Discovery and characterization of small molecules that target the GTPase Ral

Using a structure-based approach, small molecule inhibitors that selectively target the GTPase Ral are identified and characterized; these first-generation inhibitors will be valuable tools for elucidating the Ral signalling pathway and constitute a step towards developing Ral-specific agents for cancer therapy. Small molecule inhibitors against Ral GTPase Signalling through the Ras GTPase pathway is often hyperactive in cancer, and Ras pathway components are being extensively studied as targets for potential cancer therapies. Here, in a structure-based approach, Dan Theodorescu and colleagues discover small molecule inhibitors that selectively target the Ral GTPase, an important downstream mediator of Ras signalling. The inhibitors bind Ral in its inactive state and are characterised biochemically, in cellular assays and in mice. These first-generation inhibitors will be valuable tools for elucidating Ral signalling and a step towards developing Ral-specific agents for cancer therapy. The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis 1 . Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and 1 H– 15 N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.