RESUMEN
Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.
RESUMEN
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CLpro), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CLpro, and validate our results both in yeast and in authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CLpro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CLpro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.
RESUMEN
We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.
RESUMEN
We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.
