Abstract
Tumor resistance to antitubulin drugs resulting from P-glycoprotein (Pgp) drug-efflux activity, increased expression of the βIII tubulin isotype, and alterations in the drug-binding sites are major obstacles in cancer therapy. Consequently, novel antitubulin drugs that overcome these challenges are of substantial interest. Here, we study a novel chemotype named furan metotica that localizes to the colchicine-binding site in β-tubulin, inhibits tubulin polymerization, and is not antagonized by Pgp. To elucidate the structure-activity properties of this chiral chemotype, the enantiomers of its most potent member were separated and their absolute configurations determined by X-ray crystallography. Both isomers were active and inhibited all 60 primary cancer cell lines tested at the U.S. National Cancer Institute. They also efficiently killed drug-resistant cancer cells that overexpressed the Pgp drug-efflux pump 10(6)-fold. In vitro, the R-isomer inhibited tubulin polymerization at least 4-fold more potently than the S-isomer, whereas in human cells the difference was 30-fold. Molecular modeling showed that the two isomers bind to β-tubulin in distinct manners: the R-isomer binds in a colchicine-like mode and the S-isomer in a podophyllotoxin-like fashion. In addition, the dynamic binding trajectory and occupancy state of the R-isomer were energetically more favorable then those of the S-isomer, explaining the observed differences in biologic activities. The ability of a racemic drug to assume the binding modes of two prototypical colchicine-site binders represents a novel mechanistic basis for antitubulin activity and paves the way toward a comprehensive design of novel anticancer agents.