Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase

Abstract

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (M^pro, 3CL^pro) and papain-like protease (PL^pro) 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 PL^pro and M^pro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PL^pro and four M^pro 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.

Figure 1

Simplified life cycle of SARS-CoV-2. The virus enters a host’s cell and releases its genome in cytoplasm. Viral RNA is translated into two polyproteins: pp1a and pp1ab. Then, due to autocleavage, two viral proteases (PL^pro and M^pro) are liberated. Their main role is to further process polyproteins, what results in a release of other nsps. Next, a group of nsps form replication and transcription complexes (RTCs). RTCs are further involved in a generation of copies of viral genomic RNA (g-RNA), as well as a set of sub-genomic RNAs (sg-RNA) responsible for synthesis of viral structural and accessory proteins. Virions are assembled in endoplasmic reticulum-Golgi intermediate compartments (ERGICs). g-RNA is coated with structural N-protein and enters the ERGIC containing M, E, and S glycoproteins. Then, the virions are released by exocytosis^,. Inhibition of M^pro, PL^pro, and N7-MTase may lead to suppression of virus replication. Protease inhibition stops the generation of nsps, while N7-MTase inhibition prevents the synthesis of stable transcripts of the viral RNA. Created with BioRender.com.

Figure 2

Plausible catalytic cycle of ebselen involving hydrogen peroxide reduction, including formation of the ebselen open form (dark blue color)^,.

Figure 3

Preparation of ebselen, its derivatives and their ‘dimeric’ form analogues 1–33. Reagents and conditions: (a) (i) aq. HCl, (ii) NaNO₂, − 7 to + 7 °C, (b) (i) NaSeSeNa, MeOH, NaOH or LiSeSeLi, THF, HMPTA, − 7 to + 5 °C, (ii) aq. HCl, (c) 7 equiv SOCl₂, cat. (DMF), benzene, reflux, (d) RNH₂, Et₃N, MeCN or DCM, (e) 3.5 equiv SOCl₂, cat. (DMF), benzene, reflux, (f) RNH₂, Na₂CO₃, DCM, (g) H₂N-NH₂∙H₂O, MeOH, reflux. (Carried out in accordance with Refs.^,–.