title: Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3.
abstract: Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [(35)S]guanosine 5'-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.
authors.affiliation: Molecular, Cellular, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schmidt
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Smith
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Christiansen
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.forename: Manuel
authors.initials: M
authors.lastname: Grundmann
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: Christel
authors.initials: C
authors.lastname: Drewke
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
mesh_terms: D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D011422:Propionates / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D011422:Propionates; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C473103:FFAR2 protein, human; C106262:FFAR3 protein, human