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Updated: Feb 10, 2026
Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Assays for Nucleotide Competitive Reversible and Irreversible Inhibitors of Ras GTPases
Sadasivam Jeganathan1, Matthias P Müller2, Imtiaz Ali1
1Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11 , 44227 , Dortmund , Germany.
Abstract:
Although the Ras protein has been seen as a potential target for cancer therapy for the past 30 years, there was a tendency to consider it undruggable until recently. This has changed with the demonstration that small molecules with a specificity for (disease related mutants of) Ras can indeed be found, and some of these molecules form covalent adducts. A subgroup of these molecules can be characterized as competing with binding of the natural ligands GTP and GDP. Because of the distinct properties of Ras and related GTPases, in particular the very high nucleotide affinities and associated very low dissociation rates, assays for characterizing such molecules are not trivial. This is compounded by the fact that Ras family GTPases tend to be thermally unstable in the absence of a bound nucleotide. Here, we show that instead of using the unstable nucleotide-free Ras, the protein can be isolated as a 1:1 complex with a modified nucleotide (GDP-β-methyl ester) with low affinity to Ras. With this nucleotide analogue bound to the protein, testing of inhibitors is made experimentally more convenient and we present assays that allow the rapid assessment of the kinetic constants describing the inhibition process.
Insights
Developing novel assays for Ras inhibitors is crucial for cancer therapy. Researchers created a stable Ras-nucleotide complex, simplifying the testing of potential cancer drugs targeting Ras proteins.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Ras proteins are key targets in cancer therapy, but historically considered undruggable.
- Recent advances show specific small molecules can target Ras mutants, with some forming covalent adducts.
- A subset of these molecules inhibits Ras by competing with natural ligands GTP and GDP.
Purpose of the Study:
- To address challenges in assay development for Ras inhibitors due to Ras GTPase properties.
- To present a novel method for stabilizing Ras proteins for inhibitor screening.
- To enable convenient and rapid assessment of Ras inhibitor kinetic constants.
Main Methods:
- Isolating Ras GTPases as a stable 1:1 complex with GDP-β-methyl ester, a low-affinity nucleotide analogue.
- Utilizing this stable complex instead of thermally unstable nucleotide-free Ras.
- Developing and presenting new assays for rapid kinetic analysis of inhibitor binding.
Main Results:
- Demonstrated that Ras can be stabilized by complexing with GDP-β-methyl ester.
- Showcased that this stable complex facilitates experimental testing of Ras inhibitors.
- Presented assays for the rapid assessment of inhibition kinetics.
Conclusions:
- The developed method provides a more convenient experimental approach for studying Ras inhibitors.
- This stabilization strategy overcomes the thermal instability of nucleotide-free Ras.
- The assays enable efficient characterization of Ras-targeted cancer therapies.
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