The use of impedance assays to profile small molecules in a cell line natively expressing GPR43

T. Cheung, L. Dickson, P. Mitchell. in vitro Pharmacology, Takeda Cambridge Ltd, Cambridge, UNITED KINGDOM.

Introduction: GPR43 is a Gαi/q coupled GPCR that is expressed in a range of tissues, including the GI tract, adipose tissue and immune cells. The receptor is activated by SCFA, e.g. acetate, produced in the gut through bacterial fermentation (1). In line with its expression profile, GPR43 is suggested to be a potential drug target for metabolic disorders and immune/inflammatory conditions and may be of particular interest in intestinal inflammatory conditions e.g. colitis (2). The aim of this study was to investigate GPR43 pharmacology in a native immune cell system.

Method: Gene transcription studies were used to identify a relevant immune cell line endogenously expressing GPR43. Compounds were profiled in Gαi cAMP (HTRF), Gαq calcium flux and ‘capture all’ cell impedance assays. Data was compared to results from a recombinant CHO line expressing hGPR43.

Results: The macrophage-like PMA differentiated human U937 cell line (U937-PMA) was identified as a potential GPR43 expressing cell. However these cells did not respond to acetate in cAMP or calcium flux assays; unlike recombinant hGPR43 cells, in which acetate produced large responses in both assays. Consequently a cell impedance assay was used to examine acetate activity in U937-PMA cells, in which a small Gαi, GPR43 mediated response was detected. Takeda’s Chemotype 1 (CT1) compounds produced no agonist responses in this assay; however clear responses were observed in the presence of acetate, indicating positive allosteric modulator (PAM) activity (e.g. PGM044859 EC50 0.31 µM n = 3 ). CT4/7 compounds produced no agonist or PAM responses in this assay. Again these results are in contrast to the GPR43 recombinant cell data, in which all chemotypes showed Gαi agonist (HTRF) and Gαq agonist / PAM activity (flux). Agonist and PAM activity was confirmed for CT1 compounds in the recombinant GPR43 impedance assay (e.g. PGM044859 EC50 values (µM): agonist 0.4 µM, PAM 0.04 n = 3).

Conclusion: This work highlights differences in GPR43 pharmacology and signalling in native and recombinant cell lines. SCFA activated distinct signalling pathways in the native cell line and differences between Takeda chemotypes could be identified in the native line that were not evident in the stable line. Further work using pathway inhibitors in the impedance assay will help to define the signalling pathways engaged by GPR43 in the macrophage-like cell line.

References: 1. Bindels L et al. (2013). Trends Pharmacol. Sci. 34: 226-232. 2. Ulven T (2012). Front. Endocrinol. 3: 111.