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To design novel inhibitors of the SARS-CoV-2 main protease (Mpro), we investigated the binding mode of the recently reported α-ketoamide inhibitors of this enzyme. Following, we utilized in-silico screening to identify 168 peptidomimetic and non-peptidomimetic compounds that are high probability Mpro binding candidates. The compounds were synthesized in 5 to 10 mg for initial screening for their potential inhibition of Mpro using Fluorescence Resonance Energy Transfer (FRET) assay. The study was conducted using the main protease, MBP-tagged (SARS-CoV-2) Assay Kit (BPS Bioscience, #79955-2), and the fluorescence due to enzymatic cleavage of substrate measured using BMG LABTECH CLARIOstar™, a fluorescent microplate reader, with an excited/emission wavelength of 360 nm/460 nm, respectively. The FRET assay showed 29 compounds to exhibit lower fluorescence compared to the positive control, indicating inhibitory activity, with three of the compounds exhibiting over 50% enzymatic inhibition. The assay average scores were plotted as dose inhibition curves using variable parameter nonlinear regression to calculate the IC50 values. To design more potent inhibitors, an in-silico molecular docking simulation using the SARS-CoV-2 Mpro crystal structure was conducted to investigate on a molecular level the key binding residues at the active site, as well as the possible binding modes and affinity of the lead inhibitors. Additionally, an in-silico study of the compounds' molecular properties and physicochemical profiles was performed to predict their pharmacokinetic properties and assess their suitability as potential orally active drug candidates.
COVID-19, FRET, X-Ray Crystallography, Modelling
Medicinal and Pharmaceutical Chemistry
Prof. Martin K Safo
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VCU Graduate Research Posters