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

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.

Taccalonolides: The Potential Next Generation of Microtubule-Stabilizing Anticancer Agents

“…These SARs are observed as well by computational analysis such as i) taccalonolides covalently bind to β-tubulin D226 via the C22-C23 epoxide group, and ii) the bulky group at C-1 is involved in interactions with β-tubulin residues through hydrogen bonds [17,18,44]. According to established SARs, a group of taccalonolides with a fluorescein group at C-6 such as Flu-tacca-4, Flu-tacca-5, Flu-tacca-7, and Flu-tacca-8 were designed and generated to facilitate identification of binding site interactions, among which Flu-tacca-7 showed comparable potencies in the proliferation of HeLa and SK-OV-3 cells (Figure 6) [18,45].…”

“…In addition, among the other changes in the B ring such as keto-enol tautomerization at C6-C7 (taccalonolide B vs taccalonolide I), dehydrogenation (taccalonolide A vs taccalonolides AD and H 2 ) or the formation of a geminal diol www.videleaf.com group (taccalonolides AE), the 6,7-double bond (taccalonolides H 2 ) moderately increase the activity [16,30]. These SARs are observed as well by computational analysis such as i) taccalonolides covalently bind to β-tubulin D226 via the C22-C23 epoxide group, and ii) the bulky group at C-1 is involved in interactions with β-tubulin residues through hydrogen bonds [17,18,44]. According to established SARs, a group of taccalonolides with a fluorescein group at C-6 such as Flu-tacca-4, Flu-tacca-5, Flu-tacca-7, and Flu-tacca-8 were designed and generated to facilitate identification of binding site interactions, among which Flu-tacca-7 showed comparable potencies in the proliferation of HeLa and SK-OV-3 cells (Figure 6) [18,45].…”

“…Taccalonolide AF marked a milestone in understanding the structure-activity relationships (SARs) of taccalonolides and the mechanism of action of this class of compounds. The absolute configuration of the C22-C23 epoxide of taccalonolides AF and AJ was determined to be R,R, by single-crystal X-ray diffraction analysis [17,18].…”

Efficacy of a Covalent Microtubule Stabilizer in Taxane-Resistant Ovarian Cancer Models

“…Paclitaxel was obtained from Sigma-Aldrich (T1912-25MG, St. Louis, MO, USA) and brought up in 200 proof EtOH. Taccalonolide AF was semi-synthesized as previously described and brought up in 200 proof EtOH [ 16 ]. (±)-Verapamil hydrochloride was obtained from Sigma (V4629, St. Louis, MO, USA) and brought up in water.…”

“…The epoxytaccalonolides are a novel class of plant-derived microtubule stabilizers that bind covalently and irreversibly to tubulin through a specific interaction between their C-22,23 epoxide with Asp226 on β-tubulin [ 16 , 17 ]. Although the epoxytaccalonolide AF ( Figure 1 A) has demonstrated antitumor efficacy in flank xenograft breast cancer models with systemic administration, it has a narrow therapeutic index and short serum half-life of 45 min [ 18 , 19 ].…”