Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer

Abstract

The free fatty acid receptor 1 (FFA1/GPR40) is a potential target for treatment of type 2 diabetes. Although several potent agonists have been described, there remains a strong need for suitable tracers to interrogate ligand binding to this receptor. We address this by exploring fluorophore-tethering to known potent FFA1 agonists. This led to the development of 4, a high affinity FFA1 tracer with favorable and polarity-dependent fluorescent properties. A close to ideal overlap between the emission spectrum of the NanoLuciferase receptor tag and the excitation spectrum of 4 enabled the establishment of a homogenous BRET-based binding assay suitable for both detailed kinetic studies and high throughput competition binding studies. Using 4 as a tracer demonstrated that the compound acts fully competitively with selected synthetic agonists but not with lauric acid and allowed for the characterization of binding affinities of a diverse selection of known FFA1 agonists, indicating that 4 will be a valuable tool for future studies at FFA1.3

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors

“…In summary, new mutagenesis efforts will be instrumental in mapping the precise binding mode of orthosteric and allosteric agonists and antagonists. The recent development of a florescent agonist could further assist in distinguishing between orthosteric and allosteric sites at FFA1 (Christiansen et al 2016). The crystal structure is also helpful in initiation of MD simulation studies to probe flexibility of the receptor and explore the dynamics of the arginine pairing and the surrounding H-bonding network in relation to ligand binding and receptor activation.…”

NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

“…Furthermore, the assay is truly homogenous with no wash or lysis step necessary, enabling kinetic measurements of ligand binding to be performed with relative ease which would be time-consuming and cumbersome in traditional radio-ligand binding studies. Indeed, due to its relative ease of use and adaptability, since the initial description of NanoBRET ligand binding, this method has been used in a variety of binding modes (saturation, kinetic or competition) to investigate fluorescent ligand binding at a number of GPCRs including: adenosine A1 and A3, as well as angiotensin II receptor type 1 17 ; β1 and β2-adrenoceptors 17,19 ; free fatty acid receptors 1 and 2 20,21 ; histamine H1, H3 and H4 receptors 22,23 ; relaxin family peptide receptor 3 24 ; and P2Y2 receptor 25 . It has also been used to study the receptor tyrosine kinase vascular endothelial growth factor 2 26 and its co-receptor neuropilin-1 27 .…”

Structural basis for the agonist action at free fatty acid receptor 1 (FFA1R or GPR40)

“…The conformational constraint not only can improve the pharmacodynamic profile but also can improve the pharmacokinetic profile of derivatives as it can lead to blocking metabolic soft sites, as described for various agonists of GPR40. Additionally, the conformational constraint of using a triple bond can give rise to a new series of agonists for GPR40 (path F), as reported by various studies by the Ulven group in Denmark …”