Unlocking the potential of phytochemicals in inhibiting SARS-CoV-2 M Pro protein - An in-silico and cell-based approach
Khushboo Singh, J J Patten, Andrea Dimet, Robert A Davey, Stanley J Watowich, Amit Chandra, Jesse Leverett
doi:10.21203/rs.3.rs-3888947/v1
The main protease (M Pro ) of SARS-CoV-2 plays a crucial role in viral replication and is a prime target for therapeutic interventions. Phytochemicals, known for their antiviral properties, have been previously identi ed as potential M Pro inhibitors in several in silico studies. However, the e cacy of these remains in question owing to the inherent exibility of the M Pro binding site, posing challenges in selecting suitable protein structures for virtual screening. In this study, we conducted an extensive analysis of the M Pro binding pocket, utilizing molecular dynamics (MD) simulations to explore its conformational diversity. Based on pocket volume and shape-based clustering, ve representative protein conformations were selected for virtual screening. Virtual screening of a library of ~ 48,000 phytochemicals suggested 39 phytochemicals as potential M Pro inhibitors. Based on subsequent MM-GBSA binding energy calculations and ADMET property predictions, ve compounds were advanced to cell-based viral replication inhibition assays, with three compounds (demethoxycurcumin, shikonin, and withaferin A) exhibiting signi cant (EC50 < 10 uM) inhibition of SARS-CoV-2 replication. Our study provides an understanding of the binding interactions between these phytochemicals and M Pro , contributing signi cantly to the identi cation of promising M Pro inhibitors. Furthermore, beyond its impact on therapeutic development against SARS-CoV-2, this research highlights a crucial role of proper nutrition in the ght against viral infections. Phytochemical Name Docking scores Conformation 1 Conformation 2 Conformation 3 Conformation 4 Conformation 5 1,3,6-Tri-O-Galloyl-Beta-D-Glucose -7.6 -8.8 -11.1 -7.5 -10.1 2'-Acetylacteoside -8.6 -9.5 -12.1 -13.4 -7.6 2''-O-Acetylrutin -10.3 -9.6 -12.2 -10.8 -10.4 *AHDPH -8.1 -9.0 -11.6 -7.5 -9.0 Balanophotannin E -7.5 -11.0 -12.9 -9.9 -8.4 **DDHHG -9.7 -8.3 -10.9 -11.3 -8.6 ***DHMMP-TRTH-TMMO-Chr-One -9.7 -10.5 -10.7 -9.1 -9.9 Eriodictyol 7-O-Sophoroside -12.6 -9.3 -10.0 -11.1 -10.0 Forsythiaside -10.3 -12.6 -14.3 -14.6 -9.2 Hyperin 6''-[glucosyl-(1->3)-rhamnoside] -9.7 -10.9 -15.9 -12.1 -11.9 Kaempferol 3-(3R-glucosylrutinoside) -10.0 -10.6 -12.0 -11.1 -8.5 Luteolin 7-rutinoside -9.8 -9.4 -14.4 -12.0 -9.9 Narcissin -9.7 -10.5 -10.7 -9.1 -9.9 Pectolinarin -8.9 -7.7 -13.9 -8.5 -7.5 Plantagineoside C -9.4 -10.3 -13.3 -10.4 -9.3 Quercetin 3-glucoside2''-gallate -7.8 -9.2 -12.1 -10.6 -7.5 Quercetin-3-o-rutinose -12.2 -11.0 -11.1 -11.5 -11.4 Salvianolic Acid L (SAL) -9.1 -8.2 -13.3 -11.3 -7.6 Shikonin -8.1 -8.4 -8.6 -8.9 -9.5 Shimobashiric Acid C (SAC) -8.2 -8.7 -10.5 -9.6 -10. 2 *AHDPH = (3R,5R)-3-Acetoxy-5-Hydroxy-1,7-Bis(3,4-Dihydroxyphenyl)Heptane. **DDHHG = (3R,5R)-3,5-Dihydroxy-1-(3,4-Dihydroxyphenyl)-7-(4-Hydroxyphenyl)-Heptane 3-O-Beta-D-Glucopyranoside.
For a comprehensive understanding of the model speci cations, validation, and performance, please refer to the AP11.0 user manual and relevant publications 74, 75 .
Cytoxicity Assay Vero cells were seeded using a multiDrop combi liquid dispenser (Thermo) into 384-well plates at a density of 500 cells/well suspended in 50 µL of media. Cells were allowed to recover and fully attach overnight (approximately 16 hours), at which point library compounds were dispensed into wells using an Echo 550 acoustic dispenser (Labcyte). A total of six nal concentrations where tested (50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, and 1.5625 µM) and wells were back lled with DMSO such that all wells contained a xed ratio of DMSO. Compounds were incubated with cells for 1 hour prior to addition of virus and then for an additional 24 hours, then xed with 10% formalin, permeabilized 0.1% Triton X-100, washed, and stained for SARS-CoV-2 N protein using a speci c antibody (Sino Biological # MM05) and uorescently labelled secondary antibody. Cells were counter stained with Hoechst 33342 to detect cell nuclei, washed, and imaged with a Cytation 1 (Biotek) automated. Each image was then analyzed with a custom work ow in Cell Pro ler (Broad Inst., Boston, MA) which involved counting of cell nuclei and infected cells. At least 4 replicates were used to construct dose response curves.
Statistics and data normalization The rate index is calculated from cell counts using the following formula: Where X c..
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