Structural insights of the coronavirus main protease in complex with the non-covalent inhibitor CCF0058981

The highly pathogenic SARS-CoV-2 causes COVID-19, which threatens global public health and socio-economic stability through persistent transmission and mutation. Effective therapeutics against SARS-CoV-2 and its variants are urgently needed. The main protease (M^pro), highly conserved among coronaviruses and lacking human homologs, is pivotal for viral replication, making it an attractive antiviral target. CCF0058981, a novel non-covalent inhibitor developed based on the ML300 scaffold, demonstrates potent low-nanomolar inhibitory activity against SARS-CoV-2 M^pro and sub-micromolar antiviral efficacy against SARS-CoV-2. Its non-covalent binding mechanism effectively mitigates the off-target risks commonly associated with traditional covalent inhibitors, thereby providing a versatile scaffold for the development of highly safe and effective anti-coronavirus therapeutics. However, the structural basis underlying CCF0058981's inhibitory mechanism against SARS-CoV-2 M^pro remains to be elucidated. Here, we report for the first time two crystal structures of M^pro from SARS-CoV-2 and SARS-CoV in complex with the inhibitor CCF0058981. Detailed crystal structure analysis reveals that CCF0058981 occupies the catalytic pocket of M^pro via conserved hydrogen bonds and hydrophobic interactions. The superimposition analysis of the reported crystal structures also reveals that CCF0058981 maintains stable binding to the M^pro mutants (M49I and V186F), demonstrating its potential to combat drug resistance, demonstrating its potential to counteract drug resistance. Molecular dynamics simulations further validate the stability of the inhibitor-protease complex. These findings provide mechanistic insights into CCF0058981's inhibition and support developing broad-spectrum coronavirus therapeutics.