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.
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom .
Karen
K
Coopman
Yan
Y
Huang
Neil
N
Johnston
Sophie J
SJ
Bradley
Graeme F
GF
Wilkinson
Gary B
GB
Willars
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