Drug discovery opportunities and challenges at g protein coupled receptors for long chain free Fatty acids

Abstract

Discovery of G protein coupled receptors for long chain free fatty acids (FFAs), FFA1 (GPR40) and GPR120, has expanded our understanding of these nutrients as signaling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signaling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focused on agonists to replicate acute benefits of FFA receptor signaling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signaling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further pre-clinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes.

Keywords: FFA1; G protein coupled receptor; GPR120; GPR40; adipocytes; free fatty acid; pancreas; type 2 diabetes.

Figure 1

Amino acid sequences for human long chain FFA GPCRs. Diagrams represent FFA1 (A), GenBank NM_005303) and the short isoform of GPR120S [(B), BC101175] with the position of the 16 amino acid insert in GPR120L (NM_181745) indicated in the inset. Putative glycosylation sites are indicated on the extracellular Asn residues by white on black, while the conserved disulfide bridge from extracellular loop 2, and two unproven palmitoylation sites in GPR120, are also indicated by black on gray Cys residues. Basic Arg residues (white on red) have been implicated in recognizing the carboxylate anions of FFA and other agonists (Sum et al., ; Suzuki et al., 2008). A number of other residues have been identified as important for GW9508 binding to FFA1, some of which are indicated here (black on blue; Sum et al., 2007); however the specificity with which these mutations alter GW9508 binding, compared to more general effects on FFA1 activation has since been questioned (Smith et al., 2009).

Figure 2

Chemical structures of example FFA GPCR agonists (A) and three reported FFA1 antagonists (B). Information on agonist pharmacology, with references, is provided in Table 1. The structure of Metabolex example 36 is reproduced from the relevant GPR120 agonist patent (Ma et al., 2010) and two Banyu compounds are shown from isoindolin-1-one derivatives^a (cpd 2; Arakawa et al., 2010) and the phenyl-isoxazol-3-ol series^b (cpd 15; Hashimoto et al., 2010). Both GW1100 (Briscoe et al., 2006), Pfizer compound 15i (Humphries et al., 2009), and DC260126 (Hu et al., 2009) inhibited agonist stimulated FFA1 receptor calcium responses in transfected cells with respective pIC₅₀ values of 6.0, 7.7, and 6.0.

Figure 3

Summary of functional assays available to investigate FFA G protein coupled receptor pharmacology in cell lines. The indirect approaches to assessing ligand binding indicated here are discussed further in Section “Determining Ligand Affinity at FFA1 and GPR120.” Several signaling endpoints related to the G protein (FFA1, GPR120) or β-arrestin pathways (GPR120) can be measured using the example assays in italics (see text, Functional Assessment of FFA1 and GPR120 Pharmacology) culminating in assessment of function at the cellular level. Example data from our own laboratory illustrates (i) calcium responses in HEK293 cells expressing human GPR40 and stimulated with the C18:2 FFA linoleic acid, and (ii) internalization of human GPR120S–YFP receptors (green), following vehicle or oleic acid treatment, also in transfected HEK293 cells. In the image panels nuclei are counterstained with the dye H33342 (blue).