Eribulin Binds at Microtubule Ends to a Single Site on Tubulin To Suppress Dynamic Instability

Abstract

Eribulin mesylate (E7389), a synthetic analog of the marine natural product halichondrin B, is in Phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening. Using [ 3 H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K d of 46 μM, but also showing a real or apparent very high affinity (K d , 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (K d , 3.5 μM), strongly suggesting the presence of a relatively high affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per 2 microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Pre-incubation of microtubules with 2 or 4 μM vinblastine induced additional lower affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability. Keywords eribulin; halichondrin; tubulin; microtubule; binding Eribulin mesylate is a tubulin/microtubule-targeting chemotherapeutic drug that inhibits the proliferation of multiple cancer cell types (1,2). It is a synthetic analog of the natural compound, halichondrin B (Fig. 1A), initially isolated from the sea sponge Halichondria okadai (3). Eribulin is currently in Phase III clinical trials for the treatment of metastatic breast cancer. Phase I and Phase II clinical trials have demonstrated that eribulin is active in heavily pretreated individuals while maintaining a tolerable therapeutic index, with the most frequent adverse effects being neutropenia and fatigue (4-6). Neuropathy, a common dose-limiting toxicity of other microtubule-targeting drugs like paclitaxel and some vinca alkaloids (7-9), has a low incidence in eribulin-treated patients, and no grade 4 neuropathy occurred (4)(5)(6) Eribulin exerts its anticancer properties through a novel action on tubulin and microtubules (1,(10)(11)(12). In MCF7 cells, eribulin inhibited microtubule dynamic instability at low concentrations and induced depolymerization of the microtubule network at high concentrations (10 × IC 50 for inhibition of cell proliferation) (11). At significantly lower eribulin concentrations, eribulin potently inhibited microtubule dynamics, resulting in prolonged mitotic arrest and subsequent apoptosis [for a review of microtubule structure and dynamic instability, see (13,14)]. Eribulin binds at or near the vinca domain, a region that is located at the interface of two tubulin heterodimers when arranged end-to-end and overlaps the ex...

Examination of the clinical efficacy of eribulin and trastuzumab in HER2-positive recurrent breast cancer

“…The sites of recurrence were the lung in 3 patients, liver in 2, bone in 1, brain in 1, supraclavicular lymph nodes in 1, infraclavicular lymph nodes in 1 and mediastinal lymph nodes in 1. The median number of prior treatment regimens was 5 (range, [5][6][7][8][9][10][11].…”

“…The drug induces an irreversible mitotic block, which leads to cell cycle arrest in the G 2 /M phase and apoptosis (8). It is distinguished mechanistically from other antimicrotubule agents, such as paclitaxel, ixabepilone and vinblastine (9).…”

Comparison of Neuropathy-Inducing Effects of Eribulin Mesylate, Paclitaxel, and Ixabepilone in Mice

“…Eribulin mesylate is a structurally simplified, macrocyclic, ketone analogue of halichondrin B, and it inhibits microtubule dynamics through a novel mechanism relative to other tubulin-targeting agents, including the taxanes, Vinca alkaloids, and epothilones. It binds with high affinity and specificity to positively charged ends of microtubules, thereby suppressing microtubule dynamics (16). Preclinically, eribulin mesylate has shown potent anticancer activity in vitro and in vivo (17).…”

Eribulin monotherapy in a patient with heavily pretreated metastatic breast cancer: Case study and review of the literature

“…Eribulin is a non-taxane microtubule dynamics inhibitor with a mechanism of action distinct from that of other tubulin-targeting agents. Eribulin binds exclusively to the growing plus ends of the microtubule, thereby suppressing dynamic instability [2] through an inhibition of microtubule growth (with little to no effect on shortening), and causing sequestration of tubulin into nonproductive aggregates [3] .…”