抄録
Background: Understanding how a molecule interacts with its target is the basis of pharmacology. The use of fluorescent ligand probes in combination with Nanoluc-based bioluminescence resonance energy transfer (NanoBRET) technology has been successfully applied (L. Stoddart, 2015). This method represents a powerful pharmacological tool to investigate drug-target binding kinetics in real-time, using whole-living cells. In this study we show the use of NanoBRET to investigate ligand-beta-2-adrenergic receptor (beta2AR) binding kinetics in a highly metastatic breast cancer cell model.
Methods: A triple--negative human breast cancer cell line (MDA-MB-231HM) was stably transfected with N-terminally-tagged-NanoLuc-beta-2AR using fugene, and a reagent:DNA ratio of 3:1. Pharmacological characterisation of this cell line was performed using NanoBRET-based assays, using a fluorescently-labelled Propranolol analogue (Propranolol-BODIPY630/650) and unlabelled antagonists. A Clariostar (BMG Labtek) plate-reader was used to perform sequential fluorescence (emission collected at 635-735nm; BRET acceptor) and luminescence (emission collected at 445-475nm; BRET donor) reads. BRET ratios were calculated as fluorescence/luminescence ratios. Localisation of ligand-receptor interactions was investigated using a luminescence Olympus LV200 microscope (donor - 488/10nm bandpass; acceptor - 600/50nm bandpass), and BRET ratios were measured using ImageJ (Fiji) time series analyser.
Results: Measurement of ligand binding using NanoBRET yielded a binding affinity (pKD) of 7.16 +/- 0.08 (n=5) for Propranolol-BODIPY630/650. A low level of non-specific binding was observed, which was defined by co-incubating cells with 10 micromolar of unlabelled ICI118551 (beta-2AR selective antagonist). Competition binding assay showed pKi values of 8.68 +/- 0.17 (n=5) for Propranolol, and 8.55 +/- 0.05 (n=5) for ICI118551. Binding kinetics under non-equilibrium conditions showed similar binding affinity to measurements taken under equilibrium conditions (pKD = 7.24 +/- 0.14), and a residence time (RT) of 44.1+/- 0.14 min., for the fluorescent ligand studied..
Conclusion: In this study we show the use of NanoBRET technology to measure drug-receptor binding kinetics, under equilibrium and dynamic conditions, using a physiologically relevant whole-cell system.
References:
L. Stoddart et al. (2015). Application of BRET to monitor ligand binding to GPCRs. Nature Methods 12(7):661-633.
