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 al., is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μM with IC(50) values of 2.2±0.6 and 2.3±0.7 μM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μM with IC(50) values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μM or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μM, with an IC(50) of 1.2 μM, whereas VLB was only effective at 0.3–3 μM, with an IC(50) of 0.6 μM, and caused so‐called “aggregation” of tubulin at 10 μM. Scatchard analysis of the binding data for [(3)H]VLB suggested one binding site (K(d) 0.2±0.04 μM and B(max) 6.0±0.26 nM/mg protein), while that for [(3)H]TZT‐1027 suggested two binding sites, one of high affinity (K(d) 0.2±0.01 μM and B(max) 1.7±0.012 nM/mg protein) and the other of low affinity (K(d) 10.3±1.46 μM, and B(max) 11.6±0.83 nM/mg protein). [(3)H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [(3)H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [(3)H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [(3)H]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 [(3)H]GTP binding to tubulin was evident even at 100 μM. Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.