Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alpha beta(II), alpha beta(III) and alpha beta(IV)

Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties. and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alpha beta(II) and alpha beta(IV) isoforms than to the alpha beta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alpha beta(II), alpha beta(III) and alpha beta(IV) were 13358, 4558 and 10828 M-1 s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33X10(5), 1.56X10(5) and 5.34X10(5) M-1, respectively. The differences in kinetic parameters among different P-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.