Development of Selective Small Molecular Inhibitors of Heterotrimeric Galpha subunits

Overactive signaling at the level of hormone, receptor, or G-protein can initiate and potentiate diseases. Heterotrimeric G-proteins, the immediate downstream effectors of GPCRs, regulate GPCR signaling cascades. Pertussis toxin (PTX) has long been the standard laboratory tool for inhibition of Gα i. As an enzyme modifier, PTX has shortcomings when used as a pharmacologic inhibitor. Endogenous guanine nucleotide dissociation inhibitors (GDIs) such as AGS3/4 and RGS12/14 function through the GPR/GoLoco GDI domain, best exemplified to date by the cell permeable GPR consensus peptide TAT-GPR. Here we describe the development of novel selective inhibitors for Gαi. Using molecular docking, we identified potential small molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gag-GDP was used as a computational and biological counter screen. 41 molecules and the TAT-GPR were tested using nucleotide exchange, fluorescent polarization, and STD-NMR assays with one compound showing efficacy on all assays. In SKOV3 cells, the lead compound showed Gαi specific cytotoxicity and increased cAMP while inhibiting ERK and c-Src phosphorylation and cell viability. The compound also moderately enhanced phosphorylated Akt.;As a potential therapeutic application of a Gαi-GDI, we examined the effects of the lead compound in prostate cancer. Treatment for prostate cancer involves anti-androgen hormonal therapies. Success of this treatment, however, is usually undone by the return of the cancer as androgen-insensitive (AI). Human data has shown that GNAI2 (the gene for Gα i2) message levels tend to increase with the transition from androgen sensitive (AS) to AI prostate cancer. We found that Gαi2 protein levels were reduced 54, 64, and 37% in AI PC3, DU 145, and C4-2B cells, respectively, compared to AS LNCaP cells. We tested the effects of Gαi inhibition upon cell viability in LNCaP and C4-2B cells using PTX, the TAT-GPR peptide, Taxol, and our lead Gαi-GDI compound. 48 hour MTS assay results showed that Gαi inhibition was more effective in the AI C4-2B cells for all toxins tested. We conclude that targeting Gαi with a small molecule is feasible and could have therapeutic potential in a cancer stage dependent manner.