Elucidating target specificity of the taccalonolide covalent microtubule stabilizers employing a combinatorial chemical approach

The taccalonolide microtubule stabilizers covalently bind β-tubulin and overcome clinically relevant taxane resistance mechanisms. Evaluations of the target specificity and detailed drug–target interactions of taccalonolides, however, have been limited in part by their irreversible target engagement. In this study, we report the synthesis of fluorogenic taccalonolide probes that maintain the native biological properties of the potent taccalonolide, AJ. These carefully optimized, cell-permeable probes outperform commercial taxane-based probes and enable direct visualization of taccalonolides in both live and fixed cells with dramatic microtubule colocalization. The specificity of taccalonolide binding to β-tubulin is demonstrated by immunoblotting, which allows for determination of the relative contribution of key tubulin residues and taccalonolide moieties for drug–target interactions by activity-based protein profiling utilizing site-directed mutagenesis and computational modeling. This combinatorial approach provides a generally applicable strategy for investigating the binding specificity and molecular interactions of covalent binding drugs in a cellular environment.


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No randomization was used in these studies as there was no group allocation used in this study No blinding was used in these studies as there was no group allocation used in this study !-tubulin at 1:1000 (Sigma T-4026, lot 107M4801V), Texas Red-X at 1:200 (Invitrogen T-862, lot 732914), !-tubulin at 1:1000 (abcam, ab6046, lot GR3204950-1), fluorescein at 1:500 (abcam, ab19491, lot GR3209552-4), IRDye 680 or 800 goat anti-rabbit secondary antibodies at 1:10,000 (LI-COR Biosciences, 680-926-68071, lot C50706-03 & 800-925-32211, lot C70926-01), !IIItubulin 1:400 (Sigma-Aldrich, T8660) clone SDL.3D10 ascites fluid All antibodies were obtained from commercial sources and have been heavily referenced. The antibodies were validated by immunoblotting by molecular weight and by detection of additional higher molecular weight bands with tubulin antibodies only when the tubulin fusion protein was expressed. Similarly, the specificity of the fluorescein antibody was confirmed by presence only when the fluorescein tagged taccalonolide was added to cells. Validation by immunofluorescence was determined by observing microtubule bundling or loss when cells were treated with microtubule stabilizing or destabilizing drugs, respectively.