Design and Evaluation of Andrographolide Analogues as SARS-CoV-2 Main Protease Inhibitors: Molecular Modeling and in vitro Studies

Abstract

Background: The COVID-19 pandemic, caused by SARS-CoV-2, highlights the urgent need for novel antiviral agents targeting key viral proteins. The main protease (Mpro) is a crucial enzyme for viral replication, making it an attractive drug target. Andrographolide, a natural compound with known antiviral properties, serves as a promising scaffold for inhibitor development. Objective: This study aimed to design, synthesize, and evaluate C-12 dithiocarbamate andrographolide analogues as potential SARS-CoV-2 Mpro inhibitors using computational and experimental approaches. Methods: A structure-based drug design approach was employed to design andrographolide derivatives. Molecular dynamics simulations were conducted to assess binding interactions and stability. The hit compound was synthesized and evaluated using an enzyme inhibition assay against SARS-CoV-2 Mpro. Cytotoxicity was assessed in HepG2, HaCaT, and HEK293T cells to determine safety profiles. Results: Among the designed compounds, compound 1, incorporating a 2,4,5-trifluorobenzene moiety, exhibited the strongest binding affinity and stable interactions with key Mpro residues (H41, M49 and M165). Enzyme inhibition assay confirmed ~70% inhibition at 100 µM, with moderate to low cytotoxicity (CC50 values comparable to andrographolide). Conclusion: Compound 1 represents a promising non-covalent SARS-CoV-2 Mpro inhibitor. Further structural optimization is necessary to enhance potency, selectivity, and safety for therapeutic applications.