Development of inhibitors of heterotrimeric Gαi subunits

Abstract

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.

The GAPs, GEFs, GDIs and…now, GEMs: New kids on the heterotrimeric G protein signaling block

“…In the years since, a growing body of work by us and others indicates that genetic or epigenetic factors that deregulate the intricate network of "accessory proteins" in a variety of disease states are just as significant as those that directly affect the G proteins and GPCRs, if not more so. 7,[12][13][14] Non-canonical G protein signaling by GEMs; The serendipitous discovery of a family of proteins based on homology to a synthetic peptide…”

Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells

“…Recently, Appleton et al identified small molecule GDIs which weakly inhibit Gα subunits at high micromolar concentrations while maintaining intact the stimulation of the Gβγ signaling [21]. In the present study, we have designed, synthesized and profiled the effects of four compounds (9a, 9b, 13 and 14) on the migration of selected cancer cell lines.…”

“…In the present study, we have designed, synthesized and profiled the effects of four compounds (9a, 9b, 13 and 14) on the migration of selected cancer cell lines. These compounds were designed based on compound 12 (ketamine 9827), the more synthetically tractable compound, as disclosed by Appleton et al [21]. At this time, there is no suitable crystal structure of Gα i 2 that can be used for our purpose.…”

“…Previously, it has been shown that small molecule inhibitors, targeting Gα i and Gα q subunits, act as guanine nucleotide dissociation inhibitors (GDI). These molecules were able to partially restore cyclic adenosine monophosphate (cAMP) levels in forskolin-stimulated cells [ 21 ]. These compounds are, however, weakly active, showing maximum inhibition of approximately 38% at 300 μM [ 21 ].…”

“…These molecules were able to partially restore cyclic adenosine monophosphate (cAMP) levels in forskolin-stimulated cells [ 21 ]. These compounds are, however, weakly active, showing maximum inhibition of approximately 38% at 300 μM [ 21 ]. To demonstrate the feasibility of small molecule disruption of the function of Gα i 2 protein as a strategy for mitigating cancer cell migration, we used one of the more synthetically tractable lead compounds, ketimine 9827 (referred to 12 in the following sections), as a template to develop more potent Gα i 2 inhibitors.…”