The interaction of baccatin III with the taxol binding site of microtubules determined by a homogeneous assay with fluorescent taxoid

The ubiquitous Taxol binding site of microtubules also binds newly discover ed ligands. We have designed a homogeneous assay for the high throughput de tection of Taxol biomimetics, based on the displacement of 7-O-[N-(2,7-difl uoro-4 ' -fluoreseeincarbonyl)-L-alanyl]Taxol from its binding site in dilu ted solutions of preserved microtubules. The state of this reference ligand is measured by fluorescence anisotropy in a microplate reader, with varyin g concentrations of nonfluorescent competitors. The binding equilibrium con stant of Taxol has a value K-b = 3.7 x 10(7) M-1. We have found that baccat in III, an analogue of Taxol without the C-13 side chain, binds with K-b = 1.5 x 10(5) M-1, whereas the side chain methyl ester is inactive. This was unexpected from the structure- activity relationship of taxoids but compati ble with models of Taxol docked at the microtubule site. Baccatin III bindi ng has been confirmed by displacement of [H-3]Taxol and by direct HPLC meas urements of its cosedimentation with microtubules, among other methods. Con sequently, baccatin III induces microtubule bundles and multipolar spindles in PtK2 and U937 cells, and mitotic arrest and apoptotic death of the U937 cells, at concentrations 200-500-fold larger than Taxol. The simplest anal ysis of these results strongly suggests that the interaction of the C-2 C-4 substituted taxane ring system with the microtubule binding site provides most (ca. 75%) of the free energy change of Taxol binding and is sufficient to activate microtubule stabilization and transmit the antitumor effects o f Taxol, whereas the C-13 side chain provides a weak specific anchor.