Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage-a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues-bis(2-carbamoylaryl)phenyl diselenides-in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.

Centre of New Technologies University of Warsaw Banacha 2C 02 097 Warsaw Poland

College of Inter Faculty Individual Studies in Mathematics and Natural Sciences University of Warsaw Banacha 2C 02 097 Warsaw Poland

Department of Biochemistry and Structural Biology University of Texas Health Science Center at San Antonio San Antonio TX 78229 USA

Department of Chemical Biology and Bioimaging Wroclaw University of Science and Technology Wyb Wyspianskiego 27 50 370 Wroclaw Poland

Department of Organic and Medicinal Chemistry Faculty of Chemistry Wroclaw University of Science and Technology Wyb Wyspianskiego 27 50 370 Wroclaw Poland

German Center for Infection Research Hamburg Lübeck Borstel Riems Site University of Lübeck 23562 Lübeck Germany

Institute of Molecular Medicine University of Lübeck Ratzeburger Allee 160 23562 Lübeck Germany

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo Nám 2 16610 Prague Czech Republic

National Medicines Institute Ul Chełmska 30 34 00 725 Warsaw Poland

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Discovery of highly potent SARS-CoV-2 nsp14 methyltransferase inhibitors based on adenosine 5'-carboxamides