NBD-isocolcemid-tubulin interaction: A novel one-step reaction involving no conformational adjustment of reactants

Isocolcemid, a colcemid analogue in which the positions of the C-ring metho xy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that th e substitution of a NBD group in the side chain of the B-ring of isocolcemi d can reverse the effect of these structural alterations (at the C-ring) an d the newly synthesized NBD-isocolcemid restores the lost biological activi ty. It inhibits microtubule assembly with an IC50 of 12 mu M and competes e fficiently,with [H-3]colchicine, for banding to tubulin. NBD-isocolcemid ha s two binding sites on tubulin; one is characterized by fast binding, where as the binding to the other site is slow. These two sites are independent a nd unrelated to each other. Colchicine and its analogues compete with NBD-i socolcemid for the slow site. Association and dissociation rate constants f or the fast site, obtained from the stopped-flow measurements, are (7.37 +/ - 0.70) x 10(5) M-1 s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps , NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the obse rved rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentra tions. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubul in complex suggest an unaltered conformation of tubulin upon NBD-isocolcemi d binding to it as well. Implications of this on the mechanism of polymeriz ation inhibition have been discussed.