Project Summary/Abstract This multi-PI basic science project aims to transform understanding of G-protein function in physiology and disease, and provide broadly applicable routes for developing therapeutics to treat currently untreatable diseases caused by mutant constitutively active G protein α-subunits. It does so by developing knowledge required to design and synthesize, at scales required for preclinical and eventual clinical studies, a family of bioavailable inhibitors, each of which selectively targets closely related groups of heterotrimeric G proteins. The translational/clinical potential of this approach is based on recent studies indicating that a bioavailable inhibitor that targets G protein α-subunits of the Gq/11 class is therapeutically effective in mouse models of uveal melanoma, an untreatable disease that is driven by mutant constitutively active Gq/11. Similar approaches could have broad impact, because many other untreatable diseases are driven by various types of mutant constitutively active G protein α-subunits, including hormone-secreting pituitary tumors, mucosal melanoma, choroidal hemangiomas, hepatic small-vessel neoplasms, ~10-15% of all cancers, Sturge-Weber syndrome, autosomal dominant hypoparathyroidism, and certain forms of hyper- and hypocalcemia. Moreover, this approach could be used to modulate G-protein activity in diseases where GPCR-targeted drugs are ineffective due to receptor redundancy or to G proteins that cause dose-limiting side effects such as respiratory suppression or drug tolerance. Bioavailable inhibitors that directly target specific subclasses of G proteins would be extremely valuable for basic science. They would provide simple, fast, cheap and reliable chemical probes with which to identify novel functions of G proteins in normal physiology and in animal models of disease, in contrast to conventional knockout or knockdown strategies, which are slow and expensive, and can suffer from compensatory or off- target effects. The foundation of this project is a pair of nearly identical, bioavailable, cyclic depsipeptide natural products that potently and selectively inhibit the Gq/11 subfamily of G protein α-subunits. The Specific Aims of this project will address four crucial challenges: 1) limited availability of these inhibitors; 2) lack of inhibitor derivatives that could be targeted to disease tissues for chronic therapy; 3) limited understanding of the inhibitory mechanism, which has precluded design of inhibitors that target other subtypes of G proteins; and 4) absence of inhibitors that selectively target G protein subtypes other than Gq/11. These Aims will be pursued by using innovative synthetic organic chemistry, computational-based docking and inhibitor design, single- molecule FRET, NMR, and biochemical and cell-based assays of G protein function.