Small molecule interactions with the SARS-CoV-2 main protease: In silico all-atom microsecond MD simulations, PELE Monte Carlo simulations, and determination of in vitro activity inhibition

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing COVID-19 pandemic. With some notable exceptions, safe and effective vaccines, which are now being widely distributed globally, have largely begun to stabilise the situation. However, emerging variants of concern and vaccine hesitancy are apparent obstacles to eradication. Therefore, the need for the development of potent antivirals is still of importance. In this context, the SARS-CoV-2 main protease (Mpro) is a critical target and numerous clinical trials, predominantly in the private domain, are currently in progress. Here, our aim was to extend our previous studies, with hypericin and cyanidin-3-O-glucoside, as potential inhibitors of the SARS-CoV-2 Mpro. Firstly, we performed all-atom microsecond molecular dynamics simulations, which highlight the stability of the ligands in the Mpro active site over the duration of the trajectories. We also invoked PELE Monte Carlo simulations which indicate that both hypericin and cyanidin-3-O-glucoside preferentially interact with the Mpro active site and known allosteric sites. For further validation, we performed an in vitro enzymatic activity assay that demonstrated that hypericin and cyanidin-3-O-glucoside inhibit Mpro activity in a dose-dependent manner at biologically relevant (μM) concentrations. However, both ligands are much less potent than the well-known covalent antiviral GC376, which was used as a positive control in our experiments. Nevertheless, the biologically relevant activity of hypericin and cyanidin-3-O-glucoside is encouraging. In particular, a synthetic version of hypericin has FDA orphan drug designation, which could simplify potential clinical evaluation in the context of COVID-19.