Assays for Nucleotide Competitive Reversible and Irreversible Inhibitors of Ras GTPases.

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-ß-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.