diazonamide-a and dolastatin-10

The marine ascidian Diazona angulata was the source organism for the complex cytotoxic peptide diazonamide A. The molecular structure of this peptide was recently revised after synthesis of a biologically active analog of diazonamide A in which a single nitrogen atom was replaced by an oxygen atom. Diazonamide A causes cells to arrest in mitosis, and, after exposure to the drug, treated cells lose both interphase and spindle microtubules. Both diazonamide A and the oxygen analog are potent inhibitors of microtubule assembly, equivalent in activity to dolastatin 10 and therefore far more potent than dolastatin 15. This inhibition of microtubule assembly is accompanied by potent inhibition of tubulin-dependent GTP hydrolysis, also comparable with the effects observed with dolastatin 10. However, the remaining biochemical properties of diazonamide A and its analog differ markedly from those of dolastatin 10 and closely resemble the properties of dolastatin 15. Neither diazonamide A nor the analog inhibited the binding of [3H]vinblastine, [3H]dolastatin 10, or [8-14C]GTP to tubulin. Nor were they able to stabilize the colchicine binding activity of tubulin. These observations indicate either that diazonamide A and the analog have a unique binding site on tubulin differing from the vinca alkaloid and dolastatin 10 binding sites, or that diazonamide A and the analog bind weakly to unpolymerized tubulin but strongly to microtubule ends. If the latter is correct, diazonamide A and its oxygen analog should have uniquely potent inhibitory effects on the dynamic properties of microtubules.

Topics: Animals; Antineoplastic Agents; Cell Division; Depsipeptides; Drug Screening Assays, Antitumor; Guanosine Triphosphate; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrolysis; Microtubules; Mitosis; Oligopeptides; Oxazoles; Tubulin; Tumor Cells, Cultured

The antimitotic depsipeptide dolastatin 15 was radiolabeled with tritium in its amino-terminal dolavaline residue. Dolastatin 15, although potently cytotoxic, is a relatively weak inhibitor of tubulin assembly and does not inhibit the binding of any other ligand to tubulin. The only methodology found to demonstrate an interaction between the depsipeptide and tubulin was Hummel-Dreyer equilibrium chromatography on Sephadex G-50 superfine. The average apparent Kd value obtained in these studies was about 30 microM, with no difference observed when column size or tubulin concentration was varied. This relatively high dissociation constant is consistent with the apparent weak interaction of dolastatin 15 with tubulin demonstrated indirectly in the assembly assay. We attempted to gain insight into the binding site for dolastatin 15 on tubulin by studying inhibitory effects of other drugs when the gel filtration column was equilibrated with both [3H]dolastatin 15 and a second, nonradiolabeled drug. No inhibition was detected with either the colchicine site agent combretastatin A-4 or with an analog of the antimitotic marine peptide diazonamide A (both the analog and diazonamide A are potent inhibitors of tubulin assembly). Weak inhibition was observed with cemadotin, a structural analog of dolastatin 15, and with the depsipeptide cryptophycin 1. Moderate inhibition occurred with vinblastine and vincristine, and strong inhibition with maytansine, halichondrin B, and the peptides dolastatin 10 and phomopsin A. These observations suggest that the binding site(s) for peptide and depsipeptide antimitotic drugs may consist of a series of overlapping domains rather than a well-defined locus on the surface of beta-tubulin.

Topics: Animals; Binding Sites; Cattle; Chromatography, Gel; Colchicine; Depsipeptides; Heterocyclic Compounds, 4 or More Rings; Kinetics; Maytansine; Oligopeptides; Oxazoles; Protein Binding; Protein Structure, Tertiary; Tritium; Tubulin; Vinblastine; Vincristine