INTERACTION OF TUBULIN WITH BIFUNCTIONAL COLCHICINE ANALOGS - AN EQUILIBRIUM STUDY

The interaction of tubulin with simple analogs of colchicine that contain both its tropolone and trimethoxyphenyl rings was characterized, and the results were analyzed in terms of the simple bifunctional ligand model developed for the binding of colchicine on the basis of interactions of tubulin with single-ring analogs. The compound 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one binds reversibly to 0.86 .+-. 0.06 site of purified calf brain tubulin with an equilibrium constant of (4.9 .+-. 0.3) .times. 105 M-1 (25.degree. C), .DELTA.H.degree.app = -1.6 .+-. 0.7 kcal/mol, and .DELTA.S.degree.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-trimethyoxyphenyl)-propionyl]amino]-2,4,6-cycloneheptatrien-1-one interacts weakly with tubulin. Binding of the 1st analog is accompanied by ligand fluorescence appearance, quenching of protein fluorescence, perturbaton of the far-UV circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Substoichiometric concentrations of the analog inhibit microtubule assembly in vitro. Excess analog concentration under microtubule-promoting conditions induces and abnormal cooperative polymerization of tubulin, similar to that of the tubulin-colchicine complex.