Background

G protein-coupled receptors (GPCRs) play critical roles in driving cell signaling pathways and remain a prominent target class for drug development (Santos et al., 2017). Accordingly, GPCR ligand binding assays that can quantify biophysical properties critical to drug action are essential for both basic research and drug discovery campaigns (Fang, 2012; Stoddart et al., 2016). Biochemical assessment of ligand binding affinity under equilibrium conditions has traditionally been viewed as an acceptable indicator for in vivo pharmacology of drug candidates (Hulme and Trevethick, 2010). However, there is growing understanding that this approach might be too simplistic. The kinetics of ligand binding provides insights into duration of drug action including residence time and potential for rapid rebinding, which have been postulated to be relevant predictors for in vivo efficacy (Fang, 2012; Copeland, 2016; Sykes et al., 2019). In addition, many GPCRs function as parts of multi-protein signaling complexes, which might influence distinct characteristics of ligand binding (Kenakin, 2010; Fang, 2012). As a result, there is increasing interest in comprehensive measurements of GPCR ligand engagement in the physiological context of living cells in both equilibrium and real-time.

GPCR ligand binding assays routinely employ ligands labeled with either radioisotopes or fluorophores in order to track the binding to a target (Fang, 2012; Stoddart et al., 2016). The most common biochemical GPCR ligand binding assays utilize radioligands in a competitive binding format to measure interactions between ligands and their unmodified cognate GPCRs (Flanagan, 2016). The specificity of these assays relies on the use of highly selective radioligands in combination with overexpressed GPCRs within membrane preparations. In addition, these non-homogenous assays require separation of free from bound radioligand, which limits the resolution and throughput of kinetic analyses (Sykes et al., 2019). Furthermore, the wash steps are more difficult to perform in high throughput screenings.

Proximity binding assays relying on time resolved fluorescence resonance energy transfer (TR-FRET) or bioluminescent resonance energy transfer (BRET) have emerged as attractive alternatives to radioligand binding assays, particularly for high throughput screenings (Stoddart et al., 2016; Sykes et al., 2019). These assays typically use fluorescently labeled ligands (fluorescent Tracers) in a competitive binding format to measure interactions between unmodified ligands and their cognate GPCRs genetically fused to an energy donor (i.e., a lanthanide-labeled protein tag or a luciferase). Although both ligand and GPCR need to be modified, these approaches provide high specificity as signal is generated only when the fluorescent Tracer and tagged GPCR are in close proximity. The inherent distance constraints of energy transfer (Ciruela, 2008) eliminates the requirement for highly selective labeled ligands and permits homogeneous cell-based assays. The homogeneous format allows for real-time kinetic measurements and is generally more compatible with high throughput settings.

The development of the bright NanoLuc luciferase (Hall et al., 2012) enabled BRET-based GPCR ligand binding assays previously impossible with other luciferases (Stoddart et al., 2015; Soave et al., 2016). Still, due to the cell permeability of the NanoLuc substrate furimazine, the use of full length NanoLuc as the energy donor results in signal generation regardless of NanoLuc’s cellular localization. This could lead to an elevated background from NanoLuc-tagged receptors that are present in intracellular compartments and are unable to engage the Tracer. This issue was recently addressed by genetically fusing GPCRs to the luminescent peptide HiBiT rather than the full length NanoLuc (Soave et al., 2019; Boursier et al., 2020). HiBiT is a small 11-amino acid peptide that produces bright luminescence upon high affinity complementation with LgBiT, an 18 kDa subunit derived from NanoLuc (Schwinn et al., 2018). The use of HiBiT restricts signal generation in endpoint assays to the cell surface. This is because LgBiT is cell impermeable and therefore incapable to complement any HiBiT-tagged receptors located in intracellular compartments. An added benefit is the use of a small peptide tag, which is less likely to interfere with protein function and trafficking to the cell surface.

The binding protocols described here couple the specificity of BRET with the sensitivity and inherent cell-surface localization of the HiBiT/LgBiT reporter for quantification of binding interactions with selective GPCRs on the surface of living cells (Figure 1). They include four principal types of assays using β₂-AR ligand binding as an example. Two of these assays utilize increasing concentrations of a fluorescent Tracer to measure its binding characteristics in equilibrium and real-time. The other two assays employ increasing concentrations of an unmodified test compound to derive its binding properties through the competition with a fixed concentration of a fluorescent Tracer. These competition analyses can be performed under equilibrium or in a kinetic format, which take advantage of a method reported by Motulsky and Mahan (1984).


Figure 1. Monitoring cell surface ligand engagement with selective HiBiT-tagged GPCRs via BRET. BRET assay utilizing fluorescent Tracers in a competitive binding format to quantify dynamic cell-surface interactions between ligands and their cognate HiBiT-tagged GPCRs. This approach benefits from high specificity as signal is generated only when the fluorescent Tracer and tagged GPCR are in close proximity.