Development of Fluorescent AF64394 Analogues Enables Real-Time Binding Studies for the Orphan Class A GPCR GPR3

Abstract

The orphan G protein-coupled receptor (oGPCR) GPR3 represents a potential drug target for the treatment of Alzheimer’s disease and metabolic disorders. However, the limited toolbox of pharmacological assays hampers the development of advanced ligands. Here, we developed a signaling pathway-independent readout of compound–GPR3 interaction. Starting from computational binding pose predictions of the most potent GPR3 ligand, we designed a series of fluorescent AF64394 analogues and assessed their suitability for BRET-based binding studies. The most potent ligand, 45 (UR-MB-355), bound to GPR3 and closely related receptors, GPR6 and GPR12, with similar submicromolar affinities. Furthermore, we found that 45 engages GPR3 in a distinct mode compared to AF64394, and coincubation studies with the GPR3 agonist diphenyleneiodonium chloride revealed allosteric modulation of 45 binding. These insights provide new cues for the pharmacological manipulation of GPR3 activity. This novel binding assay will foster the development of future drugs acting through these pharmacologically attractive oGPCRs.

Shedding Light on the D1‐Like Receptors: A Fluorescence‐Based Toolbox for Visualization of the D1 and D5 Receptors**

“…All three precursors were labelled with both fluorescent dyes (5-TAMRA-NHS ester and DY549-P1-NHS ester) in DMF with an excess of NEt 3 as a base (Scheme 3). [61] Purification by preparative HPLC afforded the final fluorescent ligands (23)(24)(25)(26)(27)(28) in moderate to good yields, great purity, and stability (Figures S1-S6; SI).…”

Fluorescent Tools for the Imaging of Dopamine D2‐Like Receptors**

“…In addition, TAMRA ligands have already repeatedly demonstrated their suitability in NanoBRET assays, which opens up another possible application for our fluorescent ligands. [13,22,39] Chemistry One of our aims was to find out how the selection of the respective dye and variation of linker length between the ligand scaffold and the dye influence binding characteristics. Therefore, three different fluorescent ligands (16, 17, and 20) differing in either the dye and/or linker length were designed.…”

“…were coupled in DMF in the presence of triethylamine. [22,39] Purification with preparative HPLC afforded highly pure fluorescent ligands 16, 17, and 20 (> 98 %) with great stability (Figure S1-S4; SI) in good to excellent yields (60-90 %).…”

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1

“…Their complex pharmacology and probe dependence often require labor-intensive testing in various functional assays, which complicate high-throughput screening. Moreover, well-established target engagement methodologies such as radioligand binding may not be available, e.g., because of the lack of high-affinity probes. , Here, the emerging fluorescence-based binding assays represent a valuable alternative, as they do not require extremely high affinities and can be performed in a mix-and-measure setup. , Bioluminescence resonance energy transfer (BRET) ligand binding assays based on fluorescent probes and the optimized nanoluciferase (Nluc) have been established for structurally diverse GPCRs. − Especially, the combination of a receptor tagged to Nluc at its C-terminus and a TAMRA- or bodipy-labeled ligand was shown to be suitable for nanoBRET target engagement assays at the intracellular binding pockets of the chemokine receptor family. − …”