Development of inhibitors of heterotrimeric Gαi subunits

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 G alpha(i) by competing for the GPCR and G beta gamma 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 alpha(2)-adrenoceptor (alpha(2)AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for G alpha(i). Molecular docking was used to identify potential molecules that bind to and stabilize the G alpha(i)-GDP complex by directly interacting with both G alpha(i) and GDP. G alpha(i)-GTP and G alpha(q)-GDP were used as a computational counter screen and G alpha(q)-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 G alpha(i). Several compounds showed G alpha(1) specific inhibition and were able to block alpha(2)AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of G alpha subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of over-stimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease. (C) 2014 Elsevier Ltd. All rights reserved.