Abstract 2764A: Elucidating the pharmacological effects of covalent microtubule stabilizing taccalonolides through functional tagging

Abstract

Abstract Microtubule stabilizing taxanes are highly effective in the treatment of solid tumors and continue to be used as single agents and in combination with targeted therapies and immunotherapies. However, limitations of taxane therapy include acquired and inherent drug resistance. The taccalonolides are a novel class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance to the taxanes in vitro and in vivo. The taccalonolides are efficacious in taxane-resistant models due to their distinct mechanism of microtubule stabilization that involves covalent binding to β-tubulin. Pharmacological studies of the taccalonolides have been complicated by their irreversible binding to tubulin. A functional tagged taccalonolide would facilitate a greater understanding of the pharmacological, cellular, and physiological effects of this novel drug class. We have generated and characterized a series of fluorescently tagged taccalonolides that retain microtubule binding and stabilizing activities to serve as functional pharmacological taccalonolide probes. Biochemical tubulin polymerization, live cell fluorescence, immunofluorescence imaging, and the sulforhodamine B cytotoxicity assay were conducted to evaluate the microtubule binding and stabilizing effects of these taccalonolide probes. We identified stable taccalonolide probes that retain the biological activities of untagged taccalonolides and facilitate their detection in live cells and in cell lysates. The rates of taccalonolide uptake and tubulin binding in cells, tumors, and normal tissues were determined. These studies have also allowed for an evaluation of the anatomical distribution of the taccalonolides in vivo, which can inform on their potential for antitumor efficacy in distinct tumor sites. Furthermore, we have generated a cellular model to evaluate the kinetics of taccalonolide binding to β-tubulin mutants that are predicted to influence drug binding based on the crystal structure. These studies have confirmed the covalent interaction between the taccalonolides and D224 on β-tubulin and informed on other residues critical for drug binding. The generation of a functional tagged taccalonolide will continue to be a valuable tool in evaluating the pharmacokinetics of these agents and inform on drug targeting strategies that will facilitate their clinical development. Citation Format: Samantha S. Yee, Lin Du, April L. Risinger. Elucidating the pharmacological effects of covalent microtubule stabilizing taccalonolides through functional tagging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2764A.