An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CLpro (also known as the main protease or Mpro) and PLpro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CLpro and PLpro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PLpro and 3CLpro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CLpro and PLpro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2

“…As expected, GC376, used as a control, fully inhibited 3CL pro in the low nanomolar range and was moderately active against PL pro . Moreover, our studies revealed the inhibition of the viral protease PL pro by Camostat, a compound previously accepted as a specific inhibitor of the cellular protease TMPRSS2 [ 36 , 37 ]. Consequently, our findings propose a dual mechanism of action for Camostat, involving the inhibition of both viral PL pro and host TMPRSS2.…”

Pseudovirus-Based Systems for Screening Natural Antiviral Agents: A Comprehensive Review

“…Pseudovirusbased assays have facilitated the screening of natural compounds for their ability to inhibit SARS-CoV-2 entry into host cells. Furthermore, pseudovirus technology has been crucial in studying the mechanisms of action of potential antiviral compounds against SARS-CoV-2 and its variants [60][61][62][63][64][65][66][67][68][69][72][73][74][75][76][77][78][79][80][82][83][84][85]. Luteolin-7-O-glucuronide (L7OG) and folic acid (FA) showed promising entry inhibitory activity against SARS-CoV-2 pseudotypes harboring alpha and omicron spike proteins [60].…”

“…Furthermore, pseudovirus technology has been crucial in studying the mechanisms of action of potential antiviral compounds against SARS-CoV-2 and its variants [60][61][62][63][64][65][66][67][68][69][72][73][74][75][76][77][78][79][80][82][83][84][85]. Luteolin-7-O-glucuronide (L7OG) and folic acid (FA) showed promising entry inhibitory activity against SARS-CoV-2 pseudotypes harboring alpha and omicron spike proteins [60]. Similarly, dihydrotanshinone, E-64-C, and E-64-D were identified as effective against MERS-CoV by targeting the spike protein [61,62].…”

“…The authors reported that both compounds, L7OG and FA, were effective in inhibiting the entry of both variants of SARS-CoV-2. However, L7OG displayed a higher level of entry inhibition against the alpha variant, while FA showed activity against both variants [60]. In addition, Ji Yeun Kim and coauthors identified three natural compounds, dihydrotanshinone, E-64-C, and E-64-D, which act against MERS-CoV by utilizing a pseudovirus that carries S protein from MERS-CoV [61].…”