hemiasterlin and dolastatin-10

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cattle; Cytosol; Depsipeptides; Growth Inhibitors; Guanosine Triphosphate; HeLa Cells; Humans; KB Cells; Molecular Sequence Data; Oligopeptides; Peptide Mapping; Photoaffinity Labels; Protein Binding; Protein Subunits; Tubulin; Tubulin Modulators; Vinblastine

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

Fluorescence correlation spectroscopy (FCS) was applied to investigate the stability of tubulin rings that result from the interaction of alpha beta-tubulin dimers with three vinca domain-binding peptides--cryptophycin 1, hemiasterlin, and dolastatin 10. These peptides inhibit tubulin polymerization into microtubules and, instead, induce the formation of single-walled tubulin rings of 23.8 nm mean diameter for cryptophycin and 44.6 nm mean diameter for hemiasterlin and dolastatin, as revealed by electron microscopy on micromolar drug-tubulin samples. However, the hydrodynamic diameter and the apparent number of fluorescent particles, determined from analysis of FCS measurements obtained from nanomolar drug-tubulin samples, indicate variation in the stability of the rings depending on the drug and the tubulin concentration. Cryptophycin-tubulin rings appear to be the most stable even with tubulin concentration as low as 1 nM, whereas hemiasterlin-tubulin rings are the least, depolymerizing even at relatively high concentrations (100 nM). In contrast, the dolastatin-tubulin rings demonstrate an intermediate level of stability, depolymerizing significantly only at tubulin concentrations below 10 nM. We also compare the stability results with those of cytotoxicity measurements taken on several cell lines and note a rough correlation between the cytotoxicity of the drugs in cell cultures and the stability of the corresponding drug-induced rings.

Topics: Animals; Antineoplastic Agents; Cattle; Depsipeptides; Dimerization; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Nanotechnology; Oligopeptides; Paclitaxel; Peptides, Cyclic; Polymers; Rhodamines; Spectrometry, Fluorescence; Tubulin; Tumor Cells, Cultured; Vinblastine