Poisson poisoning as the mechanism of action of the microtubule-targeting agent colchicine

Abstract

AbstractMicrotubule-directed anti-cancer drugs, such as paclitaxel, vinblastine, and colchicine, disrupt cell mitosis through suppression of microtubule dynamics (“kinetic stabilization”). However, while the molecular mechanisms of paclitaxel and vinblastine act as pseudo- and true-kinetic stabilizers, respectively, the molecular mechanism of colchicine has remained enigmatic since it requires explanation of both the slow kinetics of the drug and suppression of microtubule dynamics. In this work, we applied an integrated multi-scale modeling-experimental approach to systematically characterize the microtubule targeting agent (MTA) colchicine. We found that colchicine stabilizes microtubule dynamics significantly bothin vivoandin vitroin a time and concentration-dependent manner. Molecular modeling results suggest that tubulin’s binding pocket is accessible to the drug for only 15% of the simulation trajectory time in straight and 82% in curved conformation on average, confirming that colchicine mainly binds to free tubulin. Molecular dynamics simulations show that there are conformational changes at longitudinal and lateral residues of GTP-tubulin-colchicine compared to a lattice tubulin structure, explaining why further incorporation of tubulin dimers to a tubulin-colchicine complex at a protofilament tip is unfavorable. Thermokinetic modeling of microtubule assembly shows that colchicine bound at fractions as low as ∼0.008 to free tubulin can poison the ends of protofilaments with a Poisson distribution and thus, reduce the microtubule growth rate, while stabilizing the tubulin lateral bond and reducing the microtubule shortening rate, i.e. true kinetic stabilization. This study suggests new strategies for colchicine administration to improve the therapeutic window in the treatment of cancer and inflammatory diseases.Significance StatementColchicine is an ancient microtubule targeting agent (MTA) known to attenuate microtubule (MT) dynamics but its cancer treatment efficacy is often limited by lack of a detailed understanding of the drug’s mechanism of action. The primary goal of this study was to perform a multi-scale systematic analysis of molecular mechanism of action of colchicine. The analysis indicates that unlike paclitaxel and vinblastine, colchicine poisons the ends of protofilaments of MTs at low fractions bound to tubulin, in a time-dependent manner. Our results suggest new insights into improvement of the clinical administration of colchicine and new colchicine-site inhibitors.