Pathological mitochondrial fission driven by hyperactivation of the Drp1-MiD49 interaction contributes majorly to several cardiovascular diseases. Existing synthetic Drp1 inhibitors often lack selectivity between pathological and physiological mitochondrial fission, leading to poor bioavailability and off-target effects. Allosteric modulation therefore represents a more promising strategy to fine-tune Drp1 activity than complete inhibition and our study investigated whether natural antioxidants could function as selective allosteric modulators of Drp1 by targeting a characterized allosteric site at the dimer interface using molecular docking, long-timescale (1000 ns) molecular dynamics simulations and MMPBSA analysis. Docking analysis demonstrated favourable binding of the selected phytochemicals at the Drp1 allosteric interface, with Astaxanthin exhibiting the highest docking score, while Baicalin, Luteolin, and Resveratrol formed stable interactions with critical interface residues. Molecular dynamics simulations revealed that these compounds remained consistently associated with the allosteric site and promoted compact conformations of Drp1 characterized by reduced RMSD, radius of gyration (Rg) and solvent-accessible surface area (SASA) relative to apo-Drp1. RMSF, secondary structure and principal component analyses further demonstrated ligand-induced conformational transitions in functionally important regions including the G1/P-loop, G3/Switch II, G5/G-cap and the 80-loop and progressive restriction of conformational space for the Baicalin-, Luteolin-, and Resveratrol-bound complexes. MM-PBSA calculations identified Baicalin as the most energetically favourable complex, followed by Resveratrol. Independent replica simulations of the Baicalin-, Resveratrol-, and Mdivi-1-bound complexes further supported the reproducibility of the observed molecular dynamics trends. Collectively, these findings highlight the potential of Baicalin, Resveratrol, and Luteolin as promising Drp1 allosteric modulators, supporting future development of mitochondrial fission-targeted cardiac therapeutics.
