The interaction of tubulin with simple analogues of colchicine that contain both its tropolone and trimethoxyphenyl rings has been characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine [Andreu, J. M., & Timasheff, S. N. (1982) Biochemistry 21, 534-543] on the basis of interactions of tubulin with single-ring analogues. The compound 2-methoxy-5-(2,3,4-tnmethoxyphenyl)-2,4,6-cycloheptatrien-l-one has been found to bind reversibly to 0.86 ± 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 ± 0.3) × 105 M-1 (25 °C), ΔH°app = -1.6 ± 0.7 kcal mol-1, and ΔS°app = 20.5 ± 2.5 eu. The binding appears specific for the colchicine site. The closely related compound 2-methoxy-5-[[3-(3,4,5-trimethoxyphenyl)-propionyl]amino]-2,4,6-cycloheptatrien-1-one interacts weakly with tubulin. Binding of the first analogue is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analogue inhibit microtubule assembly in vitro. Excess analogue concentration under microtubule-promoting conditions induces an abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.
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