title: Analysis of mebendazole binding to its target biomolecule by laser flash photolysis.
abstract: Mebendazole (MBZ) and related anticancer benzimidazoles act binding the β-subunit of Tubulin (TU) before dimerization with α-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, (3)MBZ(⁎) showed a transient absorption spectrum with maxima at 520 and 375 nm and a lifetime much longer in acetonitrile (12.5 μs) than in water (260 ns). The binding of MBZ to TU produces a greater increase of the lifetime of (3)MBZ(⁎) (25 μs). 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 × 10(5)M(-1) at 298K) 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.
authors.affiliation: Departamento de Química/Instituto Universitario Mixto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, Avenida de los Naranjos s/n, E-46022 Valencia, Spain.
authors.forename: Dolors
authors.initials: D
authors.lastname: Jornet
authors.affiliation: Departamento de Química/Instituto Universitario Mixto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, Avenida de los Naranjos s/n, E-46022 Valencia, Spain. Electronic address: fbosca@itq.upv.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Bosca
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9 E-28040 Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Andreu
authors.affiliation: Departamento de Química Orgánica, Universidad de Valencia, Dr. Moliner 50, E 46100 Burjasot, Spain.
authors.forename: Luis R
authors.initials: LR
authors.lastname: Domingo
authors.affiliation: Departamento de Química/Instituto Universitario Mixto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, Avenida de los Naranjos s/n, E-46022 Valencia, Spain. Electronic address: rtormos@qim.upv.es.
authors.forename: Rosa
authors.initials: R
authors.lastname: Tormos
authors.affiliation: Departamento de Química/Instituto Universitario Mixto de Tecnología Química UPV-CSIC, Universitat Politècnica de València, Avenida de los Naranjos s/n, E-46022 Valencia, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Miranda
mesh_terms: D007834:Lasers; D008463:Mebendazole / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis / Q000528:radiation effects; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014404:Tubulin; D008463:Mebendazole
keywords: Anticancer drugs; Binding constant; Laser flash photolysis; Mebendazole triplet excited state; Phosphorescence; Tubulin