Mdivi-1 as a putative direct antagonist of TLR2 and TLR4: A multiscale computational study with experimentally testable predictions

Toll-like receptors TLR2 and TLR4 are central sentinels of innate immunity, yet their aberrant activation drives deleterious inflammation in sepsis and autoimmune disorders. Mdivi-1, a well-known inhibitor of mitochondrial fission, exerts anti-inflammatory effects conventionally attributed to indirect modulation of reactive oxygen species via Drp1 inhibition. Whether Mdivi-1 can directly engage TLRs at the extracellular level remains unexplored. Here, we systematically evaluated this hypothesis using an integrative computational pipeline encompassing molecular docking, 200 ns all-atom molecular dynamics (MD) simulations, principal component analysis (PCA), dynamic cross-correlation matrix (DCCM) analysis, free energy landscape (FEL) mapping, and MM/PBSA binding free energy calculations benchmarked against established positive controls (CU-CPT22 and T5342126), complemented by Density Functional Theory (DFT) analyses including Molecular Electrostatic Potential (MEP), Interaction Region Indicator (IRI), and Frontier Molecular Orbital (FMO) evaluations. Our MD simulations suggested that Mdivi-1 forms stable complexes with both TLR2 and TLR4-MD-2, as evidenced by converged RMSD profiles (0.20 ± 0.03 nm and 0.45 ± 0.05 nm, respectively) and reduced solvent accessible surface areas. PCA and FEL analyses revealed substantial conformational space restriction upon ligand binding (cluster radius reduction from 18.5-4.2 nm for TLR2 and from 22.3 to 6.8 nm for TLR4), suggesting potential thermodynamic trapping into low energy rigidified states with an estimated entropic penalty of 1.4-1.7 kcal/mol. DCCM analysis indicated perturbation of intrinsic inter-chain communication, particularly with TLR4-MD-2, with interface correlation coefficients decreasing from the 0.65-0.80 range down to 0.40-0.60. Interaction profiling identified a critical hydrogen bond with SER346 in TLR2 (92.3% occupancy) and extensive π-π stacking networks with PHE76, PHE104, and PHE147 in the MD-2 cavity, corroborated by DFT derived electronic properties. MM/PBSA calculations yielded favorable binding free energies of -28.16 kcal/mol (TLR2) and -25.34 kcal/mol (TLR4-MD-2), predominantly driven by van der Waals forces and comparable to the reference inhibitors. Per-residue decomposition quantitatively assessed the hotspot contributions of SER346 (-1.48 kcal/mol) in TLR2 and the phenylalanine triad (-1.11 to -1.68 kcal/mol) in TLR4-MD-2, which are highly conserved across species and critical for native ligand recognition. Based on these findings, we generated experimentally testable hypotheses regarding relative binding potential, candidate binding hotspots, and functional consequences that require future empirical validation. Collectively, these multiscale computational data provide a coherent theoretical framework supporting the plausibility of Mdivi-1 acting as a computationally predicted ligand with putative TLR2 and TLR4-MD-2 binding modes, complementing its known intracellular mechanisms. These findings establish a foundation for future experimental validation through surface plasmon resonance and TLR-specific reporter assays.