60 results on '"Magdalena M. Szewczyk"'
Search Results
2. PRMT5 regulates ATF4 transcript splicing and oxidative stress response
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Magdalena M. Szewczyk, Genna M. Luciani, Victoria Vu, Alex Murison, David Dilworth, Samir H. Barghout, Mathieu Lupien, Cheryl H. Arrowsmith, Mark D. Minden, and Dalia Barsyte-Lovejoy
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Epigenetics ,PRMT5 ,Oxidative stress ,Splicing ,Intron retention ,ATF4 ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.
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- 2022
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3. Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response
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Magdalena M. Szewczyk, Yoshinori Ishikawa, Shawna Organ, Nozomu Sakai, Fengling Li, Levon Halabelian, Suzanne Ackloo, Amber L. Couzens, Mohammad Eram, David Dilworth, Hideto Fukushi, Rachel Harding, Carlo C. dela Seña, Tsukasa Sugo, Kozo Hayashi, David McLeod, Carlos Zepeda, Ahmed Aman, Maria Sánchez-Osuna, Eric Bonneil, Shinji Takagi, Rima Al-Awar, Mike Tyers, Stephane Richard, Masayuki Takizawa, Anne-Claude Gingras, Cheryl H. Arrowsmith, Masoud Vedadi, Peter J. Brown, Hiroshi Nara, and Dalia Barsyte-Lovejoy
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Science - Abstract
Protein arginine methyltransferases (PRMTs) are increasingly recognized as potential therapeutic targets but PRMT7 remains an understudied member of this enzyme family. Here, the authors develop a chemical probe for PRMT7 and apply it to elucidate the role of PRMT7 in the cellular stress response.
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- 2020
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4. Discovery of a chemical probe for PRDM9
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Abdellah Allali-Hassani, Magdalena M. Szewczyk, Danton Ivanochko, Shawna L. Organ, Jabez Bok, Jessica Sook Yuin Ho, Florence P. H. Gay, Fengling Li, Levi Blazer, Mohammad S. Eram, Levon Halabelian, David Dilworth, Genna M. Luciani, Evelyne Lima-Fernandes, Qin Wu, Peter Loppnau, Nathan Palmer, S. Zakiah A. Talib, Peter J. Brown, Matthieu Schapira, Philipp Kaldis, Ronan C. O’Hagan, Ernesto Guccione, Dalia Barsyte-Lovejoy, Cheryl H. Arrowsmith, John M. Sanders, Solomon D. Kattar, D. Jonathan Bennett, Benjamin Nicholson, and Masoud Vedadi
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Science - Abstract
PRDM9 is a PR domain containing histone methyl transferase which expression is normally restricted to the germline that has also been linked to a number of somatic cancers. Here the authors describe the identification of a small molecule that selectivity inhibits the methyltransferase activity of PRDM9 in biochemical and cellular assays
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- 2019
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5. Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors
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Jayme L. Dahlin, Kathryn M. Nelson, Jessica M. Strasser, Dalia Barsyte-Lovejoy, Magdalena M. Szewczyk, Shawna Organ, Matthew Cuellar, Gurpreet Singh, Jonathan H. Shrimp, Nghi Nguyen, Jordan L. Meier, Cheryl H. Arrowsmith, Peter J. Brown, Jonathan B. Baell, and Michael A. Walters
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Science - Abstract
A substantial obstacle in basic research is the use of poorly validated tool compounds with purported useful biological functions. Here, the authors systematically profile widely used histone acetyltransferase inhibitors and find that the majority are nonselective interference compounds.
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- 2017
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6. Author Correction: Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response
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Magdalena M. Szewczyk, Yoshinori Ishikawa, Shawna Organ, Nozomu Sakai, Fengling Li, Levon Halabelian, Suzanne Ackloo, Amber L. Couzens, Mohammad Eram, David Dilworth, Hideto Fukushi, Rachel Harding, Carlo C. dela Seña, Tsukasa Sugo, Kozo Hayashi, David McLeod, Carlos Zepeda, Ahmed Aman, Maria Sánchez-Osuna, Eric Bonneil, Shinji Takagi, Rima Al-Awar, Mike Tyers, Stephane Richard, Masayuki Takizawa, Anne-Claude Gingras, Cheryl H. Arrowsmith, Masoud Vedadi, Peter J. Brown, Hiroshi Nara, and Dalia Barsyte-Lovejoy
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Science - Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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- 2020
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7. Discovery of Nanomolar DCAF1 Small Molecule Ligands
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Alice Shi Ming Li, Serah Kimani, Brian Wilson, Mahmoud Noureldin, Héctor González-Álvarez, Ahmed Mamai, Laurent Hoffer, John P. Guilinger, Ying Zhang, Moritz von Rechenberg, Jeremy S. Disch, Christopher J. Mulhern, Belinda L. Slakman, John W. Cuozzo, Aiping Dong, Gennady Poda, Mohammed Mohammed, Punit Saraon, Manish Mittal, Pratik Modh, Vaibhavi Rathod, Bhashant Patel, Suzanne Ackloo, Vijayaratnam Santhakumar, Magdalena M Szewczyk, Dalia Barsyte-Lovejoy, Cheryl H. Arrowsmith, Richard Marcellus, Marie-Aude Guié, Anthony D. Keefe, Peter J. Brown, Levon Halabelian, Rima Al-awar, and Masoud Vedadi
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Drug Discovery ,Molecular Medicine - Published
- 2023
8. Discovery and characterization of a chemical probe targeting the zinc-finger ubiquitin-binding domain of HDAC6
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Rachel J. Harding, Ivan Franzoni, Mandeep K. Mann, Magdalena M. Szewczyk, Bijan Mirabi, Dominic D.G Owens, Suzanne Ackloo, Alexej Scheremetjew, Kevin A. Juarez-Ornelas, Randy Sanichar, Rachel J. Baker, Christian Dank, Peter J. Brown, Dalia Barsyte-Lovejoy, Vijayaratnam Santhakumar, Matthieu Schapira, Mark Lautens, and Cheryl H. Arrowsmith
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open source ,ubiquitin binding domain ,chemical tool ,ubiquitin ,chemical probe ,UBD ,HDAC6 - Abstract
These data are the R scripts, environments, output used for analyzing chemoproteomics data. The volcano plot shown in the manuscript is based on the DEP R package. To validate the enrichment output from DEP, the proteomics data was also processed with the proDA package. Filename (7 files) Description combined_protein_Int.txt Output from database search hdac6_dep_submitted.R script used to process data (from DEP package) hdac6_dep_submitted.R.RData workspace environment from DEP package hdac6probecontrol_dep.csv exported list of p-value and fold-change from DEP hdac6probecontrol_proda.csv exported list of p-value and fold-change from proDA hdac6proda_submitted.R proDA script hdac6proda_submitted.RData proDA workspace environment, PXD039880 - the raw data is also uploaded to PRIDE.
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- 2023
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9. A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization
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Marcus A. Cheek, Aiping Dong, Renato Ferreira de Freitas, Dalia Barsyte-Lovejoy, Aliakbar Khalili Yazdi, Jinrong Min, Fengling Li, Victoria Vu, Albina Bolotokova, Lindsey I. James, Jack Greenblatt, Naimee Mehta, Irina K. Popova, David Dilworth, Ming Lei, Raquel Arminda Carvalho Machado, Ronan P Hanley, David Y Nie, Matthieu Schapira, Mengqi Zhou, Elisa Gibson, Cheryl H. Arrowsmith, Michael-Christopher Keogh, Suzanne Ackloo, Matthew R. Marunde, Mona Alqazzaz, Dmitri Kireev, Nathan W. Hall, Peter Brown, Abdellah Allali-Hassani, Sina Kazemzadeh, Edyta Marcon, Tigran M. Abramyan, Dominic D G Owens, Yanli Liu, Magdalena M. Szewczyk, Matthew J. Meiners, Irene Chau, Su Qin, and Masoud Vedadi
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Gene isoform ,Methyltransferase ,Nucleolus ,Lysine ,Methylation ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Histone-Lysine N-Methyltransferase ,Cell Biology ,Nucleosomes ,Chromatin ,Cell biology ,Repressor Proteins ,Histone ,Enzyme ,Molecular Probes ,030220 oncology & carcinogenesis ,biology.protein ,Multiple Myeloma ,Cell Nucleolus - Abstract
Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
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- 2021
10. A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6
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Jian Jin, Levon Halabelian, Fengling Li, Masoud Vedadi, Rima Al-awar, Carlo C. dela Seña, Mohammad S. Eram, Aiping Dong, Carlos Zepeda-Velázquez, He Chen, Matthieu Schapira, Robert M. Campbell, Dalia Barsyte-Lovejoy, David McLeod, Cheryl H. Arrowsmith, Hong Zeng, Hong Wu, Yudao Shen, Mary M. Mader, Viacheslav V. Trush, Brian Morgan Watson, Kwang-Su Park, Irene Chau, Fanye Meng, Peter Brown, H. Ümit Kaniskan, and Magdalena M. Szewczyk
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Protein-Arginine N-Methyltransferases ,Methyltransferase ,Arginine ,Stereochemistry ,Cell ,Allosteric regulation ,Crystallography, X-Ray ,01 natural sciences ,Article ,03 medical and health sciences ,Allosteric Regulation ,Drug Discovery ,medicine ,Humans ,Enzyme Inhibitors ,IC50 ,030304 developmental biology ,Benzodiazepinones ,0303 health sciences ,Chemistry ,Nuclear Proteins ,Stereoisomerism ,Highly selective ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,HEK293 Cells ,medicine.anatomical_structure ,Molecular Medicine ,Enantiomer ,Selectivity ,Allosteric Site ,Protein Binding - Abstract
Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC(50) = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6−(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2’s enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.
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- 2021
11. Discovery of a First-in-Class Protein Arginine Methyltransferase 6 (PRMT6) Covalent Inhibitor
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Jing Liu, Dalia Barsyte-Lovejoy, Aiping Dong, Kwang-Su Park, Fanye Meng, Irene Chau, Jian Jin, Peter Brown, Levon Halabelian, Yudao Shen, He Chen, Fengling Li, Cheryl H. Arrowsmith, Masoud Vedadi, Magdalena M. Szewczyk, and Hong Zeng
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Protein-Arginine N-Methyltransferases ,Methyltransferase ,Dose-Response Relationship, Drug ,Chemistry ,Protein-arginine methyltransferase ,Nuclear Proteins ,Covalent binding ,Cocrystal ,Article ,Protein Structure, Secondary ,HEK293 Cells ,Biochemistry ,Covalent bond ,Drug Discovery ,MCF-7 Cells ,Humans ,Molecular Medicine ,Enzyme Inhibitors - Abstract
Protein arginine methyltransferase 6 (PRMT6) plays important roles in several biological processes associated with multiple cancers. Well-characterized potent, selective, and cell-active PRMT6 inhibitors are invaluable tools for testing biological and therapeutic hypotheses. Although there are several known reversible PRMT6 inhibitors, covalent PRMT6 inhibitors have not been reported. Based on a cocrystal structure of PRMT6-MS023 (a type I PRMT inhibitor), we discovered the first potent and cell-active irreversible PRMT6 inhibitor, 4 (MS117). The covalent binding mode of compound 4 to PRMT6 was confirmed by mass spectrometry and kinetic studies and by a cocrystal structure. Compound 4 did not covalently modify other closely related PRMTs, potently inhibited PRMT6 in cells, and was selective for PRMT6 over other methyltransferases. We also developed two structurally similar control compounds, 5 (MS167) and 7 (MS168). We provide these valuable chemical tools to the scientific community for further studying PRMT6 physiological and pathophysiological functions.
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- 2020
12. Altered RNA splicing initiates the viral mimicry response from inverted SINEs following type I PRMT inhibition in triple-negative breast cancer
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Cheryl H. Arrowsmith, Jocelyn Chen, Nergiz Artun, Jian Jin, Magdalena M. Szewczyk, Jing Liu, Wail Ba alawi, Qin Wu, David W. Cescon, Felipe Ciamponi, Geneviève Deblois, Daniel D. De Carvalho, Lan Xin Zhang, Jennifer Cruickshank, Parinaz Mehdipour, Katlin Maussirer, Dalia Barsyte-Lovejoy, Panagiotis Prinos, Sajid A. Marhon, Benjamin Haibe-Kains, Shili Duan, Wenjun Chen, Mathieu Lupien, David Y Nie, YiShuai Ji, Yudao Shen, and Jillian Haight
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RNA splicing ,Cancer research ,Mimicry ,Biology ,Triple-negative breast cancer - Abstract
Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here, we undertook a screen of epigenetic chemical probes to systematically uncover the epigenetic regulators critical for TNBC growth. We identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), as having anti-tumor growth activity in TNBC in vitro and in vivo. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses pre- and post-MS023 treatment is a functional biomarker and determinant of response; and these observations extend to a panel of patient-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA (dsRNA). The observed dsRNA accumulation is derived, at least in part, from inverted-repeat Alus (IR-Alus), many of which are expressed from retained introns induced by MS023, which inhibits arginine methylation of RNA-binding proteins and alters mRNA splicing machinery. Together, our results represent a shift in understanding the anti-tumor mechanism of type I PRMT inhibitors and provide a novel rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
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- 2021
13. Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells
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Dalia Barsyte-Lovejoy, Magdalena M. Szewczyk, and Victoria Vu
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chemistry.chemical_classification ,Protein-Arginine N-Methyltransferases ,Gene knockdown ,General Immunology and Microbiology ,Arginine ,biology ,medicine.diagnostic_test ,Chemistry ,General Chemical Engineering ,General Neuroscience ,Reproducibility of Results ,Methylation ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity ,Enzyme ,Western blot ,Biochemistry ,Cell culture ,biology.protein ,medicine ,Biomarker (medicine) ,Antibody - Abstract
Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.
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- 2021
14. PRMT inhibition induces a viral mimicry response in triple-negative breast cancer
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Qin Wu, David Y. Nie, Wail Ba-alawi, YiShuai Ji, ZiWen Zhang, Jennifer Cruickshank, Jillian Haight, Felipe E. Ciamponi, Jocelyn Chen, Shili Duan, Yudao Shen, Jing Liu, Sajid A. Marhon, Parinaz Mehdipour, Magdalena M. Szewczyk, Nergiz Dogan-Artun, WenJun Chen, Lan Xin Zhang, Genevieve Deblois, Panagiotis Prinos, Katlin B. Massirer, Dalia Barsyte-Lovejoy, Jian Jin, Daniel D. De Carvalho, Benjamin Haibe-Kains, XiaoJia Wang, David W. Cescon, Mathieu Lupien, and Cheryl H. Arrowsmith
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Protein-Arginine N-Methyltransferases ,Cell Line, Tumor ,Humans ,Triple Negative Breast Neoplasms ,Cell Biology ,Interferons ,Molecular Biology ,Biomarkers - Abstract
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
- Published
- 2021
15. Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3
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Xiao-Ling Cockcroft, Stephan Karl Zahn, Mark Petronczki, Catherine M. Rogers, Fengling Li, Helmut Berger, Bernadette Sharps, Markus Zeeb, Ralph A. Neumüller, Mark Pearson, Dalia Barsyte-Lovejoy, Teresa Krammer, Oleg Fedorov, Barbara Müllauer, Alexander Weiss-Puxbaum, Kilian Huber, Masoud Vedadi, Magdalena M. Szewczyk, Christopher R. Vakoc, Abdellah Allali-Hassani, Tobias Wunberg, Steven Kennedy, Christoph Reiser, Michael Schleicher, Julian E. Fuchs, Cheryl H. Arrowsmith, Moriz Mayer, Jark Böttcher, Guido Boehmelt, Dietrich Böse, Alexandra Hörmann, Andreas Zoephel, Heribert Arnhof, Sandra Winkler, Darryl B. McConnell, Peter Brown, David Dilworth, Maja Corcokovic, Klaus Rumpel, Thomas Gerstberger, Nikolai Mischerikow, Carrow I. Wells, Ulrich Reiser, and Daniela Häring
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Messenger RNA ,Cell Survival ,Chemistry ,Protein domain ,Nuclear Proteins ,Myeloid leukemia ,Histone-Lysine N-Methyltransferase ,Cell Biology ,Amplicon ,medicine.disease ,In vitro ,Cell Line ,Chromatin ,Cell biology ,Proto-Oncogene Proteins c-myc ,Leukemia ,Gene Expression Regulation ,Protein Domains ,medicine ,Humans ,CRISPR-Cas Systems ,Binding site ,Molecular Biology ,Cell Proliferation - Abstract
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains. A chemical probe BI-9321 for the PWWP1 domain of NSD3 and its inactive analog were identified. BI-9321 binds to the methyl-lysine binding site, reduces the association of NSD3 with chromatin and inhibits proliferation of acute myeloid leukemia cells.
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- 2019
16. HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1
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Matthieu Schapira, Alexander Lemak, Alma Seitova, Justin C. Deme, Peter Loppnau, Cheryl H. Arrowsmith, Ashley Hutchinson, Sem Tamara, Rachel Harding, Albert J. R. Heck, Susan M. Lea, Jeffrey P. Cantle, Xiaobing Zuo, Magdalena M. Szewczyk, Johannes F. Hevler, Jeffrey B. Carroll, and Lixin Fan
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congenital, hereditary, and neonatal diseases and abnormalities ,Huntingtin ,Architecture domain ,Drug discovery ,Chemistry ,Mutant ,Wild type ,Polyglutamine tract ,nervous system diseases ,Cell biology ,Exon ,nervous system ,mental disorders ,Gene - Abstract
Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease.
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- 2021
17. Discovery of Small-Molecule Antagonists of the PWWP Domain of NSD2
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Dalia Barsyte-Lovejoy, Masoud Vedadi, Cheryl H. Arrowsmith, Jinrong Min, Ronan P Hanley, Rima Al-awar, Matthieu Schapira, Lindsey I. James, Renato Ferreira de Freitas, Magdalena M. Szewczyk, Abdellah Allali-Hassani, Naimee Mehta, Fengling Li, Carlos Zepeda-Velázquez, Elisa Gibson, David McLeod, Yanli Liu, Peter Brown, and David Dilworth
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Models, Molecular ,Methyltransferase ,Protein domain ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Crystallography, X-Ray ,Ligands ,01 natural sciences ,Article ,Small Molecule Libraries ,03 medical and health sciences ,Structure-Activity Relationship ,Protein Domains ,Drug Discovery ,Structure–activity relationship ,Humans ,Computer Simulation ,030304 developmental biology ,0303 health sciences ,Virtual screening ,biology ,Chemistry ,Antagonist ,Histone-Lysine N-Methyltransferase ,Small molecule ,0104 chemical sciences ,3. Good health ,Cell biology ,Molecular Docking Simulation ,Repressor Proteins ,010404 medicinal & biomolecular chemistry ,Histone ,biology.protein ,Molecular Medicine ,Drug Screening Assays, Antitumor - Abstract
Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein–protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a K(d) of 3.4 μM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.
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- 2021
18. Rational Design and Synthesis of Selective PRMT4 Inhibitors: a New Chemotype for Development of Cancer Therapeutics
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Cheryl H. Arrowsmith, Cora Scholten, Léa Bouché, Masoud Vedadi, Mathew Sutherland, Ingo Hartung, Alice Li, Vijayaratnam Santhakumar, Dimitrios Panagopoulos, Magdalena M. Szewczyk, Fengling Li, Robert Britton, Timo Stellfeld, Holger Steuber, Dalia Barsyte, David Smil, and Anissa Kaghad
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Protein-Arginine N-Methyltransferases ,Indoles ,Arginine ,Antineoplastic Agents ,01 natural sciences ,Biochemistry ,Structure-Activity Relationship ,Histone H3 ,Non-histone protein ,Neoplasms ,Drug Discovery ,medicine ,Humans ,Transferase ,ddc:610 ,General Pharmacology, Toxicology and Pharmaceutics ,Enzyme Inhibitors ,Pharmacology ,Alanine ,Dose-Response Relationship, Drug ,Molecular Structure ,Chemotype ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Rational design ,Cancer ,Methylation ,medicine.disease ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,HEK293 Cells ,Drug Design ,Molecular Medicine - Abstract
ChemMedChem 16(7), 1116 - 1125 (2021). doi:10.1002/cmdc.202100018, Protein arginine N-methyl transferase 4 (PRMT4) asymmetricallydimethylates the arginine residues of histone H3 and nonhistoneproteins. The overexpression of PRMT4 in several cancershas stimulated interest in the discovery of inhibitors as biologicaltools and, potentially, therapeutics. Although severalPRMT4 inhibitors have been reported, most display poorselectivity against other members of the PRMT family of methyltransferases. Herein, we report the structure-based design of anew class of alanine-containing 3-arylindoles as potent andselective PRMT4 inhibitors, and describe key structure–activityrelationships for this class of compounds., Published by Wiley-VCH, Weinheim [u.a.]
- Published
- 2020
19. Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response
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Hideto Fukushi, Masayuki Takizawa, Shawna Organ, Masoud Vedadi, Peter Brown, Rima Al-awar, Carlo C. dela Seña, Mike Tyers, Carlos Zepeda, Levon Halabelian, David Dilworth, Suzanne Ackloo, Ahmed Aman, Amber L. Couzens, Yoshinori Ishikawa, David Macleod, Nozomu Sakai, Rachel Harding, Stéphane Richard, Tsukasa Sugo, Anne-Claude Gingras, Hiroshi Nara, Fengling Li, Eric Bonneil, Mohammad S. Eram, Cheryl H. Arrowsmith, Shinji Takagi, Dalia Barsyte-Lovejoy, María Sánchez-Osuna, Kozo Hayashi, and Magdalena M. Szewczyk
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0301 basic medicine ,Methyltransferase ,Arginine ,Science ,Cell ,General Physics and Astronomy ,Methylation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Target validation ,03 medical and health sciences ,Stress signalling ,0302 clinical medicine ,Transferases ,Cellular stress response ,medicine ,lcsh:Science ,Multidisciplinary ,Chemistry ,General Chemistry ,Hsp70 ,Cell biology ,030104 developmental biology ,Proteostasis ,medicine.anatomical_structure ,Proteasome ,030220 oncology & carcinogenesis ,lcsh:Q - Abstract
Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response., Protein arginine methyltransferases (PRMTs) are increasingly recognized as potential therapeutic targets but PRMT7 remains an understudied member of this enzyme family. Here, the authors develop a chemical probe for PRMT7 and apply it to elucidate the role of PRMT7 in the cellular stress response.
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- 2020
20. Author Correction: Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response
- Author
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Levon Halabelian, Nozomu Sakai, David McLeod, Anne-Claude Gingras, Rima Al-awar, Hiroshi Nara, Dalia Barsyte-Lovejoy, Masoud Vedadi, David Dilworth, Hideto Fukushi, Stéphane Richard, Carlos Zepeda, Cheryl H. Arrowsmith, María Sánchez-Osuna, Ahmed Aman, Carlo C. dela Seña, Kozo Hayashi, Suzanne Ackloo, Magdalena M. Szewczyk, Yoshinori Ishikawa, Peter Brown, Fengling Li, Eric Bonneil, Mike Tyers, Mohammad S. Eram, Masayuki Takizawa, Amber L. Couzens, Shawna Organ, Rachel Harding, Shinji Takagi, and Tsukasa Sugo
- Subjects
Protein-Arginine N-Methyltransferases ,Arginine ,Science ,General Physics and Astronomy ,Pharmacology ,Biology ,Methylation ,General Biochemistry, Genetics and Molecular Biology ,Target validation ,Stress signalling ,Text mining ,Transferases ,Stress, Physiological ,Cellular stress response ,Sf9 Cells ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,lcsh:Science ,Author Correction ,Multidisciplinary ,business.industry ,General Chemistry ,HCT116 Cells ,Recombinant Proteins ,Gene Knockdown Techniques ,lcsh:Q ,business ,Protein Processing, Post-Translational - Abstract
Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.
- Published
- 2020
21. Discovery of a chemical probe for PRDM9
- Author
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Ernesto Guccione, Philipp Kaldis, Dalia Barsyte-Lovejoy, Danton Ivanochko, Magdalena M. Szewczyk, David Dilworth, Matthieu Schapira, Peter Loppnau, S. Zakiah A. Talib, Fengling Li, Solomon Kattar, Levon Halabelian, Ronan C. O'Hagan, Jabez Bok, Levi L. Blazer, Evelyne Lima-Fernandes, Genna M. Luciani, Nathan Palmer, Mohammad S. Eram, Cheryl H. Arrowsmith, Benjamin Nicholson, John M. Sanders, Peter Brown, Abdellah Allali-Hassani, Masoud Vedadi, Shawna Organ, Jessica Sook Yuin Ho, D. Jonathan Bennett, and Qin Wu
- Subjects
0301 basic medicine ,Genome instability ,S-Adenosylmethionine ,Subfamily ,Methyltransferase ,Science ,Protein domain ,General Physics and Astronomy ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,medicine.disease_cause ,Gene ,Article ,General Biochemistry, Genetics and Molecular Biology ,PR Domain ,Histones ,Inhibitory Concentration 50 ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Transferases ,Histone post-translational modifications ,medicine ,Humans ,Enzyme Inhibitors ,lcsh:Science ,PRDM9 ,Multidisciplinary ,biology ,Drug discovery ,Chemistry ,HEK 293 cells ,Histone-Lysine N-Methyltransferase ,General Chemistry ,DNA Methylation ,3. Good health ,Cell biology ,Histone ,HEK293 Cells ,030104 developmental biology ,Molecular Probes ,030220 oncology & carcinogenesis ,biology.protein ,lcsh:Q ,Chemical tools ,Carcinogenesis - Abstract
PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases., PRDM9 is a PR domain containing histone methyl transferase which expression is normally restricted to the germline that has also been linked to a number of somatic cancers. Here the authors describe the identification of a small molecule that selectivity inhibits the methyltransferase activity of PRDM9 in biochemical and cellular assays
- Published
- 2019
22. TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma
- Author
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Dalia Barsyte-Lovejoy, Yuji Baba, Peter Brown, Atsushi Kiba, Daisuke Tomita, Douglas R. Cary, Aiping Dong, Hong Zeng, Mihoko Kunitomo, Kazuhide Nakayama, Cheryl H. Arrowsmith, Fengling Li, Matthieu Schapira, Mohammad S. Eram, Charles E. Grimshaw, Yasuhiro Imaeda, Carlo C. dela Seña, Kumar Singh Saikatendu, Hong Wu, Akihiro Ohashi, Michiko Tawada, Renato Ferreira de Freitas, Magdalena M. Szewczyk, and Masoud Vedadi
- Subjects
0301 basic medicine ,crystal structure ,Methyltransferase ,Arginine ,CARM1 ,Chemistry ,Protein subunit ,small molecule inhibitor ,Cell cycle ,TP-064 ,Small molecule ,3. Good health ,multiple myeloma ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Oncology ,Biochemistry ,Cell culture ,030220 oncology & carcinogenesis ,PRMT4 ,IC50 ,Research Paper - Abstract
Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.
- Published
- 2018
23. Discovery of Potent and Selective Allosteric Inhibitors of Protein Arginine Methyltransferase 3 (PRMT3)
- Author
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Zhengtian Yu, Sean Xiao, Dalia Barsyte-Lovejoy, Xiaobao Yang, Peter Atadja, Sun-Joon Min, Kehao Zhao, Matthieu Schapira, Jian Jin, Fengling Li, En Li, Mohammad S. Eram, Xiao Luo, Elena Dobrovetsky, Magdalena M. Szewczyk, Jennifer Lin-Jones, Keith Schmidt, H. Ümit Kaniskan, Miao Dai, Ying Lin, Feng He, David Smil, Feng Liu, Peter Brown, Cheryl H. Arrowsmith, Masoud Vedadi, and Irene Zang
- Subjects
0301 basic medicine ,Models, Molecular ,Protein-Arginine N-Methyltransferases ,Arginine ,Protein Conformation ,Allosteric regulation ,Ribosome ,Article ,03 medical and health sciences ,Inhibitory Concentration 50 ,Structure-Activity Relationship ,Protein structure ,Allosteric Regulation ,In vivo ,Drug Discovery ,Structure–activity relationship ,Transferase ,Humans ,030102 biochemistry & molecular biology ,Chemistry ,HEK 293 cells ,Hydrogen Bonding ,Bridged Bicyclo Compounds, Heterocyclic ,3. Good health ,030104 developmental biology ,HEK293 Cells ,Biochemistry ,Drug Design ,Molecular Medicine - Abstract
PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is crucial for maturation of ribosomes and has been implicated in several diseases. We recently disclosed a highly potent, selective, and cell-active allosteric inhibitor of PRMT3, compound 4. Here, we report comprehensive structure–activity relationship studies that target the allosteric binding site of PRMT3. We conducted design, synthesis, and evaluation of novel compounds in biochemical, selectivity, and cellular assays that culminated in the discovery of 4 and other highly potent (IC50 values: ∼10–36 nM), selective, and cell-active allosteric inhibitors of PRMT3 (compounds 29, 30, 36, and 37). In addition, we generated compounds that are very close analogs of these potent inhibitors but displayed drastically reduced potency as negative controls (compounds 49–51). These inhibitors and negative controls are valuable chemical tools for the biomedical community to further investigate biological functions and disease associations of PRMT3.
- Published
- 2017
24. Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors
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Dalia Barsyte-Lovejoy, Jonathan H. Shrimp, Jonathan B. Baell, Jayme L. Dahlin, Gurpreet Singh, Nghi H. Nguyen, Jessica M. Strasser, Matthew E. Cuellar, Kathryn M. Nelson, Magdalena M. Szewczyk, Jordan L. Meier, Michael A. Walters, Cheryl H. Arrowsmith, Peter Brown, and Shawna Organ
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Cell Survival ,High-throughput screening ,Science ,Chemical biology ,General Physics and Astronomy ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Drug Discovery ,Bioassay ,Humans ,Epigenetics ,Sulfhydryl Compounds ,Enzyme Inhibitors ,lcsh:Science ,Histone Acetyltransferases ,Multidisciplinary ,biology ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Drug discovery ,HEK 293 cells ,General Chemistry ,Histone acetyltransferase ,In vitro ,0104 chemical sciences ,3. Good health ,High-Throughput Screening Assays ,030104 developmental biology ,HEK293 Cells ,Biochemistry ,biology.protein ,MCF-7 Cells ,lcsh:Q - Abstract
Many compounds with potentially reactive chemical motifs and poor physicochemical properties are published as selective modulators of biomolecules without sufficient validation and then propagated in the scientific literature as useful chemical probes. Several histone acetyltransferase (HAT) inhibitors with these liabilities are now routinely used to probe epigenetic pathways. We profile the most commonly used HAT inhibitors and confirm that the majority of them are nonselective interference compounds. Most (15 out of 23, 65%) of the inhibitors are flagged by ALARM NMR, an industry-developed counter-screen for promiscuous compounds. Biochemical counter-screens confirm that most of these compounds are either thiol-reactive or aggregators. Selectivity panels show many of these compounds modulate unrelated targets in vitro, while several also demonstrate nonspecific effects in cell assays. These data demonstrate the usefulness of performing counter-screens for bioassay promiscuity and assay interference, and raise caution about the utility of many widely used, but insufficiently validated, compounds employed in chemical biology., A substantial obstacle in basic research is the use of poorly validated tool compounds with purported useful biological functions. Here, the authors systematically profile widely used histone acetyltransferase inhibitors and find that the majority are nonselective interference compounds.
- Published
- 2017
25. Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation
- Author
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Tiziana Bonaldi, Cheryl M. Koh, Cheryl H. Arrowsmith, Christine Thompson, Heike Wollmann, Bas J. Wouters, Luca Pignata, Jia Yi Fong, Magdalena M. Szewczyk, Ari Melnick, Dalia Barsyte-Lovejoy, Daniele Musiani, Cheng Mun Wun, Alex Penson, Pierre-Alexis Goy, Jian Jin, Eirini P. Papapetrou, Mark D. Minden, Enrico Massignani, Genna M. Luciani, Omar Abdel-Wahab, Olena Barbash, Ernesto Guccione, Ruud Delwel, Diana Hp. Low, Michelle Ki, Alexander Rialdi, Kimihito Cojin Kawabata, Andriana G. Kotini, Timothy K. Hart, Megan C. Schwarz, Slim Mzoughi, Yudao Shen, Stanley Chun-Wei Lee, and Hematology
- Subjects
0301 basic medicine ,RNA Splicing Factors ,Protein-Arginine N-Methyltransferases ,Cancer Research ,Spliceosome ,Methyltransferase ,Arginine ,THP-1 Cells ,RNA Splicing ,Antineoplastic Agents ,Mice, Transgenic ,Article ,Catalysis ,03 medical and health sciences ,0302 clinical medicine ,Tumor Cells, Cultured ,Animals ,Humans ,Gene Regulatory Networks ,Pyrroles ,RNA, Neoplasm ,Enzyme Inhibitors ,Gene ,Chemistry ,Protein arginine methyltransferase 5 ,U937 Cells ,Methylation ,Ethylenediamines ,Xenograft Model Antitumor Assays ,Cell biology ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,Repressor Proteins ,Leukemia, Myeloid, Acute ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,RNA splicing ,K562 Cells - Abstract
Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.
- Published
- 2019
26. Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress responses
- Author
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Stéphane Richard, Cheryl H. Arrowsmith, Hiroshi Nara, Carlo C. dela Seña, Shinji Takagi, Carlos Zepeda, Rima Al-awar, Yoshinori Ishikawa, Dalia Barsyte-Lovejoy, David Macleod, Shawna Organ, Suzanne Ackloo, Peter Brown, Fengling Li, Tsukasa Sugo, Masoud Vedadi, Nozomu Sakai, David Dilworth, Mohammad S. Eram, Hideto Fukushi, Kozo Hayashi, Magdalena M. Szewczyk, Masayuki Takizawa, and Rachel Harding
- Subjects
0303 health sciences ,Methyltransferase ,Arginine ,Chemistry ,Cell ,Methylation ,3. Good health ,Hsp70 ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Proteostasis ,medicine.anatomical_structure ,Proteasome ,030220 oncology & carcinogenesis ,Cellular stress response ,medicine ,030304 developmental biology - Abstract
Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective and cell active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells, is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 resulted in drastically reduced levels of arginine monomethylation of HSP70 family members and other stress-associated proteins. Structural and biochemical analysis revealed that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibited methylation of both constitutive and inducible forms of HSP70, and led to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.
- Published
- 2018
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27. Discovery of a Potent, Selective, and Cell-Active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6
- Author
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H. Ümit Kaniskan, Renato Ferreira de Freitas, David Smil, Dalia Barsyte-Lovejoy, Cheryl H. Arrowsmith, Jian Jin, Matthieu Schapira, Magdalena M. Szewczyk, Jing Liu, Fengling Li, Mohammad S. Eram, Guillermo Senisterra, Masoud Vedadi, Yudao Shen, and Peter Brown
- Subjects
Models, Molecular ,0301 basic medicine ,Protein-Arginine N-Methyltransferases ,Methyltransferase ,Arginine ,Cell ,Chemical biology ,Crystallography, X-Ray ,Article ,03 medical and health sciences ,Drug Discovery ,medicine ,Humans ,Epigenetics ,Enzyme Inhibitors ,Drug discovery ,Chemistry ,HEK 293 cells ,Dual inhibitor ,Nuclear Proteins ,3. Good health ,HEK293 Cells ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,Molecular Medicine - Abstract
Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure–activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and non-epigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of PRMT4 and PRMT6 in health and disease.
- Published
- 2016
28. A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases
- Author
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Brandon A. Speed, Fengling Li, Mohammad S. Eram, Jing Liu, Kyle V. Butler, Magdalena M. Szewczyk, H. Uemit Kaniskan, Hong Zeng, Peter Brown, Jian Jin, Matthieu Schapira, Hong Wu, Masoud Vedadi, Aiping Dong, Dalia Barsyte-Lovejoy, Guillermo Senisterra, Carlo C. dela Seña, Cheryl H. Arrowsmith, and Yudao Shen
- Subjects
Models, Molecular ,0301 basic medicine ,Protein-Arginine N-Methyltransferases ,Methyltransferase ,Arginine ,Cell ,Antineoplastic Agents ,Human type ,Biology ,Biochemistry ,Article ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Humans ,Transferase ,Pyrroles ,Cell Proliferation ,Molecular Structure ,Extramural ,General Medicine ,Ethylenediamines ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,Ethanolamines ,Cell culture ,Molecular Medicine - Abstract
Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.
- Published
- 2015
29. Characterization of inv(3) cell line OCI-AML-20 with stroma-dependent CD34 expression
- Author
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David Dilworth, Lihua Xie, Anne Tierens, Dalia Barsyte-Lovejoy, Yoni Moskovitz, Cheryl H. Arrowsmith, Alex Murison, Magdalena M. Szewczyk, Amanda Mitchell, Mathieu Lupien, John E. Dick, Mark D. Minden, Liran I. Shlush, and Genna M. Luciani
- Subjects
0301 basic medicine ,Adult ,Male ,Cancer Research ,Stromal cell ,medicine.medical_treatment ,CD34 ,Antigens, CD34 ,Biology ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Stroma ,hemic and lymphatic diseases ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,neoplasms ,Molecular Biology ,Chromosome 7 (human) ,Chemotherapy ,Gene Expression Regulation, Leukemic ,Myeloid leukemia ,Cell Biology ,Hematology ,biochemical phenomena, metabolism, and nutrition ,Coculture Techniques ,Neoplasm Proteins ,Leukemia, Myeloid, Acute ,030104 developmental biology ,Cell culture ,030220 oncology & carcinogenesis ,Chromosome Inversion ,Cancer research ,Chromosomes, Human, Pair 3 ,Chromosome Deletion ,Stromal Cells ,Chromosomes, Human, Pair 7 - Abstract
Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.
- Published
- 2018
30. Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice
- Author
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Taryne Chong, Šárka Lhoták, Richard C. Austin, Magdalena M. Szewczyk, Elizabeth J. White, Suleiman A. Igdoura, Bernardo L. Trigatti, Gabriel Gyulay, Mark T Fuller, Omid Dadoo, and Alison Fox-Robichaud
- Subjects
0301 basic medicine ,Apolipoprotein E ,Male ,medicine.medical_specialty ,Very low-density lipoprotein ,Mice, Knockout, ApoE ,T-Lymphocytes ,Cholesterol, VLDL ,Down-Regulation ,Neuraminidase ,Inflammation ,Biochemistry ,Muscle, Smooth, Vascular ,03 medical and health sciences ,Mice ,Apolipoproteins E ,Internal medicine ,medicine ,Animals ,Hyaluronic Acid ,Molecular Biology ,Aorta ,Triglycerides ,Chemistry ,Monocyte ,Macrophages ,Lipid metabolism ,Molecular Bases of Disease ,Cell Biology ,Cholesterol, LDL ,Atherosclerosis ,Transplantation ,Chemotaxis, Leukocyte ,P-Selectin ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Liver ,lipids (amino acids, peptides, and proteins) ,medicine.symptom ,Lipid glycosylation ,Lipoprotein - Abstract
Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1(hypo)Apoe(−/−)). We found that the hypomorphic NEU1 expression in male Apoe(−/−) mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe(−/−) bone marrow (BM) into Neu1(hypo)Apoe(−/−) mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1(hypo)Apoe(−/−) mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe(−/−) mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.
- Published
- 2018
31. LLY-283, a Potent and Selective Inhibitor of Arginine Methyltransferase 5, PRMT5, with Antitumor Activity
- Author
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Steven Kennedy, Dalia Barsyte-Lovejoy, Zahid Quyoom Bonday, Ken Weichert, Wayne P. Bocchinfuso, Grogan Michael John, Mary M. Mader, Masoud Vedadi, Robert M. Campbell, Cheryl H. Arrowsmith, Cortez Guillermo S, Fengling Li, Stephen Antonysamy, Mohammad S. Eram, Ernesto Guccione, Binghui Li, Peter Brown, and Magdalena M. Szewczyk
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Methyltransferase ,Protein arginine methyltransferase 5 ,Organic Chemistry ,Methylation ,Biochemistry ,Molecular biology ,In vitro ,3. Good health ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Enzyme ,chemistry ,In vivo ,030220 oncology & carcinogenesis ,Drug Discovery ,Transcriptional regulation ,Signal transduction - Abstract
[Image: see text] Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine in a number of nuclear and cytoplasmic proteins. Although the cellular functions of PRMT5 have not been fully unraveled, it has been implicated in a number of cellular processes like RNA processing, signal transduction, and transcriptional regulation. PRMT5 is ubiquitously expressed in most tissues and its expression has been shown to be elevated in several cancers including breast cancer, gastric cancer, glioblastoma, and lymphoma. Here, we describe the identification and characterization of a novel and selective PRMT5 inhibitor with potent in vitro and in vivo activity. Compound 1 (also called LLY-283) inhibited PRMT5 enzymatic activity in vitro and in cells with IC(50) of 22 ± 3 and 25 ± 1 nM, respectively, while its diastereomer, compound 2 (also called LLY-284), was much less active. Compound 1 also showed antitumor activity in mouse xenografts when dosed orally and can serve as an excellent probe molecule for understanding the biological function of PRMT5 in normal and cancer cells.
- Published
- 2018
32. Discovery of a Dual PRMT5–PRMT7 Inhibitor
- Author
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Steven Kennedy, David Smil, Matthieu Schapira, Fengling Li, Mohammad S. Eram, Peter Brown, Magdalena M. Szewczyk, Masoud Vedadi, Cheryl H. Arrowsmith, and Dalia Barsyte-Lovejoy
- Subjects
MAPK/ERK pathway ,0303 health sciences ,Methyltransferase ,Arginine ,Cell growth ,Protein arginine methyltransferase 5 ,Organic Chemistry ,Biology ,Biochemistry ,3. Good health ,03 medical and health sciences ,Transduction (genetics) ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Drug Discovery ,RNA splicing ,DNA ,030304 developmental biology - Abstract
The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5-PRMT7 axis as a target for therapy.
- Published
- 2015
33. A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)
- Author
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David Smil, Ying Lin, Masoud Vedadi, Kehao Zhao, Feng He, Steven Kennedy, Jian Jin, Keith Schmidt, H. Ümit Kaniskan, Matthieu Schapira, Bryan L. Roth, Cheryl H. Arrowsmith, Dalia Barsyte-Lovejoy, Elena Dobrovetsky, Zhengtian Yu, Xiao Luo, Magdalena M. Szewczyk, Miao Dai, Xi Ping Huang, Xiaobao Yang, Peter Atadja, Fengling Li, Mohammad S. Eram, Sun-Joon Min, Aiping Dong, Jennifer Lin-Jones, Irene Zang, Peter Brown, and Melissa R. Landon
- Subjects
Protein-Arginine N-Methyltransferases ,Methyltransferase ,Arginine ,Allosteric regulation ,Plasma protein binding ,Calorimetry ,Molecular Dynamics Simulation ,Methylation ,Article ,Catalysis ,Histones ,Allosteric Regulation ,Cell Line, Tumor ,medicine ,Humans ,Transferase ,Enzyme Inhibitors ,Binding site ,Binding Sites ,Chemistry ,General Medicine ,General Chemistry ,Surface Plasmon Resonance ,Isoquinolines ,Protein Structure, Tertiary ,HEK293 Cells ,Mechanism of action ,Biochemistry ,Mutagenesis ,medicine.symptom ,Protein Binding - Abstract
PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell- active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure-based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC50 = 31 ± 2 nm, KD = 53 ± 2 nm) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non-epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3-SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well- characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.
- Published
- 2015
34. Correction to Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening
- Author
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Vijayaratnam Santhakumar, Magdalena M. Szewczyk, Dalia Barsyte-Lovejoy, Masoud Vedadi, Peter Brown, Mohammad S. Eram, Steven Kennedy, Matthieu Schapira, Cheryl H. Arrowsmith, Renato Ferreira de Freitas, and David Smil
- Subjects
Virtual screening ,Biochemistry ,CARM1 ,Chemistry ,Drug Discovery ,Coactivator ,Molecular Medicine ,Arginine methyltransferase - Published
- 2016
35. Abstract 1646: Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor
- Author
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Manfred Husemann, Fengling Li, Volker Badock, Viacheslav V. Trush, Detlef Stoeckigt, Cheryl H. Arrowsmith, Jörg Weiske, Shawna Organ, Stephan Siegel, Stefan Gradl, Amaury Ernesto Fernandez-Montalvan, Norbert Schmees, Magdalena M. Szewczyk, Holger Steuber, Masoud Vedadi, Steven Kennedy, Clara D. Christ, Marcus Bauser, Dalia Barsyte-Lovejoy, Andrea Haegebarth, Ingo Hartung, Carlo Stresemann, and Peter Brown
- Subjects
Cancer Research ,Thermal shift assay ,Methyltransferase ,Oncology ,Kinase ,Chemistry ,Transcriptional regulation ,Methylation ,MAP3K2 ,Binding site ,Protein kinase A ,Molecular biology - Abstract
SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase (PKMT) which was initially described as H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has recently been reported to methylate and regulate several non-histone cancer relevant proteins such as mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition overexpression of SMYD3 has been linked to poor prognosis in certain cancers, thus supporting a possible oncogenic role for SMYD3 and making it an attractive target for anticancer drug development. Here we report the discovery of a novel potent and selective SMYD3 inhibitor series. We performed a thermal shift assay based (TSA) high throughput screening followed by extensive biophysical validation resulting in identification of a benzodiazepine-based SMYD3 inhibitor series. The co-crystallization structures revealed that this series binds to the substrate binding site and occupies the hydrophobic pocket for lysine binding using an unprecedented hydrogen bond pattern. The competitive behavior of the inhibitor in biochemical assays was consistent with the binding mode observed in the crystal structure. Further optimization generated BAY-6035, which showed improved nanomolar potency and was selective against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibited methylation of MAP3K2 by SMYD3 in a cellular assay with similar potency. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe and will foster the exploration of the biologic role of SMYD3 in diseased and non-diseased tissues. Citation Format: Stefan Gradl, Holger Steuber, Jörg Weiske, Norbert Schmees, Stephan Siegel, Detlef Stoeckigt, Clara D. Christ, Fengling Li, Shawna Organ, Dalia Barsyte-Lovejoy, Magdalena M. Szewczyk, Steven Kennedy, Viacheslav Trush, Masoud Vedadi, Cheryl H. Arrowsmith, Peter J. Brown, Manfred Husemann, Amaury E. Fernandez-Montalvan, Volker Badock, Marcus Bauser, Andrea Haegebarth, Ingo V. Hartung, Carlo Stresemann. Discovery and characterization of BAY-6035, a novel benzodiazepine-based SMYD3 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1646.
- Published
- 2018
36. Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca2+-pump isoform 4, on coronary artery
- Author
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Jyoti Pande, Iwona Kuszczak, Emanuel Escher, Shawn Grover, Magdalena M. Szewczyk, and Ashok K. Grover
- Subjects
Gene isoform ,Swine ,Calcium pump ,duodenum ,Biology ,Protein Engineering ,plasma membrane ,Cell membrane ,vascular ,Affinity chromatography ,Peptide Library ,calcium pumps ,Extracellular ,medicine ,Animals ,Protein Isoforms ,sperm motility ,Ca(2+) Mg(2+)-ATPase ,Endoplasmic reticulum ,Cell Membrane ,Articles ,Cell Biology ,Coronary Vessels ,medicine.anatomical_structure ,Biochemistry ,Mutagenesis ,Biophysics ,Molecular Medicine ,Signal transduction ,Peptides - Abstract
Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.
- Published
- 2008
37. Ca2+-pumps and Na+–Ca2+-exchangers in coronary artery endothelium versus smooth muscle
- Author
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Kim A. Davis, Patangi K. Rangachari, Ashok K. Grover, Magdalena M. Szewczyk, Sue E. Samson, and Fiona Simpson
- Subjects
Gene isoform ,Cell type ,SERCA ,coronary artery ,Swine ,sodium–calcium exchanger ,Biology ,Sodium-Calcium Exchanger ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Contractility ,Plasma Membrane Calcium-Transporting ATPases ,PMCA ,Animals ,Sodium-calcium exchanger ,Reverse Transcriptase Polymerase Chain Reaction ,Endoplasmic reticulum ,Muscle, Smooth ,Cell Biology ,Articles ,musculoskeletal system ,Coronary Vessels ,Cell biology ,Biochemistry ,Cytoplasm ,cardiovascular system ,Molecular Medicine ,Plasma membrane Ca2+ ATPase ,Endothelium, Vascular ,tissues - Abstract
Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+–Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.
- Published
- 2007
38. Ca2+ -mediated ascorbate release from coronary artery endothelial cells
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Magdalena M. Szewczyk, Kelly Best, Ashok K. Grover, Chiu-Yin Kwan, Kim A. Davis, Sue E. Samson, Kanwaldeep K. Mallhi, and John X. Wilson
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Pharmacology ,endocrine system ,Phospholipase C ,animal diseases ,chemistry.chemical_element ,hemic and immune systems ,Calcium ,Ascorbic acid ,Molecular biology ,eye diseases ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Symporter ,Extracellular ,Dehydroascorbic acid ,Cyclopiazonic acid ,tissues ,Adenosine triphosphate - Abstract
1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.
- Published
- 2006
39. Erratum: The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex
- Author
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Yupeng, He, Sujatha, Selvaraju, Michael L, Curtin, Clarissa G, Jakob, Haizhong, Zhu, Kenneth M, Comess, Bailin, Shaw, Juliana, The, Evelyne, Lima-Fernandes, Magdalena M, Szewczyk, Dong, Cheng, Kelly L, Klinge, Huan-Qiu, Li, Marina, Pliushchev, Mikkel A, Algire, David, Maag, Jun, Guo, Justin, Dietrich, Sanjay C, Panchal, Andrew M, Petros, Ramzi F, Sweis, Maricel, Torrent, Lance J, Bigelow, Guillermo, Senisterra, Fengling, Li, Steven, Kennedy, Qin, Wu, Donald J, Osterling, David J, Lindley, Wenqing, Gao, Scott, Galasinski, Dalia, Barsyte-Lovejoy, Masoud, Vedadi, Fritz G, Buchanan, Cheryl H, Arrowsmith, Gary G, Chiang, Chaohong, Sun, and William N, Pappano
- Subjects
Cell Biology ,Molecular Biology - Published
- 2017
40. Allosteric inhibitors of plasma membrane Ca pumps: Invention and applications of caloxins
- Author
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Jyoti, Pande, Magdalena M, Szewczyk, and Ashok K, Grover
- Subjects
Topic Highlight - Abstract
Plasma membrane Ca(2+) pumps (PMCA) play a major role in Ca(2+) homeostasis and signaling by extruding cellular Ca(2+) with high affinity. PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states. Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology. We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area. A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides, caloxins may become clinically useful in cardiovascular diseases, neurological disorders, retinopathy, cancer and contraception.
- Published
- 2010
41. Phage display: concept, innovations, applications and future
- Author
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Magdalena M. Szewczyk, Jyoti Pande, and Ashok K. Grover
- Subjects
chemistry.chemical_classification ,Phage display ,viruses ,Phagemid ,Mutagenesis (molecular biology technique) ,Bioengineering ,Peptide ,Biopanning ,Computational biology ,Biology ,Applied Microbiology and Biotechnology ,Combinatorial chemistry ,Protein–protein interaction ,Epitope mapping ,chemistry ,Mutagenesis ,Peptide Library ,Bacteriophages ,Peptide library ,Peptides ,Biotechnology ,Protein Binding - Abstract
Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.
- Published
- 2010
42. Caloxin 1c2‐ A Novel Engineered Selective Inhibitor of Plasma Membrane Ca 2+ ‐ Pump Isoform 4, Effects on Coronary Artery
- Author
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Magdalena M. Szewczyk, Jyoti Pande, Iwona Kuszczak, and Ashok K. Grover
- Subjects
Gene isoform ,Membrane ,medicine.anatomical_structure ,Chemistry ,Genetics ,medicine ,Biophysics ,Molecular Biology ,Biochemistry ,Biotechnology ,Artery - Published
- 2008
43. Caloxin 1c2: Experimental Evidence That It Binds Plasma Membrane Calcium Pump
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Rajneet Kuner, Jyoti Pande, Ashok K. Grover, Magdalena M. Szewczyk, and Paromita Ghosh
- Subjects
Chemistry ,Genetics ,Biophysics ,Plasma Membrane Calcium Pump ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2008
44. Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1
- Author
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Magdalena M. Szewczyk, Kanwaldeep K. Mallhi, Ahilya N. Sawh, Jyoti Pande, Fiona Simpson, and Ashok K. Grover
- Subjects
Gene isoform ,Male ,Endothelium ,Physiology ,Calcium-Transporting ATPases ,Biology ,Rats, Inbred WKY ,Muscle, Smooth, Vascular ,Structure-Activity Relationship ,Smooth muscle ,medicine ,Extracellular ,Ca2 pump ,Animals ,Humans ,Protein Isoforms ,Gene ,Aorta ,Messenger RNA ,Dose-Response Relationship, Drug ,Cell Membrane ,Cell Biology ,Cell biology ,Rats ,medicine.anatomical_structure ,Membrane ,Biochemistry ,Endothelium, Vascular ,Peptides ,Muscle Contraction - Abstract
Plasma membrane Ca2+pumps (PMCA) that expel Ca2+from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca2+-Mg2+-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a Kivalue of 46 ± 5 μM, making it 10× more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca2+-Mg2+-ATPase in human embryonic kidney-293 cells with a higher Kivalue (105 ± 11 μM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 μM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca2+] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.
- Published
- 2006
45. Allosteric inhibitors of plasma membrane Ca2+pumps: Invention and applications of caloxins
- Author
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Ashok K. Grover, Jyoti Pande, and Magdalena M. Szewczyk
- Subjects
Gene isoform ,Cell type ,Membrane ,Allosteric regulation ,Extracellular ,Biology ,Pharmacology ,Lipid raft ,Homeostasis ,Calcium signaling ,Cell biology - Abstract
Plasma membrane Ca(2+) pumps (PMCA) play a major role in Ca(2+) homeostasis and signaling by extruding cellular Ca(2+) with high affinity. PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states. Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology. We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area. A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides, caloxins may become clinically useful in cardiovascular diseases, neurological disorders, retinopathy, cancer and contraception.
- Published
- 2011
46. Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders
- Author
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Andreas Krämer, Elmar Wolf, Frank Löhr, Dalia Barsyte-Lovejoy, Martin Eilers, Mathias Diebold, Magdalena M. Szewczyk, Christoph A. Sotriffer, Cheryl H. Arrowsmith, Lena-Marie Berger, Stephanie Heinzlmeir, Jakob Gebel, Bernhard Kuster, Volker Dötsch, Stefan Knapp, Bikash Adhikari, Anja Dölle, Janik Weckesser, and Nicola Berner
- Subjects
Male ,Scaffold protein ,Dihydropyridines ,Antineoplastic Agents ,Ligands ,01 natural sciences ,Structure-Activity Relationship ,03 medical and health sciences ,Drug Discovery ,Humans ,WDR5 ,Gene ,Transcription factor ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Chemistry ,WD Repeat-Containing Protein 5 ,Biphenyl Compounds ,Intracellular Signaling Peptides and Proteins ,0104 chemical sciences ,Cell biology ,010404 medicinal & biomolecular chemistry ,Histone ,Drug Design ,Histone methyltransferase ,biology.protein ,Molecular Medicine ,Female ,Linker - Abstract
Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.
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- View/download PDF
47. Recruitment of FBXO22 for Targeted Degradation of NSD2.
- Author
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Nie DY, Tabor JR, Li J, Kutera M, St-Germain J, Hanley RP, Wolf E, Paulakonis E, Kenney TMG, Duan S, Shrestha S, Owens DDG, Pon A, Szewczyk M, Lamberto AJ, Menes M, Li F, Barsyte-Lovejoy D, Brown NG, Barsotti AM, Stamford AW, Collins JL, Wilson DJ, Raught B, Licht JD, James LI, and Arrowsmith CH
- Abstract
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF
FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.- Published
- 2023
- Full Text
- View/download PDF
48. Multicenter Randomized Trial Assessing MRI and Image-guided Biopsy for Suspected Prostate Cancer: The MULTIPROS Study.
- Author
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Wei C, Szewczyk-Bieda M, Bates AS, Donnan PT, Rauchhaus P, Gandy S, Ragupathy SKA, Singh P, Coll K, Serhan J, Wilson J, and Nabi G
- Subjects
- Male, Humans, Aged, Magnetic Resonance Imaging, Prospective Studies, Image-Guided Biopsy, Prostatic Neoplasms, Multiparametric Magnetic Resonance Imaging
- Abstract
Background The optimal diagnostic pathway for prostate cancer (PCa) is evolving, requiring further evaluation in a randomized controlled trial. Purpose To assess the diagnostic accuracy of prebiopsy multiparametric MRI in the identification of clinically significant PCa (csPCa) using radical prostatectomy (RP) specimens as the reference standard, and to test the diagnostic accuracy of combined US and MRI fusion-targeted biopsy with systematic biopsies. Materials and Methods In a prospective randomized controlled trial including university hospitals, men with suspected PCa were recruited between January 2015 and August 2020 to assess the diagnostic accuracy of multiparametric MRI before biopsy in detection of csPCa at biopsy and RP histopathologic structure (primary outcome). Men with lesions suspicious for cancer (Prostate Imaging and Reporting Data System [PI-RADS] ≥3) at multiparametric MRI were first randomized to either systematic random prostate biopsies alone (control group) or US and MRI fusion-targeted biopsies with systematic random prostate biopsies (intervention group) at a one-to-one ratio to compare the diagnostic accuracy of systematic random versus combined fusion with systematic random biopsies (secondary outcome). A subset of recruited participants ( n = 89) underwent RP and histologic sectioning. Results There were 582 participants who were eligible to undergo multiparametric MRI (mean age, 65 years ± 6 [SD]). In total, 413 had a PI-RADS score of at least 3 and were randomized into either the intervention group (207 of 413; 50.1%) or control group (206 of 413; 49.9%). The csPCa detection rate in the intervention group was higher, with an adjusted odds ratio of 1.79 (95% CI: 1.14, 2.79; P = .01). A subgroup of 89 men underwent RP (21.5%; 89 of 413). Multiparametric MRI helped correctly identify 131 of 182 csPCa foci in 89 men (sensitivity, 72%; 95% CI: 65, 78). The specificity, positive predictive value, and negative predictive value were 71% (91 of 128), 78% (131 of 168), and 64% (91 of 142), respectively. Conclusion Prebiopsy multiparametric MRI was accurate in the depiction of clinically significant PCa. Combining US and MRI fusion-targeted biopsies with systematic biopsies helped detect more clinically significant lesions than did systematic biopsies alone. Clinical trial registration no. NCT02745496 © RSNA, 2023 Supplemental material is available for this article.
- Published
- 2023
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49. Reference compounds for characterizing cellular injury in high-content cellular morphology assays.
- Author
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Dahlin JL, Hua BK, Zucconi BE, Nelson SD Jr, Singh S, Carpenter AE, Shrimp JH, Lima-Fernandes E, Wawer MJ, Chung LPW, Agrawal A, O'Reilly M, Barsyte-Lovejoy D, Szewczyk M, Li F, Lak P, Cuellar M, Cole PA, Meier JL, Thomas T, Baell JB, Brown PJ, Walters MA, Clemons PA, Schreiber SL, and Wagner BK
- Subjects
- Oxidation-Reduction, High-Throughput Screening Assays
- Abstract
Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. Here we generate cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds that cause cellular damage produces bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting., (© 2023. The Author(s).)
- Published
- 2023
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- View/download PDF
50. Predicting the Performance of Concurrent Systematic Random Biopsies during Image Fusion Targeted Sampling of Multi-Parametric MRI Detected Prostate Cancer. A Prospective Study (PRESET Study).
- Author
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Alqahtani S, Zhang X, Wei C, Zhang Y, Szewczyk-Bieda M, Wilson J, Huang Z, and Nabi G
- Abstract
The study was aimed to develop a predictive model to identify patients who may benefit from performing systematic random biopsies (SB) in addition to targeted biopsies (TB) in men suspected of having prostate cancer. A total of 198 patients with positive pre-biopsy MRI findings and who had undergone both TB and SB were prospectively recruited into this study. The primary outcome was detection rates of clinically significant prostate cancer (csPCa) in SB and TB approaches. The secondary outcome was net clinical benefits of SB in addition to TB. A logistic regression model and nomogram construction were used to perform a multivariate analysis. The detection rate of csPCa using SB was 51.0% (101/198) compared to a rate of 56.1% (111/198) for TB, using a patient-based biopsy approach. The detection rate of csPCa was higher using a combined biopsy (64.6%; 128/198) in comparison to TB (56.1%; 111/198) alone. This was statistically significant ( p < 0.001). Age, PSA density and PIRADS score significantly predicted the detection of csPCa by SB in addition to TB. A nomogram based on the model showed good discriminative ability (C-index; 78%). The decision analysis curve confirmed a higher net clinical benefit at an acceptable threshold.
- Published
- 2021
- Full Text
- View/download PDF
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