dolastatin-10 and phomopsin

This paper summarizes published data on the interactions of tubulin with antimitotic compounds that inhibit the binding of vinca alkaloids to the protein. These are all relatively complex natural products isolated from higher plants, fungi and marine invertebrate animals. These agents are maytansine, rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B and their congeners. Effects on tubulin polymerization, ligand binding interactions and structure-activity relationships are emphasized.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Depsipeptides; Ethers, Cyclic; Lactones; Macrolides; Maytansine; Mycotoxins; Oligopeptides; Tubulin; Vinca Alkaloids

Synthetic investigations of ustiloxin natural products are described. The first total synthesis of ustiloxin F was completed in 15 steps via ethynyl aziridine ring-opening by a phenol derivative. The results of biological tests of synthetic ustiloxins D and F, and two analogs, O-Me-ustiloxin D and 6-Ile-ustiloxin, demonstrated that the free hydroxyl group ortho to the ether linkage is critical for activity and variations at the Val/Ala site produce changes in the biological activity suggesting the need for further perturbations at this site to more extensively study the tubulin binding.

Topics: Biological Products; Cell Line, Tumor; Humans; Inhibitory Concentration 50; Molecular Structure; Mycotoxins; Peptides, Cyclic; Tubulin

Several naturally occurring peptides and depsipeptides which include the cryptophycins, dolastatin 10, hemiasterlin, and phomopsin A have been found to be potent antimitotic agents, causing cell death at picomolar or low nanomolar concentrations. These compounds inhibit microtubule growth, modulate the dynamics of microtubules, and induce the self-association of tubulin dimers into single-walled rings and spirals. These peptides exhibit mutual competitive inhibition in binding to beta-tubulin, while noncompetitively inhibiting the binding of vinblastine and vincristine to beta-tubulin. Despite the abundance of biochemical information, the details of their molecular interactions with tubulin are not known. In this study, using a combination of molecular dynamics simulations and molecular docking studies, a common binding site for cryptophycin 1, cryptophycin 52, dolastatin 10, hemiasterlin, and phomopsin A on beta-tubulin has been identified. Application of these same methods to alpha-tubulin indicated no interaction between alpha-tubulin and any of the peptides. On the basis of the docking results, a model for the mechanism of microtubule disruption and formation of aberrant nonmicrotubule structures is proposed. Both the active site and mechanism of microtubule depolymerization predictions are in good agreement with experimental findings.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Cattle; Computational Biology; Computer Simulation; Depsipeptides; Lactams; Lactones; Ligands; Models, Molecular; Molecular Sequence Data; Mycotoxins; Oligopeptides; Peptides; Peptides, Cyclic; Point Mutation; Protein Binding; Protein Conformation; Protein Isoforms; Saccharomyces cerevisiae Proteins; Tubulin

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia. It inhibits microtubule assembly at concentrations below 5.0 microM (IC50, 3.0 microM) and causes formation of tubulin aggregates at higher (> 10 microM) concentrations in a somewhat different manner from that caused by vinblastine. Electron microscopical analysis showed irregular aggregates of microtubule proteins in the presence of 10 microM dolastatin 10. Dolastatin 10 inhibited the binding of both radiolabeled rhizoxin and phomopsin A to tubulin with inhibition constants (Ki) of 7 x 10(-8) M and 1 x 10(-7) M, respectively. The results suggest that at least one of the binding sites of dolastatin 10 on tubulin is the rhizoxin binding site.

Topics: Animals; Antineoplastic Agents; Binding Sites; Binding, Competitive; Brain; Depsipeptides; Lactones; Macrolides; Microscopy, Electron; Microtubules; Mollusca; Mycotoxins; Oligopeptides; Polymers; Swine; Tubulin

Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent as inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [3H]colchicine to tubulin. The combination of ustiloxin A with cholchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.

Topics: Amino Acid Sequence; Animals; Brain Chemistry; Cattle; Depsipeptides; Isoleucine; Molecular Sequence Data; Mycotoxins; Oligopeptides; Peptides, Cyclic; Structure-Activity Relationship; Tubulin; Valine; Vinblastine

Dolastatin 10 is an unusual peptide of marine origin which binds to tubulin in the vinblastine/maytansine/phomopsin-binding region and potently inhibits mitosis. Using N,N'-ethylenebis(iodoacetamide) (EBI) and iodo[14C]acetamide as probes for the effects of ligands on the thiol groups of tubulin, we found that dolastatin 10 has effects on the sulfhydryls indistinguishable from those of phomopsin A but quite different from those of vinblastine and maytansine. Using the binding of bis-5,5'-[8-(N-phenyl)aminonaphthalene-1-sulfonic acid] (BisANS) as a measure of tubulin decay, we found that dolastatin 10 resembled phomopsin A in that decay was not detectable by this assay in its presence. Interestingly, both otherwise very different peptides are among the most effective inhibitors of tubulin decay yet discovered.

Topics: Alkylation; Anilino Naphthalenesulfonates; Animals; Antineoplastic Agents; Brain Chemistry; Cattle; Cross-Linking Reagents; Cystine; Depsipeptides; Ethylenediamines; Microtubules; Mycotoxins; Oligopeptides; Protein Conformation; Sulfhydryl Compounds; Tubulin; Vinblastine