Abstract
SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.
Highlights
SARS-CoV-2 proteases Main protease (Mpro) and papain-like protease (PLpro) are promising targets for antiviral drug development
Several assays are available, including fluorescence, fluorescence resonance energy transfer (FRET), and luciferase-based assays for screening protease inhibitors, we developed this assay to measure the SARSCoV-2 protease inhibition in live cells, which can directly measure the cytotoxicity of compounds
Construct A, d cells transfected with in-cell protease assay (ICP) construct A with inactive Mpro mutant (C145A). e Cells transfected with ICP construct B, f cells transfected with ICP construct B with inactive PLpro mutant (C1651A)
Summary
SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. The COVID-19 vaccine inequity, vaccine hesitancy, and the appearance of SARS-CoV-2 variants of concern (VOC) with increased ability to spread and potential to escape from both vaccine and natural infection immunity highlight the importance of developing antiviral drugs to combat SARS-CoV-2 infections[6]. 2021, a new SARS-CoV-2 variant of concern B.1.1.529 with a large number of mutations was reported to WHO (www.who.int) from South Africa, and the VOC was named Omicron In this context, the repurposing of existing drugs may provide opportunities for relatively rapid identification of clinical candidates[8]. Even though all viral enzymes that participate in coronavirus replication are potentially druggable targets, antiviral studies with small-molecule inhibitors have been focused on the RNA-dependent RNA polymerase (RdRP) and the two viral proteases PLpro and Mpro[9,11,12]. Camostat mesylate, which inhibits the plasma membrane-associated host serine protease, TMPRSS2, has been shown to block the SARS-CoV cell entry mechanism[18]
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