Structural basis for the inhibition of the coronavirus main protease by oridonin

Currently, there are seven coronaviruses that can infect humans: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus, human coronavirus 229E (HCoV-229E), HCoV-NL63, HCoV-HKUl, and HCoV-OC43. The coronavirus pandemic, especially highly pathogenic coronaviruses such as SARS-CoV-2, has caused severe damage to human health and the social economy. Therefore, the development of broad-spectrum drugs is critical to address the current unmet urgent clinical need and to contain potential future coronaviruses. The conserved main protease (M^pro) of coronavirus is critical for virus replication and has been considered an ideal drug target. Here, we identified a natural compound, oridonin, that shows broad-spectrum inhibition of the M^pros of SARS-CoV-2, SARS-CoV, HCoV-NL63, and HCoV-229E and can also inhibit the mutant of SARS-CoV-2's M^pro (G15S: Lambda variant). The inhibitory effect of oridonin highly depends on the covalent modification of the electrophilic enone structure to the cysteine residue (Cys144/Cys145) in the active site of the M^pro. The crystal structure of the HCoV-229E complex with oridonin was first determined by X-ray crystallography, and we compared it with the previously reported structure of SARS-CoV-2 M^pro complexes with oridonin. Molecular dynamics simulations further demonstrated that oridonin stably binds to both SARS-CoV-2 Mᵖʳᵒ and HCoV-229E M^pro. Through detailed structural biological analysis, we revealed that oridonin blocks substrate binding through a unique nonpeptidomimetic covalent binding mode. Overall, our data provide evidence that oridonin can serve as a lead compound for the design of a covalent inhibitor against the coronavirus.