Abstract
RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.
Highlights
RAS is a family of GTPase proto-oncogenes, comprising four different isoforms: KRAS4A, KRAS4B, HRAS, and NRAS.[1]
The nucleotide-bound state relies on two types of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs)
We found that KRAS and PDE6D relative levels are widely variable among different cell types, with the highest levels of both proteins in human cancer cells (A549, H358, and H2009) and their lowest levels in retinal retinal pigmented epithelial (RPE) cells (Figure 7A,B)
Summary
RAS is a family of GTPase proto-oncogenes, comprising four different isoforms: KRAS4A, KRAS4B, HRAS, and NRAS.[1] All RAS isoforms are ubiquitously expressed, albeit at different quantitative ratios.[2] These proteins act as molecular switches, whose conformation and active state are coupled to their bound nucleotide, either GTP (“on”) or GDP (“off”). RAS has a weak intrinsic GTPase activity and exhibits picomolar binding affinity for nucleotides.[3] The nucleotide-bound state relies on two types of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs reduce the binding affinities of small G-proteins to nucleotides, allowing for the fast displacement of the bound nucleotide. GAPs, on the other hand, accelerate the otherwise slow intrinsic GTPase activity of G-proteins, allowing for the fast hydrolysis of GTP to GDP
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