Coronaviruses are contagious pathogens primarily responsible for respiratory and intestinal infections. Research efforts to develop antiviral agents against coronavirus demonstrated the main protease (Mpro) protein may represent effective drug target. X-ray crystallographic structure of the SARS-CoV2 Mpro protein demonstrated the significance of Glu166, Cys141, and His41 residues involved in protein dimerization and its catalytic function. We performed in silico screening of compounds from Curcuma longa L. (Zingiberaceae family) against Mpro protein inhibition. Employing a combination of molecular docking, scoring functions, and molecular dynamics simulations, 267 compounds were screened by docking on Mpro crystallographic structure. Docking score and interaction profile analysis exhibited strong binding on the Mpro catalytic domain with compounds C1 (1E,6E)-1,2,6,7-tetrahydroxy-1,7bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione) and C2 (4Z,6E)-1,5-dihydroxy-1,7-bis(4hydroxyphenyl)hepta-4,6-dien-3-one as lead agents. Compound C1 and C2 showed minimum binding score (-9.08 and -8.07 kcal/mole) against Mpro protein in comparison to shikonin and lopinavir (% À5.4 kcal/mole) a standard Mpro inhibitor. Furthermore, principal component analysis, free energy landscape and protein-ligand energy calculation studies revealed that these two compounds strongly bind to the catalytic core of the Mpro protein with higher efficacy than lopinavir, a standard antiretroviral of the protease inhibitor class. Taken together, this structure based optimization has provided lead on two natural Mpro inhibitors for further testing and development as therapeutics against human coronavirus.

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The present study explores the SARS-CoV-2 drugable target inhibition efficacy of phytochemicals from Indian medicinal plants using molecular docking, molecular dynamics (MD) simulation, and MM-PBSA analysis. A total of 130 phytochemicals were screened against SARS-CoV-2 Spike (S)-protein, RNA-dependent RNA polymerase (RdRp), and Main protease (Mpro). Result of molecular docking showed that Isoquercetin potentially binds with the active site/protein binding site of the Spike, RdRP, and Mpro targets with a docking score of -8.22, -6.86, and -9.73 kcal/mole, respectively. Further, MS 3, 7-Hydroxyaloin B, 10-Hydroxyaloin A, showed -9.57, -7.07, -8.57 kcal/mole docking score against Spike, RdRP, and Mpro targets respectively. The MD simulation was performed to study the favorable confirmation and energetically stable complex formation ability of Isoquercetin and 10-Hydroxyaloin A phytochemicals in Mpro-unbound/ligand bound/standard inhibitor bound system. The parameters such as RMSD, RMSF, Rg, SASA, Hydrogen-bond formation, energy landscape, principal component analysis showed that the lead phytochemicals form stable and energetically stabilized complex with the target protein. Further, MM-PBSA analysis was performed to compare the Gibbs free energy of the Mpro-ligand bound and standard inhibitor bound complexes. The analysis revealed that the His-41, Cys145, Met49, and Leu27 amino acid residues were majorly responsible for the lower free energy of the complex. Drug likeness and physiochemical properties of the test compounds showed satisfactory results. Taken together, the study concludes that that the Isoquercetin and 10-Hydroxyaloin A phytochemical possess significant efficacy to bind SARS-Cov-2 Mpro active site. The study necessitates further in vitro and in vivo experimental validation of these lead phytochemicals to assess their anti-SARS-CoV-2 potential.

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Breast cancer is a heterogeneous disease consisting of atypical cell populations that share stem cell‐like characteristics associated with therapeutic resistance, disease relapse, and poor clinical outcome. MicroRNAs (miRNA), and small noncoding RNA, are pivotal in the regulation of self‐renewal, stemness, and cellular differentiation. Withaferin A (WA), a steroidal lactone, is a major bioactive constituent of Withania somnifera (Solanaceae) known for its anticancer properties. In this study, the effect of WA on modulation of miRNA expression in breast cancer‐derived mammosphere was assessed utilizing small RNA sequencing. Treatment with WA inhibited MCF‐7 and T47D cells derived mammosphere formation with a significant decrease in CD44, EpCAM, Nanog, OCT4, and SOX2 as markers of self‐renewal and stemness. Small RNA sequencing demonstrated a total of 395 differentially expressed miRNAs (DEMs) including 194 upregulated and 201 downregulated miRNAs in WA‐treated MCF‐7 mammospheres. Bioinformatics analysis utilizing the KEGG pathway, Gene Ontology enrichment, protein−protein, and miRNA‐mRNA interaction network identified altered expression in a few hub genes viz. AKT1, PTEN, MYC, CCND1, VEGFA, NOTCH1, and IGFR1 associated with DEMs in WA‐treated mammospheres. Further quantitative RT‐PCR analysis validated the expression of DEMs including miR‐549a‐5p, miR‐1247‐5p, miR‐124‐5p, miR‐137‐5p, miR‐34a‐5p, miR‐146a‐5p, miR‐99a‐5p, miR‐181a‐5p, let‐7c‐5p, and let‐7a‐5p. In particular, let‐7c‐5p is designated as a tumor suppressor in breast cancer. An increase in miR‐let‐7c‐5p expression was noted after WA treatment, with a simultaneous decrease in CCND1 and c‐MYC at mRNA and protein levels. Taken together, our study demonstrated WA‐mediated miRNA expression, in particular, upregulation of miR‐let‐7c‐5p, leads to the inhibition of breast cancer cells derived mammospheres.