ANTIPROLIFERATIVE MECHANISM OF ACTION OF CRYPTOPHYCIN-52 - KINETIC STABILIZATION OF MICROTUBULE DYNAMICS BY HIGH-AFFINITY BINDING TO MICROTUBULE ENDS

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophyc in family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (greater than or equal to 10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation a t mitosis by depolymerizing spindle microtubules and disrupting chromo some organization. However, low concentrations of cryptophycin-52 inhi bited cell proliferation at mitosis (IC50 = 11 pM) without significant ly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individua l microtubules in vitro (IC50 = 20 nM), reducing the rate and extent o f shortening and growing without significantly reducing polymer mass o r mean microtubule length. Using [H-3] cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (K- d, 47 nM, maximum of approximate to 19.5 cryptophycin-52 molecules per microtubule), By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approx imate to 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%, Cryptophycin-52 became conce ntrated in cells 730-fold, and the resulting intracellular cryptophyci n-52 concentration was similar to that required to stabilize microtubu le dynamics in vitro. The data suggest that cryptophycin-52 potently p erturbs kinetic events at microtubule ends that are required for micro tubule function during mitosis and that it acts by forming a reversibl e cryptophycin-52-tubulin stabilizing cap at microtubule ends.