Molecular insights into nilvadipine-hemoglobin interactions: conformational dynamics and binding mechanisms.

Understanding the molecular basis of drug-protein interactions is essential for predicting pharmacokinetics and potential off-target effects. Here, we employ a combined experimental and computational approach to characterize the binding of Nilvadipine (a dihydropyridine calcium channel blocker) to hemoglobin (Hb). Using Soret band absorption and steady-state fluorescence spectroscopy across 298-310 K, we observed pronounced static quenching of Hb's intrinsic fluorescence, yielding Stern-Volmer constants (K SV) in the order of 104 M-1 and 1 : 1 binding stoichiometry. Thermodynamic parameters derived from van't Hoff analysis (ΔH° > 0, ΔS° > 0, and ΔG° < 0) highlighted hydrophobic interactions as the primary driving force and confirmed the spontaneity of complex formation. Förster resonance energy transfer (FRET) measurements further positioned Nilvadipine at ∼3.0 nm from Hb's fluorophores, consistent with a static, ground-state complex. Molecular docking identified a preferential binding pose stabilized by hydrogen bonds with ASN68 and ASP64, hydrophobic contacts involving ALA82, LEU83, and LEU86, and interactions with the heme group, yielding a computed binding energy of -5.50 kcal mol-1 in close agreement with spectroscopically derived ΔG°. Over 100 ns of molecular dynamics (MD) simulations, the Hb-Nilvadipine complex remained structurally robust, with backbone RMSD values <0.2 nm, minor radius of gyration (R g) reduction, limited per-residue fluctuations (RMSF < 0.3 nm), and negligible changes in solvent-accessible surface area (SASA). Together, these data demonstrate that Nilvadipine forms a stable, hydrophobically driven complex with Hb without perturbing its global fold, suggesting that Hb may serve as a transient reservoir for the drug in circulation. This integrative study provides a detailed roadmap for interrogating small-molecule binding to blood proteins and offers insights valuable for drug delivery, safety assessment, and the design of Hb-based carriers.