Probing the Pore Drug Binding Site of Microtubules with Fluorescent Taxanes: Evidence of Two Binding Poses

Abstract

The pore site in microtubules has been studied with the use of Hexaflutax, a fluorescent probe derived from paclitaxel. The compound is active in cells with similar effects to paclitaxel, indicating that the pore may be a target to microtubule stabilizing agents. While other taxanes bind microtubules in a monophasic way, thus indicating a single type of sites, Hexaflutax association is biphasic. Analysis of the phases indicates that two different binding sites are detected, reflecting two different modes of binding, which could arise from different arrangements of the taxane or fluorescein moieties in the pore. Association of the 4-4-20 antifluorescein monoclonal antibody-Hexaflutax complex to microtubules remains biphasic, thus indicating that the two phases observed arise from two different poses of the taxane moiety.

Zampanolide and dactylolide: cytotoxic tubulin-assembly agents and promising anticancer leads

“…Zampanolide strongly inhibited paclitaxel binding without interfering with peloruside A binding, and its specific binding to the taxane luminal site can impede or modify the taxane pore site. Thus hexaflutax (a fluorescent taxane probe that binds to the taxane pore site 34 ) did not bind to tubulin in the presence of zampanolide even at stoichiometric concentrations with tubulin. 23 The apparent binding constants of both 1 and 3 increased with temperature, indicating possible covalent binding to microtubules, 35,36 similar to that of cyclostreptin.…”

Recent progress with microtubule stabilizers: new compounds, binding modes and cellular activities

“…The kinetic techniques used to measure the interaction of hexaflutax with the pore site revealed it does so in two distinct ways, likely due to different rearrangements of taxane and fluorescein binding; one in which interactions are made only with β -tubulin subunits and another in which the interactions are made with both α and β -tubulin subunits. 123 This work showed that both binding interactions proposed separately by Freedman and Magnani are indeed possible. Hexaflutax led to the characteristic microtubule bundling and mitotic arrest seen with other microtubule stabilizers, albeit with much lower potency than cyclostreptin, suggesting that binding at the pore site may be sufficient for microtubule stabilization.…”

“…however, proposed that the taxane core is only bound to a single β -tubulin subunit with the side chain bound to α-tubulin. 137 Diaz aimed to better characterize the interactions of taxane-site binding agents within the pore site 123 using hexaflutax, a fluorescent taxane derivative that binds to the external pore site on microtubules with no observable modification of the interior luminal site. The kinetic techniques used to measure the interaction of hexaflutax with the pore site revealed it does so in two distinct ways, likely due to different rearrangements of taxane and fluorescein binding; one in which interactions are made only with β -tubulin subunits and another in which the interactions are made with both α and β -tubulin subunits.…”

“…Hexaflutax led to the characteristic microtubule bundling and mitotic arrest seen with other microtubule stabilizers, albeit with much lower potency than cyclostreptin, suggesting that binding at the pore site may be sufficient for microtubule stabilization. 123 This work has led to the hypothesis that the pore site on microtubules may be a new pharmacological target for microtubule stabilization.…”

Drug discovery targeting cell division proteins, microtubules and FtsZ

“…These probes exhibited potent tubulin assembly promotion and tumor cell killing activities, thus have been proven to be useful as tools for the determination of thermodynamic parameters and exploration of ligand–microtubule interactions. 66, 67 …”