title: Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.
abstract: FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, Wolfson Link Building 253, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.forename: Maria E
authors.initials: ME
authors.lastname: Due-Hansen
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Christiansen
authors.forename: Anna Mette
authors.initials: AM
authors.lastname: Hansen
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Mackenzie
authors.forename: Hannah
authors.initials: H
authors.lastname: Murdoch
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Pandey
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: Rudi
authors.initials: R
authors.lastname: Marquez
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D044482:Benzeneacetamides / Q000494:pharmacology; D001665:Binding Sites; D002087:Butyrates / Q000494:pharmacology*; D003521:Cyclopropanes / Q000737:chemistry / Q000494:pharmacology*; D019858:Enteroendocrine Cells / Q000378:metabolism; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008066:Lipolysis / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011956:Receptors, Cell Surface / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled
chemical_list: D044482:Benzeneacetamides; D002087:Butyrates; D003521:Cyclopropanes; D052216:Glucagon-Like Peptide 1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011956:Receptors, Cell Surface; D013844:Thiazoles; C582956:(R)-3-(cyclopentylmethyl)-4-(cyclopropyl-(4-(2,6-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid; C582955:3-benzyl-4-(cyclopropyl-(4-(2,5-dichlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid; C473103:FFAR2 protein, human; D043562:Receptors, G-Protein-Coupled
keywords: 7-Helix Receptor; Adipocyte; Fatty Acid; G Protein-coupled Receptors (GPCRs); Signal Transduction