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 (M^pro) 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 M^pro 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 M^pro. 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 M^pro 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 M^pro inhibitor. Further structural optimization is necessary to enhance potency, selectivity, and safety for therapeutic applications.

Keywords: COVID-19; MD simulations; SARS-CoV-2 main protease; andrographolide analogues; enzyme-based assay.

Figure 1

Rational design of novel andrographolide analogues based on ensitrelvir framework. The atomic labels of the core structure of andrographolide are also provided.

Figure 2

Chemical structures of designed andrographolide analogues 1–6.

Scheme 1

The procedure for the synthesis of 14-acetyl-3,19-isopropylidene andrographolide.

Scheme 2

The procedure for the synthesis of compound 1.

Figure 3

Analysis of structural dynamics. (A) Time evolution of the backbone root-mean-square deviation (RMSD) of the amino acids within 5 Å of the ligand. (B) The calculated radius of gyration (R_g) of the designed compounds and ensitrelvir in complex with SARS-CoV-2 M^pro.

Figure 4

Number of atomic contacts (# contacts) between the ligand and the active site residues plotted over the entire 500-ns simulation period. The average # contacts from the final 50 ns is represented as mean ± SD.

Figure 5

Time evolution of the number of intermolecular hydrogen bonds (H-bonds) between the surrounding amino acids and the ligand.

Figure 6

(A) Representation of the SARS-CoV-2 M^pro homodimer, showing protomer A (light blue) with the ligand bound and protomer B (light pink) in the unbound state. (B) Calculated solvent-accessible surface area (SASA) of the six modeled complexes during 500-ns MD simulation. Amino acids within a 5-Å radius of the ligand were selected for SASA calculations. The average SASA values (mean ± SD) for protomer A and protomer B, derived from the last 50 ns of individual MD simulations, are depicted in light blue and pink, respectively.

Figure 7

In vitro evaluation of compound 1’s inhibitory activity and analysis of key amino acid residues in SARS-CoV-2 M^pro involved in its binding. (A) Percentage of relative inhibition of SARS-CoV-2 M^pro activity in the presence of 100 μM compound 1 compared to its parent compound, andrographolide. (B) Dose-response curve of Relative Fluorescence Units (RFU) generated during the enzyme’s initial rate period over time (%RFU/s), used to calculate the IC₅₀ value of compound 1 against SARS-CoV-2 M^pro. (C) Per-residue free energy decomposition of key binding residues, presented with a surface representation indicating their contribution levels (blue to green).