Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance (preprint)

SARS-CoV-2 main protease, Mpro, is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a Mpro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.

Design of SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model (preprint)

The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PLpro) is a promising but challenging drug target. In this study, we designed and synthesized 85 noncovalent PLpro inhibitors that bind to the newly discovered Val70Ub site and the known BL2 groove pocket. Potent compounds inhibited PLpro with inhibitory constant Ki values from 13.2 to 88.2 nM. The co-crystal structures of PLpro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC50 from 0.44 to 2.02 microM. Oral treatment with Jun12682 significantly improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting PLpro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations (preprint)

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations (preprint)

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir (preprint)

The SARS-CoV-2 main protease (Mpro) is a cysteine protease and a validated antiviral drug target. Paxlovid is an FDA-approved oral COVID-19 antiviral that contains the Mpro inhibitor nirmatrelvir and the metabolic booster ritonavir. The emergence of SARS-CoV-2 variants mutations in the Mpro raised the alarm of potential drug resistance. In this study, we aim to discover Mpro drug resistant mutants from naturally observed polymorphisms. Through analyzing the SARS-CoV-2 sequences deposited in Global initiative on Sharing Avian influenza Data (GISAID) database, we identified 66 prevalent Mpro mutations located at the nirmatrelvir binding site. The Mpro mutant proteins were expressed and characterized for enzymatic activity and nirmatrelvir inhibition. While the majority of the Mpro mutants had reduced enzymatic activity (kcat/Km >10-fold decrease), 11 mutants including S144M/F/A/G/Y, M165T, E166Q, H172Q/F, and Q192T/S/V showed comparable enzymatic activity as the wild-type (kcat/Km <10-fold change) and resistance to nirmatrelvir (Ki > 10-fold increase). We further demonstrate that the enzymatic activity and inhibitor resistance of these single mutations can be enhanced by additional substitutions in a double mutant. X-ray crystal structures were determined for six of the single mutants with and/or without GC-376/nirmatrelvir. The structures illustrate how mutations can reduce ligand binding by impacting the conformational stability of the active site. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.

Invalidation of dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) as SARS-CoV-2 main protease inhibitors and the discovery of PGG as a papain-like protease inhibitor (preprint)

The COVID-19 pandemic spurred a broad interest in antiviral drug discovery. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are attractive antiviral drug targets given their vital roles in viral replication and modulation of host immune response. Structurally disparate compounds were reported as Mpro and PLpro inhibitors from either drug repurposing or rational design. Two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 main protease (Mpro) inhibitors. With our continuous interest in studying the mechanism of inhibition and resistance of Mpro inhibitors, we report herein our independent validation/invalidation of these two natural products. Our FRET-based enzymatic assay showed that neither dieckol nor PGG inhibited SARS-CoV-2 Mpro (IC50 > 20 µM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 papain-like protease (PLpro) with an IC50 of 3.90 µM. The binding of PGG to PLpro was further confirmed in the thermal shift assay. However, PGG was cytotoxic in 293T-ACE2 cells (CC50 = 7.7 µM), so its intracellular PLpro inhibitory activity could not be quantified by the cell-based Flip-GFP PLpro assay. In addition, we also invalidated ebselen, disulfiram, carmofur, PX12, and tideglusib as SARS-CoV-2 PLpro inhibitors using the Flip-GFP assay. Overall, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PLpro inhibitor might worth further pursuing. 

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays (preprint)

SARS-CoV-2 main protease (M pro ) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as M pro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed M pro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based Flip-GFP assay. Collectively, our results have shown that majority of the M pro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to M pro , while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit M pro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery. Graphical abstract Flip-GFP and Protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate the reported SARS-CoV-2 M pro inhibitors.