Publications by authors named "Suzanne L Jacques"

16 Publications

  • Page 1 of 1

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 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

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.5b01071DOI Listing
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.5b00773DOI Listing
March 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

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

Pharmacology, biodistribution, and efficacy of GPCR-based pepducins in disease models.

Methods Mol Biol 2011 ;683:259-75

Tufts Medical Center, Molecular Oncology Research Institute, Boston, MA, USA.

G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60761-919-2_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780409PMC
February 2011

Enhanced selectivity profile of pyrazole-urea based DFG-out p38alpha inhibitors.

Bioorg Med Chem Lett 2010 Aug 17;20(16):4885-91. Epub 2010 Jun 17.

Department of Chemistry, Cambridge South Laboratories, Pfizer Inc., 620 Memorial Drive, Cambridge, MA 02139, USA.

By targeting an extended region of the conventional 'DFG-out' pocket of p38alpha, while minimizing interactions with the specificity pocket and eliminating interactions with the adenine binding site, we are able to design and synthesize a number of pyrazole-urea based DFG-out p38alpha inhibitors with good potencies, and excellent selectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2010.06.073DOI Listing
August 2010

Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance.

J Biochem 2010 Apr 10;147(4):601-9. Epub 2010 Feb 10.

Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA.

The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms. Kinetic studies show that activation of c-Kit RTK occurs through an inter-molecular autophosphorylation. Phosphopeptide mapping of c-Kit reveals that 14-22 phosphates are added to each mol of wild-type (WT) c-Kit during the activation. Phosphorylation sites are found on the JM, kinase insert (KID), c-terminal domains and the activation loop (A-loop), but only the sites on the JM domain contribute to the kinase activation. The A-loop tyrosine (Y(823)) is not phosphorylated until very late in the activation (>90% completion), indicating that the A-loop phosphorylation is not required for c-Kit activation. A sunitinib-resistant mutant D816H that accelerates auto-activation by 184-fold shows no phosphorylation on the A-loop tyrosine after full activation. A loss-of-phosphorylation mutation Y823F remains fully competent in auto-activation. Similar to WT and D816H, the unactivated Y823F mutant binds sunitinib and imatinib with high affinity (K(D) = 5.9 nM). But unlike the WT and D816H where the activated enzymes lose the ability to bind the two drugs, activated Y823F binds the two inhibitors effectively. These observations suggest that the A-loop of activated Y823F remains flexible and can readily adopt unactivated conformations to accommodate DFG-out binders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jb/mvq015DOI Listing
April 2010

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Proc Natl Acad Sci U S A 2009 Feb 21;106(5):1542-7. Epub 2009 Jan 21.

Departments of Structural and Computational Biology, Cancer Biology, and Biochemical Pharmacology, Pfizer Global Research and Development, 10777 Science Center Drive, La Jolla, CA 92121, USA.

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0812413106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635778PMC
February 2009

Structure of the catalytic domain of human polo-like kinase 1.

Biochemistry 2007 May 27;46(20):5960-71. Epub 2007 Apr 27.

Pfizer Global Research and Development, Research Technology Center, 620 Memorial Drive, Cambridge, Massachusetts 02139, USA.

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi602474jDOI Listing
May 2007

Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis.

Circulation 2006 Mar 27;113(9):1244-54. Epub 2006 Feb 27.

Division of Hematology/Oncology, Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111, USA.

Background: Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors.

Methods And Results: Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1.

Conclusions: We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.105.587758DOI Listing
March 2006

Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding.

Chem Biol 2003 Nov;10(11):1033-41

Division of Hematology/Oncology and Department of Medicine, New England Medical Center, Boston, MA 02111, USA.

Protease-activated G protein-coupled receptors (PAR1-4) are tethered-ligand receptors that are activated by proteolytic cleavage of the extracellular domain (exodomain) of the receptor. PAR1, the prototypic member of the PAR family, is the high-affinity thrombin receptor of platelets and vascular endothelium and plays a critical role in blood coagulation, thrombosis, and inflammation. Here, we describe the solution structure of the thrombin-cleaved exodomain of PAR1. The side chains of a hydrophobic hirudin-like (Hir) sequence and adjacent anionic motif project into solution. Docking of the exodomain Hir sequence to exosite I of thrombin reveals that the tethered ligand in the cleaved exodomain bends away from thrombin, leaving its active site available to another large macromolecular substrate. The N-terminal ligand is longer than anticipated and forms an intramolecular complex with a region located in the C terminus of the exodomain. Mutational analysis confirmed that this C-terminal region is a ligand binding site for both intra- and intermolecular ligands. A lipidated-ligand binding site peptide was found to be an effective inhibitor of thrombin-induced platelet aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2003.10.014DOI Listing
November 2003

Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding.

Chem Biol 2003 Nov;10(11):1033-41

Division of Hematology/Oncology and Department of Medicine, New England Medical Center, Boston, MA 02111, USA.

Protease-activated G protein-coupled receptors (PAR1-4) are tethered-ligand receptors that are activated by proteolytic cleavage of the extracellular domain (exodomain) of the receptor. PAR1, the prototypic member of the PAR family, is the high-affinity thrombin receptor of platelets and vascular endothelium and plays a critical role in blood coagulation, thrombosis, and inflammation. Here, we describe the solution structure of the thrombin-cleaved exodomain of PAR1. The side chains of a hydrophobic hirudin-like (Hir) sequence and adjacent anionic motif project into solution. Docking of the exodomain Hir sequence to exosite I of thrombin reveals that the tethered ligand in the cleaved exodomain bends away from thrombin, leaving its active site available to another large macromolecular substrate. The N-terminal ligand is longer than anticipated and forms an intramolecular complex with a region located in the C terminus of the exodomain. Mutational analysis confirmed that this C-terminal region is a ligand binding site for both intra- and intermolecular ligands. A lipidated-ligand binding site peptide was found to be an effective inhibitor of thrombin-induced platelet aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2003.10.014DOI Listing
November 2003

Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage.

Biochem J 2003 Dec;376(Pt 3):733-40

Molecular Cardiology Research Institute, Division of Hematology/Oncology, Tufts-New England Medical Center and Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA.

Thrombin activation of human platelets is mediated by the high-affinity PAR1 (protease-activated receptor-1) and the low-affinity PAR4 receptor. PAR1 and PAR4 exhibit markedly disparate kinetics of activation that likely reflect differences in the macromolecular association of thrombin with their respective N-terminal extracellular domains (exodomains). Here we examine the mechanism of initial thrombin binding and cleavage of the high- and low-affinity PAR exodomains using steady-state kinetic analyses. We showed that the PAR4 exodomain lacks the functional hirudin-like sequence found in PAR1 and does not bind exosite I to cause allosteric activation or inhibition of thrombin. Instead, PAR4 contains an anionic cluster, Asp(57)...Asp(59) ...Glu(62)...Asp(65) (DDED), in its exodomain, which slows the dissociation of PAR4 from the cationic thrombin. The analogous anionic residues in the PAR1 exodomain do not influence affinity for thrombin. Although PAR4 is cleaved more slowly than PAR1 on the cell surface, peptides containing the PAR4 P(4)-P(1) active-site-interacting sequence, Pro(45)-Ala-Pro-Arg (PAPR), are efficiently cleaved due to the optimal placement of dual prolines at positions P(4) and P(2). In comparison, thrombin has low affinity and slow cleavage rates for peptides that have a P(3) proline as occurs in human PAR3. Thus, to compensate for the lack of exosite I binding, PAR4 utilizes proline residues in its P(4)-P(1) sequence to provide high-affinity interactions with the active site and an anionic cluster to slow dissociation from the cationic thrombin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20030954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223816PMC
December 2003