Binding of indanocine to the colchicine site on tubulin promotes fluorescence, and its binding parameters resemble those of the colchicine analogue AC

Abstract

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