SARS-CoV-2 Mpro inhibitors from Siphonostegia chinensis: an integrated biophysical and computational study

Background The COVID-19 pandemic caused by SARS-CoV-2 underscores the urgent need for novel antiviral agents. Preliminary screening indicated that the traditional Chinese medicine Siphonostegia chinensis Benth. (Bei Liu Ji Nu) possesses inhibitory activity against the SARS-CoV-2 main protease (Mpro), a key enzyme for viral replication. Methods To identify the active constituents responsible for its anti-SARS-CoV-2 activity, we employed an integrated strategy combining bio-layer interferometry (BLI) and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Results Two phenolic compounds, verbascoside and 3,4-dicaffeoylquinic acid, were successfully fished out and identified from the ethanol extract as the most potent binders to Mpro. The binding affinities (KD) were determined to be 2.149 × 10 −6 M and 2.487 × 10 −5 M, respectively. Subsequent in vitro enzymatic inhibition assays confirmed their inhibitory effects, with IC 50 values of 0.076 µM and 0.194 µM, respectively. To elucidate the inhibition mechanism, molecular docking and molecular dynamics simulations were performed, revealing stable binding modes of both compounds within the catalytic pocket of Mpro through key interactions. Preliminary ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) predictions were also conducted to assess their drug-likeness. Conclusion Our findings demonstrate that verbascoside and 3,4-dicaffeoylquinic acid exhibit high binding affinity and structural stability against SARS-CoV-2 Mpro, highlighting their potential as lead compounds for inhibiting viral replication. Furthermore, the comprehensive strategy established in this study provides a reliable and efficient approach for identifying bioactive components from complex herbal matrices, offering new perspectives and a scientific basis for the development of natural product-derived targeted therapeutics.