Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability

Abstract

Eribulin mesylate (E7389), a synthetic analogue 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 [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. 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 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) 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 microM), 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 two 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. Preincubation of microtubules with 2 or 4 microM 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.

Figure 1

(A) Eribulin (E7389) and its parent compound halichondrin B. The location of tritiation in [³H]eribulin is indicated by an asterisk (*). (B) Binding of eribulin to soluble tubulin. (C) Enlargement of (B) showing the binding of eribulin to soluble tubulin with the curve determined by non-linear regression. [³H]Eribulin was incubated with soluble MAP-depleted bovine brain tubulin (2 μM) for 20 min at 30 °C, followed by centrifugation through a microspin gel filtration column. Data are means ± SEM of three to six experiments.

Figure 2

Eribulin-induced depolymerization of MAP-rich microtubules. (A) Control (solid circle), 1 μM (open triangle), 5 μM (solid square), 10 μM (solid triangle), and 20 μM eribulin (open square). (B) Extent of final depolymerization at 30 min after eribulin addition. MAP-rich tubulin (3 mg/ml) was assembled to steady state, eribulin was added at time zero and depolymerization was followed by turbidity (A350 nm). Data are means ± SEM of four to five experiments.

Figure 3

Binding of eribulin to microtubules. (A) Eribulin binds microtubules at a maximum of ~15 molecules/microtubule. (B) Double-reciprocal plot of eribulin binding to microtubules of a representative experiment (R² = 0.99). MAP-rich tubulin (3 mg/ml) was assembled to steady state, then incubated with [³H]eribulin for 1-2 min, followed by centrifugation through a stabilizing cushion. Data are means ± SEM of four experiments.

Figure 4

Effects of vinblastine on eribulin binding to microtubules. (A) Vinblastine inhibited eribulin binding at low eribulin concentrations, but increased binding at high eribulin concentrations. (B) Enlargement of (A) showing effects of vinblastine at low eribulin concentrations. Vinblastine was added to microtubules and incubated for 15 min before [³H]eribulin was added. Eribulin binding in the absence of vinblastine (red circles), and in the presence of 2 μM vinblastine (green squares) and 4 μM vinblastine (blue triangles). Non-linear regression (Materials and Methods) was used to generate the curves for control and 2 μM vinblastine; a simple linear equation was used for 4 μM vinblastine.

Figure 5

Electron micrographs of microtubules after incubation with (A) no drug, (B) 50 μM eribulin or (C) 50 μM vinblastine. Ends in controls or eribulin-treated microtubules were either blunt (black triangles) or splayed (white arrows). After incubation with vinblastine, all ends were extensively splayed and many had protruding spiraled protofilaments (black arrows). Magnification 100,000 ×, scale bar 100 nm.