Mutational fitness landscape and drug resistance.

Robust technology has been developed to systematically quantify fitness landscapes that provide valuable opportunities to improve our understanding of drug resistance and define new avenues to develop drugs with reduced resistance susceptibility. We outline the critical importance of drug resistance studies and the potential for fitness landscape approaches to contribute to this effort. We describe the major technical advancements in mutational scanning, which is the primary approach used to quantify protein fitness landscapes. There are many complex steps to consider in planning and executing mutational scanning projects including developing a selection scheme, generating mutant libraries, tracking the frequency of variants using next-generation sequencing, and processing and interpreting the data. Key experimental parameters impacting each of these steps are discussed to aid in planning fitness landscape studies. There is a strong need for improved understanding of drug resistance, and fitness landscapes provide a promising new approach.

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms.

With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The 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.

Analyses of HIV proteases variants at the threshold of viability reveals relationships between processing efficiency and fitness.

Investigating the relationships between protein function and fitness provides keys for understanding biochemical mechanisms that underly evolution. Mutations with partial fitness defects can delineate the threshold of biochemical function required for viability. We utilized a previous deep mutational scan of HIV-1 protease (PR) to identify variants with 15-45 per cent defects in replication and analysed the biochemical function of eight variants (L10M, L10S, V32C, V32I, A71V, A71S, Q92I, Q92N). We purified each variant and assessed the efficiency of peptide cleavage for three cut sites (MA-CA, TF-PR, and PR-RT) as well as gel-based analyses of processing of purified Gag. The cutting activity of at least one site was perturbed relative to WT protease for all variants, consistent with cutting activity being a primary determinant of fitness effects. We examined the correlation of fitness defects with cutting activity of different sites. MA-CA showed the weakest correlation (R 2 = 0.02) with fitness, suggesting relatively weak coupling with viral replication. In contrast, cutting of the TF-PR site showed the strongest correlation with fitness (R 2 = 0.53). Cutting at the TF-PR site creates a new PR protein with a free N-terminus that is critical for activity. Our findings indicate that increasing the pool of active PR is rate limiting for viral replication, making this an ideal step to target with inhibitors.