Background : COVID-19, a disease caused by a novel coronavirus, is a major global human threat that has turned into a pandemic. Identification and development of drugs/vaccines is urgently needed for prevention and therapy of the COVID-19 virus (SARS-CoV-2) infection. SARS-CoV-2 main protease (Mpro,) plays a key role in viral protein processing for its replication and inhibition of Mpro leads to prevention of viral replication. The SARS-CoV-2 Mpro is considered a promising drug target, as it is dissimilar to human proteases. Several peptidic protease inhibitors have been developed as SARS-CoV-2 Mpro inhibitors for COVID-19 treatment. In this study, we screened naturally occurring peptidic α-hydroxy amides (PHAs) (phebestin, probestin, bestatin) for SARS-CoV-2 Mpro inhibition. Materials and methods : In vitro COVID-19 Mpro fluorometric assay was performed to evaluate its inhibitory efficacy by PHAs. Isothermal titration calorimetry technique was used to demonstrate the binding affinity between COVID-19 Mpro and PHAs. Circular dichroism technique was used to determine the structural changes in secondary structure of COVID-19 Mpro induced by PHAs. Molecular docking studies were performed to demonstrate the molecular level mechanism behind COVID-19 Mpro inhibitory efficacy of PHAs. In addition, cytotoxic effects of PHAs were evaluated on lung epithelial cells of mice for its therapeutic applications for COVID-19 through nasal delivery. Results and discussion : Phebestin (IC50 = 17.98 µM) Probestin (IC50 = 48.31 µM) and Bestatin (IC50 &gt; 100 µM) significantly inhibited the COVID-19 Mpro activity. The KD (dissociation constant) values of phebestin and probestin for COVID-19 Mpro were 31.23 and 43.51 µM, respectively. CD spectrum analysis demonstrated that phebestin and probestin (10 µM) induced changes in the secondary structure of COVID-19 Mpro. Molecular docking studies demonstrated that Interactions of phebestin and probestin with active site amino acids. Phebestin and probestin did not show any cytotoxicity on normal mouse lung epithelial cells up to 100 µM. Conclusion : Preliminary in vitro studies demonstrated that naturally occurring PHAs derivatives phebestin and probestin are SARS-CoV-2 Mpro inhibitors and warranted further studies to develop as drugs for COVID-19.
SARS-CoV-2 main protease inhibitory efficacy of natural peptidic α-hydroxy amides for therapeutic applications of COVID-19