Publications by authors named "Tim J Wigle"

34 Publications

In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement.

Cell Chem Biol 2020 07;27(7):877-887.e14

Ribon Therapeutics, 35 Cambridgepark Drive, Suite 300, Cambridge, MA 02140, USA.

Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
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http://dx.doi.org/10.1016/j.chembiol.2020.06.009DOI Listing
July 2020

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

SLAS Discov 2020 Mar 19;25(3):241-252. Epub 2019 Dec 19.

Ribon Therapeutics Inc., Cambridge, MA, USA.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.
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http://dx.doi.org/10.1177/2472555219883623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036481PMC
March 2020

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

PLoS One 2018 9;13(5):e0197082. Epub 2018 May 9.

Epizyme Inc., Cambridge, Massachusetts, United States of America.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197082PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942779PMC
August 2018

Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method.

Methods Mol Biol 2016 ;1439:33-45

Epizyme, Inc., 4th Floor, 400 Technology Square, Cambridge, MA, 02139, USA.

Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.
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http://dx.doi.org/10.1007/978-1-4939-3673-1_2DOI Listing
December 2017

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

ACS Med Chem Lett 2016 Feb 27;7(2):134-8. Epub 2015 Aug 27.

Epizyme Inc. , Fourth Floor, 400 Technology Square, Cambridge, Massachusetts 02139, United States.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753551PMC
February 2016

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.

Biochemistry 2016 Mar 11;55(11):1645-51. Epub 2016 Feb 11.

Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.
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http://dx.doi.org/10.1021/acs.biochem.5b01202DOI Listing
March 2016

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat.

J Med Chem 2016 Feb 27;59(4):1556-64. Epub 2016 Jan 27.

Epizyme , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01501DOI Listing
February 2016

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

ACS Med Chem Lett 2015 Jun 6;6(6):655-9. Epub 2015 Apr 6.

Epizyme, Inc. , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02138, United States.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468411PMC
June 2015

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity.

ACS Med Chem Lett 2015 May 4;6(5):491-5. Epub 2015 Mar 4.

Epizyme, Inc. , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434464PMC
May 2015

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Nat Chem Biol 2015 Jun 27;11(6):432-7. Epub 2015 Apr 27.

Departments of Biology and Molecular Discovery, Epizyme, Inc., Cambridge, Massachusetts, USA.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.
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http://dx.doi.org/10.1038/nchembio.1810DOI Listing
June 2015

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

J Biomol Screen 2015 Jul 9;20(6):810-20. Epub 2015 Mar 9.

Epizyme, Inc., Cambridge, MA, USA.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.
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http://dx.doi.org/10.1177/1087057115575689DOI Listing
July 2015

Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas.

PLoS One 2014 10;9(12):e111840. Epub 2014 Dec 10.

Research and Development, Epizyme Inc., Cambridge, Massachusetts, United States of America.

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone - a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111840PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262195PMC
October 2017

Reaction coupling between wild-type and disease-associated mutant EZH2.

ACS Chem Biol 2014 Nov 28;9(11):2459-64. Epub 2014 Aug 28.

Epizyme, Inc. 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.
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http://dx.doi.org/10.1021/cb500548bDOI Listing
November 2014

Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma.

Mol Cancer Ther 2014 Apr 21;13(4):842-54. Epub 2014 Feb 21.

Authors' Affiliations: Epizyme Inc., Cambridge; Eisai Inc., Andover, Massachusetts; and Eisai Co. Ltd., Tsukuba-shi, Ibaraki, Japan.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0773DOI Listing
April 2014

Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

Biochem J 2013 Jul;453(2):241-7

Epizyme Inc., 400 Technology Square, Cambridge, MA 02139, USA.

H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.
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http://dx.doi.org/10.1042/BJ20130439DOI Listing
July 2013

Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2.

Proc Natl Acad Sci U S A 2013 May 25;110(19):7922-7. Epub 2013 Apr 25.

Epizyme, Inc, Cambridge, MA 02139, USA.

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.
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http://dx.doi.org/10.1073/pnas.1303800110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651445PMC
May 2013

Drugging the human methylome: an emerging modality for reversible control of aberrant gene transcription.

Curr Opin Chem Biol 2013 Jun 23;17(3):369-78. Epub 2013 Apr 23.

Epizyme Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139, United States.

Protein and DNA methylation have emerged as critical mechanisms for the control of regulated gene transcription. In humans, the addition, recognition and removal of methyl groups are orchestrated by at least 344 proteins that we collectively refer to as the 'methylome'. The large size of the methylome likely reflects the importance of precise control over this small covalent modification. An increasing number of reports implicating the misregulation of methylation in disease make the proteins governing this modification attractive target for small molecule drug discovery. In light of the emerging opportunities for the development of therapeutics that modulate methylation-dependent pathways, this review examines the protein families that constitute the methylome, with emphasis on the methylation of arginine and lysine residues of proteins. Genetic aberrations that give rise to disease are highlighted, in addition to recent proof-of-concept successes in the development of small molecule modulators of methylome constituents.
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http://dx.doi.org/10.1016/j.cbpa.2013.03.035DOI Listing
June 2013

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells.

Nat Chem Biol 2012 Nov 30;8(11):890-6. Epub 2012 Sep 30.

Epizyme, Inc., Cambridge, MA, USA.

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.
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http://dx.doi.org/10.1038/nchembio.1084DOI Listing
November 2012

Conformational adaptation drives potent, selective and durable inhibition of the human protein methyltransferase DOT1L.

Chem Biol Drug Des 2012 Dec 9;80(6):971-80. Epub 2012 Oct 9.

Epizyme Inc., 325 Vassar Street, Cambridge, MA 02139, USA.

DOT1L is the human protein methyltransferase responsible for catalyzing the methylation of histone H3 on lysine 79 (H3K79). The ectopic activity of DOT1L, associated with the chromosomal translocation that is a universal hallmark of MLL-rearranged leukemia, is a required driver of leukemogenesis in this malignancy. Here, we present studies on the structure-activity relationship of aminonucleoside-based DOT1L inhibitors. Within this series, we find that improvements in target enzyme affinity and selectivity are driven entirely by diminution of the dissociation rate constant for the enzyme-inhibitor complex, leading to long residence times for the binary complex. The biochemical K(i) and residence times measured for these inhibitors correlate well with their effects on intracellular H3K79 methylation and MLL-rearranged leukemic cell killing. Crystallographic studies reveal a conformational adaptation mechanism associated with high-affinity inhibitor binding and prolonged residence time; these studies also suggest that conformational adaptation likewise plays a critical role in natural ligand interactions with the enzyme, hence, facilitating enzyme turnover. These results provide critical insights into the role of conformational adaptation in the enzymatic mechanism of catalysis and in pharmacologic intervention for DOT1L and other members of this enzyme class.
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http://dx.doi.org/10.1111/cbdd.12050DOI Listing
December 2012

A687V EZH2 is a gain-of-function mutation found in lymphoma patients.

FEBS Lett 2012 Sep 28;586(19):3448-51. Epub 2012 Jul 28.

Epizyme, Inc., 325 Vassar St., Cambridge, MA 02139, USA.

Heterozygous point mutations at Y641 and A677 in the EZH2 SET domain are prevalent in about 10-24% of Non-Hodgkin lymphomas (NHL). Previous studies indicate that these are gain-of-function mutations leading to the hypertrimethylation of H3K27. These EZH2 mutations may drive the proliferation of lymphoma and make EZH2 a molecular target for patients harboring these mutations. Here, another EZH2 SET domain point mutation, A687V, occurring in about 1-2% of lymphoma patients, is also shown to be a gain-of-function mutation that greatly enhances its ability to perform dimethylation relative to wild-type EZH2 and is equally proficient at catalyzing trimethylation. We propose that A687V EZH2 also leads to hypertrimethylation of H3K27 and may thus be a driver mutation in NHL.
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http://dx.doi.org/10.1016/j.febslet.2012.07.066DOI Listing
September 2012

Promoting illiteracy in epigenetics: an emerging therapeutic strategy.

Authors:
Tim J Wigle

Curr Chem Genomics 2011 22;5(Suppl 1):48-50. Epub 2011 Aug 22.

Epizyme, 840 Memorial Drive, Cambridge, MA 02139, USA.

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http://dx.doi.org/10.2174/1875397301005010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178898PMC
November 2011

The Y641C mutation of EZH2 alters substrate specificity for histone H3 lysine 27 methylation states.

FEBS Lett 2011 Oct 17;585(19):3011-4. Epub 2011 Aug 17.

Epizyme, Inc., 325 Vassar St., Cambridge, MA 02139, USA.

Mutations at tyrosine 641 (Y641F, Y641N, Y641S and Y641H) in the SET domain of EZH2 have been identified in patients with certain subtypes of non-Hodgkin lymphoma (NHL). These mutations were shown to change the substrate specificity of EZH2 for various methylation states of lysine 27 on histone H3 (H3K27). An additional mutation at EZH2 Y641 to cysteine (Y641C) was also found in one patient with NHL and in SKM-1 cells derived from a patient with myelodisplastic syndrome (MDS). The Y641C mutation has been reported to dramatically reduce enzymatic activity. Here, we demonstrate that while the Y641C mutation ablates enzymatic activity against unmethylated and monomethylated H3K27, it is superior to wild-type in catalyzing the formation of trimethylated H3K27 from the dimethylated precursor.
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http://dx.doi.org/10.1016/j.febslet.2011.08.018DOI Listing
October 2011

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells.

Nat Chem Biol 2011 Jul 10;7(8):566-74. Epub 2011 Jul 10.

Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.
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http://dx.doi.org/10.1038/nchembio.599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184254PMC
July 2011

Small-molecule ligands of methyl-lysine binding proteins.

J Med Chem 2011 Apr 18;54(7):2504-11. Epub 2011 Mar 18.

Center for Integrated Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, Division of Medicinal Chemistry and Natural Products, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.
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http://dx.doi.org/10.1021/jm200045vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109722PMC
April 2011

Epigenetics: Tools and Technologies.

Drug Discov Today Technol 2010 ;7(1):e59-e65

The Center for Integrative Chemical Biology and Drug Discovery, The University of North Carolina at Chapel Hill, Genetics Medicine Building, Campus Box 7363, Chapel Hill, NC 27599-7363, USA.

Epigenetics refers to heritable changes that control how the genome is accessed in different cell-types and during development and differentiation. Even though each cell contains essentially the same genetic code, epigenetic mechanisms permit specialization of function between cells. The state of chromatin, the complex of histone proteins, RNA and DNA that efficiently package the genome, is largely regulated by specific modifications to histone proteins and DNA, and the recognition of these marks by other proteins and protein complexes. The enzymes that produce these modifications (the 'writers'), the proteins that recognize them (the 'readers'), and the enzymes that remove them (the 'erasers') are critical targets for manipulation in order to further understand the histone code and its role in biology and human disease.
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http://dx.doi.org/10.1016/j.ddtec.2010.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018755PMC
January 2010

Identification of non-peptide malignant brain tumor (MBT) repeat antagonists by virtual screening of commercially available compounds.

J Med Chem 2010 Nov;53(21):7625-31

Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599-7363, United States.

The malignant brain tumor (MBT) repeat is an important epigenetic-code "reader" and is functionally associated with differentiation, gene silencing, and tumor suppression. (1-3) Small molecule probes of MBT domains should enable a systematic study of MBT-containing proteins and potentially reveal novel druggable targets. We designed and applied a virtual screening strategy that identified potential MBT antagonists in a large database of commercially available compounds. A small set of virtual hits was purchased and submitted to experimental testing. Nineteen of the purchased compounds showed a specific dose-dependent protein binding and will provide critical structure-activity information for subsequent lead generation and optimization.
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http://dx.doi.org/10.1021/jm1007374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974617PMC
November 2010

Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis.

Chem Biol 2010 Jul;17(7):695-704

Center for Integrative Chemical Biology and Drug Discovery Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, The University of North Carolina, CB #7363, 120 Mason Farm Road, Chapel Hill, NC 27599-7363, USA.

The discovery of small molecules targeting the >80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.
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http://dx.doi.org/10.1016/j.chembiol.2010.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914686PMC
July 2010

Novel Inhibitors of E. coli RecA ATPase Activity.

Curr Chem Genomics 2010 May 26;4:34-42. Epub 2010 May 26.

Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, 27707, USA.

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.
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http://dx.doi.org/10.2174/1875397301004010034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905775PMC
May 2010

Protein lysine methyltransferase G9a inhibitors: design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines.

J Med Chem 2010 Aug;53(15):5844-57

Center for Integrative Chemical Biology and Drug Discovery, Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Protein lysine methyltransferase G9a, which catalyzes methylation of lysine 9 of histone H3 (H3K9) and lysine 373 (K373) of p53, is overexpressed in human cancers. Genetic knockdown of G9a inhibits cancer cell growth, and the dimethylation of p53 K373 results in the inactivation of p53. Initial SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template represented by 3a (BIX01294), a selective small molecule inhibitor of G9a and GLP, led to the discovery of 10 (UNC0224) as a potent G9a inhibitor with excellent selectivity. A high resolution X-ray crystal structure of the G9a-10 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. On the basis of the structural insights revealed by this cocrystal structure, optimization of the 7-dimethylaminopropoxy side chain of 10 resulted in the discovery of 29 (UNC0321) (Morrison K(i) = 63 pM), which is the first G9a inhibitor with picomolar potency and the most potent G9a inhibitor to date.
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http://dx.doi.org/10.1021/jm100478yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920043PMC
August 2010

Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreen-based assay for antagonists of methyl-lysine binding proteins.

J Biomol Screen 2010 Jan 11;15(1):62-71. Epub 2009 Dec 11.

Center for Integrative Chemical Biology and Drug Discovery, Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.

The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.
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http://dx.doi.org/10.1177/1087057109352902DOI Listing
January 2010