Chatterjee Sabarni K, Laffray Julien, Patel Pruthvi, Ravindra Rudravajhala, Qin Yong, Kuehne Martin E, Bane Susan L
Department of Chemistry, State University of New York, Binghamton, New York 13902-6016, USA.
Biochemistry. 2002 Nov 26;41(47):14010-8. doi: 10.1021/bi026182m.
Vinblastine is an antimitotic agent that has been used extensively in cancer chemotherapy. The biological effects of the drug are believed to be the result of its interaction with tubulin, the major component of cellular microtubules. Fluorescence spectroscopy is a powerful and versatile technique for studying drug-tubulin interactions, but it rarely has been applied to studies involving vinca alkaloids. We have prepared a new fluorescent derivative of vinblastine designed to retain high affinity for tubulin while possessing a fluorophore that absorbs and emits visible light. A coumarin derivative of vinblastine, 17-deacetyl-O-(3-carbonylamino-7-diethylaminocoumarin) vinblastine (F-VLB), was prepared by reaction of 17-deacetylvinblastine with 7-diethylaminocoumarin-3-carbonyl azide. F-VLB was a potent inhibitor of in vitro microtubule assembly (IC(50) = 0.5 microM). F-VLB binding to tubulin was inhibited by vinblastine. Tubulin binding induced an increase in the F-VLB emission intensity and shifted the emission maximum to higher energy (from 500 to 480 nm). The Stokes shift of tubulin-bound F-VLB was about the same as the Stokes shift of the molecule in ethanol, indicating that the tubulin-bound fluorophore is probably on the exterior of the vinblastine binding site. Unlike vinblastine, F-VLB failed to induce self-assembly of tubulin that could be detected by light scattering or electron microscopy, although some self-association could be detected by analytical ultracentrifugation. Equilibrium binding parameters were quantitatively determined by monitoring the change in fluorescence anisotropy of F-VLB upon tubulin binding. The apparent equilibrium constant for F-VLB binding to tubulin [K(a)(app) = (7.7 +/- 0.5) x 10(4) M(-1) at 25 degrees C] was identical to the equilibrium constant for vinblastine binding to 2 microM tubulin (K(1)) measured under similar buffer and temperature conditions using ultracentrifugation [Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828-12835]. Binding allocolchicine to tubulin did not significantly affect F-VLB's affinity for the protein [K(a)(app) = (9.1 +/- 0.4) x 10(4) M(-1) at 25 degrees C]. Analysis of the steady-state emission spectra yielded a distance between the colchicine and vinca binding sites on tubulin of approximately 40 A. F-VLB bound to paclitaxel- and glutaraldehyde-stabilized microtubules, with approximately equal affinity. We conclude that F-VLB can be used to obtain information about the vinblastine binding site on tubulin under equilibrium conditions.
长春碱是一种抗有丝分裂剂，已广泛应用于癌症化疗。该药物的生物学效应被认为是其与微管蛋白相互作用的结果，微管蛋白是细胞微管的主要成分。荧光光谱法是研究药物与微管蛋白相互作用的一种强大且通用的技术，但很少应用于涉及长春花生物碱的研究。我们制备了一种新的长春碱荧光衍生物，旨在对微管蛋白保持高亲和力，同时拥有一个能吸收和发射可见光的荧光团。通过17 - 去乙酰长春碱与7 - 二乙氨基香豆素 - 3 - 羰基叠氮化物反应制备了长春碱的香豆素衍生物，17 - 去乙酰 - O -（3 - 羰基氨基 - 7 - 二乙氨基香豆素）长春碱（F - VLB）。F - VLB是体外微管组装的有效抑制剂（IC(50) = 0.5 microM）。长春碱可抑制F - VLB与微管蛋白的结合。微管蛋白结合导致F - VLB发射强度增加，并使发射最大值向更高能量移动（从500 nm移至480 nm）。与微管蛋白结合的F - VLB的斯托克斯位移与该分子在乙醇中的斯托克斯位移大致相同，表明与微管蛋白结合的荧光团可能位于长春碱结合位点的外部。与长春碱不同，F - VLB未能诱导可通过光散射或电子显微镜检测到的微管蛋白自组装，尽管通过分析超速离心可检测到一些自缔合。通过监测F - VLB与微管蛋白结合时荧光各向异性的变化来定量测定平衡结合参数。F - VLB与微管蛋白结合的表观平衡常数[在25℃时K(a)(app) = (7.7 ± 0.5) x 10(4) M(-1)]与在类似缓冲液和温度条件下使用超速离心法测量的长春碱与2 microM微管蛋白结合的平衡常数（K(1)）相同[Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828 - 12835]。将别秋水仙碱与微管蛋白结合不会显著影响F - VLB对该蛋白 的亲和力[在25℃时K(a)(app) = (9.1 ± 0.4) x 10(4) M(-1)]。对稳态发射光谱的分析得出秋水仙碱和长春花生物碱在微管蛋白上的结合位点之间的距离约为40 Å。F - VLB与紫杉醇和戊二醛稳定的微管结合，亲和力大致相等。我们得出结论，F - VLB可用于在平衡条件下获取有关长春碱在微管蛋白上结合位点的信息。
