TGR5 is a potent TGR5(GPCR19) agonist.

TGR5/GPCR19 (melanophore cells):7.5(pEC50), TGR5/GPCR19 (U2-OS cells):6.8(pEC50)

Inhibition assays are carried out in 384-well white ProxiPlates in 10 μL of reaction volume. Standard reaction mixtures consisted of the compound (in 2% DMSO final concentration), enzyme mix (0.001 μM of PHD2, 10 μM of Fe(II), 100 μM of ascorbate) and peptide mix (0.06 μM of biotinylated C-terminal oxygen dependent degradation domain (CODD) peptide, 2 μM of 2OG) in 50 mM HEPES pH 7.5, 0.01% Tween-20 and 0.1% BSA buffer. Compounds (e.g., IOX2) are preincubated with the enzyme mix for 15 min before being incubated with peptide mix for 10 min at 22°C. Each reaction is quenched with 5 μL of 30 mM EDTA. The bead mix containing AlphaScreen beads is preincubated for 1h with a rabbit monoclonal antibody selective for hydroxy-HIF1α (Pro564) and are added to the wells for a further 1 h at 22°C. The plates are then analyzed with an Envision plate reader. The IC50 values are calculated using nonlinear regression with normalized dose-response fit using Prism GraphPad (n≥3)[1].