Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors

Abstract

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]­melatonin and 2-[125I]­iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

Melatonin receptor structure and signaling

“…2-[ 125 I]iodomelatonin, [ 3 H]melatonin binding assays [91] Nano-BRET ligand binding assay [92] GTPγS 35 binding assay [93] BRET G protein activation assay [5,6] ß-arrestin recruitment assay [5,94,95] Internalization assay [96] cAMP assay [97] Impedance measurement [98] BRET receptor dimerization assay [99] future direction. MT 1 has been shown to signal from the plasma membrane and from intracellular compartments such as mitochondria.…”

Thirty‐seven years of MT1 and MT2 melatonin receptor localization in the brain: Past and future challenges

“…Several fluorescent melatonin receptor ligands have already been generated and evaluated pharmacologically. [99][100][101] However, to this day, none of these ligands has been evaluated for the detection of melatonin binding in tissue sections. Nevertheless, further development of such fluorescent binding probes will probably generate new interesting tools for the localization of melatonin receptors even in living cells and tissues.…”

“…Beyond their use for studying receptor pharmacology, such ligands can be combined with single molecule fluorescent imaging to uncover even the subcellular localization of their receptors. Several fluorescent melatonin receptor ligands have already been generated and evaluated pharmacologically 99–101 . However, to this day, none of these ligands has been evaluated for the detection of melatonin binding in tissue sections.…”

Molecular Characterization and Pharmacology of Melatonin Receptors in Animals

“…Labeled subtype-specific ligands are highly desirable to determine the specific receptor present in cells and tissues naturally expressing the receptor(s). Outside of the strict domain of labelled compounds, recent studies reported the development of fluorescent ligands at melatonin receptors [80] as well as the synthesis of light-activatable caged melatonin compounds [81]. These molecules represent key elements in the toolbox for the pharmacological studies of melatonin receptors.…”

“…"No Aff" means no affinity. b pK d values from Gbahou et al[80]. All other values are from Legros et al[77].…”