Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi–GDP complex by directly interacting with both Gαi and GDP. Gαi–GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

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Overactive signaling at the level of hormone, receptor, or G‐protein can initiate and potentiate cellular transformation and other diseases. We hypothesize that direct inhibition of overactive G‐protein signaling is cytotoxic to ovarian cancers. We describe the development of selective small molecule inhibitors which bind to and stabilize the GDP‐bound form of the Galphai1. Initial inhibitors were identified using computational high throughput docking of a 280,000 small molecule database to Galphai1‐GDP and counter‐screened against Galphai1‐GTP and Galphaq‐GDP. Nucleotide exchange assays have verified selective G‐protein inhibition. This inhibition was supported by live cell bioluminescence resonance energy transfer (BRET) experiments measuring the interaction of Galphai1 with the regulatory protein AGS4 in HEK293 cells. Inhibition of Galphai1 but not Galphaq decreased cell viability in SKOV3 ovarian cancer cells. The mechanism of Gi pathway inhibition is also explored. Pharmacologic regulation of both receptor dependent and independent signaling through specific heterotrimeric subunits will provide a unique window into a major signaling axis while providing data and compounds for the treatment of a refractory disease like ovarian cancer.