Characterization of the interaction of TZT-1027, a potent antitumor agent,with tubulin

TZT-1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit Et nl., is a potent antimicrot ubule agent, We have compared the activity of TZT-1027 with that of dolasta tin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS), TZT-1027 and dolastatin 10 inhibited microtubule polym erization concentration-dependently at 1-100 mu M with IC50 values of 2.2+/ -0.6 and 2.3+/-0.7 mu M, respectively. VLB, VCR and VDS inhibited microtubu le polymerization at 1-3 mu M with IC50 values of 2.7+/-0.6, 1.6+/-0.4 and 1.6+/-0.2 mu M, respectively, but showed a slight decrease in inhibitory ef fect at concentrations of 10 mu M or more, TZT-1027 also inhibited monosodi um glutamate-induced tubulin polymerization concentration-dependently at 0. 3-10 mu M, with an IC50 of 1.2 mu M, whereas VLB was only effective at 0.3- 3 mu M, with an IC50 of 0.6 mu M, and caused so-called "aggregation" of tub ulin at 10 mu M. Scatchard analysis of the binding data for [H-3]VLB sugges ted one binding site (K-d 0.2 +/- 0.04 mu M and B-max 6.0+/-0.26 nM/mg prot ein), while that for [H-3]TZT-1027 suggested two binding sites, one of high affinity (K-d 0.2+/-0.01 mu M and B-max 1.7+/-0.012 nM/mg protein) and the other of low affinity (K-d 10.3+/-1.46 mu M and B-max 11.6+/-0.83 nM/mg pr otein), [3H]TZT-1027 was completely displaced by dolastatin 10 but only inc ompletely by VLB, [H-3]VLB was completely displaced by dolastatin 10 and TZ T-1027, Furthermore, TZT-1027 prevented [H-3]VLB from binding to tubulin in a non-competitive manner according to Lineweaver-Burk analysis. TZT-1027 c oncentration-dependently inhibited both [H-3]guanosine 5'-triphosphate (GTP ) binding to and GTP hydrolysis on tubulin, VLB inhibited the hydrolysis of GTP on tubulin concentration-dependently to a lesser extent than TZT-1027, but no inhibitory effect of VLB on [H-3]GTP binding to tubulin was evident even at 100 mu M. Thus, TZT-1027 affected the binding of VLB to tubulin, b ut its binding site was not completely identical to that of VLB, TZT-1027 h ad a potent inhibitory effect on tubulin polymerization and differed from v inca alkaloids in its mode of action against tubulin polymerization.