The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the BIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin-and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the BIII isotype of tubulin was generated to evaluate the effect of BIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the BIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the BIII-tubulinoverexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms.
Drugs that affect microtubule dynamics, including the taxanes and vinca alkaloids, have been a mainstay in the treatment of leukemias and solid tumors for decades. New, more effective microtubule-targeting agents continue to enter into clinical trials and some, including the epothilone ixapebilone, have been approved for use. In contrast, several other drugs of this class with promising preclinical data were later shown to be ineffective or intolerable in animal models or clinical trials. In this review we discuss the molecular mechanisms as well as preclinical and clinical results for a variety of microtubule-targeting agents in various stages of development. We also offer a frank discussion of which microtubule-targeting agents are amenable to further development based on their availability, efficacy and toxic profile.
The taccalonolides are highly acetylated steroids that stabilize cellular microtubules and overcome multiple mechanisms of taxane resistance. Recently, two potent taccalonolides, AF and AJ, were identified that bind tubulin directly and enhance microtubule polymerization. Extensive studies were conducted to characterize these new taccalonolides. AF and AJ caused aberrant mitotic spindles and bundling of interphase microtubules that differed from the effects of either paclitaxel or laulimalide. AJ also distinctly affected microtubule polymerization in that it enhanced the rate and extent of polymerization in the absence of any noticeable effect on microtubule nucleation. Additionally, the resulting microtubules were found to be profoundly cold stable. These data, along with studies showing synergistic antiproliferative effects between AJ and either paclitaxel or laulimalide, suggest a distinct binding site. Direct binding studies demonstrated that AJ could not be displaced from microtubules by paclitaxel, laulimalide or denaturing conditions, suggesting irreversible binding of AJ to microtubules. Mass spectrometry confirmed a covalent interaction of AJ with a peptide of β-tubulin containing the cyclostreptin binding sites. Importantly, AJ imparts strong inter-protofilament stability in a manner different from other microtubule stabilizers that covalently bind tubulin, consistent with the distinct effects of the taccalonolides as compared to other stabilizers. AF was found to be a potent and effective antitumor agent that caused tumor regression in the MDA-MB-231 breast cancer xenograft model. The antitumor efficacy of some taccalonolides, which stabilize microtubules in a manner different from other microtubule stabilizers, provides the impetus to explore the therapeutic potential of this site.
Eribulin mesylate (eribulin), an analog of the marine natural product halichondrin B, is a microtubule-depolymerizing drug that has utility in the treatment of patients with breast cancer. Clinical trial results have demonstrated that eribulin treatment provides a survival advantage to patients with metastatic or locally advanced breast cancer previously treated with an anthracycline and a taxane. Furthermore, a pooled analysis of two pivotal phase III trials has demonstrated that eribulin also improves overall survival in several patient subgroups, including in women with human epidermal growth factor receptor 2 (HER2)-negative disease and triple-negative breast cancer. This review covers the preclinical research that led to the clinical testing and approval of eribulin, as well as subsequent research that was prompted by distinct and unexpected effects of eribulin in the clinic. Initial studies with halichondrin B, and then eribulin, demonstrated unique effects on tubulin binding that resulted in distinct microtubule-dependent events and antitumor actions. Consistent with the actions of the natural product, eribulin has potent microtubule-depolymerizing activities and properties that distinguish it from other microtubule targeting agents. Here, we review new results that further differentiate the effects of eribulin from other agents on peripheral nerves, angiogenesis, vascular remodeling and epithelial-to-mesenchymal transition. Together, these data highlight the distinct properties of eribulin and begin to delineate the mechanisms behind the increased survival benefit provided by eribulin for patients.
Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin−GB1 complex.
The taccalonolides are a class of microtubule stabilizing agents isolated from plants of the genus Tacca. In efforts to define their structure activity relationships, we isolated 5 new taccalonolides, AC-AF, and H2, from one fraction of an ethanol extract of Tacca plantaginea. The structures were elucidated using a combination of spectroscopic methods, including 1D and 2D NMR and HRESIMS. Taccalonolide AJ, an epoxidation product of taccalonolide B, was generated by semi-synthesis. Five of these taccalonolides demonstrated cellular microtubule stabilizing activities and antiproliferative actions against cancer cells, with taccalonolide AJ exhibiting the highest potency with an IC50 value of 4.2 nM. The range of potencies of these compounds, from 4.2 nM to greater than 50 µM, for the first time provides the opportunity to identify specific structural moieties crucial for potent biological activities as well as those that impede optimal cellular effects. In mechanistic assays taccalonolide AF and AJ stimulated the polymerization of purified tubulin, an activity that had not previously been observed for the taccalonolides A and B, providing the first evidence that this class of microtubule stabilizers can interact directly with tubulin/microtubules. Taccalonolides AF and AJ were able to enhance tubulin polymerization to the same extent as paclitaxel, but with a distinct kinetic profile, suggesting a distinct binding mode or the possibility of a new binding site. The potencies of taccalonolides AF and AJ, their direct interaction with tubulin, together with the previous excellent in vivo antitumor activity of this class reveal the potential of the taccalonolides as new anticancer agents.
The taccalonolides are a unique class of microtubule stabilizers that do not bind directly to tubulin. Three new taccalonolides, Z, AA and AB, along with two known compounds, taccalonolides R and T, were isolated from Tacca chantrieri and Tacca integrifolia. Taccalonolide structures were determined by 1D and 2D NMR methods. The biological activities of the new taccalonolides, as well as taccalonolides A, B, E, N, R and T, were evaluated. All nine taccalonolides display microtubule stabilizing activity, but profound differences in antiproliferative potencies were noted, with IC50 values ranging from the low nanomolar range for taccalonolide AA (32 nM) to the low micromolar range for taccalonolide R (13 µM). These studies demonstrate that diverse taccalonolides possess microtubule stabilizing properties and that significant structure-activity relationships exist. In vivo antitumor evaluations of taccalonolides A, E and N show that each of these molecules has in vivo antitumor activity.
The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p K9R,K16R , is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p K9R,K16R can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p K9R,K16R -expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.
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