Agarwala Kishan Lal, Kubara Kenji, Seletsky Boris M, Sagane Koji, Littlefield Bruce A
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki, 300-2635, Japan.
Eisai Inc., 35 Cambridgepark Drive, Cambridge, MA 02140, USA.
Arch Biochem Biophys. 2025 Sep;771:110482. doi: 10.1016/j.abb.2025.110482. Epub 2025 May 29.
The clinically approved anticancer agent eribulin (Halaven®) exerts cytotoxic antimitotic effects by binding to high affinity sites on exposed plus ends of growing microtubules (MT). Despite X-ray crystallographic mapping of eribulin's binding pocket within β-tubulin's vinca domain, the biophysical basis for eribulin's MT plus end binding exclusivity remains unknown. We performed surface plasmon resonance (SPR) studies of tubulin binding to biotinylated eribulin probes to ask if eribulin discriminates between GTP and GDP forms of β-tubulin, which characterize growing MT plus ends and MT sides, respectively. Tubulin binding to biotin-eribulin proceeded via a single state binding reaction, while binding to biotin-vinblastine occurred via a two-state reaction incorporating a conformational change. Biochemical approaches confirmed tubulin conformational changes induced by vinblastine but not eribulin. SPR competition studies using free eribulin and vinblastine confirmed tubulin binding specificity to cognate biotinylated probes, showing that eribulin binding within the β-tubulin vinca domain is physically and functionally distinct from vinblastine. SPR studies using tubulin containing only GTP or GDP forms of β-tubulin showed that while biotin-eribulin has only slightly higher overall affinity for GTP-tubulin, dissociation from GTP-tubulin was ∼7-fold slower than from GDP-tubulin, establishing that eribulin discriminates between GTP and GDP forms of β-tubulin. In contrast, vinblastine fails to discriminate between these two tubulin forms, consistent with its known binding to both MT ends and sides. Overall, our results establish, for the first time, the biophysical basis for eribulin's MT plus end binding exclusivity as resulting from the ability to discriminate between GTP and GDP forms of β-tubulin.
临床批准的抗癌药物艾瑞布林（Halaven®）通过与生长中的微管（MT）暴露的正端上的高亲和力位点结合发挥细胞毒性抗有丝分裂作用。尽管通过X射线晶体学绘制了艾瑞布林在β-微管蛋白的长春花碱结构域内的结合口袋，但艾瑞布林对MT正端结合的排他性的生物物理基础仍然未知。我们进行了微管蛋白与生物素化艾瑞布林探针结合的表面等离子体共振（SPR）研究，以探究艾瑞布林是否能区分β-微管蛋白的GTP和GDP形式，这两种形式分别表征生长中的MT正端和MT侧面。微管蛋白与生物素-艾瑞布林的结合通过单态结合反应进行，而与生物素-长春花碱的结合通过包含构象变化的双态反应发生。生化方法证实长春花碱可诱导微管蛋白构象变化，而艾瑞布林则不能。使用游离艾瑞布林和长春花碱的SPR竞争研究证实了微管蛋白与同源生物素化探针的结合特异性，表明艾瑞布林在β-微管蛋白长春花碱结构域内的结合在物理和功能上与长春花碱不同。使用仅含有β-微管蛋白的GTP或GDP形式的微管蛋白进行的SPR研究表明，虽然生物素-艾瑞布林对GTP-微管蛋白的总体亲和力仅略高，但从GTP-微管蛋白上的解离比从GDP-微管蛋白上慢约7倍，这表明艾瑞布林能区分β-微管蛋白的GTP和GDP形式。相比之下，长春花碱无法区分这两种微管蛋白形式，这与其已知的与MT两端和侧面的结合情况一致。总体而言，我们的结果首次确立了艾瑞布林对MT正端结合排他性的生物物理基础，即其能够区分β-微管蛋白的GTP和GDP形式。
