Study reports an orally available SARS-CoV-2 Mpro inhibitor with potent in vivo antiviral activity

By Bhavana KunkalikarReviewed by Danielle Ellis, B.Sc.Apr 29 2022

In a recent study published in the Nature Microbiology journal, researchers examined the efficiency of the main protease (M^pro) inhibitor against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Coronavirus disease 2019 (COVID-19) vaccine has been instrumental in curbing the morbidity and mortality caused by SARS-CoV-2 infections. This has necessitated the development of efficient treatment options against COVID-19.

About the study

In the present study, researchers assessed the efficiency of a SARS-CoV-2 M^pro inhibitor in exhibiting potent antiviral activity in vivo against the SARS-CoV-2 wild-type (WT) strain, and B.1.1.7 (Alpha), B.1.617.1 (Kappa) and P.3 (Theta) variants.

The team used the Ugi four-component reaction (Ugi-4CR) to generate covalent M^pro inhibitors. First, the researchers selected clinically approved α-ketoamide (R=acetyl) and acrylamide (R=vinyl) as the candidates for the electrophilic group at the R position that would bind to a cysteine residue. This results in the generation of compounds 1a and 1b, with R as acetyl or vinyl groups, and R1, R2, and R3 as pyridine, tert-butylbenzene, and tert-butyl, respectively. Furthermore, the bioactivities of 1a and 1b against M^pro were evaluated by a differential scanning fluorimetry (DSF) assay and a fluorescence resonance energy transfer (FRET) assay.

The team subsequently optimized R1, R2, and R3 of 1a, leading to the generation of 16 compounds having several combinations of R2 and R3. The epimeric mixtures comprising the 16 compounds were separated via chiral high-performance liquid chromatography (HPLC). The team later attempted to add deuterium to the resulting compounds and generated 11 compounds with deuterium.

The equilibrium-binding constant K_i and the inactivation rate constant k_inac values were estimated for the candidate Y180 to verify its binding ability to M^pro. Furthermore, the in vitro activity of Y180 against M^pro was evaluated using the human lung epithelial cell line, Calu-3, and VeroE6 cells that expressed transmembrane serine protease 2 (VeroE6-TMPRSS2). Subsequently, the cells were exposed to SARS-CoV-2 WT strain and B.1.17, B.1.617.1, and P.3 variants.    

Results

The study results showed that the racemic mixture of 1a showed good activity with a thermal shift (∆T_m) of 4.7 °C in the DSF assay and 3.11 μM in the FRET assay at a 50% inhibitory concentration (IC50), while 1b showed undetectable activity. This led to the selection of α-ketoamide with R=acetyl for the electrophilic group at the R position.

All 16 compounds comprised epimeric mixtures with a fixed S-configuration at R3 and potent antiviral activity against M^pro. Separation of the epimeric mixtures generated (R)-epimers with higher antiviral potency than the corresponding (S)-epimers. Among these, (R)-5b had the highest antiviral activity against M^pro, while the corresponding (S)-5b showed weaker activity. Notably, (R)-5b could undergo rapid configuration conversion to form (S)-5b.

This configuration conversion was prevented by replacing the exchangeable hydrogen with deuterium, generating the compound d-(R)-6a, which had antiviral potency similar to that of (R)-5b. After further generation of compounds with deuterium, the researchers selected d-(R)-6c (Y180) that showed highly potent antiviral activity in enzymatic as well as cellular antiviral assays.

The K_i and K_inac values of Y180 were 1 nM and 2.6×10⁻⁴ s⁻¹, respectively, which indicated the high potency of Y180 as a M^pro inhibitor. Furthermore, Y180 showed no activity against several mammalian proteases with structures similar to that of M^pro, such as caspase 2, chymotrypsin, thrombin, cathepsin B, cathepsin D, and cathepsin L, suggesting the high selectivity of Y180 against M^pro.     

The in vitro antiviral potency of Y180 showed no cytotoxicity in the Calu-3 and VeroE6-TMPRSS2 cells. Moreover, 20µM of Y180 reduced viral replication by 4.44-, 5.83-, 4.74- and 6.03-log against the SARS-CoV-2 WT strain and the B.1.1.7, B.1.617.1, and P.3 variants, respectively, 24 hours after infection in VeroE6-TMPRSS2 cells. On the other hand, in Calu-3 cells, the same concentration of 20µM of Y180 showed potent antiviral activity with 2.14-, 3.13-, 1.77- and 2.29-log decline in the number of viral copies against the SARS-CoV-2 WT strain and the B.1.1.7, B.1.617.1 and P.3 variants.

Y180 also showed a significant increase in cell viability at 0.8 µM concentration along with potent protective activity against SARS-CoV-2-induced cell death. Notably, this protection was more efficient than that of remdesivir (RDV) for the SARS-CoV-2 WT strain and the emerging variants.       

In vivo assessment of Y180 antiviral activity showed that in K18-human angiotensin-converting enzyme 2 (K18-hACE2) mice infected with B.1.1.7, Y180 reduced the number of viral copies in the nasal turbinate and the lungs. Infectious viral titers in the nasal turbinate and the lungs were significantly lower in mice treated with Y180 as compared to untreated mice.

Conclusion

Overall, the study findings showed that the orally available M^pro inhibitor demonstrated in the present study had potent in vivo as well as in vitro antiviral activity against the SARS-CoV-2 WT strain and the B.1.1.7, B.1.617.1, and P.3 variants.