Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs

Coordination Chemistry of Anticrowns. Interaction of the Perfluorinated Three-Mercury Anticrown (o-C₆F₄Hg)₃ with Azacrowns

The interaction of the three-mercury anticrown (o-C6F4Hg)3 (1) with various azacrowns such as 2,9,16,23-tetra-tert-butylphthalocyanine (TBPC), octaazapentacyclooctacosane (OAOC), 1,4,8,11-tetraazacyclotetradecane (cyclam), and N,N,N-trimethyl-1,3,5-triazacyclohexane (TACH) has been studied. It has been shown that the reaction of 1 with TBPC in CH2Cl2 at 20 °C affords 1:1 complex {[(o-C6F4Hg)3](TBPC)} (2) according to elemental analysis, and the interaction of 1 with OAOC·0.5H2O in ether results in the formation of complex {[(o-C6F4Hg)3]2(OAOC)(H2O)2} (3). The molecules of azacrown and water in 3 are located between the parallel planes of two mercuramacrocycles strongly laterally shifted relative to one another and are bonded to them through Hg-N and Hg-O secondary interactions. An especially interesting complex, [(CH2)10(NH2)2(NH)2]{[(o-C6F4Hg)3]2Cl2} (4), has been obtained by the reaction of 1 with cyclam [(CH2)10(NH)4] in CH2Cl2 at 20 °C. The complex has a structure of a double-decker sandwich wherein two chloride anions and the diprotonated molecule of cyclam as a counterdication are disposed between the parallel planes of two molecules of 1. Each chloride anion in 4 is η3-coordinated with the Hg centers of the neighboring anticrown, whereas the NH and NH2+ groups of the diprotonated cyclam form three H-bonds with each of these chloride anions. The mechanism of the formation of this unusual sandwich is discussed. The synthesis and structure of an analogous complex, [(CH2)10(NH2)2(NH)2]{[(o-C6F4Hg)3]Cl2}·2(CH3)2CO, which forms polydecker sandwiches in the crystal are also described. The interaction of 1 with cyclam in aqueous THF at 20 °C gives a complex, {[(o-C6F4Hg)3][(CH2)10(NH)4](THF)(H2O)} (6), also forming infinite polymeric chains in the crystal. A characteristic structural feature of 6 is the presence in each its monomeric unit of a bipyramidal fragment formed by 1 and two different η3-coordinated Lewis bases (THF and water). From the reaction of 1 with TACH, a complex having a unique double-cage structure, {[(o-C6F4Hg)3](TACH)2} (7), has been isolated