Characterization of the Interaction of TZT‐1027, a Potent Antitumor Agent, with Tubulin

Abstract

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 μ M with IC 50 values of 2.2±0.6 and 2.3±0.7 μ M , respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μ M with IC 50 values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μ M , respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μ M or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μ M , with an IC 50 of 1.2 μ M , whereas VLB was only effective at 0.3–3 μ M , with an IC 50 of 0.6 μ M , and caused so‐called “aggregation” of tubulin at 10 μ M. Scatchard analysis of the binding data for [ 3 H]VLB suggested one binding site (K d 0.2±0.04 μ M and B max 6.0±0.26 n M /mg protein), while that for [ 3 H]TZT‐1027 suggested two binding sites, one of high affinity (K d 0.2±0.01 μ M and B max 1.7±0.012 n M /mg protein) and the other of low affinity (K d 10.3±1.46 μ M , and B max 11.6±0.83 n M /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 μ M . 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 mo...

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

“…One of the more interesting, which we also prepared in a radiolabeled form, is auristatin-PE (24) (Fig. 1), also prepared independently as TZT-1027 (25). We began to study the potential of direct photoaffinity labeling with the [ 3 H]dolastatin 10 1 and the [ 3 H]auristatin-PE to provide information about the binding site for these peptides on tubulin.…”

The novel microtubule-interfering agent TZT-1027 enhances the anticancer effect of radiation in vitro and in vivo

“…TZT-1027 is a novel antitumour agent that inhibits microtubule polymerisation and exhibits potent antitumour activity in preclinical models (Miyazaki et al, 1995;Kobayashi et al, 1997;Natsume et al, 2000Natsume et al, , 2003Natsume et al, , 2006Otani et al, 2000;Watanabe et al, 2000Watanabe et al, , 2006a. We investigated the effect of TZT-1027 on cell cycle progression with the use of tsFT210 cells, which can be synchronised in G 2 phase by incubation at 39.41C and consequent inactivation of Cdc2 (Osada et al, 1997;Tamura et al, 1999).…”

“…TZT-1027 inhibits microtubule assembly by binding to tubulin (Kobayashi et al, 1997;Natsume et al, 2000). In vitro, it inhibits the growth of various human cancer cells at low concentrations .…”

Reference profiling of the genomic response induced by an antimicrotubule agent, TZT-1027 (Soblidotin), in vitro

“…25 They share the same binding site and have similar affinities, 14,28 whereas additional binding sites have either high affinities (Kd: 1-2 mmol) or low affinities (Kd: 0.25-3 mmol). 24 Previous studies have also reported that dolastatins can also bind at different sites from those used by Vinca alkaloids 14,29 These additional binding sites might be responsible for the differences in genomic response induced by the dolastatins and Vinca alkaloids.…”

“…TZT-1027 is a mitotic spindle poison that interacts with tubulin at the Vinca alkaloids binding site, 14 but the spectra of antitumor activity of TZT-1027 and the Vinca alkaloids are different in vivo. 7,10,15 TZT-1027 also showed potent antitumor activity against a vincristine-resistant cell line.…”