Small-Molecule Antagonists of Ral GTPases in Cancer

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 DESCRIPTION (provided by applicant): Ral GTPase-regulated signaling networks control diverse set of cellular processes such as exocytosis, endocytosis, transcription factor activation, and actin cytoskeletal reorganization. Substantial evidence, including from our own work, supports a role for Ral in Ras-driven cancers such as non-small cell lung cancer (NSCLC). We found that a small molecule (BQU57) that inactivates Ral by promoting its GDP-bound state significantly impaired NSCLC proliferation in anchorage-dependent and independent assays. BQU57 was selective for Ral relative to the GTPases Ras and RhoA and inhibited tumor xenograft growth to a similar extent to the depletion of Ral using RNA interference. In addition to BQU57, we identified a small-molecule orthosteric antagonist of the protein-protein interaction between Ral and its effector protein RalBP1. The compound impairs Ral activation in NSCLC cells and inhibits proliferation in anchorage-dependent and independent assays. Our long-term objective is to develop small-molecule Ral antagonists that are suitable for treating NSCLC in the clinic. Our short-term objective is to develop derivatives of our two parental structures to probe Ral function in vivo and set the stage for clinical investigation of promising compounds. Our central hypothesis is that structure-guided computational design of derivatives of Ral antagonists will lead to small molecules that exhibit higher affinity, better pharmacokinetic (PK) properties and greater efficacy in vivo. Our preliminary data strongly positions us to test our hypothesis. BQU57 already exhibits efficacy in vivo and our orthosteric inhibitors show promising inhibition of Ral activity and cell viability in NSCLC. In our first aim, we employ structure-based computational methods and absorption, distribution, metabolism, and excretion (ADME) predictions to design derivatives for each class of small molecules. In the second aim, we synthesize 20-25 derivatives every year for each of the two structural classes and evaluate these compounds for binding and inhibition using biochemical and biophysical approaches. 1H-15N transverse relaxation-optimized (TROSY) NMR spectroscopy and X-ray crystallography are used to provide structural evidence of direct binding. In the third aim, we evaluate compounds for their effect on Ral activation, and assess their activity in a panel of NSCLC cell lines using anchorage-dependent and independent assays. We select the most promising compound for each of the two classes of Ral antagonists and characterize stability in vitro and PK parameters in vivo. Efficacy studies will be carried out first using human xenografts, followed by evaluation in transgenic mouse models for the most promising compounds. We also perform combination studies of our Ral antagonists with inhibitors of Ras signaling pathways. We expect that this work will lead to small-molecule Ral antagonists with nanomolar range affinity that are orally bioavailable, possess suitable PK properties, and exhibit substantial efficacy in blocking Ral driven tumor formation and growth.