Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules.
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.
Krishnamurthy
Girija
G
Biophysics/Enzymology, Screening Sciences, Oncology Research, and Chemical Sciences, Wyeth Research, Pearl River, New York 10965, USA. krishng@wyeth.com
Cheng
Wendy
W
Lo
Mei-Chu
MC
Aulabaugh
Ann
A
Razinkov
Vladimir
V
Ding
WeiDong
W
Loganzo
Frank
F
Zask
Arie
A
Ellestad
George
G
D000818:Animals; D002151:Calorimetry / Q000379:methods; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*; D050257:Tubulin Modulators; D014461:Ultracentrifugation / Q000379:methods
C473896:HTI-286; D009842:Oligopeptides; D014404:Tubulin; D050257:Tubulin Modulators; D017239:Paclitaxel; D005990:Glycerol