cryptophycin and hemiasterlin

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