Interactions of the sponge-derived antimitotic tripeptide hemiasterlin with tubulin: Comparison with dolastatin 10 and cryptophycin 1

The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiaster lin resembles most other antimitotic peptides in noncompetitively inhibitin g the binding of vinblastine to tubulin (apparent K-i value, 7.0 mu M), com petitively inhibiting the binding of dolastatin 10 to tubulin (apparent K-i value, 2.0 mu M), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of fr ee drug, even at low drug concentrations. The tubulin oligomerization react ion induced by hemiasterlin was compared to the reactions induced by dolast atin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced format ion of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morpholog y of the cryptophycin 1 aggregate consisted primarily of smaller rings (dia meter about 30 nm). However, the hemiasterlin aggregate differed from the d olastatin 10 aggregate in that its formation was not associated with turbid ity development, and the morphology of the hemiasterlin aggregate (as oppos ed to the dolastatin 10 aggregate) did not change greatly when microtubule- associated proteins were present (tight coils and pinwheels are observed wi th dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacificatio n of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 deg rees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory a t both reaction temperatures.