Use of a p120 RasGAP glue to inhibit KRas in NF1-null cells

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder that is associated with a spectrum of pathologies including glioma, autism spectrum disorder, Lisch nodules of the iris and neurofibroma. The NF1 gene encodes neurofibromin, a Ras GTPase-activating protein. Deletion or disabling mutations in NF1 gene result in an absence of neurofibromin and sustained Ras activation, driving uncontrolled cell proliferation and tumorigenesis. Targeting the Ras protein directly has until recently proved challenging due to the lack of binding cavities on molecular surface and due to its high affinity for GTP/GDP. Purpose: Our study aims to evaluate the efficacy of novel molecular glues designed to modulate Ras activity by increasing its binding to p120RasGAP, a ubiquitously expressed negative regulator of Ras that, like NF1, augments the GTPase activity of Ras, assisting its conversion from active Ras-GTP to inactive, Ras-GDP. The goal is to use these compounds to enable p120RasGAP to functionally replace neurofibromin in NF1 -deficient cells, restoring normal Ras signaling. Methods: We used an artificial intelligence/machine learning (AI/ML) protocol to identify potential molecular glues that should stabilize the p120RasGAP/Ras complex. To assess the impact of these compounds, we used NF1-deficient cell line and employed Western Blot analysis to measure changes in phosphorylated ERK (p-ERK) levels as a downstream marker of Ras activity. In addition, we used shRNA-mediated knockdown of the RASA1 gene, which encodes p120RasGAP protein, to verify whether the observed effects are dependent on the proposed Ras/RasGAP interaction mechanism. To assess direct interaction, purified recombinant KRAS and p120RasGAP were used in AlphaScreen binding assay. Compound binding was further validated by surface plasmon resonance (SPR). Results: Preliminary data indicate a reduction in p-ERK levels upon treatment with novel compounds, suggesting partial suppression of Ras signaling. Furthermore, in p120RasGAP knockdown cells, the molecular glues had a less potent effect in reducing p-ERK levels, suggesting that our compounds act through the proposed mechanism rather than producing off-target effects. Of the three most perspective candidates, one promoted significant binding of KRAS to p120RasGAP in an AlphaScreen assay. This result was also confirmed by SPR. Conclusion: AL/ML methods can be used to design molecular glues that stabilize the interaction of p120RasGAP to Ras, providing a promising avenue for targeted NF1 therapies. Such compounds might be useful in reducing Ras activity in NF1-mutant cells. We aim to further characterize this interaction using STD NMR and initiate crystallization trials to determine the structural basis of binding.