potter-homogenizer
  • ultrasonic desintegrators e.g. Potter-homogenizer
  • enzymatic lysis e.g. NP-40, Triton X-100, CHAPS, SDS
  • detergents e.g. NP-40, Triton X-100, CHAPS, SDS
  • osmotic shock repeated freezing and thawing, or a combination of these methods.
  • 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.
  • 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.
  • 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 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.
  • the binding between the immobilization product and the histone demethylase is, e.g., via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • "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 can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (volume/volume).
  • 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.
  • "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
  • the buffer is selected from the group consisting of Tris-HCl or HEPES.
  • 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.
  • 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).
  • 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.
  • 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.
  • the compound is presumably a strong interactor with the histone demethylase, which is indicative for its therapeutic potential.
  • the amount of the complex formed during the method is determined.
  • 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.
  • the complex between one histone demethylase and the immobilization product is detected by determining its amount.
  • the histone demethylase are separated from the immobilization product in order to determine the amount of said complex.
  • 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).
  • ionic strength, pH value, detergents ionic strength, pH value, detergents.
  • it can be tested whether a compound of interest can specifically elute the histone demethylase from the immobilization compound.
  • 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).
  • extremes of pH value high or low pH; e.g. lowering pH by
  • 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.
  • magnetic beads can be used as solid support so that the beads can be eliminated from the sample by using a magnetic device.
  • 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.
  • 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.
  • an antibody in order to detect a histone demethylase or in order to determine its amount (e.g. via ELISA)
  • 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).
  • such antibodies are known in the art.
  • the skilled person is aware of methods for producing the same.
  • a histone demethylase is detected or the amount of a histone demethylase is determined by mass spectrometry or immunodetection methods.
  • mass spectrometry mass spectrometry, MS
  • MS mass spectrometry
  • 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.
  • 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).
  • 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).
  • SILAC amino acids in cell culture
  • 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).
  • the characterization by mass spectrometry 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the identification methods of the invention involve the use of compounds which are tested for their ability to be a histone demethylase interacting compound.
  • 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.
  • the compound has an effect on the histone demethylase, e.g. a stimulatory or inhibitory effect.
  • 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.
  • 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.
  • 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.
  • 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” 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.
  • 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.
  • 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.
  • the histone demethylase containing protein preparation is first incubated with the compound and then with the immobilization product.
  • 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).
  • 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.
  • compounds are used at concentrations ranging from 1 nM to 1 mM, preferably from 1 nM to 100 ⁇ , preferably from 1 nM to 10 ⁇ .
  • the second step, contacting with the immobilized ligand, is preferably performed for 10 to 60 minutes at 4°C.
  • 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.
  • compounds are used at concentrations ranging from 1 nM to 1 mM, preferably from 1 nM to 100 ⁇ , preferably from 1 nM to 10 ⁇ .
  • 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.
  • the amount of the complex formed in step c) is compared to the amount formed in step b)
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the embodiments as defined above for the identification methods of the invention also apply to the purification method of the invention.
  • the purification method of the invention further comprises after step c) the identification of proteins being capable of binding to said histone demethylases.
  • 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.
  • 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.
  • 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.
  • 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.
  • the histone demethylase contains at least one mutation.
  • 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.
  • 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).
  • 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.
  • 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).
  • FIG. 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).
  • FIG. 2 Amino acid sequence of human JMJD6 (IPI00604598.1). Peptides identified by mass spectrometry are underlined (Experiment X01 1753; HeLa and Jurkat cells).
  • FIG. 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).
  • 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 GeminiTM 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.
  • LC/MS Method D was conducted on an Acquity UPLC BEH CI 8 column (50 mm x 4.6 mm i.d. 2.7 ⁇ 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 UV detection was from wavelength 214 nm to 254 nm.
  • Example 4 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%)
  • 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.
  • 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.
  • immobilized compound 1 (example 1) for the capturing and identification of histone demethylases from cell lysate in a competition binding assay.
  • the identified histone demethylases are shown in Table 6 including the percent competition values for the sample to which 200 ⁇ 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).
  • IPI International Protein Index
  • 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.
  • 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 ⁇ 50.2, 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.
  • Sepharose-beads with the immobilized compound 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 ⁇ 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).
  • 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 ⁇ 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/ ⁇ in 5mM TEAB. Digestion was allowed to proceed for 4 hours at 37°C and the reaction was subsequently stopped using 5 ⁇ 5% formic acid.
  • TEAB triethylammonium bicarbonate buffer
  • the peptide extracts of samples treated with 200 ⁇ 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 ⁇ 50 mM TEAB solution, pH 8.5 and 10 ⁇ ethanol were added.
  • the iTRAQ reagent was dissolved in 120 ⁇ ethanol and 10 ⁇ 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 ⁇ 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 ⁇ of 0.1% formic acid in water.
  • 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
  • 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.
  • IPI International Protein Index
  • 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).
  • Table 6 Identified histone demethylases with compound 1 from mixed HeLa and Jurkat cell lysates
  • the 5x-DP buffer was filtered through a 0.22 ⁇ 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).
  • 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.
  • immobilized compound 1 (example 1) for the capturing and identification of histone demethylases from cell lysate in (nuclear extract) a competition binding assay.
  • the identified histone demethylases are shown in Table 8 including the percent competition values for the sample to which 200 ⁇ 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).
  • IPI International Protein Index
  • 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.
  • 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).
  • 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 homo
  • 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.
  • 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
  • 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)).
  • 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)
  • the lysate was centrifuged for one hour at 33,500 rpm in a ⁇ 50.2 rotor (Beckman Coulter LE
  • 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.

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Abstract

Cette invention concerne des composés d'immobilisation, des produits d'immobilisation et leurs préparations ainsi que des procédés et des utilisations pour identifier des composés ayant une interaction avec les histones déméthylases ou pour purifier ou pour identifier des protéines d'histone déméthylase.
PCT/EP2012/001926 2011-05-04 2012-05-04 Composés et procédés pour identifier des molécules ayant une interaction avec les histones déméthylases et pour purifier des protéines d'histone déméthylase WO2012150042A1 (fr)

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WO2018106984A1 (fr) 2016-12-09 2018-06-14 Constellation Pharmaceuticals, Inc. Marqueurs pour un traitement personnalisé du cancer avec des inhibiteurs de lsd1

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018106984A1 (fr) 2016-12-09 2018-06-14 Constellation Pharmaceuticals, Inc. Marqueurs pour un traitement personnalisé du cancer avec des inhibiteurs de lsd1

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