A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

Abstract

Microtubule-targeting agents (MTAs) like taxol and vinblastine are amongst the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine-site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine-site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine-site on -tubulin. The high resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of a novel sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next generation MTAs for the treatment of cancer.