HTI-286 is a synthetic analogue of the natural product hemiasterlin and is a potent antimitoticagent. HTI-286 inhibits the proliferation of tumor cells during mitosis. The observed antimitotic activityis due to the binding of HTI-286 to tubulin. This report details the effects of HTI-286 on soluble tubulinand preassembled microtubules. HTI-286 binds tubulin monomer and oligomerizes it to an 18.5 S speciescorresponding to a discrete ring structure consisting of about 13 tubulin units as determined by sedimentationequilibrium 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. Theinteractions of HTI-286 with tubulin were studied by isothermal titration calorimetry. HTI-286 bindstubulin rapidly, and the initial association of HTI-286 with tubulin is enthalpically driven with a
Hvalue of -14 kcal/mol at 25
C and a dissociation constant of ca. 100 nM. However, the accompanyingtubulin oligomerization event does not produce measurable heats at 25
C. The dissociation constantestimated from the changes in the intrinsic fluorescence of tubulin was found to be consistent with thecalorimetric 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 chromatographicconditions. The tubulin oligomers formed in the presence of HTI-286 dissociate on the column, while thecorresponding oligomers formed in the presence of hemiasterlin are stable. Tubulin undergoes a changein the secondary structure in the presence of HTI-286, which is evidenced by changes in the circulardichroic absorption spectrum of tubulin. In contrast to the microtubule-stabilizing effects of paclitaxel,both HTI-286 and hemiasterlin depolymerize preassembled microtubules at micromolar concentrations.
