Protease Inhibitors: Developing evolution-resistant drugs for COVID-19
Analyzing how mutations affect the main protease of SARS-CoV-2 may help researchers develop drugs that are effective against current and future variants of the virus.
- Department of Ecology, Evolution and Organismal Biology, Brown University, United States
- Center for Computational Molecular Biology, Brown University, United States
As of mid-July 2022, more than 12 billion doses of vaccines against SARS-CoV-2, the virus responsible for COVID-19, have been administered to over 5 billion individuals (WHO, 2022). However, mutations that allow the virus to evade vaccines are spreading globally, leading to a need for boosters and updated vaccines (Callaway, 2021; Vogel, 2022). This immunity ‘arms race’ illustrates how efforts to control the COVID-19 pandemic could benefit from additional pharmaceutical approaches.
One of these approaches is the use of inhibitor drugs to block the action of viral proteases, the enzymes that cleave polyproteins encoded in the viral genome to yield several functional proteins. If these proteases are inactivated, the virus cannot synthesize the proteins it requires to reproduce, so inhibiting these enzymes may be an effective way of treating viral diseases. For example, protease inhibitors are key components of highly active antiretroviral therapy (HAART), which renders HIV a chronic disease.
In the case of SARS-CoV-2, many inhibitor candidates have been identified for its main protease, which is called Mpro (Jin et al., 2020; Narayanan et al., 2022). One of these inhibitors, with the trade name Paxlovid, has been authorized for emergency use by the United States Federal Drugs Administration (FDA), and pharmacists have been allowed to prescribe it since July 2022 (Owen et al., 2021; FDA, 2022). But can Mpro inhibitors be designed to avoid the rapid obsolescence we have observed in vaccines as a result of viral evolution? Now, in eLife, Julia Flynn, Daniel Bolon and colleagues at the University of Massachusetts (UMass) Chan Medical School and the Novartis Institutes for Biomedical Research report the results of experiments assessing the effects of different mutations on the proteolytic activity of MPro (Flynn et al., 2022).
Using a method called deep mutational scanning (Fowler and Fields, 2014), Flynn et al. generated a ‘library’ that contained almost every single missense mutation of the Mpro enzyme relative to the sequence found in the original Wuhan isolate (Wu et al., 2020). Missense mutations are changes to a single nucleotide in the sequence that alter the resulting amino acid. Assessing the activity of the MPro variants that result from each conceivable missense mutation revealed how they each affect the activity of the enzyme. To do this, Flynn et al. placed the library into yeast cells, and assessed how well each variant of the protease worked in three different environments.
The results showed that the proteolytic activity of each variant was correlated across the three assays, implying that the assays capture the same properties of MPro. Flynn et al. also found a correlation between the results of their assays and previous measurements of the catalytic rates for individual variants. Additionally, the vast majority of the 290 missense mutations of MPro observed most frequently (at least 100 times) in COVID-19 patients exhibited activity comparable to that of the wild type. This further confirmed that Flynn et al.’s assay captures the biologically relevant function of the enzyme. Finally, the team found that all of the mutation-intolerant residues in MPro (the sites at which at least 17 out of the 20 alternative amino acids block the protein’s activity) are highly conserved in other coronaviruses. This suggests that inhibitors designed against the current SARS-CoV-2 form of the enzyme will likely be active against future, emergent outbreaks.
Most protease inhibitors act by competitively occupying the enzyme’s binding pocket to the exclusion of its native substrate. Therefore, Flynn et al. decided to further analyze missense mutations in the vicinity of the binding pocket that nevertheless preserve enzyme activity, reasoning that these mutations might represent opportunities for the virus to evolve resistance to inhibitors. Unfortunately, they found that mutations at many of those residues preserve the proteolytic ability of Mpro (Cho et al., 2021).
Similarly, and perhaps of more immediate concern, Flynn et al. identified three missense mutations that reduced the binding stability to Paxlovid by at least 1 kcal/mol, corresponding to an approximately five-fold reduction in the drug’s binding affinity, while maintaining proteolytic activity. These represent likely resistance mutations; indeed, a subset of these mutations have now been observed in laboratory populations of SARS-CoV-2 challenged with this version of Paxlovid, as well as in currently circulating isolates (Service, 2022). It will be important to monitor the frequency of these three mutations in patients being treated with Paxlovid as its use becomes more widespread.
But the potential implications of the findings of Flynn et al. go further than providing insight into prospects for resistance evolution against existing Mpro inhibitors. Some years ago, the notion of the ‘substrate envelope’ of a viral protease – the overlapping volume occupied by all of its native substrates – was introduced (King et al., 2004). The inspiration was that the substrate specificity of the HIV protease seemed to be determined by the shape of the substrate rather than by a specific amino acid sequence. Because resistance mutations must allow an enzyme to distinguish between its inhibitors and its substrates, they might work by introducing atomic overlaps or disfavored electrostatics in the region where the inhibitor protrudes beyond the substrate envelope. In a recent companion study, researchers at UMass and Novartis (including many authors from Flynn et al.) have reported a high-resolution structure of the SARS-CoV-2 Mpro substrate envelope (Shaqra et al., 2022).
Together, these two articles provide clear guidance for the development of evolution-resistant protease inhibitors against SARS-CoV-2. To find clinically durable candidates, compounds should be screened first to identify those that lie entirely within the enzyme’s substrate envelope. Of these, care should be taken to select those that interact with one or more mutation-intolerant residues. This will make it more difficult for the protease to evolve the ability to discriminate between its native substrates and the drug without blocking its activity. Of course, Darwinian evolution is a tremendously powerful force, and resistance has evolved against every antimicrobial class ever deployed in the clinic. Nevertheless, the functional and structural resolution now available for Mpro-inhibitors offers new optimism 30 months into the COVID-19 pandemic.
References
-
Dynamic profiling of β-coronavirus 3CL Mpro protease ligand-binding sitesJournal of Chemical Information and Modeling 61:3058–3073.https://doi.org/10.1021/acs.jcim.1c00449
-
Deep mutational scanning: a new style of protein scienceNature Methods 11:801–807.https://doi.org/10.1038/nmeth.3027
-
Combating susceptibility to drug resistance: lessons from HIV-1 proteaseChemistry & Biology 11:1333–1338.https://doi.org/10.1016/j.chembiol.2004.08.010
Article and author information
Author details
Daniel M Weinreich
Publication history
- Version of Record published: July 26, 2022 (version 1)
Copyright
© 2022, Weinreich
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 681
- views
-
- 158
- downloads
-
- 1
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Protease Inhibitors: Developing evolution-resistant drugs for COVID-19
eLife 11:e81334.
https://doi.org/10.7554/eLife.81334
Further reading
-
- Developmental Biology
- Evolutionary Biology
Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.
-
- Evolutionary Biology
- Immunology and Inflammation
CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.
