Comparative Docking Suggests Peripheral Association of Tirapazamine with Bovine Serum Albumin

Molecular docking was used to investigate the interactions between tirapazamine (a bioreductive anticancer drug) and bovine serum albumin (BSA), with emphasis on binding site localization and geometry. Docking simulations were performed using SwissDock with the AutoDock Vina 1.2.0 scoring engine. Warfarin (a canonical Sudlow Site I ligand) was docked under the same conditions as a reference to provide a relative comparison of binding orientation within Subdomain IIA. Docking results indicate that tirapazamine occupies a peripheral entrance-region position within Subdomain IIA, with limited polar interactions and substantial solvent exposure. In contrast, warfarin adopts a more enclosed binding orientation consistent with canonical Site I burial. UV–visible measurements across three independent titration experiments yielded apparent association constants of 2.30 ± 1.05 × 104 M−1 (mean ± SD), consistent with a weak interaction regime and supporting the docking results. Geometric analysis further shows that tirapazamine maintains greater minimum ligand–residue distances from core Site I residues than warfarin, consistent with reduced burial. This interpretation is further supported by the physicochemical profile of tirapazamine, which is characterized by low lipophilicity and high polarity, properties that are incompatible with deep binding in hydrophobic pockets (as seen with warfarin). Short-timescale molecular dynamics simulations (2 ns, triplicate) confirmed the stability of the docked poses, with tirapazamine exhibiting markedly lower ligand RMSD variability (0.247 ± 0.002 Å) than warfarin (0.578 ± 0.163 Å), consistent with a stable peripheral binding geometry. Together, these findings support a model in which tirapazamine associates weakly and peripherally with serum albumin, consistent with non-canonical Site I binding.