Hudson, Brian D2; Due-Hansen, Maria E4; Christiansen, Elisabeth4; Hansen, Anna3; Mackenzie, Amanda E2; Murdoch, Hannah2; Pandey, Sunil K4; Ward, Richard J2; Marquez, Rudi2; Tikhonova, Irina G2; Ulven, Trond4; Milligan, Graeme2
1 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU2 unknown3 Catalysis Research Group (Interface Chemistry)4 Department of Physics, Chemistry and Pharmacy, Faculty of Science, SDU
FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) 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 either 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 FFA2 agonists that interact with the orthosteric binding site. 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 (ECL2) and the transmembrane domain (TM) 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 the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.
Journal of Biological Chemistry, 2013, Vol 288, p. 17296-17312