Abstract
Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRasG12C using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 104 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.
Highlights
It has been known for many years that mutations in Ras proteins, KRas, are important causative factors in many human cancers[1]
Strategies involving direct competition with GTP binding in analogy to kinase inhibitors that compete with ATP binding were not considered seriously for many years, since it has been apparent for over 25 years that GTP and GDP bind with extremely high affinity to Ras proteins[3, 4], meaning that inhibitors would have to compete with several hundred μM concentrations of GTP/ GDP binding with sub-nM affinity to their targets
A method which leads to estimates of the kinetic parameters as well as the affinity involves stopped-flow measurements of association of fluorescent nucleotides with nucleotide-free Ras followed by competiton between fluorescent and non-fluorescent nucleotides to obtain the parameters for the non-labelled substances[3]
Summary
It has been known for many years that mutations in Ras proteins, KRas, are important causative factors in many human cancers[1]. A further approach is less direct and is aimed at the prenyl-binding protein PDEδ, which plays an important role in the regulation of Ras localization at the plasma membrane Selective inhibitors of this factor offer a possible approach to modulation of Ras activity[7]. We proceeded to assess the properties of the covalent inhibitor SML-8-73-111 in terms of its kinetics of interaction with KRas and its rate of reaction with KRasG12C Using this data, we simulated and compared the likely effect of a β-phosphate modified covalent inhibitor with that of a putative covalent inhibitor that retains the high affinity of GDP to Ras proteins in the cellular situation. Our data indicate that a nucleotide competitive inhibitor can only work if it retains the high reversible affinity of GDP/GTP, even if the inhibitor can react covalently with the Ras protein
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