Abstract
Glucagon-like peptide-1 (GLP-1) mediates anti-diabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 'compound 2' (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) has been described as a GLP-1R allosteric modulator and agonist. Using HEK-293 cells expressing human GLP-1Rs we extended this work to consider the impact of compound 2 on G-protein activation, Ca2+ signaling and receptor internalisation and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Gαs in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist respectively. GLP-1 increased intracellular [Ca2+] by release from intracellular stores, which was mimicked by compound 2 with slower kinetics. In either intact cells or membranes, the orthosteric antagonist, exendin 9-39, inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalised EGFP-tagged GLP-1Rs but the speed and magnitude evoked by compound 2 were less. Exendin 9-39 inhibited internalisation by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalisation. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer-term assays with intact cells or acute assays with membranes.
Footnotes
- Received January 14, 2010.
- Revision received May 26, 2010.
- Accepted May 26, 2010.
- The American Society for Pharmacology and Experimental Therapeutics