A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available protease inhibitors (nirmatrelvir and ensitrelvir) with cell-based, biochemical and SARS-CoV-2 replicon assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease inhibitor resistance mechanisms and show the relevance of specific mutations, thereby informing treatment decisions.

Highly specific SARS-CoV-2 main protease (Mpro) mutations against the clinical antiviral ensitrelvir selected in a safe, VSV-based system.

In the SARS-CoV-2 pandemic, the so far two most effective approved antivirals are the protease inhibitors nirmatrelvir, in combination with ritonavir (Paxlovid) and ensitrelvir (Xocova). However, antivirals and indeed all antimicrobial drugs are sooner or later challenged by resistance mutations. Studying such mutations is essential for treatment decisions and pandemic preparedness. At the same time, generating resistant viruses to assess mutants is controversial, especially with pathogens of pandemic potential like SARS-CoV-2. To circumvent gain-of-function research with non-attenuated SARS-CoV-2, a previously developed safe system based on a chimeric vesicular stomatitis virus dependent on the SARS-CoV-2 main protease (VSV-Mpro) was used to select mutations against ensitrelvir. Ensitrelvir is clinically especially relevant due to its single-substance formulation, avoiding drug-drug interactions by the co-formulated CYP3A4 inhibitor ritonavir in Paxlovid. By treating VSV-Mpro with ensitrelvir, highly-specific resistant mutants against this inhibitor were selected, while being still fully or largely susceptible to nirmatrelvir. We then confirmed several ensitrelvir-specific mutants in gold standard enzymatic assays and SARS-CoV-2 replicons. These findings indicate that the two inhibitors can have distinct viral resistance profiles, which could determine treatment decisions.

Study of key residues in MERS-CoV and SARS-CoV-2 main proteases for resistance against clinically applied inhibitors nirmatrelvir and ensitrelvir.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35% and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of SARS-CoV-2, MERS-CoV and other main proteases. Nirmatrelvir inhibited several of these proteases, whereas ensitrelvir was less broadly active. To simulate nirmatrelvir's clinical use against MERS-CoV and subsequent resistance development, we applied a safe, surrogate virus-based system. Using the surrogate virus, we previously selected hallmark mutations of SARS-CoV-2-Mpro, such as T21I, M49L, S144A, E166A/K/V and L167F. In the current study, we selected a pool of MERS-CoV-Mpro mutants, characterized the resistance and modelled the steric effect of catalytic site mutants S142G, S142R, S147Y and A171S.

A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions.

Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors.

Vaccines and drugs have helped reduce disease severity and blunt the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease [Mpro; 3C-like protease (3CLpro)] of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy.

SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376.

Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors.

Vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy.

A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition.

Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and inhibition in living cells. Here, we engineer a safe VSV-based system to report both gain- and loss-of-function of coronavirus main protease (Mpro/3CLpro/Nsp5) activity in living cells. We use SARS-CoV-2 3CLpro in this system to confirm susceptibility to known inhibitors (boceprevir, GC376, PF-00835231, and PF-07321332/nirmatrelvir) and reevaluate other reported inhibitors (baicalein, ebselen, carmofur, ethacridine, ivermectin, masitinib, darunavir, and atazanavir). Moreover, we show that the system can be adapted to report both the function and the chemical inhibition of proteases from different coronavirus species as well as from distantly related viruses. Together with the fact that live cell assays also reflect compound permeability and toxicity, we anticipate that this system will be useful for both identification and optimization of additional coronavirus protease inhibitors.