Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry

[Image: see text] Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (K(d) = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular K(i) = 22 nM), docetaxel (cellular K(i) = 16 nM), cabazitaxel (cellular K(i) = 6 nM), and ixabepilone (cellular K(i) = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (K(b)) of the microtubule destabilizers colchicine (K(b) = 80 nM, α = 0.08), vinblastine (K(b) = 7 nM, α = 0.18), and maytansine (K(b) = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (K(b) = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.