Publications by authors named "P Ann Boriack-Sjodin"

46 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2472555219878408DOI Listing
April 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2017.11.003DOI Listing
February 2018

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

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.5b01202DOI Listing
March 2016

Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of Enzymes.

Biochemistry 2016 Mar 22;55(11):1557-69. Epub 2015 Dec 22.

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

Methyltransferase proteins make up a superfamily of enzymes that add one or more methyl groups to substrates that include protein, DNA, RNA, and small molecules. The subset of proteins that act upon arginine and lysine side chains are characterized as epigenetic targets because of their activity on histone molecules and their ability to affect transcriptional regulation. However, it is now clear that these enzymes target other protein substrates, as well, greatly expanding their potential impact on normal and disease biology. Protein methyltransferases are well-characterized structurally. In addition to revealing the overall architecture of the subfamilies of enzymes, structures of complexes with substrates and ligands have permitted detailed analysis of biochemical mechanism, substrate recognition, and design of potent and selective inhibitors. This review focuses on how knowledge gained from structural studies has impacted the understanding of this large class of epigenetic enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.5b01129DOI 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

Negishi cross-coupling enabled synthesis of novel NAD(+)-dependent DNA ligase inhibitors and SAR development.

Bioorg Med Chem Lett 2015 Nov 14;25(22):5172-7. Epub 2015 Oct 14.

Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.

Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2015.09.075DOI Listing
November 2015

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

Pyrimidinone nicotinamide mimetics as selective tankyrase and wnt pathway inhibitors suitable for in vivo pharmacology.

ACS Med Chem Lett 2015 Mar 13;6(3):254-9. Epub 2015 Jan 13.

AstraZeneca R&D Boston , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ß-catenin, can lead to oncogenesis. Inhibition of the poly(ADP-ribose) polymerase (PARP) catalytic domain of the tankyrases (TNKS1 and TNKS2) is known to inhibit the Wnt pathway via increased stabilization of Axin. In order to explore the consequences of tankyrase and Wnt pathway inhibition in preclinical models of cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound 1), we discovered the pyrrolopyrimidinone compound 25 (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound 25 can be formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ml5003663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4360163PMC
March 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

Rational design of inhibitors of the bacterial cell wall synthetic enzyme GlmU using virtual screening and lead-hopping.

Bioorg Med Chem 2014 Nov 16;22(21):6256-69. Epub 2014 Sep 16.

Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, United States.

An aminoquinazoline series targeting the essential bacterial enzyme GlmU (uridyltransferase) were previously reported (Biochem. J.2012, 446, 405). In this study, we further explored SAR through a combination of traditional medicinal chemistry and structure-based drug design, resulting in a novel scaffold (benzamide) with selectivity against protein kinases. Virtual screening identified fragments that could be fused into the core scaffold, exploiting additional binding interactions and thus improving potency. These efforts resulted in a hybrid compound with target potency increased by a 1000-fold, while maintaining selectivity against selected protein kinases and an improved level of solubility and protein binding. Despite these significant improvements no significant antibacterial activity was yet observed within this class.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2014.08.017DOI Listing
November 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

Thiazolopyridone ureas as DNA gyrase B inhibitors: optimization of antitubercular activity and efficacy.

Bioorg Med Chem Lett 2014 Feb 25;24(3):870-9. Epub 2013 Dec 25.

Department of Medicinal Chemistry, AstraZeneca India Pvt. Ltd, Bellary Road, Hebbal, Bangalore 560024, India. Electronic address:

Scaffold hopping from the thiazolopyridine ureas led to thiazolopyridone ureas with potent antitubercular activity acting through inhibition of DNA GyrB ATPase activity. Structural diversity was introduced, by extension of substituents from the thiazolopyridone N-4 position, to access hydrophobic interactions in the ribose pocket of the ATP binding region of GyrB. Further optimization of hydrogen bond interactions with arginines in site-2 of GyrB active site pocket led to potent inhibition of the enzyme (IC50 2 nM) along with potent cellular activity (MIC=0.1 μM) against Mycobacterium tuberculosis (Mtb). Efficacy was demonstrated in an acute mouse model of tuberculosis on oral administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2013.12.080DOI Listing
February 2014

Identification through structure-based methods of a bacterial NAD(+)-dependent DNA ligase inhibitor that avoids known resistance mutations.

Bioorg Med Chem Lett 2014 Jan 15;24(1):360-6. Epub 2013 Nov 15.

Department of Bioscience, Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Dr., Waltham, MA 02451, United States.

In an attempt to identify novel inhibitors of NAD(+)-dependent DNA ligase (LigA) that are not affected by a known resistance mutation in the adenosine binding pocket, a detailed analysis of the binding sites of a variety of bacterial ligases was performed. This analysis revealed several similarities to the adenine binding region of kinases, which enabled a virtual screen of known kinase inhibitors. From this screen, a thienopyridine scaffold was identified that was shown to inhibit bacterial ligase. Further characterization through structure and enzymology revealed the compound was not affected by a previously disclosed resistance mutation in Streptococcus pneumoniae LigA, Leu75Phe. A subsequent medicinal chemistry program identified substitutions that resulted in an inhibitor with moderate activity across various Gram-positive bacterial LigA enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2013.11.007DOI Listing
January 2014

Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents.

J Med Chem 2013 Nov 29;56(21):8712-35. Epub 2013 Oct 29.

Infection Innovative Medicines, AstraZeneca R&D Boston , 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.

The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 μM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm401208bDOI Listing
November 2013

Thiazolopyridine ureas as novel antitubercular agents acting through inhibition of DNA Gyrase B.

J Med Chem 2013 Nov 18;56(21):8834-48. Epub 2013 Oct 18.

Department of Medicinal Chemistry, ‡Department of Biosciences, and §DMPK and Animal Sciences, AstraZeneca India Pvt. Ltd. , Bellary Road, Hebbal, Bangalore 560024, India.

A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 μM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm401268fDOI Listing
November 2013

Antibody humanization by redesign of complementarity-determining region residues proximate to the acceptor framework.

Methods 2014 Jan 28;65(1):68-76. Epub 2013 Jun 28.

Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142, United States. Electronic address:

Antibodies are key components of the adaptive immune system and are well-established protein therapeutic agents. Typically high-affinity antibodies are obtained by immunization of rodent species that need to be humanized to reduce their immunogenicity. The complementarity-determining regions (CDRs) contain the residues in a defined loop structure that confer antigen binding, which must be retained in the humanized antibody. To design a humanized antibody, we graft the mature murine CDRs onto a germline human acceptor framework. Structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to murine, or by mutating some residues on the CDRs' backside to human or to a de novo designed sequence. The first approach, framework redesign, can yield an antibody with binding better than the CDR graft and one equivalent to the mature murine, and reduced immunogenicity. The second approach, CDR redesign, is presented here as a new approach, yielding an antibody with binding better than the CDR graft, and immunogenicity potentially less than that from framework redesign. Application of both approaches to the humanization of anti-α4 integrin antibody HP1/2 is presented and the concept of the hybrid humanization approach that retains "difficult to match" murine framework amino acids and uses de novo CDR design to minimize murine amino acid content and reduce cell-mediated cytotoxicity liabilities is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2013.06.024DOI Listing
January 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

Sulfonylpiperidines as novel, antibacterial inhibitors of Gram-positive thymidylate kinase (TMK).

Bioorg Med Chem Lett 2013 Jan 5;23(1):169-73. Epub 2012 Nov 5.

AstraZeneca Infection Innovative Medicines, 35 Gatehouse Drive, Waltham, MA 02451, USA.

Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK. Formation of hydrogen bonds with Arg48 in Staphylococcus aureus TMK was key to obtaining excellent enzyme affinity, as verified by protein crystallography. Replacement of a methylene linker in the series by a sulfonamide was accomplished with retention of binding conformation. Further optimization of logD yielded phenol derivative 11, a potent inhibitor of TMK showing excellent MICs against a broad spectrum of Gram-positive bacteria and >10(5) selectivity versus the human TMK homologue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2012.10.128DOI Listing
January 2013

In vivo validation of thymidylate kinase (TMK) with a rationally designed, selective antibacterial compound.

ACS Chem Biol 2012 Nov 28;7(11):1866-72. Epub 2012 Aug 28.

AstraZeneca Infection Innovative Medicines, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cb300316nDOI Listing
November 2012

Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

Bioorg Med Chem Lett 2012 Aug 19;22(15):5150-6. Epub 2012 Jun 19.

Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.

We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2012.05.128DOI Listing
August 2012

An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode.

Biochem J 2012 Sep;446(3):405-13

Discovery Sciences Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 Å. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P (N-acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure-activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20120596DOI Listing
September 2012

Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents.

Antimicrob Agents Chemother 2012 Mar 19;56(3):1240-6. Epub 2011 Dec 19.

Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA.

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.05485-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294908PMC
March 2012