Uncovering the Naturally Occurring Covalent Inhibitors of Sars-Cov-2 Mpro from the Chinese Medicine Sappanwood and Deciphering Their Synergistic Anti-Mpro Effects

Abstract

Ethnopharmacological relevance : The Chinese medicine sappanwood is primarily sourced from the dried heartwood of the medicinal plant Caesalpinia sappan Linn., has been found with a variety of valuable properties including anti-inflammatory, anti-oxidant and anti-viral effects. Preliminary investigations have demonstrated that sappanwood showed strong anti-SARS-CoV-2 Mpro effects, but the key constituents responsible for SARS-CoV-2 Mpro inhibition and their anti-Mpro mechanisms have not been uncovered.Aim of the study: This study aims to uncover the naturally occurring covalent inhibitors of SARS-CoV-2 Mpro from the sappanwood extract (SWE), to characterize the anti-Mpro mechanisms of the newly identified inhibitors in SWE, as well as to elucidate their synergistic anti-Mpro mechanisms.Materials and Methods: The proteolytic activity of SARS-CoV-2 Mpro was monitored using a fluorescence-based biochemical assay. Comprehensive phytochemical profiling of SWE was conducted by UHPLC-Q-Exactive Orbitrap HRMS. The nanoLC-MS/MS-based chemoproteomic profiling was employed to characterize the phytochemical-modified peptides of SARS-CoV-2 Mpro. Inactivation kinetics, surface plasmon resonance, and molecular dynamics (MD) simulations were utilized to investigate the binding affinity and binding modes of the newly identified SARS-CoV-2 Mpro inhibitors.Results: SWE was found with strong anti-Mpro effect in both dose- and time-dependent manners. Twenty-three constituents in SWE were subsequently identified by utilizing UHPLC-Q-Exactive Orbitrap HRMS, while chemoproteomic profiling revealed that 14 constituents in SWE could covalently modify SARS-CoV-2 Mpro. The anti-SARS-CoV-2 Mpro effects of these newly identified Mpro binders were then tested one by one. The results showed that most of the tested phytochemicals in SWE exhibited time-dependent inhibition on SARS-CoV-2 Mpro, while hematoxylin, brazilin, sappanchalcone, and protosappanin B were identified as the potent time-dependent inhibitors against SARS-CoV-2 Mpro. Furthermore, the combination of hematoxylin and protosappanin B could synergistically block the formation of catalytic active Mpro dimers and then significantly inhibit the catalytic activity of Mpro. MD simulations provided further insight into the synergistic effects between two identified natural Mpro inhibitors (hematoxylin and protosappanin B).Conclusions: The naturally occurring covalent inhibitors of SARS-CoV-2 Mpro from the SWE were identified using an integrated approach. Among all identified covalent inhibitors of SARS-CoV-2 Mpro, hematoxylin, brazilin, sappanchalcone, and protosappanin B emerged as the efficacious Mpro inactivators, which offers powerful evidence to support the anti-coronavirus effects of the Chinese medicine sappanwood.

Keywords: SARS-CoV-2 Mpro, sappanwood extract (SWE), covalent inhibitors, hematoxylin, brazilin