Structural and mechanistic insights into Main protease (Mpro) dimer interface destabilization inhibitor: Unveiling new therapeutic avenues against SARS-CoV-2

SARS-CoV-2 variants recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral target. Mpro features several druggable sites, including active site and allosteric sites near the dimerization interface that regulate its catalytic activity. This study has identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to target Mpro was validated. FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC50 values in the range from 0.08 to 7.31 μM. Additionally, in-vitro cell-based antiviral assays showed high efficacy with EC50 values in the range of 1.8 to 18.92 μM. Crystal structure of Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for Mpro proteolytic activity. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and directs the rational design of minocycline derivatives as antiviral drugs.