Abstract 4493: Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors.
Authors: Agarwala Kishan Lal , Kubara Kenji , Sagane Koji , Seletsky Boris M. , Littlefield Bruce A. 
Published: August 2014

Eribulin (E7389) is an antimitotic drug that inhibits microtubule assembly in vitro and in vivo by binding to tubulin. Several studies have been performed on eribulin mechanism of action; however, the mode of interaction with soluble tubulin (αβ-heterodimer) and binding site differences with vinca alkaloids (vinblastine and vincristine) and paclitaxel have not been well characterized. To understand binding differences, we performed an in vitro conformational change assays and established a surface plasmon resonance (SPR)-based direct binding assay using chemical probes and soluble tubulin. Unlike vinca alkaloids and paclitaxel, binding of eribulin with tubulin has little or no effect on conformational change of tubulin which is consistent with previous observations in the following three independent assays: 1) limited proteolysis pattern, 2) intrinsic tryptophan fluorescence and 3) exposure of hydrophobic area on the tubulin molecule. We therefore examined whether eribulin binds to the vinca or paclitaxel site of tubulin in competitive inhibition assay on SPR. Vinca alkaloids and paclitaxel failed to inhibit the binding of tubulin to immobilized eribulin chemical probe. Similarly, eribulin and paclitaxel did not inhibit the binding of tubulin to immobilized vinblastine chemical probe. Thus, the eribulin binding site on tubulin is different from that of vinca alkaloids and paclitaxel. In addition, we further investigated the binding affinities and characteristics of interaction of eribulin with GTP- or GDP-forms of tubulin, respectively. Eribulin chemical probe binds both forms of tubulin specifically with significantly higher affinity (∼40 nM) as compared with vinblastine chemical probe (∼1 μM). Interestingly, we found that eribulin chemical probe preferentially recognizes GTP- over GDP-tubulin and dissociation of GTP-form from eribulin chemical probe was slower than that of GDP-form of tubulin. In contrast, vinblastine chemical probe did not show significant difference in binding to the two nucleotide states of tubulin. This indicates that eribulin distinguishes different nucleotide state of tubulin and binds to GTP-tubulin more tightly. These characteristic features of eribulin differ from those of vinca alkaloids and paclitaxel. Overall, our results suggest that eribulin exerts its unique antimitotic activity by inhibiting microtubule assembly by binding with high affinity at a novel tubulin site at the plus-end of the growing microtubule. This may potentially relate to the mechanism of eribulin clinical efficacy and diminished neuronal toxicity.

Citation Format: Kishan Lal Agarwala, Kenji Kubara, Koji Sagane, Boris M. Seletsky, Bruce A. Littlefield. Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2013-4493