title: Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles.
abstract: Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and characterization of small molecule GLP-1R ligands as possible therapeutics.
authors.affiliation: From the Centro de Investigación Lilly, Eli Lilly and Co., Alcobendas 28108, Spain and.
authors.forename: Ana B
authors.identifier: http://orcid.org/0000-0002-4190-6891
authors.initials: AB
authors.lastname: Bueno
authors.affiliation: Endocrine Discovery and.
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Showalter
authors.affiliation: Quantitative Biology, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285.
authors.forename: David B
authors.initials: DB
authors.lastname: Wainscott
authors.affiliation: Endocrine Discovery and.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Stutsman
authors.affiliation: From the Centro de Investigación Lilly, Eli Lilly and Co., Alcobendas 28108, Spain and.
authors.forename: Aranzazu
authors.initials: A
authors.lastname: Marín
authors.affiliation: Endocrine Discovery and.
authors.forename: James
authors.initials: J
authors.lastname: Ficorilli
authors.affiliation: Endocrine Discovery and.
authors.forename: Over
authors.initials: O
authors.lastname: Cabrera
authors.affiliation: Quantitative Biology, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285.
authors.forename: Francis S
authors.identifier: http://orcid.org/0000-0002-4260-2451
authors.initials: FS
authors.lastname: Willard
authors.affiliation: Endocrine Discovery and sloop_kyle_w@lilly.com.
authors.forename: Kyle W
authors.identifier: http://orcid.org/0000-0001-6748-9929
authors.initials: KW
authors.lastname: Sloop
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D000067757:Glucagon-Like Peptide-1 Receptor / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D000097789:Glucagon-Like Peptide-1 Receptor Agonists
chemical_list: C570819:4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine; C000593833:GLP1R protein, human; C000593834:Glp1r protein, mouse; D000067757:Glucagon-Like Peptide-1 Receptor; D007328:Insulin; D050505:Mutant Proteins; D011743:Pyrimidines; D000242:Cyclic AMP; D005947:Glucose; D000097789:Glucagon-Like Peptide-1 Receptor Agonists
keywords: G protein-coupled receptor (GPCR); allosteric regulation; diabetes; glucagon-like peptide-1; insulin secretion; pancreatic islet