Dolastatin 15 binds in the vinca domain of tubulin as demonstrated by Hummel-Dreyer chromatography.
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.
Cruz-Monserrate
Zobeida
Z
National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA.
Mullaney
Jeffrey T
JT
Harran
Patrick G
PG
Pettit
George R
GR
Hamel
Ernest
E
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D003078:Colchicine / Q000494:pharmacology; D047630:Depsipeptides; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008453:Maytansine / Q000378:metabolism / Q000494:pharmacology; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010080:Oxazoles / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014316:Tritium; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology; D014750:Vincristine / Q000378:metabolism / Q000494:pharmacology
D047630:Depsipeptides; D006576:Heterocyclic Compounds, 4 or More Rings; D009842:Oligopeptides; D010080:Oxazoles; D014404:Tubulin; D014316:Tritium; C075610:dolastatin 15; C408862:diazonamide A; D008453:Maytansine; D014750:Vincristine; D014747:Vinblastine; C094621:cemadotin; C064570:dolastatin 10; D003078:Colchicine