HTI-286 is a synthetic analogue of the
natural product hemiasterlin and is a potent
antimitotic agent.
HTI-286 inhibits the proliferation of
tumor cells during mitosis. The observed
antimitotic activity is due to the binding of
HTI-286 to
tubulin. This report details the effects of
HTI-286 on soluble
tubulin and preassembled microtubules.
HTI-286 binds
tubulin monomer and oligomerizes it to an 18.5 S species corresponding to a discrete ring structure consisting of about 13
tubulin units as determined by sedimentation equilibrium analyses. The rate of formation of the oligomers is dependent on the concentration of
HTI-286 and the time of incubation.
Tubulin oligomers, specifically the 18.5 S species, form slowly. The interactions of
HTI-286 with
tubulin were studied by isothermal titration calorimetry.
HTI-286 binds
tubulin rapidly, and the initial association of
HTI-286 with
tubulin is enthalpically driven with a DeltaH value of -14 kcal/mol at 25 degrees C and a dissociation constant of ca. 100 nM. However, the accompanying
tubulin oligomerization event does not produce measurable heats at 25 degrees C. The dissociation constant estimated from the changes in the intrinsic fluorescence of
tubulin was found to be consistent with the calorimetric results. Both
HTI-286 and
hemiasterlin bind
tubulin with nearly equal potency. However, the stability of the
tubulin oligomers is not identical under size-exclusion column chromatographic conditions. The
tubulin oligomers formed in the presence of
HTI-286 dissociate on the column, while the corresponding oligomers formed in the presence of
hemiasterlin are stable.
Tubulin undergoes a change in the secondary structure in the presence of
HTI-286, which is evidenced by changes in the circular dichroic absorption spectrum of
tubulin. In contrast to the microtubule-stabilizing effects of
paclitaxel, both
HTI-286 and
hemiasterlin depolymerize preassembled microtubules at micromolar concentrations.