Antiproliferative mechanism of action of cryptophycin-52: Kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Abstract

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>10 times the IC 50 ), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC 50 ‫؍‬ 11 pM) without significantly 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 individual microtubules in vitro (IC 50 ‫؍‬ 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [ 3 H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (K d , 47 nM, maximum of Ϸ19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that Ϸ5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.

Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

“…Microtubules are unusual receptors for drugs in that an individual microtubule contains ϳ1690 tubulin dimers/m of length and, thus, very large numbers of binding sites. In addition, occupancy of just a few sites on an individual microtubule exerts powerful suppression of dynamics (17,25). The two drugs did exert different effects on the length-based catastrophe frequency.…”

The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth

“…Many microtubule-targeted drugs, including vinblastine, colchicine, cryptophycin 52, and paclitaxel, can bind with relatively high affinity directly to microtubules, either to their ends, as with the first three drugs, or along their lengths, as with paclitaxel (10,23,32,41,42). Many drugs that bind directly to microtubules can stabilize the microtubule against shortening and significantly alter the catastrophe and rescue frequencies.…”

Total Synthesis of Cryptophycin A and Analogs

“…7 -8 For Cryptophycin A, the ratio of cryptophycin concentration to the concentration of tubulin was calculated to be approximately 1:400. 8 Cryptophycin-52 is even more potent, having a ratio of approximately 1:2,500, 7 making it the most potent stabilizer of microtubulin dynamics currently known.…”

“…Within the past year, it was discovered that Cryptophycin A and Cryptophycin-52 interact with tubulin to stabilize the dynamics at picomolar concentrations, without altering the microtubule content or organization. 7,8 Earlier, it was known that micromolar concentrations of cryptophycin inhibited tubulin polymerization 1 ,9 by targeting tubulin, altering the microtubule/tubulin equilibrium, and causing it to depolymerize and form aggregates within cells. 9,10 More recent studies have indicated that the mechanism of action may involve the interaction of cryptophycin at the microtubule ends.…”

“…Cryptophycin-52 is currently undergoing clinical evaluation for cancer chemotherapy because of its wide range of activity against a variety of tumor models, coupled with its increased stability and its similar potency to Cryptophycin A. 7 To date, no extensive structure-activity relationship (SAR) studies have been published regarding the CIO side chain, although a variety of analogs of the C16 side chain have been synthesized 12 along with numerous variations on the C5 to N8, ß-alanine portion of the molecule. 13 Al-awar et al synthesized a wide variety of C16 analogs that have helped provide insight about the SAR of the aryl ring.…”