Evaluating Potential Broad-Spectrum Antiviral Activity using ColabFold and Docking (preprint)

Broad-spectrum antivirals that work against many viruses provide an immediate treatment for diseases caused by novel pathogenic viruses. Notably, there is no universal drug against all four genera of the coronaviridae family, in particular d-coronaviruses, which have recently spilled over from pigs to humans. Here, we present and illustrate an in-silico strategy to evaluate potential broad-spectrum activity of an EUA-approved drug; viz., nirmatrelvir, for the porcine d-coronavirus (PDCoV) that has infected humans. First, we show that the sequence-based protein structure prediction method, ColabFold, can provide structures for the M dimer of a-, b-, and g-coronaviruses that are highly similar to the respective X-ray structures. Next, we validated the performance of the docking software, AutoDock Vina 1.2.3 on ColabFold-predicted SARS-CoV-2 and MERS-CoV M structures by showing that AutoDock Vina 1.2.3 can yield poses of nirmatrelvir that are near the catalytic Cys, as seen in the respective nirmatrelvir-bound X-ray structures. By using AutoDock Vina 1.2.3 to dock nirmatrelvir to the ColabFold-predicted M structure of PDCoV, we provide evidence that nirmatrelvir may inhibit PDCoV M . These results show the feasibility of using state-of-the-art sequence-based protein structure prediction and docking methods to assess broad-spectrum antivirals for known viruses against novel viruses lacking solved structures but sharing highly similar conserved viral domains.

A strategy for evaluating antiviral resistance: Application to small molecule drugs/inhibitors against SARS-CoV-2 (preprint)

Alterations in viral fitness cannot be inferred from only mutagenesis studies of an isolated viral protein. To-date, no systematic analysis has been performed to identify mutations that improve virus fitness and reduce drug efficacy. We present a generic strategy to evaluate which viral mutations will diminish drug efficacy and applied it to assess how SARS-CoV-2 evolution may affect the efficacy of current approved/candidate small-molecule antivirals for Mpro, PLpro, and RdRp. For each drug target, we determined the drug-interacting virus residues from available structures and the selection pressure of the virus residues from the SARS-CoV-2 genomes. This enabled the identification of promising drug target regions and small-molecule antivirals that the virus can develop resistance. Our strategy of utilizing sequence and structural information from genomic sequence and protein structure databanks can rapidly assess the fitness of any emerging virus variants and can aid antiviral drug design for future pathogens.

Synergistic Inhibition of SARS-CoV-2 Replication using Disulfiram/Ebselen and Remdesivir (preprint)

The SARS-CoV-2 replication and transcription complex (RTC) comprising nonstructural protein (nsp) 2-16 plays crucial roles in viral replication, reducing the efficacy of broad-spectrum nucleoside analog drugs such as remdesivir and in evading innate immune responses. Most studies target a specific viral component of the RTC such as the main protease or the RNA-dependent RNA polymerase. In contrast, our strategy is to target multiple conserved domains of the RTC to prevent SARS-CoV-2 genome replication and to create a high barrier to viral resistance and/or evasion of antiviral drugs. We show that clinically-safe Zn-ejector drugs, disulfiram/ebselen, can target conserved Zn2+-sites in SARS-CoV-2 nsp13 and nsp14 and inhibit nsp13 ATPase and nsp14 exoribonuclease activities. As the SARS-CoV-2 nsp14 domain targeted by disulfiram/ebselen is involved in RNA fidelity control, our strategy allows coupling of the Zn-ejector drug with a broad-spectrum nucleoside analog that would otherwise be excised by the nsp14 proofreading domain. As proof-of-concept, we show that disulfiram/ebselen, when combined with remdesivir, can synergistically inhibit SARS-CoV-2 replication in Vero E6 cells. We present a mechanism of action and the advantages of our multi-targeting strategy, which can be applied to any type of coronavirus with conserved Zn2+-sites.

Multi-Targeting of Functional Cysteines in Multiple Conserved SARS-CoV-2 Domains by Clinically Safe Zn-ejectors (preprint)

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 (PLpro), 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 viral replication. We show that five Zn-ejectors can release Zn2+ from PLpro and nsp10, and clinically-safe disulfiram and ebselen can not only covalently bind to the Zn-bound/catalytic cysteines in both proteins, but also inhibit PLpro protease activity. 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.