Comparative effects of the endogenous agonist glucagon-like peptide-1 (GLP-1)-(7-36) amide and the small-molecule ago-allosteric agent "compound 2" at the GLP-1 receptor.
Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic 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, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) 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 [Ca(2+)] 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 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization 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 internalization. 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 in acute assays with membranes.
Coopman
Karen
K
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom .
Huang
Yan
Y
Johnston
Neil
N
Bradley
Sophie J
SJ
Wilkinson
Graeme F
GF
Willars
Gary B
GB
D001711:Biotransformation / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival; D000242:Cyclic AMP / Q000378:metabolism; D003627:Data Interpretation, Statistical; D019204:GTP-Binding Proteins / Q000378:metabolism; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis / Q000494:pharmacology*; D000067757:Glucagon-Like Peptide-1 Receptor; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D010446:Peptide Fragments / Q000096:biosynthesis / Q000494:pharmacology*; D011810:Quinoxalines / Q000494:pharmacology*; D018027:Receptors, Glucagon / Q000096:biosynthesis / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013450:Sulfones / Q000494:pharmacology*; D014343:Trypan Blue
C517397:3-(tert-butylamino)-6,7-dichloro-2-(methylsulfonyl)quinoxaline; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D010446:Peptide Fragments; D011810:Quinoxalines; D018027:Receptors, Glucagon; D013450:Sulfones; C055453:glucagon-like peptide 1 (7-36)amide; D049452:Green Fluorescent Proteins; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D014343:Trypan Blue; D002118:Calcium