Comprehensive fitness landscape of SARS-CoV-2 M^pro reveals insights into viral resistance mechanisms

Abstract

With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

Bat coronaviruses related to SARS-CoV-2: what about their 3CL proteases (MPro)?

“…Altogether, our structural analysis reveals that neither of these two structural differences between SARS-CoV-2 and bat coronaviruses M pro should determine any relevant structural alteration of the main protease. Notably, this observation is in agreement with a recent article that characterised the effect of each possible M pro mutation on its functionality: both Pro96 and Asn180 are marked as highly tolerant to mutations 25 .…”

Learning from pre-pandemic data to forecast viral escape

“…We benchmark our models on a series of viral protein deep mutational scans [1][2][3][4][5][6][7][8][9][10][11][12][13][51][52][53][54][55][56][57][58] (Table S2, Table S4). For each viral mutational scan, we select the variable or variables of protein fitness or antibody escape treated as primary in the publications.…”

From the Wuhan-Hu-1 strain to the XD and XE variants: is targeting the SARS-CoV-2 spike protein still a pharmaceutically relevant option against COVID-19?

“…As can be seen in Figure 8 , indeed, the proline residue is not involved in any intermolecular interaction relevant to the structural stability of the protease, suggesting that its only role could be limited to a joint between more relevant residues such as R131, which mediates several interactions through its sidechain guanidium group (specifically, a salt bridge with both D289 and D197, and a hydrogen bond with the backbone of T198) and its backbone (a hydrogen bond between its backbone amide proton and the amide carbonyl oxygen of T135 and another one between its carbonyl oxygen and the amide proton of F134), and N133, which is itself involved in a network of intermolecular interactions with both its backbone (hydrogen bond between its amide proton and the carboxyl oxygen of D197) and its sidechain (the amide proton donates to the carbonyl oxygen of G195 while the carbonyl oxygen receives from the hydroxyl group of T135). These structural insights are confirmed also by a functional screening performed by Flynn et al., which showed that mutations at this position, especially the P132H found in these viral variants, are generally well-tolerated, while mutations of both R131 and N133 drastically reduce or even abolish the catalytic activity of the protease 86 .…”