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Thesis
Kinetic Analysis of Small Molecule Inhibitors Targeting Protein Arginine Methyltransferase-5 (PRMT5) using Surface Plasmon Resonance (SPR)
Master of Science (M.S.), Drexel University
May 2020
DOI:
https://doi.org/10.17918/00000234
Abstract
Surface Plasmon Resonance (SPR) is a biophysical technique that is able to measure molecular binding interactions in real time. Utilizing a Biacore 8K SPR instrument and Insight Evaluation Software (GE Healthcare), it is possible to analyze the dynamics of the molecular interactions between small molecule inhibitors to protein arginine methyltransferase-5 (PRMT5); an enzyme responsible for the symmetric dimethylation of arginine residues of both histone and non-histone proteins. PRMT5 has been implicated in a wide variety of cancers including ovarian, lung, and lymphoid cancers. Studies have also linked the overexpression of PRMT5 with poorer prognosis and survival. PRMT5's methyltransferase function has attracted interest in developing selective inhibitors targeting the SAM-binding pocket and the substrate-binding pocket of PRMT5 in developing drugs to treat certain cancers. Reaction Biology Corp. has produced three small molecule inhibitors,VY-5-173, VY-5-177, and VY-5-178, designed to target the substrate-binding pocket of PRMT5 in developing proteolysis-targeting chimeras (PROTACs) molecules. PROTACs are an emerging platform that mediates the selective degradation of target proteins. In this work, the binding behaviors and kinetics of VY-5-173, VY-5-177, and VY-5-178 were compared to known small molecule inhibitors, EPZ015666, LLY-283, and JNJ-64619178, against PRMT5 using distinct binding modes in four different conditions. Measurements were conducted on the Biacore 8K in the absence and presence of cofactors MTA, SAH, and SAM to probe for competition and/or cooperativity that would provide insight into the binding modes of the small molecule inhibitors. Based on the SPR data, VY-5-173, VY-5-177, and VY-5-178 bind preferentially to cofactor bound PRMT5 with a difference of 100-fold: low- micromolar KD in MTA-bound PRMT5, mid-nanomolar KD in SAH-bound PRMT5, and high-picomolar KD in SAM-bound PRMT5. Among all of the buffer conditions assayed using SPR, the results demonstrate that compounds VY-5-173, VY-5-177, and VY-5-178 each bind SAM-bound PRMT5 with the highest affinity denoted by picomolar KDs, slow off-rates, and nanomolar highest starting concentrations. The kinetics and binding behaviors of VY-5-173, VY-5-177, and VY-5-178 closely resemble the binding modes of EPZ015666 as SAM-cooperative compounds; an analogue of EPZ015666 is currently in clinical trials for the treatment of solid tumors and non-Hodgkin's Lymphoma.
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Details
- Title
- Kinetic Analysis of Small Molecule Inhibitors Targeting Protein Arginine Methyltransferase-5 (PRMT5) using Surface Plasmon Resonance (SPR)
- Creators
- Lynn Kim Baker
- Contributors
- Rebecca Eells (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- x, 73, VI pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- College of Medicine; Pharmacology and Physiology; Drexel University
- Other Identifier
- 991014695542704721