Binding of Indanocine to the Colchicine Site on Tubulin Promotes Fluorescence, and Its Binding Parameters Resemble Those of the Colchicine Analogue AC

摘要

Indanocine, a synthetic indanone, has shown potential antiproliferative activity against severalntumor types. It is different from many other microtubule-disrupting drugs, because it displays toxicityntoward multidrug resistance cells. We have examined the interaction of indanocine with tubulin andndetermined their binding and thermodynamic parameters using isothermal titration calorimetry (ITC).nIndanocine is weakly fluorescent in aqueous solution, and the binding to tubulin enhances fluorescencenwith a large blue shift in the emission maxima. Indanocine binds to the colchicine site of tubulin, althoughnit bears no structural similarity with colchicine. Nevertheless, like colchicine analogue AC, indanocine isna flexible molecule in which two halves of the molecule are connected through a single bond. Also, likenAC, indanocine binds to the colchicine binding site of tubulin in a reversible manner and the associationnreaction occurs at a faster rate compared to that of colchicine-tubulin binding. The binding kinetics wasnstudied using stopped-flow fluorescence. The association process follows biphasic kinetics similar to thatnof the colchicine-tubulin interaction. The activation energy of the reaction was 10.5 ( 0.81 kcal/mol.nFurther investigation using ITC revealed that the enthalpy of association of indanocine with tubulin isnnegative and occurs with a negative heat capacity change (ΔCp ) -175.1 cal mol-1 K-1). The bindingnis unique with a simultaneous participation of both hydrophobic and hydrogen bonding forces. Finally,nwe conclude that even though indanocine possesses no structural similarity with colchicine, it recognizesnthe colchicine binding site of tubulin and its binding properties resemble those of the colchicine analoguenAC.