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In vitro TGR5 agonist assay protocol

SM Sachiho Miyata
YK Yuji Kawashima
MS Miku Sakai
MM Masaya Matsubayashi
KM Keisuke Motoki
YM Yui Miyajima
YW Yousuke Watanabe
NC Noriko Chikamatsu
TT Tetsuya Taniguchi
RT Ryukou Tokuyama
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TGR5 agonist activity was evaluated by transfecting CHO cells with TGR5 using the Discover X expression kit (cAMP Hunter eXpress Assay Kit, Discover X Fremont, CA, USA). Cells were incubated with the test compounds for 0.5 h, and intracellular cAMP levels were measured using Multi-Mode Microplate Reader (FlexStation 3, Molecular Devices Inc., San Jose, CA, USA). The response to 10 µM INT-767 3 was set as 100% TGR5 activation37.

Each compound was tested at concentrations of 10, 1, or 0.1 µM in duplicate (n = 2), with the average value shown. EC50 value of compounds 20p and 20q were determined by using a 4 parameter logistic equation, Y = min + (max − min)/(1 + 10^(n * (log10(EC50) − log10(conc)))), in nonlinear least squares fitting using the Microsoft Excel Solver55.

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