WO2013023770A1 - Essai de profilage de chromatine - Google Patents

Essai de profilage de chromatine Download PDF

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WO2013023770A1
WO2013023770A1 PCT/EP2012/003421 EP2012003421W WO2013023770A1 WO 2013023770 A1 WO2013023770 A1 WO 2013023770A1 EP 2012003421 W EP2012003421 W EP 2012003421W WO 2013023770 A1 WO2013023770 A1 WO 2013023770A1
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protein
compound
chromatin
histone
proteins
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Gerard Drewes
Marcus Bantscheff
Antje Dittmann
Philipp RATHERT
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Cellzome Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2522/00—Reaction characterised by the use of non-enzymatic proteins
    • C12Q2522/10—Nucleic acid binding proteins
    • C12Q2522/101—Single or double stranded nucleic acid binding proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • the present invention relates to methods for the identification and characterization of proteins that are associated with or bound to chromatin as well as to the identification and characterization of compounds interacting with said proteins.
  • Chromatin is the state in which DNA is packaged within the cell.
  • the nucleosome is the unit of chromatin and it consists of an octamer of the four core histone proteins (dimers of histone H3, H4, H2A and H2B) around which 147 base pairs of DNA are wrapped.
  • the histones are small, basic proteins rich in arginine and lysine residues resulting in a high affinity for DNA.
  • Histone H3 and H4 are among the most conserved proteins known.
  • Each of the core histones has a histone fold domain and a flexible N-terminal tail (H2A and H2B also have C-terminal tails) which contains sites for covalent modification that are important for chromatin function. Most of these modifications occur on the N-terminal unstructured part of the histone - particularly on histone H3 and H4 - that protrudes like a tail from the nucleosome, hence the name of "histone tails" (Kouzarides, 2007. Cell 128(4):693-705).
  • H3K4me3 plant homeodomain (PHD) finger-containing proteins have recently been identified as readers of trimethylation of H3K4 (H3K4me3). Misinterpretation of H3K4me3 by leukemia-associated translocations of PHD finger factors (NUP98-JARID1A or NUP98-PHF23) is crucial for the induction of leukemia. Somatic mutations of ING1, a PHD finger factor, interfere with the reading of H3K4me3 and associate with the development of squamous cell carcinoma, head and neck squamous cell carcinoma and melanoma (Chi et al., 2010. Nat. Rev. Cancer. 10(7):457-69).
  • histone modifying enzymes and proteins that recognize these modifications
  • HDACs histone deacetylases
  • HDACs typically require the availability of purified or recombinant HDACs. However, not all HDACs can be produced will sufficient enzymatic activity to allow for inhibitor screening (Blackwell et al., 2008. Life Sciences 82(21-22): 1050- 1058). In addition, some preparations of HDACs expressed in insect cells are contaminated with endogenous insect HDACs making the interpretation of assay results ambiguous.
  • HDAC inhibitors Another, although not in all instances necessary prerequisite for the identification of selective HDAC inhibitors is a method that allows for the determination of the target selectivity of these molecules. For example, it can be intended to provide molecules that bind to and inhibit a particular drug target but do not interact with a closely related target, inhibition of which could lead to unwanted side effects. Conventionally, large panels of individual enzyme assays are used to assess the inhibitory effect of a compound for HDACs (Khan et al., 2008. Biochem. J. 409(2):581-9; Blackwell et al., 2008. Life Sci. 82(21-22): 1050-1058).
  • 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).
  • 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).
  • the present invention relates to a method for the identification of a protein being capable of interacting with a given compound, comprising the steps of a) providing a chromatin preparation, b) incubating the chromatin preparation with a buffer comprising a given
  • the present invention it is possible to very efficiently identify proteins being capable of interacting with a given compound, as shown in the examples of the present invention.
  • the given compound is a compound identified in a phenotypic assay. Otherwise, the compound may be as defined below.
  • phenotypic screening assay relates to any kind of assay in which the response of an organism or live cells to a test compound is measured (Clemons et al., 2004. Curr. Opin. Chem. Biol. 8(3):334-338).
  • the cell viability may be measured after treatment with a test compound.
  • the present invention relates to a method for the identification of a compound being capable of interacting with a chromatin-associated protein, comprising the steps of a) providing a chromatin preparation, b) incubating the chromatin preparation with a buffer comprising a compound of interest and a solvent under conditions allowing for the displacement of at least one protein associated with said chromatin preparation, and c) detecting the at least one protein displaced in step b).
  • chromatin preparation refers to any kind of composition containing chromatin.
  • the chromatin preparation of the present invention is preferably an aqueous composition comprising chromatin isolated from a tissue or a cell.
  • providing a chromatin preparation refers to all sorts of procedures suitable to prepare the chromatin preparation of the present invention. These procedures include, but are not limited to, the preparation of whole cell extracts derived from any kind of organism containing chromatin, the preparation of total cell extracts from particular species of cells, e.g., grown in culture, and containing chromatin, including, but not limited to, the preparation of total cell extracts derived from a particular host cell expressing a recombinant protein of interest, or the preparation of a protein composition by any other means.
  • the preparation of a cell extract may include all necessary steps of harvesting a cell from a culture by means of, e.g., diverse centrifugation and/or fractionation steps, or the steps of lysing the cells by either mechanical or chemical means including, but not limited to, e.g., multiple freezing and/or thawing cycles and/or ultrasonic treatment of the cells.
  • provision of the chromatin preparation according to the present invention includes the steps of harvesting at least one cell and isolating the nucleus thereof.
  • Cell lysates or partial cell lysates can e.g. 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 (Castle, J. D. 2004. Purification of Organelles from Mammalian Cells. Current Protocols in Protein Science. 4.2.1 ⁇ 1.2.57).
  • cell organelles e.g. nucleus, mitochondria, ribosomes, golgi etc.
  • 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 lysis is performed simultaneously.
  • the cell is first harvested and then separately lysed.
  • the at least one cell is pharmacologically treated with a substance before the chromatin preparation is carried out.
  • said substance is an HDAC inhibitor, a methyltransferase inhibitor, a peptide, a growth factor, or an antibody.
  • HDAC inhibitor refers to any kind of compound that interferes with the function of a histone acetylase. HDAC inhibitors are known in the art (Hauser et al., 2009. Curr. Top. Med. Chem. 9(3):227-234).
  • SAHA a HDAC inhibitor such as SAHA
  • the treatment with a substance may include serum starvation of cells and subsequent stimulation by adding serum or defined growth factors.
  • Serum starvation of cells is carried out in a cell culture medium that contains low serum concentrations relative to normal growth medium, typically 0.1 to 0.5% serum.
  • step b) of the claimed methods the chromatin preparation is incubated with a buffer comprising the compound and a solvent.
  • buffer generally refers to any kind of water-based or aqueous solution containing water.
  • Water-based solution or aqueous solution means that the solvent is predominantly water (more that 50 %, 60 %, 75 %, 80 %, preferably more than 90 %, more preferably more than 95 %, or even 100 % of the solvent is water).
  • the solvent is preferably water.
  • the solvent may comprise other solvents as preferably DMSO. The other solvents may e.g. be present at an amount of 1 %, 2 %, 5 %, or 10 %.
  • composition of the present invention may be present in the composition of the present invention such as salts, proteins, cellular components, ingredients of a culture media etc..
  • the buffer used in the context the present invention comprises in some steps a given compound and a solvent.
  • the buffer comprises a solvent but not a given compound.
  • steps a) to c) of the methods of the invention are repeated but using in step b) a buffer comprising the solvent but not the compound.
  • said buffer is used as a control.
  • steps a) to c) of the methods of the invention are repeated using in step b) a buffer comprising the given compound in a different concentration and the solvent. This enables inter alia the determination of the affinity of the compound for the protein or the determination of the selectivity profile of the compound.
  • the term "compound” generally refers to any molecule which is able to interact with a chromatin-associated protein and able to modulate its association or binding to the chromatin.
  • the compound is able to partially or completely inhibit the association or binding of at least one protein with chromatin.
  • the compound is able to enhance the binding of the protein to chromatin leading to a stabilization of the chromatin - protein complex.
  • 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.
  • 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.
  • the compound of the invention may also be a peptide.
  • the peptide may further modified, for example the peptide may be acetylated or methylated.
  • the peptide is a histone tail.
  • histone tail denotes the flexible aminoterminal regions of the core histones (H2A, H2B, H3, H4) and the flexible carboxyterminal regions of histones H2A and H2B that extend beyond the surface of the nucleosome (Jufvas et al., 201 1. PLoS One 6(l):el5960).
  • the peptide is located within the 40 aminoterminal amino acid residues of a histone tail.
  • this peptide comprises 5 to 20 amino acid residues.
  • the histone tail comprises the carboxyterminal amino acid residues of histones H2A and H2B.
  • At least one of the amino acids of the histone tail has been further modified (Jufvas et al., 201 1. PLoS One 6(1 ):el 5960).
  • At least one lysine is acetylated (Kac) on the free epsilon amino group.
  • At least one lysine is mono-methylated (Kmel), di-methylated (Kme2) or tri- methylated ( me3).
  • At least one arginine is mono-methylated (Rmel), asymmetrically di- methylated (Rme2a) or symmetrically di-methylated (Rme2s).
  • Equally preferred is a combination of several modifications as defined above on one histone tail peptide.
  • the methods of the invention do not include any purification step after step b) involving the given compound.
  • the term "displacement” relates to the fact that the respective protein is dissociated from the chromatin preparation. It can be tested whether a given compound of interest can specifically displace a protein from its associated chromatin.
  • the term "under conditions allowing for the displacement of at least one protein” includes all conditions under which a displacement of a protein from an associated structure such as chromatin is possible. Such conditions are known to the person skilled in the art.
  • the incubation of the chromatin preparation with the buffer according to the methods of the present invention is performed under essentially physiological 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.
  • 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.
  • 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 C1, pH 6.5-8.5, 20-37°C, and 0.001-10 mM divalent cation (e.g.
  • a non-ionic detergent (Tween, NP-40, Triton-XlOO) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (volume/volume).
  • the following buffered aequous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HC1, pH5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents.
  • "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.
  • the fact that a protein is displaced from the chromatin preparation indicates that the compound interacts with the protein.
  • the term "interacting" includes both direct or indirect binding of the compound to the chromatin-associated protein.
  • the term “interacting” is to be understood that the interactions occurs via a further chromatin-associated protein.
  • the compound binds directly or indirectly to the protein.
  • the compound binds directly to the protein.
  • step c) includes the determination of the amount of the at least one protein.
  • an increased amount of the at least one protein displaced in the presence of the compound in comparison to the amount of the at least one protein displaced in the absence of the compound indicates that the protein interacts with the compound.
  • the detecting or the determining of the amount of the at least one displaced protein is carried out by mass spectrometry
  • mass spectrometry The identification of proteins with mass spectrometric analysis (mass spectrometry) is known in the art (Shevchenko et al., 1996. Analytical Chemistry 68: 850-858; Mann et al., 2001. Annual Review of Biochemistry 70: 437-473) and is further illustrated in the Example section.
  • bottom-up mass spectrometric approaches are employed to identify displaced proteins. These approaches use enzymatic proteolysis of proteins and subsequent mass spectrometric identification of the generated peptides and are known in the art (Shevchenko et al., 1996.
  • the displaced proteins are separated prior to mass spectrometric analysis by using methods known in the field such as: SDS-gel electrophoresis, isoelectric focussing and chromatography (Capriotti et al., 201 1. J. Chromatogr. A., 1218(49):8760- 76; Ly and Wasinger, 201 1. Proteomics 1 1(4):513-534).
  • proteolytically generated peptides are separated prior to mass spectrometric analysis by using reversed-phase chromatography and other chromatographic or electrophoretic methods known in the art. (Donato et al., 201 1. J. Chromatogr. A., 1218(49):8777-90; Ly and Wasinger, 201 1. Proteomics 1 1(4):513-534).
  • 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. 1(5): 376-386).
  • SILAC stable isotope labeling by 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 quantification) or TMT isobaric tagging reagent can be used.
  • the iTRAQ and TMT reagents are sets of multiplexed, amine-specific, stable isotope reagents that can label peptides in up to six (TMT) or eight (iTRAQ) different biological samples enabling simultaneous identification and quantification of peptides.
  • TMT six
  • iTRAQ eight
  • the samples are analyzed with a nano-flow liquid chromatography system coupled online to a tandem mass spectrometer (LC-MS/MS) experiment followed by reporter ion quantification in the MS/MS spectra (Ross et al., 2004. Mol. Cell. Proteomics 3(12): 1154-1 169; Dayon et al., 2008.
  • clCAT cleavable isotope-coded affinity tags
  • any other stable isotope containing chemical labeling reagent can be used (Bantscheff et al., 2007; Anal. Bioanal. Chem. 389(4): 1017-1031).
  • label-free mass spectrometric methods for relative or absolute protein quantification including but not limited to spectrum counting, S/MRM and extracted ion chromatogram (XIC) based approaches might be used (Neilson et al., 201 1. Proteomics l l(4):535-553).
  • the mass spectrometric analysis is carried out in a non-directed way ("shot gun approach").
  • the instrument is programmed to analyse as many peptide ion signals as possible, thus enabling the unbiased analysis of displaced proteins (Domon and Aebersold, 2010. Nat. Biotechnology 28(7):710-721).
  • the mass spectrometric analysis is carried out in a directed or targeted way by either programming the instrument to specifically analyse only a subset of proteins by methods including but not restricted to accurate inclusion mass screening, accurate mass and retention time tags, selected reaction monitoring and other methods known in the art (Domon and Aebersold, 2010. Nat. Biotechnology 28(7):710-721).
  • the displaced protein (including binding partners such as regulatory subunits), can be detected or its amount can be determined by using a specific antibody directed against said protein.
  • Suitable antibody-based assays include but are not limited to Western blots, ELISA assays, sandwich ELISA assays and antibody arrays or any 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).
  • the at least one protein is a protein modifying enzyme.
  • protein modifying enzyme generally includes all enzymes that are capable of transferring a post-translational modification (PTM) to a protein.
  • PTM post-translational modification
  • a protein modifying enzyme is preferably a histone modifying enzyme.
  • histone modifications are acetylation of lysines, methylation of lysines and arginines, phosphorylation of serines, threonines and tyrosines, ubiquitinylation of lysines and transformation of arginine to citrulline. With the exception of the citrullination, all these modifications are reversible.
  • Acetylation by the creation of a negative charge has a tendency to displace the tail from the nucleosome and the DNA and favours gene transcription. Methylation does not create a new charge and can promote or inhibit transcription. Phosphorylation has often a negative impact on the role of nearby histone marks.
  • PTMs covalent posttranslational modifications
  • enzymes classified as "writers” e.g. acetyltransferases
  • erasers e.g. deacetylases
  • Proteins binding to and recognizing these modifications are referred to as "readers” (e.g. by bromodomain proteins that bind to acetylated lysine).
  • HATs histone acetyl transferases
  • HDACs histone deacetylases
  • Methyl groups are added by protein methyl transferases (PTMs) and removed by protein demethylases (PDMs).
  • Lysine residues can be mono-, di- or tri-methylated by protein lysine methyltransferases (PKMTs) and arginine residues can be mono- or di-methylated by protein arginine methyl transferases (PRMTs).
  • the dimethylation on arginine can be asymmetrical or symmetrical. Lysine and arginine methylation of histone tails can either activate or repress gene transcription.
  • PADs Protein arginine deiminases catalyze the conversion of arginine residues to citrulline residues in proteins. In humans five highly related calcium-dependent PADs exist (PAD1-PAD4 and PAD6).
  • H3S10 histone 3
  • Ubiquitin moieties can be added to histones (mono- or poly-ubiquitination) by protein ubiquitin ligases (E3 ligases) and removed by protein ubiquitin carboxyl-terminal hydrolases (UCHs).
  • E3 ligases protein ubiquitin ligases
  • UCHs protein ubiquitin carboxyl-terminal hydrolases
  • sumoylation is a large modification and shows some similarity to ubiquitylation. All four core histones can be sumoylated and specific sites have been identified on H4, H2A, and H2B. Sumoylation antagonizes both acetylation and ubiquitylation, which occurs on the same lysine residue, and consequently represses transcription in yeast (Nathan et al., 2006. Genes Dev. 20(8):966-976).
  • the protein modifying enzyme is a histone methyltransferase, a histone acetyltransferase, a histone demethylase, a histone deacetylase or a protein arginine deiminase.
  • the expressions "histone methyltransferase”, “histone acetyltransferase”, “histone demethylase”, “histone deacetylase” and “protein arginine deiminase” inter alia 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.
  • the at least one protein is a protein recognizing a post- translational modification.
  • Posttranslational histone modifications are recognized ("read") by proteins containing domains that bind specifically to a particular mark. Bromodomain containing proteins recognize acetylated lysine. MBT domains recognize mono- and di-methylated lysines. Di- and tri-methylated lysines are read by TUDOR, CHROMO and PHD domains. TUDOR domains also recognize symmetrically dimethylated arginine whereas PHD domains also are able to bind to mono-methylated, unmethylated and acetylated lysines. These different proteins interact with transcription factors and regulators to control transcriptional initiation, elongation and termination as well as other functions like DNA methylation or DNA repair.
  • the protein recognizing a post-translational modification is a protein that recognizes a histone modification.
  • said protein is a BRD-protein.
  • BBD-protein generally refers to a protein containing a bromodomain (Wu and Chiang, 2007. J. Biol. Chem. 282, 13141- 13145).
  • the amount of the at least one displaced protein is determined after separation of the chromatin preparation. This means that the chromatin preparation is separated from the fraction containing the displaced protein and only after this separation the amount of the protein is determined.
  • the term "separating" generally includes all procedures known to the person skilled in that art by which a biochemical molecule may be purified from insoluble material, including, but not limited to, standard biochemical methods such as centrifugation or filtration.
  • the affinity of the compound for the protein is determined.
  • the term "determining the affinity” as used in the context of the present invention generally refers to the analysis of the binding affinity of the compound to its interacting protein.
  • determining the affinity according to the present invention means determining the dissociation constant KD of the compound for its protein.
  • the determination of the affinity of the compound to the interacting protein requires that the amount of the compound is varied, resulting in the generation of a concentration-response curve from which the affinity of compound can be derived.
  • Such methods are known in the art (Cheng and Prusoff, 1973. Biochem. Pharmacol. 22(23): 3099-4108; Bantscheff et al., 2007. Nat. Biotechnol. 25(9): 1035-1044 especially the online supplement; Sharma et al., 2009. Nat. Methods 6(10): 741-744).
  • the selectivity profile of the compound is determined.
  • selectivity profile generally refers to the comparison of the affinity or activity of a given compound for a group of proteins, for example the members of a protein target family. Examples of selectivity profiles for HDAC inhibitors have been published previously (Bantscheff et al., 201 1. Nat. Biotechnol. 29(3):255-265).
  • Example 1 The determination of the selectivity profile of the compound in respect to proteins identified by the method of the present invention is exemplified in Example 1 and displayed in Figure 5.
  • 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.
  • Figure 1 Amino acid sequence of human EHMT1 (IPI00015526.3). Peptides identified by mass spectrometry are underlined.
  • Figure 2 Amino acid sequence of human EHMT2 (IPI00893814.1). Peptides identified by mass spectrometry are underlined.
  • FIG. 3 Display of proteins eluted with two different concentrations of BIX01294.
  • BIX01294 was used at a concentration of 500 ⁇ and 20 ⁇ . Elution efficiency is calculated as fold change relative to the DMSO control (logarithmic scale). Only proteins which show a concentration dependent elution response are depicted.
  • Figure 4 Display of proteins eluted with three different concentrations of UNC0638.
  • UNC0638 was used at 300 ⁇ , 60 ⁇ and 12 ⁇ . Elution efficiency is calulated as fold change relative to the DMSO control (logarithmic scale). Only proteins which show a concentration dependent elution response are depicted.
  • Figure 5 Display of a selected set of eluted proteins identified via mass spectrometry. Proteins are grouped into different classes. The result demonstrates selective elution of EHMT1 and EHMT2 with 500 ⁇ BIX01294. In total 1259 proteins were identified in this experiment.
  • Nuclei are isolated from cells and chromatin is prepared. A chromatin preparation is contacted with a test compound at various concentrations (or DMSO as solvent control), thus causing displacement of target proteins from the chromatin. Eluted proteins are identified and quantified by quantitative HPLC-mass spectrometry. Protocols
  • HL-60 cells ATCC CCL-240, Manassas, VA, USA were grown in spinner flasks (Integra Bioscience 182101, Zizers, Switzerland) in IMDM medium (Invitrogen 21980.065, Carlsbad, CA, USA) supplemented with 20% fetal calf serum (PAA Laboratories 15/101, Pasching, Austria) up to a cell concentration of lxlO 6 cells/mL. Cells were harvested by centrifugation and washed once with 1 x PBS buffer (Invitrogen, 14190-094). Cells were either used immediately for the isolation of nuclei or frozen and stored at -80 °C.
  • hypotonic cell homogenization buffer A (10 niM Tris-Cl, pH 7.4, 1.5 mM MgCl 2 , 10 mM KC1, 25 tnM NaF, 1 mM Na 3 V0 4 , 1 mM DTT). After 3 minutes incubation on ice cells were centrifuged at 2350 rpm, 4 °C. One volume of cell pellet was resuspended in two volumes of hypotonic cell homogenization buffer A supplemented with protease inhibitors (Complete, EDTA-free Protease Inhibitor Cocktail Tablets, Roche, #1 1873580001 , 1 tablet/25 mL).
  • protease inhibitors Complete, EDTA-free Protease Inhibitor Cocktail Tablets, Roche, #1 1873580001 , 1 tablet/25 mL.
  • nuclei were resuspended in 2x volumes DP buffer (50 mM Tris, pH 7.4, 5 % (w/v) glycerol, 1.5 mM MgCl 2 , 150 mM NaCl, 25 mM NaF, 1 mM DTT, 1 mM Na 3 V0 4 ) supplemented with 0.4% (w/v) Nonidet-P40 and protease inhibitors and homogenized with 20 strokes in a Dounce glass homogenizer.
  • a volume of nuclei suspension corresponding to 5-200 ⁇ , of chromatin pellet was aliquoted into 1.5 mL tubes and centrifuged 5 min at 1700 x g, 4 °C.
  • 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 5 mM TEAB.
  • porcine trypsin Promega
  • the peptide extracts corresponding to the different aliquots treated with different concentrations of compound 1 were labeled with variants of the isobaric tagging reagent as shown in Table 2 (TMT sixplex Label Reagent Set, part number 90066, Thermo Fisher Scientific Inc., Rockford, IL 61 105 USA).
  • the TMT reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides on amino groups in up to six different biological samples enabling simultaneous identification and quantification of peptides.
  • the TMT 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 ⁇ acetonitrile were added.
  • the TMT reagent was dissolved in acetonitrile to a final concentration of 24 mM 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 labelled samples were then combined, dried in a vacuum centrifuge and resuspended in 60% 200mM TEAB / 40% acetonitrile.
  • Peptide samples were injected into a capillary LC system (CapLC, Waters) and separated using a reversed phase CI 8 column (X-Bridge 1 mm x 150 mm, Waters, USA). Gradient elution was performed at a flow-rate of 50 ⁇ 7 ⁇ .
  • Solvent A 20 mM ammoniumformiate, pHl l
  • solvent B 20 mM ammoniumformiate, pHl l, 60% acetonitrile and 1 min fractions were automatically collected throughout the separation range (Micro-fraction collector, Sunchrom, Germany) and pooled to yield a total of 16 peptide fractions.
  • LTQ-Orbitrap XL and Orbitrap Velos instruments were operated with XCalibur 2.0/2.1 software. Intact peptides were detected in the Orbitrap at 30.000 resolution. Internal calibration was performed using the ion signal from (Si(CH 3 ) 2 0) 6 H + at m/z 445.120025 (Olsen et al., 2005. Mol. Cell Proteomics 4, 2010-2021). Data dependent tandem mass spectra were generated for up to six peptide precursors using a combined CID/HCD approach (Kocher et al., 2009. J. Proteome Res. 8, 4743-4752). For CID up to 5000 ions (Orbitrap XL) or up to 3000 ions (Orbitrap Velos) were accumulated in the ion trap within a maximum ion accumulation time of 200 msec.
  • MascotTM 2.0 (Matrix Science) was used for protein identification using 10 ppm mass tolerance for peptide precursors and 0.8 Da (CID) tolerance for fragment ions. Carbamidomethylation of cysteine residues and TMT modification of lysine residues were set as fixed modifications and S,T,Y phosphorylation, methionine oxidation, N-terminal acetylation of proteins and iTRAQ/TMT modification of peptide N-termini were set as variable modifications.
  • the search database consisted of a customized version of the IPI protein sequence database combined with a decoy version of this database created using a script supplied by Matrix Science (Elias et al., 2005. Nat. Methods 2, 667-675).
  • Protein identifications were accepted as follows: i) For single spectrum to sequence assignments, we required this assignment to be the best match and a minimum Mascot score of 31 and a lOx difference of this assignment over the next best assignment. Based on these criteria, the decoy search results indicated ⁇ 1% false discovery rate (FDR); ii) For multiple spectrum to sequence assignments and using the same parameters, the decoy search results indicate ⁇ 0.1% false discovery rate. For protein quantification a minimum of 2 sequence assignments matching to unique peptides was required. FDR for quantified proteins was «0.1%. 4.6 Peptide and protein quantification
  • Centroided iTRAQ/TMT reporter ion signals were computed by the XCalibur software operating and extracted from MS data files using customized scripts. Only peptides unique for identified proteins were used for relative protein quantification. Further spectra used for quantification were filtered according to the following criteria: Mascot ion score > 15, signal to background ratio of the precursor ion > 4, s2i > 0.5 (Savitski et al., 2010. J. Am. Soc. Mass Spectrom. 21 (10): 1668-79). Reporter ion intensities were multiplied with the ion accumulation time yielding an area value proportional to the number of reporter ions present in the mass analyzer.
  • Proteins identified after displacement from chromatin with compound UNC0638 and BIX01294 are shown in Table 4.
  • the identified peptides for EHMTl and EHMT2 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).
  • Concentration- dependence of proteins displaced from chromatin with BIX01294 is shown in Figure 3.
  • the methyltransferases EHMTl and EHMT2 were eluted in a concentration-dependent fashion, as was their known interactor WIZ.

Abstract

La présente invention concerne des méthodes pour l'identification et la caractérisation de protéines interagissant avec la chromatine ainsi que l'identification de composés interagissant avec lesdites protéines.
PCT/EP2012/003421 2011-08-18 2012-08-08 Essai de profilage de chromatine WO2013023770A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109641933A (zh) * 2016-09-02 2019-04-16 路德维格癌症研究有限公司 染色质相互作用的全基因组鉴定

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008033992A2 (fr) * 2006-09-13 2008-03-20 University Of Medicine And Dentistry Of New Jersey Essai de criblage à haut débit de modulateurs d'enzymes modifiant les histones
US20110021362A1 (en) * 2009-07-20 2011-01-27 Constellation Pharmaceuticals Agents for stimulating activity of methyl modifying enzymes and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008033992A2 (fr) * 2006-09-13 2008-03-20 University Of Medicine And Dentistry Of New Jersey Essai de criblage à haut débit de modulateurs d'enzymes modifiant les histones
US20110021362A1 (en) * 2009-07-20 2011-01-27 Constellation Pharmaceuticals Agents for stimulating activity of methyl modifying enzymes and methods of use thereof

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
"Short Protocols in Molecular Biology", 1999, WILEY, article "Immunology", pages: 11 - 1,11-30
ABMAYR, S. M.; YAO, T.; PARMELY, T.; WORKMAN, J. L.: "Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells", CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 75, 2006, pages 12.1.1 - 12.1.10
AGGER ET AL., CURR. OPIN. GENET. DEV., vol. 18, no. 2, 2008, pages 159 - 168
ARMIROTTI; DAMONTE, PROTEOMICS, vol. 10, no. 20, 2010, pages 3566 - 76
BANTSCHEFF ET AL., ANAL. BIOANAL. CHEM., vol. 389, no. 4, 2007, pages 1017 - 1031
BANTSCHEFF ET AL., NAT. BIOTECHNOL., vol. 25, no. 9, 2007, pages 1035 - 1044
BANTSCHEFF ET AL., NAT. BIOTECHNOL., vol. 29, no. 3, 2011, pages 255 - 265
BARSKI ET AL., CELL, vol. 129, no. 4, 2007, pages 823 - 837
BLACKWELL ET AL., LIFE SCI., vol. 82, no. 21-22, 2008, pages 1050 - 1058
BLACKWELL ET AL., LIFE SCIENCES, vol. 82, no. 21-22, 2008, pages 1050 - 1058
CAPRIOTTI ET AL., J. CHROMATOGR. A., vol. 1218, no. 49, 2011, pages 8760 - 76
CASTLE, J. D.: "Purification of Organelles from Mammalian Cells", CURRENT PROTOCOLS IN PROTEIN SCIENCE, 2004, pages 4.2.1 - 4.2.57
CHENG; PRUSOFF, BIOCHEM. PHARMACOL., vol. 22, no. 23, 1973, pages 3099 - 4108
CHI ET AL., NAT. REV. CANCER., vol. 10, no. 7, 2010, pages 457 - 69
CLEMONS ET AL., CURR. OPIN. CHEM. BIOL., vol. 8, no. 3, 2004, pages 334 - 338
COPELAND ET AL., CURR. OPIN. CHEM. BIOL., vol. 14, no. 4, 2010, pages 505 - 510
DAYON ET AL., ANAL. CHEM., vol. 80, no. 8, 2008, pages 2921 - 2931
DIGNAM ET AL., NUCLEIC ACIDS RES., vol. 11, no. 5, 1983, pages 1475 - 89
DOMON; AEBERSOLD, NAT. BIOTECHNOLOGY, vol. 28, no. 7, 2010, pages 710 - 721
DONATO ET AL., J. CHROMATOGR. A., vol. 1218, no. 49, 2011, pages 8777 - 90
ELIAS ET AL., NAT. METHODS, vol. 2, 2005, pages 667 - 675
HAUSER ET AL., CURR. TOP. MED. CHEM., vol. 9, no. 3, 2009, pages 227 - 234
HOWARD R. PETTY: "Current Protocols in Cell Biology", October 1998, JOHN WILEY & SONS, INC.
JUFVAS ET AL., PLOS ONE, vol. 6, no. 1, 2011, pages E15960
JUFVAS ET AL., PLOS ONE, vol. 6, no. L, 2011, pages E 15960
KAMPRANIS; TSICHLIS, ADV. CANCER RES., vol. 102, 2009, pages 103 - 169
KARWA; MITRA: "Sample Preparation Techniques in Analytical Chemistry", 2003, WILEY, article "Sample preparation for the extraction, isolation, and purification of Nuclei Acids"
KERSEY ET AL., PROTEOMICS, vol. 4, no. 7, 2004, pages 1985 - 1988
KHAN ET AL., BIOCHEM. J., vol. 409, no. 2, 2008, pages 581 - 9
KOCHER ET AL., J. PROTEOME RES., vol. 8, 2009, pages 4743 - 4752
KOUZARIDES, CELL, vol. 128, no. 4, 2007, pages 693 - 705
KUBICEK ET AL., MOL. CELL, vol. 25, 2007, pages 473 - 481
LY; WASINGER, PROTEOMICS, vol. 11, no. 4, 2011, pages 513 - 534
MANN ET AL., ANNUAL REVIEW OF BIOCHEMISTRY, vol. 70, 2001, pages 437 - 473
NATHAN ET AL., GENES DEV., vol. 20, no. 8, 2006, pages 966 - 976
NEILSON ET AL., PROTEOMICS, vol. 11, no. 4, 2011, pages 535 - 553
OLSEN ET AL., MOL. CELL PROTEOMICS, vol. 4, 2005, pages 2010 - 2021
ONG ET AL., MOL. CELL. PROTEOMICS., vol. 1, no. 5, 2002, pages 376 - 386
ROSS ET AL., MOL. CELL. PROTEOMICS, vol. 3, no. 12, 2004, pages 1154 - 1169
RUTHENBURG ET AL., NAT. REV. MOL. CELL BIOL., vol. 8, no. 12, 2007, pages 983 - 994
SAVITSKI ET AL., J. AM. SOC. MASS SPECTROM., vol. 21, no. 10, 2010, pages 1668 - 79
SHARMA ET AL., NAT. METHODS, vol. 6, no. 10, 2009, pages 741 - 744
SHEVCHENKO ET AL., ANAL. CHEM., vol. 68, 1996, pages 850 - 858
SHEVCHENKO ET AL., ANALYTICAL CHEMISTRY, vol. 68, 1996, pages 850 - 858
STEFAN KUBICEK ET AL: "Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase", MOLECULAR CELL, CELL PRESS, CAMBRIDGE, MA, US, vol. 25, no. 3, 9 February 2007 (2007-02-09), pages 473 - 481, XP002635011, ISSN: 1097-2765, [retrieved on 20070208], DOI: 10.1016/J.MOLCEL.2007.01.017 *
THOMPSON ET AL., ANAL. CHEM., vol. 75, no. 8, 2003, pages 1895 - 1904
VEDADI ET AL., NATURE CHEMICAL BIOLOGY, vol. 7, no. 8, 2011, pages 566 - 574
VEDADI MASOUD ET AL: "A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells", NATURE CHEMICAL BIOLOGY, vol. 7, no. 8, 10 July 2011 (2011-07-10), pages 566 - 574, XP002667515 *
WU ET AL., J. PROTEOME RES., vol. 5, 2006, pages 651 - 658
WU; CHIANG, J. BIOL. CHEM., vol. 282, 2007, pages 13141 - 13145

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109641933A (zh) * 2016-09-02 2019-04-16 路德维格癌症研究有限公司 染色质相互作用的全基因组鉴定
CN109641933B (zh) * 2016-09-02 2023-09-29 路德维格癌症研究有限公司 染色质相互作用的全基因组鉴定

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