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 (DeltaC(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.