Silibinin as potential tool against Sars-Cov-2: in silico spike receptorbinding domain and main protease molecular docking analysis and in vitro endothelial protective effects

Introduction. The etiological agent of 2019 Severe Acute Respiratory Syndrome (SARS) has been identified as a novel human coronavirus-2 (SARS-CoV-2). Due to lack of effective treatment, an intensive research that has been undertaken pointing targets involved in virus entry and replication. In particular CoV-2 spike protein that binds human ACE2 cellular receptor to facilitate fusion and entry into cells and the 3C-like protease (3CLpro), vital for virus replication. In addition, SARS-CoV-2 causes massive inflammation boosting cytokines which induce endothelial dysfunction contributing to the high mortality rate. Activation of endothelial cells is thought to be the primary driver for the increasingly recognised complication of thrombosis and endothelial dysfunction leads to detrimental shifts in the vascular equilibrium towards vasoconstriction (manifesting as organ ischemia, infarction and intrapulmonary shunting), inflammation, and a pro-coagulant state resulting in thrombosis. In the present study we focussed on the flavonolignan Silibinin, the major bioactive component of the silymarin extract from the seeds of the milk thistle herb (Silybum marianum), in dual targeting of virus lifecycle and host response to cytokine storm in term of endothelial dysfunctional state. Materials and methods. Through in silico analysis, the best docking poses of the silibinin vs two different targets, SARS-CoV-2 spike receptor-binding domain (RBD) bound with ACE2 (6M0J) and 3CLpro main protease (6LU7 and 7BQY), were evaluated. In addition, the potential protective in vitro effects on human umbilical vein endothelial cells (HUVECs) exposed to TNF- was assessed evaluating two main genes, MCP-1 involved in macrophages infiltration/activation and Endothelin-1 (ET-1) able to shift the vascular equilibrium towards more vasoconstriction. Results. The interaction of silibinin with the RBD showed a high affinity interaction (-8.97 kcal/mol) forming four hydrogen bounds. Furthermore, the silibinin docking pose was found in close contact with the key amino acid residues of the ACE2 receptor involved in the binding of SARS-CoV-2 S-protein RBD. Silibinin showed good negative binding affinity (-10.17 kcal/mol) and estimated inhibition constant (Ki = 35.07 nM) with 3CLpro main protease and it formed five hydrogen bonds particularly with key amino acid residues, and hydrophobic interaction with His41 in the catalytic site. Finally, Silibinin inhibited TNF-a induced MCP-1 and ET-1 in HUVECs in a dose dependent way starting from 5μM. Discussions and conclusions. The in silico analysis suggests that silibinin can affect SARS-CoV-2 entry and replication into the host cells so supporting a potential treatment option. Finally, silibinin reduce endothelial dysfunction induced by proinflammatory mediators restoring vascular equilibrium.