Over the last few years, the SARS-CoV-2 virus, responsible for COVID-19, has undergone significant changes, evolving from the original wild-type strains to the highly transmissible Omicron variant. Omicron has been particularly concerning due to its ability to evade immune protection from vaccines and monoclonal antibodies developed against earlier strains. A critical player in the virus’s life cycle is the main protease (M^pro), also known as NSP5 or 3CL protease, which plays a crucial role in the cleavage and maturation of SARS-CoV-2 proteins within the host cells. This makes M^pro a key target for antiviral drug development.
In 2021, the FDA approved nirmatrelvir (NTV) as the first oral antiviral drug targeting M^pro, marketed under the brand name Paxlovid. Despite its initial success, some patients treated with NTV have developed mutations in M^pro, such as the E166V mutation, making the virus up to 100 times less sensitive to the drug. Two additional M^pro inhibitors, Ensitrelvir (ETV) and Leritrelvir (LTV), have also been approved for use. Importantly, LTV, approved in China in March 2024, does not require co-administration with ritonavir, a drug used with some antivirals to enhance their effectiveness.
However, the effectiveness of these drugs against different M^pro variants is not fully understood. In a recent study published in the Quantitative Biology journal, research groups led by Nan Li and Xuefei Li at the Shenzhen Institutes of Advanced Technology (SIAT) at the Chinese Academy of Sciences (CAS) examined how well these drugs work against SARS-CoV-2 variants and other coronaviruses. Their article titled, “Assessing the Inhibition Efficacy of Clinical Drugs Against the Main Proteases of SARS-CoV-2 Variants and Other Coronaviruses,” provides valuable insights.
The authors firstly compared the chemical structures and binding modes of four M^pro inhibitors. Among these, ETV is the only non-covalent inhibitor, while the other three (NTV, GC376, and LTV) are all covalent inhibitors. NTV shares structural similarities with GC376, primarily occupying the P1-P3 pockets of M^pro, whereas ETV binds to the P1′-P2 pocket. Notably, LTV features a unique α-ketoamide warhead structure that forms hydrophobic interactions with the P1′ pocket, a characteristic absent in the nitrile-based warhead of NTV.
The research team also looked at six specific locations in the M^pro protein that frequently develop mutations. By analyzing global data from the GISAID database, they found a yearly increase in mutations at these sites . After purifying the M^pro mutant protein in E. coli BL21, the researchers assessed the fold change in inhibitory activity (IC₅₀) of the four inhibitors against the wild-type and mutant M^pro. They found that NTV and ETV exhibited resistance to the E166 and S144 mutants, respectively, while LTV maintained superior inhibitory activity.
Finally, the study examined how well these drugs worked against M^pro from other pathogenic α- and β- coronaviruses, which share sequence homology with SARS-CoV-2 . Their results show LTV maintains a strong inhibitory activity (IC50 pro from various coronaviruses, whereas ETV had limited impact on the α-coronaviruses 229E and NL63.
Overall, the study highlights that LTV is more effective than NTV and ETV against M^pro mutants, making it a promising candidate for treating drug-resistant strains of SARS-CoV-2. Additionally, LTV shows potential as a broad-spectrum treatment for different coronaviruses. Future research should focus on tracking SARS-CoV-2 mutations and testing LTV’s effectiveness in living organisms (in vivo) to further evaluate its potential.