Interaction of Vinca Alkaloids with Tubulin: A Comparison of Vinblastine, Vincristine, and Vinorelbine

Abstract

Vinca alkaloids are antimitotic drugs that inhibit microtubule assembly and induce tubulin self-association into coiled spiral aggregates. Previous sedimentation velocity results with vinblastine have been interpreted by a mechanism involving isodesmic ligand-mediated or ligand-mediated plus ligand-facilitated self-association [Lobert et al. (1995) Biochemistry 34, 8050−8060]. In this study, we compare the vincristine- or vinorelbine-induced self-association of porcine brain tubulin with our prior vinblastine studies in the presence of 50 μM GDP or 50 μM GTP. Vincristine demonstrates the highest overall affinity for tubulin, K₁K₂, and vinorelbine the lowest (vincristine > vinblastine > vinorelbine). These are the first quantitative studies comparing the interaction of a new vinca alkaloid derivative, vinorelbine (Navelbine), with other vinca alkaloids. The relative binding affinities reported here correlate with the weekly drug doses used clinically in cancer chemotherapy, where vincristine is used at the lowest dosages and vinorelbine at the highest. Surprisingly, K₁, the affinity of drug for tubulin heterodimers, is identical for all three drugs. When data are fit with the ligand-mediated model, the differences in overall affinity are due to effects on K₂, the affinity of liganded heterodimers for spiral polymers. When data are fit with the ligand-mediated plus -facilitated model, affinity differences are also reflected in K₃, the binding of the drug to unliganded polymers. We find that GDP enhances self-association in the presence of all three drugs 3−5-fold over GTP. The enhancement is manifested in K₂ and K₃ and amounts to an average of 0.90 ± 0.17 kcal/mol. Thus, nucleotide enhancement is linked to the self-association step. Data collected at 5, 25, and 36 °C for all three drugs show increased maximum s̄_20,w values with increasing temperature and are consistent with an entropically driven reaction for the overall process. To investigate these results further, stopped-flow light scattering experiments have been conducted. Relaxation times are longest for the largest vincristine polymers and shortest for the smallest vinorelbine polymers, consistent with a cascade of events corresponding to successive dissociation events from spiral polymers, the larger the polymers the longer the relaxation time. Relaxation times for any single drug decrease with increasing tubulin concentration, consistent with the occurrence of oligomer annealing in addition to the association of liganded heterodimers to the ends of the growing spirals. Relaxation times were used to estimate on and off rates for liganded heterodimer association with spirals, and their ratio gives affinity constants (K_app) that are independently consistent with K₂ estimates from sedimentation velocity results for vinblastine and vinorelbine. For vincristine-induced tubulin polymers, a two-step process is observed with a second relaxation time more than 20-fold longer than times observed for vinblastine or vinorelbine. Sedimentation velocity experiments at low speeds and electron microscopy are consistent with the presence of a small amount of larger polymers (≥40S) in the vincristine samples, possibly involving alignment of spirals. Under our experimental conditions, these larger polymers appear to have a minimal effect on the estimated energetics of the vincristine-induced self-association of tubulin.