Interaction of tubulin with single ring analogues of colchicine.

Simple analogues of the tropolone and trimethoxyphenyl moieties of colchicine have been used as probes for the colchicine binding site of purified calf brain tubulin. [3H]Tropolone methyl ether was found to bind to one site per tubulin molecule with equilibrium constant of (2.2 +/- 0.2) x 10(3) M-1 at 0 degree C, with the interaction having delta H0app = -8.3 +/- 1.0 kcal mol-1 and delta S0app = -15.2 +/- 3.6 eu. The binding of tropolone methyl ether and colchicine was inhibited by each other. Both tropolone and its methyl ether inhibited tubulin polymerization into microtubules in vitro. N-[3H]Acetylmescaline bound to tubulin with a K congruent to 4 x 10(2) M-1 at 37 degrees C. This interaction was inhibited by colchicine and at lower temperatures was below the sensitivity of the measuring method employed. [14C]Mescaline interacted with higher affinity site(s) not related to the colchicine site. Both mescaline and N-acetylmescaline inhibited partially the microtubule assembly at 10(-3) M concentrations. No linkage was observed between the binding of tropolone methyl ether and N-acetylmescaline. The relatively weak interactions of both the two separate parts of colchicine can account quantitatively for the much tighter binding of the complete drug to tubulin within a proposed model which takes into account the entropic advantage of colchicine as a bifunctional ligand.