Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules
Girija Krishnamurthy1, Wendy Cheng, Mei-Chu Lo
1Biophysics/Enzymology, Screening Sciences, Oncology Research, and Chemical Sciences, Wyeth Research, Pearl River, New York 10965, USA. krishng@wyeth.com
Abstract:
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.
Insights
HTI-286, a hemiasterlin analogue, potently inhibits tumor cell proliferation by binding to tubulin and forming ring-like structures. This antimitotic agent depolymerizes microtubules, offering a novel cancer therapeutic strategy.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- HTI-286 is a synthetic analogue of hemiasterlin, a natural antimitotic agent.
- Antimitotic agents target cell division, offering potential cancer therapies.
Purpose of the Study:
- To investigate the molecular mechanisms of HTI-286's antimitotic activity.
- To characterize the interaction of HTI-286 with tubulin and microtubules.
Main Methods:
- Sedimentation equilibrium analyses to determine tubulin oligomer structure.
- Isothermal titration calorimetry and intrinsic fluorescence to study binding kinetics and thermodynamics.
- Circular dichroism spectroscopy to assess changes in tubulin secondary structure.
Main Results:
- HTI-286 binds tubulin monomer, inducing oligomerization into a discrete 18.5 S ring structure.
- Binding is rapid, enthalpically driven (ΔH = -14 kcal/mol), with a dissociation constant of ~100 nM.
- HTI-286 and hemiasterlin depolymerize preassembled microtubules, unlike paclitaxel.
Conclusions:
- HTI-286 functions as a potent antimitotic agent by disrupting tubulin dynamics.
- The distinct stability of HTI-286-induced tubulin oligomers compared to hemiasterlin warrants further investigation.
- HTI-286 represents a promising lead compound for anticancer drug development.
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