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A GLP‐1R Positive Allosteric Modulator Acts through Covalent Modification
Author(s) -
Nolte Whitney
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb514
Subject(s) - allosteric regulation , chemistry , potentiator , allosteric modulator , receptor , g protein coupled receptor , long term potentiation , mechanism of action , biochemistry , pharmacology , biology , in vitro
The glucagon‐like peptide‐1 receptor (GLP‐1R) is an important target for the treatment of type 2 diabetes. Recent studies have identified several small molecule potentiators of the GLP‐1R. The focus of these studies is to investigate the mechanism of action of one of the most studied GLP‐1R potentiators, 4‐(3‐benzyloxyphenyl)‐2‐ethylsulfinyl‐6‐(trifluoromethyl)pyrimidine (BETP), which potentiates the signaling of weak agonists at the receptor. BETP is a reactive electrophile, making covalent modification of the receptor a potential mechanism for its action. The potentiation of weak agonists by BETP is resistant to washout, providing evidence that it acts by covalently modifying the GLP‐1R. Clickable analogs of BETP modify active, membrane‐bound GLP‐1R in living cells. Studies using mass spectrometry‐based proteomics identified Cys347 and Cys438 as sites at which BETP modifies the GLP‐1R. The functional relevance of the modification at Cys347 and not Cys438 was established through site‐directed mutagenesis. Full agonists retained activity at the Cys347Ala mutant of GLP‐1R; however, BETP no longer potentiated the function of the weak agonist, GLP‐1(9‐36)NH 2 . These results illustrate that BETP covalently modifies Cys347 in intracellular loop 3 (ICL3) of GLP‐1R and that this modification is necessary for its potentiation of GLP‐1R activity. We further show that introduction of a cysteine residue in ICL3 of two other GPCRs, the glucagon receptor and the gastric inhibitory polypeptide receptor, sensitizes them to ligand potentiation by BETP and GLP‐1R ago‐allosteric modulator, compound 2. These results highlight the potential relevance of this site in ICL3 to enhancing the activity of class B GPCRs and may facilitate the discovery of new small molecule modulators