The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

Department of Biochemistry and Immunology Federal University of Minas Gerais Belo Horizonte Minas Gerais 31270 901 Brazil

Department of Medicine Division of Infectious Diseases University of California San Diego La Jolla California 92093 United States

Department of Oncology and Pneumonology Internal Medicine 8 University Hospital Tübingen Otfried Müller Straße 10 DE72076 Tübingen Germany

Department of Pharmaceutical Chemistry University of California San Francisco San Francisco California 94143 United States

Institute of Exact Sciences Department of Chemistry Federal University of Minas Gerais Belo Horizonte Minas Gerais 31270 901 Brazil

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic 16610 Prague Czech Republic

Institute of Pharmacy Pharmaceutical Medicinal Chemistry and Tübingen Center for Academic Drug Discovery Eberhard Karls University Tübingen Auf der Morgenstelle 8 72076 Tübingen Germany

School of Pharmacy Faculty of Health Sciences University of Eastern Finland 70211 Kuopio Finland

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego 9500 Gilman Drive La Jolla California 92093 0657 United States

bioRxiv. 2022 Jan 05:2022.01.05.475095. doi: 10.1101/2022.01.05.475095. PubMed

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