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.

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.

Exploration of plant-derived natural polyphenols toward COVID-19 main protease inhibitors: DFT, molecular docking approach, and molecular dynamics simulations† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra07364h

Recent outbreaks of coronavirus have brought serious challenges to public health around the world, and it is essential to find effective treatments. In this study, the 3C-like proteinase (3CLpro) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has been considered as an important drug target because of its role in viral replication. We initially optimized 251 compounds at the PM7 level of theory for docking with 3CLpro, and then we selected the top 12 compounds for further optimization with the B3LYP-D3/6-311G** method and obtained the top four compounds by further molecular docking. Quantum chemistry calculations were performed to predict molecular properties, such as the electrostatic potential and some CDFT descriptors. We also performed molecular dynamics simulations and free energy calculations to determine the relative stability of the selected four potential compounds. We have identified key residues controlling the 3CLpro/ligand binding from per-residue based decomposition of the binding free energy. Convincingly, the comprehensive results support the conclusion that the compounds have the potential to become a candidate for anti-coronavirus treatment. The combination of molecular dynamics simulations and quantitative calculations as a powerful tool for screening molecules.

The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition (preprint)

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and is considered to be one of the most promising SARS-CoV-2 drug targets. While Mpro mutations have occurred in many variants, including Omicron (B.1.1.529), their structural and biochemical importance have, until this point, remained uncharacterized. Using X-ray crystallography, we show the Omicron Mpro mutant (P132H) induces a small rearrangement of residues distal from the active site. While enzymatic activity and small-molecule inhibition appears unchanged, the melting temperature (Tm) of Omicron Mpro is 2.6 {degrees} C lower than Alpha and Delta Mpro. Yet, when incubated with inhibitors, these enzymes have nearly identical Tm values. The physiological importance of the P132H mutant is unclear; however, we show residue 132 is located at the interface of the dimerization and catalytic domains and is frequently mutated in other variants. Lower thermal stability may indicate increased flexibility that can potentially broaden substrate profile or alter inhibitor binding. Therefore, structural insights are key to anticipating future mutations that may promote drug-resistance and are especially important at present, given the recent approval of nirmatrelvir (PAXLOVID), an oral SARS-CoV-2 Mpro inhibitor.

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay (preprint)

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity (preprint)

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 microM and EC50 of 1.27 microM, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L (preprint)

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a {gamma}-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1 pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1 pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the -ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease (preprint)

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 M. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 [A] resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.