Analysis of mebendazole binding to its target biomolecule by laser flash photolysis

Mebendazole (MBZ) and related anticancer benzimidazoles act binding the beta-subunit of Tubulin (TU) before dimerization with alpha-TU with subsequent blocking microtubule formation. Laser flash photolysis (LFP) is a new tool to investigate drug-albumin interactions and to determine binding parameters such as affinity constant or population of binding sites. The aim of this study was to evaluate the interactions between the nonfluorescent mebendazole (MBZ) and its target biomolecule TU using this technique. Before analyzing the MBZ@TU complex it was needed to determine the photophysical properties of MBZ triplet excited state ((3)MBZ*) in different media. Hence, 3MBZ* showed a transient absorption spectrum with maxima at 520 and 375 nm and a lifetime much longer in acetonitrile (12.5 mu s) than in water (260 ns). The binding of MBZ to TU produces a greater increase of the lifetime of 3MBZ* (25 mu s). This fact and the strong electron acceptor capability observed for (3)MBZ* evidence that MBZ must not be located close to any electron donor amino acid of TU such as its tryptophan or cysteine residues. Adding increasing amounts of MBZ to aqueous TU was determined the MBZ-TU binding constant (2.0 +/- 0.5 x 10(5) M-1 at 298 K) which decreased with increasing temperature. The LFP technique has proven to be a powerful tool to analyze the binding of drug-TU systems when the drug has a detectable triplet excited state. Results indicate that LFP could be the technique of choice to study the interactions of non-fluorescent drugs with their target biomolecules. (C) 2015 Elsevier B.V. All rights reserved.