key: cord-0764741-3j3n1kqn
authors: Sargsyan, Karen; Lin, Chien-Chu; Chen, Ting; Grauffel, Cédric; Chen, Yi-Ping; Yang, Wei-Zen; Yuan, Hanna S.; Lim, Carmay
title: Multi-targeting of functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-ejectors
date: 2020-09-01
journal: Chem Sci
DOI: 10.1039/d0sc02646h
sha: 14f7f5ab2a6bf1dd8a441180872e2938c316d087
doc_id: 764741
cord_uid: 3j3n1kqn

We present a near-term treatment strategy to tackle pandemic outbreaks of coronaviruses with no specific drugs/vaccines by combining evolutionary and physical principles to identify conserved viral domains containing druggable Zn-sites that can be targeted by clinically safe Zn-ejecting compounds. By applying this strategy to SARS-CoV-2 polyprotein-1ab, we predicted multiple labile Zn-sites in papain-like cysteine protease (PL(pro)), nsp10 transcription factor, and nsp13 helicase. These are attractive drug targets because they are highly conserved among coronaviruses and play vital structural/catalytic roles in viral proteins indispensable for virus replication. We show that five Zn-ejectors can release Zn(2+) from PL(pro) and nsp10, and clinically-safe disulfiram and ebselen can not only covalently bind to the Zn-bound cysteines in both proteins, but also inhibit PL(pro) protease. We propose combining disulfiram/ebselen with broad-spectrum antivirals/drugs to target different conserved domains acting at various stages of the virus life cycle to synergistically inhibit SARS-CoV-2 replication and reduce the emergence of drug resistance.

CoV sequences by one and two residues whose C a atoms are > 16 and 75 Å from Zn 2+ , respectively. The structure of SARS-CoV-2 papain-like protease (PLpro) was derived from that of the SARS-CoV PLpro (4m0w_A) using MODELLER. 7 The % numbers in parentheses is % sequence identity between the SARS-CoV-2 and template sequences. Each structure is depicted using a color gradient from blue (Nterminus) to red (C-terminus). Cell-based assays. Vero E6 cells were pretreated with the disulfiram/ebselen and/or hydroxychloroquine (HCQ) at various concentrations for 1 hr at 37°C and then adsorbed with SARS-CoV-2 (TCDC#4) at MOI 0.005 (100 PFU/well) for 1 hr at 37°C.

After virus adsorption, the cells were replenished with fresh medium and compounds at the indicated concentrations for 1-day incubation. The cells were fixed with 10% formaldehyde, permeabilized with 0.5% Triton X-100, and stained with anti-SARS-CoV-2 N protein antibody (provided by Dr. An-Suei Yang, Genomic Research Center, Academia Sinica, Taiwan) and anti-human IgG-488 (green). The N protein expression was measured using a high-content image analysis system (Molecular Devices) and the average infection rate of no-drug treatment was set as 100%. The percentage of inhibition was calculated as 100%infection rate. For cell viability test, Vero E6 cells were treated with each compound for 24 hrs and cell viability was determined by Cell

Counting Kit-8 (CCK-8). IC50 and CC50 were calculated by Prism software.

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