Publications by authors named "Robert A Copeland"

114 Publications

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

SLAS Discov 2020 04 4;25(4):361-371. Epub 2019 Oct 4.

Accent Therapeutics, Lexington, MA, USA.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (mA) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, mA can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I -adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC values of mechanism-based inhibitors -adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.
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http://dx.doi.org/10.1177/2472555219878408DOI Listing
April 2020

Rebuttal to Response to the Article "Enzyme-Inhibitor Interactions and a Simple, Rapid Method for Determining Inhibition Modality".

SLAS Discov 2020 01 12;25(1). Epub 2019 Sep 12.

Accent Therapeutics, Inc., Lexington, MA, USA.

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http://dx.doi.org/10.1177/2472555219875106DOI Listing
January 2020

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Cancer Cell 2019 07 27;36(1):100-114.e25. Epub 2019 Jun 27.

Epigenetics Research Unit, GlaxoSmithKline, Collegeville, PA 19426, USA. Electronic address:

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.
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http://dx.doi.org/10.1016/j.ccell.2019.05.014DOI Listing
July 2019

Enzyme-Inhibitor Interactions and a Simple, Rapid Method for Determining Inhibition Modality.

SLAS Discov 2019 06 27;24(5):515-522. Epub 2019 Feb 27.

1 Accent Therapeutics, Inc., Lexington, MA, USA.

Contemporary chemical biology and drug discovery are increasingly focused on the discovery of inhibitory molecules that interact with enzyme targets in specific ways, such as allosteric or orthosteric binding. Hence, there is increasing interest in evaluating hit compounds from high-throughput diversity screening to determine their mode of interaction with the target. In this work, the common inhibition modalities are reviewed and clarified. The impact of substrate concentration, relative to substrate K, for each common inhibition modality is also reviewed. The pattern of changes in IC that accompany increasing substrate concentration are shown to be diagnostic of specific inhibition modalities. Thus, replots of IC as a function of the ratio [S]/K are recommended as a simple and rapid means of assessing inhibition modality. Finally, specific recommendations are offered for ideal experimental conditions for the determination of inhibition modality through the use of IC replots.
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http://dx.doi.org/10.1177/2472555219829898DOI Listing
June 2019

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

PLoS One 2018 1;13(6):e0197372. Epub 2018 Jun 1.

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

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983452PMC
December 2018

RNA-modifying proteins as anticancer drug targets.

Nat Rev Drug Discov 2018 06 18;17(6):435-453. Epub 2018 May 18.

Accent Therapeutics, Inc., Cambridge, MA, USA.

All major biological macromolecules (DNA, RNA, proteins and lipids) undergo enzyme-catalysed covalent modifications that impact their structure, function and stability. A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA stability and translation to proteins; these mechanisms of translational control have been termed epitranscriptomics. Emerging data suggest that some epitranscriptomic mechanisms are altered in human cancers as well as other human diseases. In this Review, we examine the current understanding of RNA modifications with a focus on mRNA methylation, highlight their possible roles in specific cancer indications and discuss the emerging potential of RNA-modifying proteins as therapeutic targets.
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http://dx.doi.org/10.1038/nrd.2018.71DOI Listing
June 2018

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

Protein methyltransferase inhibitors as precision cancer therapeutics: a decade of discovery.

Philos Trans R Soc Lond B Biol Sci 2018 06;373(1748)

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

The protein methyltransferases (PMTs) represent a large class of enzymes that catalyse the methylation of side chain nitrogen atoms of the amino acids lysine or arginine at specific locations along the primary sequence of target proteins. These enzymes play a key role in the spatio-temporal control of gene transcription by performing site-specific methylation of lysine or arginine residues within the histone proteins of chromatin, thus effecting chromatin conformational changes that activate or repress gene transcription. Over the past decade, it has become clear that the dysregulated activity of some PMTs plays an oncogenic role in a number of human cancers. Here we review research of the past decade that has identified specific PMTs as oncogenic drivers of cancers and progress toward the discovery and development of selective, small molecule inhibitors of these enzymes as precision cancer therapeutics.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
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http://dx.doi.org/10.1098/rstb.2017.0080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915721PMC
June 2018

Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study.

Lancet Oncol 2018 05 9;19(5):649-659. Epub 2018 Apr 9.

DITEP Gustave Roussy, Villejuif, Paris, France; INSERM U1170, Villejuif, Paris, France.

Background: Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2.

Methods: We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonié (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing.

Findings: Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours.

Interpretation: Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours.

Funding: Epizyme and Eisai.
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http://dx.doi.org/10.1016/S1470-2045(18)30145-1DOI Listing
May 2018

Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

Sci Rep 2017 12 21;7(1):17993. Epub 2017 Dec 21.

Epizyme, Inc., Cambridge, Massachusetts, USA.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.
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http://dx.doi.org/10.1038/s41598-017-18446-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740082PMC
December 2017

The Elements of Translational Chemical Biology.

Cell Chem Biol 2018 02 7;25(2):128-134. Epub 2017 Dec 7.

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

A causal relationship between target activity modulation by small molecules and phenotypic consequence is the cornerstone of chemical biology and drug discovery. Here we articulate elements of translational chemical biology, as guideposts to ensure the appropriate use of chemical probes and the conclusions drawn from cellular studies with these molecules.
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http://dx.doi.org/10.1016/j.chembiol.2017.11.003DOI Listing
February 2018

Drug Discovery and Chemical Biology of Cancer Epigenetics.

Cell Chem Biol 2017 Sep;24(9):1120-1147

Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, USA. Electronic address:

Comprehensive whole-exome sequencing, DNA copy-number determination, and transcriptomic analyses of diverse cancers have greatly expanded our understanding of the biology of many tumor types. In addition to mutations in the common cell-of-origin specific driver mutations, these studies have also revealed a large number of loss-of-function and gain-of-function mutations in chromatin-modifying proteins (CMPs). This has revealed that epigenetic dysregulation is a common feature of most pediatric and adult cancers. Many specific and potent inhibitors have been developed for multiple CMP classes, which have assisted in elucidating the role of epigenetics as well as epigenetic vulnerabilities in these cancer types. Clinical trials with numerous CMP inhibitors are also currently in progress to evaluate the therapeutic potential of epigenetic inhibitors. In this review, we aim to provide a summary of genetic mutations in epigenetic genes and a review of CMP inhibitors suitable for preclinical studies or currently in clinical trials. Additionally, we highlight the CMPs for which potent inhibitors have not been developed and additional research focus should be dedicated.
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http://dx.doi.org/10.1016/j.chembiol.2017.08.020DOI Listing
September 2017

EZH2 Inhibition by Tazemetostat Results in Altered Dependency on B-cell Activation Signaling in DLBCL.

Mol Cancer Ther 2017 11 23;16(11):2586-2597. Epub 2017 Aug 23.

Epizyme Inc., Cambridge, Massachusetts.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) Here, we demonstrate that cell lines bearing mutations show a cytotoxic response, while cell lines with WT- show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT- models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT- backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, /BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0840DOI Listing
November 2017

Mechanisms of Pinometostat (EPZ-5676) Treatment-Emergent Resistance in -Rearranged Leukemia.

Mol Cancer Ther 2017 08 20;16(8):1669-1679. Epub 2017 Apr 20.

Epizyme Inc., Cambridge, Massachusetts.

DOT1L is a protein methyltransferase involved in the development and maintenance of -rearranged (-r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the gene. The observation of responses and subsequent relapses in the adult trial treating -r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have -r. TER was achieved in five -r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0693DOI Listing
August 2017

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: and Preclinical Models.

Mol Cancer Ther 2017 05 14;16(5):850-860. Epub 2017 Mar 14.

Epizyme Inc., Cambridge, Massachusetts.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0678DOI Listing
May 2017

Drug-target interaction kinetics: underutilized in drug optimization?

Future Med Chem 2016 12 15;8(18):2173-2175. Epub 2016 Nov 15.

Research & Development, Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, USA.

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http://dx.doi.org/10.4155/fmc-2016-0183DOI Listing
December 2016

Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

PLoS One 2016 8;11(7):e0158888. Epub 2016 Jul 8.

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

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938529PMC
July 2017

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

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666.

ACS Med Chem Lett 2016 Feb 2;7(2):162-6. Epub 2015 Dec 2.

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

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).
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http://dx.doi.org/10.1021/acsmedchemlett.5b00380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753547PMC
February 2016

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

Epigenetic Medicinal Chemistry.

ACS Med Chem Lett 2016 Feb 10;7(2):124-7. Epub 2015 Dec 10.

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

The past decade has seen tremendous growth in our understanding of epigenetics and chromatin remodeling. Small, organic molecule modulators of a number of chromatin modifying proteins (CMPs) have been reported over this time frame and several of these have advanced to human clinical trials. In this Viewpoint, I summarize the current state of medicinal chemistry efforts focused on epigenetic targets and attempt to provide some insight into future directions on which the community may wish to focus.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753546PMC
February 2016

CARM1 Preferentially Methylates H3R17 over H3R26 through a Random Kinetic Mechanism.

Biochemistry 2016 Mar 5;55(11):1635-44. Epub 2016 Feb 5.

Epizyme Inc. , Cambridge, Massachusetts 02139, United States.

CARM1 is a type I arginine methyltransferase involved in the regulation of transcription, pre-mRNA splicing, cell cycle progression, and the DNA damage response. CARM1 overexpression has been implicated in breast, prostate, and liver cancers and therefore is an attractive target for cancer therapy. To date, little about the kinetic properties of CARM1 is known. In this study, substrate specificity and the kinetic mechanism of the human enzyme were determined. Substrate specificity was examined by testing CARM1 activity with several histone H3-based peptides in a radiometric assay. Comparison of kcat/KM values reveals that methylation of H3R17 is preferred over that of H3R26. These effects are KM-driven as kcat values remain relatively constant for the peptides tested. Shortening the peptide at the C-terminus by five amino acid residues greatly reduced binding affinity, indicating distal residues may contribute to substrate binding. CARM1 appears to bind monomethylated peptides with an affinity similar to that of unmethylated peptides. Monitoring of the CARM1-dependent production of monomethylated and dimethylated peptides over time by self-assembled monolayer and matrix-assisted laser desorption ionization mass spectrometry revealed that methylation by CARM1 is distributive. Additionally, dead-end and product inhibition studies suggest CARM1 conforms to a random sequential kinetic mechanism. By defining the kinetic properties and mechanism of CARM1, these studies may aid in the development of small molecule CARM1 inhibitors.
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http://dx.doi.org/10.1021/acs.biochem.5b01071DOI Listing
March 2016

PRC2 and SWI/SNF Chromatin Remodeling Complexes in Health and Disease.

Biochemistry 2016 Mar 17;55(11):1600-14. Epub 2016 Feb 17.

Epizyme Inc. , 400 Technology Square, 4th floor, Cambridge, Massachusetts 02139, United States.

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.
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http://dx.doi.org/10.1021/acs.biochem.5b01191DOI Listing
March 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

The Biochemistry of Chromatin Remodeling.

Biochemistry 2016 Mar 22;55(11):1555-6. Epub 2016 Jan 22.

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http://dx.doi.org/10.1021/acs.biochem.6b00014DOI Listing
March 2016

The drug-target residence time model: a 10-year retrospective.

Nat Rev Drug Discov 2016 Feb 18;15(2):87-95. Epub 2015 Dec 18.

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

The drug-target residence time model was first introduced in 2006 and has been broadly adopted across the chemical biology, biotechnology and pharmaceutical communities. While traditional in vitro methods view drug-target interactions exclusively in terms of equilibrium affinity, the residence time model takes into account the conformational dynamics of target macromolecules that affect drug binding and dissociation. The key tenet of this model is that the lifetime (or residence time) of the binary drug-target complex, and not the binding affinity per se, dictates much of the in vivo pharmacological activity. Here, this model is revisited and key applications of it over the past 10 years are highlighted.
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http://dx.doi.org/10.1038/nrd.2015.18DOI Listing
February 2016

Structural Insights into Ternary Complex Formation of Human CARM1 with Various Substrates.

ACS Chem Biol 2016 Mar 23;11(3):763-71. Epub 2015 Nov 23.

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

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers. CARM1 is known to methylate histone H3 and nonhistone substrates. To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors, but no ternary structures with nucleoside and peptide substrates have been reported. Here, the crystal structures of human CARM1 with the S-adenosylmethione (SAM) mimic sinefungin and three different peptide sequences from histone H3 and PABP1 are presented, with both nonmethylated and singly methylated arginine residues exemplified. This is the first example of multiple substrate sequences solved in a single PRMT enzyme and demonstrates how the CARM1 binding site is capable of accommodating a variety of peptide sequences while maintaining a core binding mode for the unmethylated and monomethylated substrates. Comparison of these with other PRMT enzyme-peptide structures shows hydrogen bonding patterns that may be thematic of these binding sites.
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http://dx.doi.org/10.1021/acschembio.5b00773DOI Listing
March 2016