Publications by authors named "Richard Chesworth"

28 Publications

  • Page 1 of 1

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2019.05.014DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983452PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197082PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942779PMC
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18446-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740082PMC
December 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. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-16-0678DOI Listing
May 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).
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.5b00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753551PMC
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b01501DOI Listing
February 2016

Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human.

Cancer Chemother Pharmacol 2016 Jan 8;77(1):43-62. Epub 2015 Dec 8.

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

Purpose: The metabolism and disposition of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was investigated in rat and dog. Metabolite profiles were compared with those from adult patients in the first-in-man phase 1 study as well as the cross-species metabolism observed in vitro.

Methods: EPZ-5676 was administered to rat and dog as a 24-h IV infusion of [(14)C]-EPZ-5676 for determination of pharmacokinetics, mass balance, metabolite profiling and biodistribution by quantitative whole-body autoradiography (QWBA). Metabolite profiling and identification was performed by radiometric and LC-MS/MS analysis.

Results: Fecal excretion was the major route of elimination, representing 79 and 81% of the total dose in and rat and dog, respectively. QWBA in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 168 h. Fecal recovery of dose-related material in bile duct-cannulated animals as well as higher radioactivity concentrations in the wall of the large intestine relative to liver implicated intestinal secretion as well as biliary elimination. EPZ-5676 underwent extensive oxidative metabolism with the major metabolic pathways being hydroxylation of the t-butyl group (EPZ007769) and N-dealkylation of the central nitrogen. Loss of adenine from parent EPZ-5676 (M7) was observed only in rat and dog feces, suggesting the involvement of gut microbiota. In rat and dog, steady-state plasma levels of total radioactivity and parent EPZ-5676 were attained rapidly and maintained through the infusion period before declining rapidly on cessation of dosing. Unchanged EPZ-5676 was the predominant circulating species in rat, dog and man.

Conclusions: The excretory and metabolic pathways for EPZ-5676 were very similar across species. Renal excretion of both parent EPZ-5676 and EPZ-5676-related material was low, and in preclinical species fecal excretion of parent EPZ-5676 and EPZ007769 accounted for the majority of drug-related elimination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00280-015-2929-yDOI Listing
January 2016

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

Xenobiotica 2016 ;46(3):268-77

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/00498254.2015.1072253DOI Listing
October 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.1810DOI Listing
June 2015

Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652.

Drug Metab Dispos 2015 Jul 17;43(7):936-43. Epub 2015 Apr 17.

Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.)

Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl}methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography - mass spectrometry (LC-MS(n)) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylene-diamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent Km values of >1000 μM for NAT1 and 165 ± 14.1 µM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 μL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/dmd.115.064014DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057115575689DOI Listing
July 2015

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0773DOI Listing
April 2014

Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor.

Biopharm Drug Dispos 2014 May 14;35(4):237-52. Epub 2014 Feb 14.

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

(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bdd.1889DOI Listing
May 2014

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia.

Blood 2013 Aug 25;122(6):1017-25. Epub 2013 Jun 25.

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

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2013-04-497644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739029PMC
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1303800110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651445PMC
May 2013

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

Mol Neurodegener 2012 Dec 18;7:61. Epub 2012 Dec 18.

EnVivo Pharmaceuticals, Inc, 500 Arsenal Street, Watertown, MA 02472, USA.

Background: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aβ42 and Aβ40. Many drug discovery efforts have focused on decreasing the production of Aβ42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aβ production to favor shorter, less amyloidogenic peptides than Aβ42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer's disease.

Results: Here we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aβ42 in H4 cells (IC50 = 67 nM) and increased the shorter Aβ38 by 1.7 fold at the IC50 for lowering of Aβ42. AβTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aβ42 and did not alter AβTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aβ deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aβ aggregates, amyloid plaques, inflammatory markers, and cognitive deficits.

Conclusions: EVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aβ42, attenuated memory deficits, and reduced Aβ plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1750-1326-7-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573960PMC
December 2012

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/cbdd.12050DOI Listing
December 2012

EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors.

Neuropharmacology 2012 Feb 10;62(2):1099-110. Epub 2011 Nov 10.

Maastricht University, School for Mental Health and Neuroscience, 6200 MD Maastricht, The Netherlands.

EVP-6124, (R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was evaluated here in vitro and in vivo. In binding and functional experiments, EVP-6124 showed selectivity for α7 nAChRs and did not activate or inhibit heteromeric α4β2 nAChRs. EVP-6124 had good brain penetration and an adequate exposure time. EVP-6124 (0.3 mg/kg, p.o.) significantly restored memory function in scopolamine-treated rats (0.1 mg/kg, i.p.) in an object recognition task (ORT). Although donepezil at 0.1 mg/kg, p.o. or EVP-6124 at 0.03 mg/kg, p.o. did not improve memory in this task, co-administration of these sub-efficacious doses fully restored memory. In a natural forgetting test, an ORT with a 24 h retention time, EVP-6124 improved memory at 0.3 mg/kg, p.o. This improvement was blocked by the selective α7 nAChR antagonist methyllycaconitine (0.3 mg/kg, i.p. or 10 μg, i.c.v.). In co-application experiments of EVP-6124 with acetylcholine, sustained exposure to EVP-6124 in functional investigations in oocytes caused desensitization at concentrations greater than 3 nM, while lower concentrations (0.3-1 nM) caused an increase in the acetylcholine-evoked response. These actions were interpreted as representing a co-agonist activity of EVP-6124 with acetylcholine on α7 nAChRs. The concentrations of EVP-6124 that resulted in physiological potentiation were consistent with the free drug concentrations in brain that improved memory performance in the ORT. These data suggest that the selective partial agonist EVP-6124 improves memory performance by potentiating the acetylcholine response of α7 nAChRs and support new therapeutic strategies for the treatment of cognitive impairment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuropharm.2011.10.024DOI Listing
February 2012

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

Cancer Cell 2011 Jul;20(1):53-65

Epizyme, Inc., Cambridge, MA 02139, USA.

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccr.2011.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046888PMC
July 2011

Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment.

Chem Biol 2011 Jun;18(6):777-93

Department of Physiology, UT Southwestern Medical Center, Dallas, TX 75390, USA.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2011.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124661PMC
June 2011

Regiospecific and stereoselective syntheses of (+/-) morphine, codeine, and thebaine via a highly stereocontrolled intramolecular 4 + 2 cycloaddition leading to a phenanthrofuran system.

J Am Chem Soc 2009 Aug;131(32):11402-6

Department of Chemistry, Columbia University, New York, New York, USA.

Total syntheses of the morphine alkaloids are described that use a direct stereoselective formation of the phenanthrofuran system via an intramolecular 4 + 2 cycloaddition of a diene tethered to the 4-position of a 7-methoxybenzofuran-3-carboxylic acid ester.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja9038505DOI Listing
August 2009

Estrogen receptor beta selective ligands: discovery and SAR of novel heterocyclic ligands.

Bioorg Med Chem Lett 2005 Dec 10;15(24):5562-6. Epub 2005 Oct 10.

Cardiovascular and Metabolic Diseases, Medicinal Chemistry, Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA.

A series of ligands with varying heterocyclic cores and substituents that display a range of selectivity's (up to >100x) for ER-beta over ER-alpha are reported.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2005.08.010DOI Listing
December 2005

Tetrahydroisoquinolines as subtype selective estrogen agonists/antagonists.

Bioorg Med Chem Lett 2004 Jun;14(11):2729-33

Pfizer Global Research and Development, CVMD Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA.

Two series of 6-hydroxy and 7-hydroxy tetrahydroisoquinolines were prepared. Evaluating a range of C-1, C-4, and N-substituents led to the discovery of ER alpha and ER beta selective analogs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2004.03.077DOI Listing
June 2004