Interaction of the Antitumor Compound Cryptophycin-52 with Tubulin

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Abstract
Cryptophycin-52 (LY355703) is currently undergoing clinical evaluation for cancer chemotherapy. It is a potent suppresser of microtubule dynamics in vitro, and low picomolar concentrations appear to inhibit cancer cell proliferation at mitosis by stabilizing spindle microtubules. In the present study, using [3H]cryptophycin-52, we found that the compound bound to tubulin at a single high-affinity site [apparent Ka (3.6 ± 1) × 106 L/mol, 34 °C]. The binding of cryptophycin-52 to tubulin was rapid, not appreciably temperature-dependent, and very poorly reversible. However, we could remove [3H]cryptophycin-52 from [3H]cryptophycin-52−tubulin complex by denaturing the complex with either urea treatment or boiling. These data suggest that the binding of cryptophycin-52 to tubulin is not covalent. A van't Hoff plot of the binding data indicated that the binding of cryptophycin-52 to tubulin is primarily entropy-driven with a minimum enthalpy contribution. In addition, cryptophycin-52 perturbed the far-ultraviolet circular dichroic spectrum of tubulin and it inhibited the colchicine-induced guanosine triphosphatase activity of tubulin, indicating that its binding to tubulin induces a conformational change in the tubulin. Competition experiments with vinblastine suggest that the binding site for crytophycin-52 may overlap with the vinblastine binding site.