Targeting Arginine Methyltransferases: Identification of First-In-Class PRMT5 Inhibitor EPZ015666
Kenneth W. Duncan, PhD, Epizyme, Inc., Cambridge
Protein Arginine Methyltransferase-5 (PRMT5) has been reported to play a role in multiple diverse cellular processes including tumorigenesis. Overexpression of PRMT5 has been observed in cell lines and primary patient samples derived from lymphomas, particularly Mantle Cell Lymphoma (MCL). Here, we will describe the identification and characterization of EPZ015666 (GSK3235025), a potent, selective and orally available inhibitor of PRMT5. This novel inhibitor is SAM-uncompetitive, peptide-competitive and interacts with the PRMT5:MEP50 complex through a unique inhibition mode not previously observed for any SAM-dependent enzyme. Treatment of MCL cell lines with EPZ015666 led to robust inhibition of PRMT5 mediated methylation and cell killing, with IC50 values in the nM range. Oral dosing of EPZ015666 demonstrated dose-dependent anti-tumor activity in multiple MCL xenograft models. In a Z-138 MCL xenograft model 93% tumor growth inhibition was observed after 21 days of dosing accompanied by a corresponding decrease in symmetrically dimethylated levels of PRMT5 substrates. In summary, we have developed the first potent and selective small molecule inhibitor of PRMT5 that has cellular activity as well as in vivo efficacy and used it as a chemical probe to confirm reports that MCL cells are dependent on PRMT5 activity for their survival. EPZ015666 represents a validated PRMT5:MEP50 chemical probe for the further study of PRMT5 biology and arginine methylation in cancer and other diseases.
Coauthors: Elayne Chan-Penebre1, Kristy G. Kuplast1, Christina R. Majer1, P. Ann Boriack-Sjodin1, Tim J. Wigle1, L. Danielle Johnston1, Nathalie Rioux1, Michael J. Munchhof1, Lei Jin1, Suzanne L. Jacques1, Kip A. West1, Trupti Lingaraj1, Kimberly Stickland1, Scott A. Ribich1, Alejandra Raimondi1, Margaret Porter-Scott1, Nigel J. Waters1, Roy M. Pollock1, Jesse J. Smith1, Olena Barbash2, Melissa Pappalardi2, Thau F. Ho3, Kelvin Nurse3, Khyati P. Oza3, Kathleen T. Gallagher4, Ryan Kruger2, Mikel P. Moyer1, Robert A. Copeland1, Richard Chesworth1.
1. Epizyme, Inc., Cambridge.
2. Cancer Epigenetics DPU, GlaxoSmithKline, Collegeville, Pennsylvania.
3. Department of Biological Reagents and Assay Development, GlaxoSmithKline.
4. Discovery Core Technologies and Capabilities, GlaxoSmithKline.
Development of 'Clickable' Fluorescent Sensors for Targeted Mg2+ Detection in Cellular Organelles
Jessica Gruskos, New York University
Organelle-targeted fluorescent sensors enable the study of intracellular ion compartmentalization and trafficking in the context of both physiological and pathological cellular processes. We describe a new molecular tool for site-specific anchoring and activation of ratiometric Mg2+ sensors for subcellular detection by fluorescence microscopy. Our strategy capitalizes on the fast response of small-molecule indicators and the ease of localization of genetically encoded fusion proteins for subcellular targeting of probes. Organelle-targeted self-labeling protein tags are covalently labeled with ligands bearing a reactive moiety for click chemistry. The anchored reactive moiety serves as an attachment point to site-specifically anchor and activate, in a fluorogenic fashion, sensors decorated with tetrazines through the inverse electron demand Diels–Alder reaction. We demonstrate the utility of our tool to anchor APTRA-based Mg2+ sensors in intracellular compartments for the study of subcellular Mg2+ distribution and mobilization. Our strategy can be easily adapted to other metal ion indicators for subcellular imaging.
Coauthors: Guangqian Zhang and Daniela Buccella, New York University.
Synthesis and Photophysical Studies of Azetidinyl Rhodamines Tailored for Biological Imaging
Anand K. Muthusamy, BA, Janelia Research Campus, Howard Hughes Medical Institute
As biological imaging modalities such as super-resolution imaging and single-particle tracking increase in sophistication, fluorophore chemistry should provide robust tools to interrogate living systems. We found that replacing the ubiquitous N, N-dimethylamino group in rhodamine dyes with azetidines affords significant increases in quantum yield and photostability. Moreover, substitutions on the azetidine ring tune photophysical properties and offer handles for further modification. Heteroatom substitutions for oxygen in the xanthene moiety offer more dramatic tuning. Combining these two findings has yielded a fluorinated azetidine-carbon/silicon rhodamine conjugated to Halo/SNAP-tag that transitions to its quinoid/fluorescent form only when bound to its target. Additionally, the 3-carboxy azetidine offers a handle to append photochemically active molecules which can alter photostability. The synthesis of these fluorophores has been accessed in a modular fashion, using palladium-catalyzed cross couplings to install the key azetidine moiety. We have created a library of dyes spanning the visible spectrum combined with tags such as SNAP/CLIP/Halo-Tag ligands and reactive chemical handles. With our fluorogenic probes, this library enables robust multi-color, no-wash imaging with a large photon budget. The utility of these dyes has been demonstrated in neurobiology imaging experiments. We freely share our dyes with the academic community and have received positive feedback. We have begun computational studies and spectroscopic experiments for modeling non-radiative decay to explain the "azetidine effect" and for modeling solvation to explain fluorogenic properties aforementioned. Our goal is to build a physical organic intuition connecting probe properties to the parameters of biological experiments.
Coauthors: Jonathan B. Grimm, Timothy A. Brown, Luke D. Lavis, Janelia Research Campus, Howard Hughes Medical Institute.
siRNA Nanotechnology: A Self-Assembled Platform for Cancer Gene Therapy
Mayurbhai R. Patel, MSc, Program in Molecular Pharmacology & Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center; Department of Chemistry and Biochemistry, Seton Hall University
The emerging field of RNA nanotechnology has ushered-in a new wave of programmable self-assembled nanostructures. Among the most significant examples, are the nanoparticle formulations that incorporate multiple short-interfering RNA (siRNA). These discrete higher-ordered nanostructures function to synergize the RNA interference (RNAi) gene knockdown effect, leading to significant biological responses in cells. Significantly, siRNA nanotechnology has been successfully applied in the treatment of cancer. In this presentation, a library of self-assembled siRNA nanostructures have been formulated for potent cancer gene therapy applications in endometrial cancer. The siRNAs were designed to target the Glucose Regulated Protein of 78 kilodalton (GRP78) oncogene. The solid-phase synthesis of linear, V-shape and Y-shape RNA templates effectively formed functional scaffolds for siRNA hybridization and self-assembly. The higher-order siRNA nanostructures were found to display distinct sizes and shapes which effected potent anti-cancer activity within a GRP78-overexpressing AN3CA endometrial cancer cell line. Thus, the development of this novel siRNA nanotechnology strategy has effectively expanded the repertoire of functional siRNAs for fruitful applications in cancer gene therapy and for potentially screening important oncogene targets.
Coauthors: Stephen D. Kozuch3, Christopher N. Cultrara3, Reeta Yadav2, Suiying Huang2, Uri Samuni2, John Koren, 3rd1, Gabriela Chiosis1, and David Sabatino3.
1. Program in Molecular Pharmacology & Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center.
2. Department of Chemistry and Biochemistry, Queens College, City University of New York; and the PhD Programs in Chemistry and Biochemistry, The Graduate Center of the City University of New York.
3. Department of Chemistry and Biochemistry, Seton Hall University.
Targeting Nucleic Acid Junctions Using Triptycene-Based Molecules
Stephanie A. Barros, PhD, University of Pennsylvania
Small molecule modulation of nucleic acid structure is critical to several processes in biological systems. Targeting nucleic acids specifically with small molecules remains a significant challenge in chemical biology. The selective modulation of a subset of nucleic acid structures using small molecules would allow for precise chemical control of cellular processes. Nucleic acid junctions are ubiquitous structures found in DNA and RNA involved in several biological processes and viral genomes. We demonstrate a new class of structure-specific nucleic acid junction binders based on triptycene. These triptycene molecules significantly stabilize DNA three-way junctions over double helical DNA. This new class of molecules also has the ability to modulate junctions in trinucleotide repeat expansions implicated in neurodegenerative diseases as well as bacterial mRNA temperature sensors. Triptycene is a versatile scaffold to target higher-order nucleic acid junctions.
Coauthor: David M. Chenoweth, University of Pennsylvania.
Small-Molecule-Induced Oxidation of Protein Disulfide Isomerase is Neuroprotective
Anna Kaplan, PhD, Department of Biological Sciences, Columbia University
Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is upregulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective compound, LOC14, as a modulator of PDI. LOC14 was identified using a high-throughput screen of ~10,000 lead-optimized compounds for potent rescue of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of effects of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 61.7 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using chemical shift perturbations from 2D-NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site, and to observe by HSQC NMR that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.
Coauthors: Michael M. Gaschler2, Denise E. Dunn3, Ryan Colligan1,4, Lewis M. Brown1,4, Arthur G. Palmer III5, Donald C. Lo3, and Brent R. Stockwell1,2.
1. Department of Biological Sciences, Columbia University.
2. Department of Chemistry, Columbia University.
3. Duke University Medical Center.
4. Quantitative Proteomics Center, Columbia University.
5. Department of Biochemistry and Molecular Biophysics, Columbia University.
Chemical Approaches to Sorting Out Reversible Protein Lys Modifications
Philip A. Cole, MD, PhD, Johns Hopkins University School of Medicine
The histone lysine demethylase LSD1 is a flavin-dependent amine oxidase that selectively cleaves one or two methyl groups from histone H3 that is mono- or di-methylated at the Lys4 position. Cellular LSD1 is prominently located in the gene silencing CoREST complex that includes the scaffolding protein CoREST and the histone deacetylase HDAC1. LSD1, HDAC1, and the CoREST complex are considered anti-tumor targets for cancer drug discovery. We will discuss our approaches and progress to design selective inhibitors of LSD1 and the CoREST complex using suicide inactivation strategies.