Fluorescent ligands: Bringing light to emerging GPCR paradigms

Abstract

In recent years, several novel aspects of GPCR pharmacology have been described, which are thought to play a role in determining the in vivo efficacy of a compound. Fluorescent ligands have been used to study many of these, which have also required the development of new experimental approaches. Fluorescent ligands offer the potential to use the same fluorescent probe to perform a broad range of experiments, from single‐molecule microscopy to in vivo BRET. This review provides an overview of the in vitro use of fluorescent ligands in further understanding emerging pharmacological paradigms within the GPCR field, including ligand‐binding kinetics, allosterism and intracellular signalling, along with the use of fluorescent ligands to study physiologically relevant therapeutic agents.

Pharmacological characterisation of novel adenosine A3 receptor antagonists

“…fully used to determine the real-time kinetics of ligand binding to GPCRs [23][24][25] . Here, we investigated the ability of the selective A 3 R antagonists MRS 1220, K17 or K18 to inhibit specific binding of the fluorescent A 3 R antagonist CA200645 to Nluc-A 3 R 26,27 .…”

Fluorescent ligands for dopamine D2/D3 receptors

“…In contrast, NanoBRET binding assays 11 make use of a small and bright luciferase variant (Nluc) 19 , that is fused to the extracellular part of the investigated GPCR. In both cases, binding of the fluorescent ligand is detected based on RET between the receptor as light emitting donor and the fluorescent ligand serving as the acceptor 10 . Both RET-assays have been proven extremely useful for the characterization of ligand binding at GPCRs, especially if high affinity radioligands are not available or if binding kinetics 10 , 23 , 24 are central to the investigated research question.…”

“…In both cases, binding of the fluorescent ligand is detected based on RET between the receptor as light emitting donor and the fluorescent ligand serving as the acceptor 10 . Both RET-assays have been proven extremely useful for the characterization of ligand binding at GPCRs, especially if high affinity radioligands are not available or if binding kinetics 10 , 23 , 24 are central to the investigated research question. Typical saturation hyperbolas were obtained for all three ligands when we performed NanoBRET assays with living HEK293T cells expressing an Nluc-D 3 R fusion protein.…”

“…Over recent years, fluorescence-based technologies have emerged as exciting alternative to study ligand affinity 10 – 12 . Similar to radioactive probes, fluorescent molecules can be detected at very low concentration with high specificity.…”

“…Although the synthesis of small-molecule fluorescent ligands is far from trivial, it requires lower level regulatory and safety precautions compared to the preparation of radioligands. Moreover, several fluorescence-based technologies like resonance energy transfer (RET) and fluorescence anisotropy (also known as fluorescence polarization) do not require separation of unbound ligands and assays can thus be run in a homogenous, fast “mix-and-measure” setup 10 , 13 . In combination with sophisticated techniques like total internal reflection fluorescence (TIRF) microscopy, fluorescent ligands allow to detect and track GPCRs with single molecule resolution 14 – 16 .…”