Abstract
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.
Highlights
The taccalonolide microtubule stabilizers covalently bind β-tubulin and overcome clinically relevant taxane resistance mechanisms
We describe the synthesis and optimization of a fluorogenic taccalonolide probe, Flu-tacca-7 (11). This stable, cell-permeable probe is used for activity-based protein profiling (ABPP) in human cancer cell lines to confirm the specificity of covalent binding of the taccalonolides to β-tubulin and evaluate key β-tubulin residues and taccalonolide moieties that mediate taccalonolide-tubulin binding
The careful examination of the chemical environment in the binding pocket revealed that the C-6 ketone group of 2 was positioned relatively remote from all βtubulin residues and was not involved in any inter- and intramolecular interactions (Fig. 1e)
Summary
The taccalonolide microtubule stabilizers covalently bind β-tubulin and overcome clinically relevant taxane resistance mechanisms. 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. The taccalonolides are a class of microtubule stabilizers that covalently bind β-tubulin[17,18] and effectively circumvent clinically relevant models of resistance to taxanes both in vitro and in vivo[19,20,21] Despite their promising therapeutic potential, the covalent nature of taccalonolide binding has hampered our ability to perform detailed binding studies using conventional approaches[22,23]. Flu-tacca-7 (11) represents a class of irreversible microtubule labeling probes that are superior to commercially available options, providing a valuable tool for cellular evaluations of this important drug target
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