Taccalonolide Microtubule Stabilizers

Abstract

Microtubule stabilizers are a mainstay in the treatment of many solid cancers and continue to find utility in combination therapy with molecularly targeted anticancer agents and immunotherapeutics. However, innate and acquired resistance to microtubule stabilizers can limit their clinical efficacy. The taccalonolides are a unique class of microtubule stabilizers isolated from plants of Tacca that circumvent clinically relevant mechanisms of drug resistance. Although initial reports suggested that the microtubule-stabilizing activity of the taccalonolides was independent of direct tubulin binding, additional studies have identified that potent C-22, C-23 epoxidized taccalonolides covalently bind the Aspartate 226 residue of β-tubulin and that this interaction is critical for their microtubule-stabilizing activity. The taccalonolides have distinct properties as compared to other microtubule stabilizers with regard to their biochemical effects on tubulin structure and dynamics that promote distinct cellular phenotypes. Some taccalonolides have demonstrated in vivo antitumor efficacy in drug-resistant tumor models with exquisite potency and long-lasting antitumor efficacy as a result of their irreversible target engagement. The recent identification of a site on the taccalonolide scaffold that is amenable to modification has provided evidence of the specificity of the taccalonolide-tubulin interaction. This also affords an opportunity to further optimize the targeted delivery of the taccalonolides to further improve their anticancer efficacy and potential for clinical development.

Keywords: Antitumor natural product; Microtubule stabilizer; Microtubule-targeted agent; Taccalonolide.

Figure 1.

Tacca Chantrieri

Figure 2.

The effect of taccalonolide-enriched Tacca fractions on microtubule structures in HeLa cervical cancer cells expressing GFP-tagged tubulin. The taccalonolides promote bundling of interphase microtubules (top panels) as well as the formation of multiple asters in mitotic cells that are markedly distinct from the microtubule spindle in normal mitotic cells (bottom panels).

Figure 3.

Comparison of the biochemical tubulin polymerization activities of paclitaxel and taccalonolide AJ A. Paclitaxel (10 μM) promotes the immediate polymerization of purified tubulin (20 μM) as compared to a vehicle control. B. In contrast, taccalonolide AJ (10 μM) dependent polymerization of purified tubulin (20 μM) is associated with a lag time of 8 – 10 minutes.

Figure 4.

Taccalonolide AJ binding covalently to Asp226 on β-tubulin as determined by x-ray crystallography of the T2R-TTL-taccalonolide AJ complex.

Figure 5.

Distinct mitotic spindle structures in normal cells (left), paclitaxel-treated cells (middle), and taccalonolide-treated cells (right).

Figure 6.

Fluorescein-tagged taccalonolide (green) colocalized with β-tubulin immunofluorescence (orange) in fixed HCC1937 triple-negative breast cancer cells after 24 h treatment.

Figure 7.

Flu-tacca-7 is a cell permeable fluorescent taccalonolide containing pivalate protective groups on the fluorophore that prevent fluorescence as well as target engagement prior to intracellular esterase cleavage. Upon cellular entry and pivalate deprotection to generate flu-tacca-8, the probe can directly bind tubulin and fluorescently label intracellular microtubules. The quenching provided by the pivalate groups permits live cellular imaging without the need to remove excess probe from the medium, providing a no-wash, irreversible fluorogenic labeling system for cellular microtubules.

Scheme 1.

Synthesis of taccalonolides AF (28) and AJ (29) via epoxidation of taccalonolides A (1) and B (2), respectively.

Scheme 2.

Reaction mechanism of the covalent binding of taccalonolide AJ to β-tubulin Asp226.