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G Protein-Coupled Receptor

GPCRs (G protein-coupled receptors) are the largest and most diverse family of membrane proteins that transduce extracellular stimuli into intracellular signals. They regulate a wide variety of physiological processes and are also well-validated ‘druggable’ targets.

We have used Promega’s NanoBRETTM GPCR reagents to examine the binding kinetics of fluorescently tagged propranolol to β2MAR tracer in a 3D scaffold format. We studied the use of this tracer to measure the competitive binding of known β2MAR compounds and measured the rate at which these compounds displace the tracer.

This format has significant advantages over traditional methods of compound profiling in 2D well plates:

  • Cells adhere to scaffolds and thus, can be effectively moved between plates, replacing buffers without washing and providing the ability to use ‘assay ready compound plates’
  • Cells can be cryo-preserved on scaffolds, then defrosted and used repeatedly from the same stock or batch, therefore improving data consistency

Fig. Bioluminescence Energy Transfer (BRET) to measure compound engagement with the β2 Adrenergic Receptor (β2MAR)

This technology can be applied to other GPCR’s with the benefits of

  • Highly specific detection of binding interactions due to the inherent distance constrains of BRET
  • Small and minimally interfering bioluminescent peptide tag
  • Selective cell surface detection
  • Non-radioactive and homogeneous live cell assay
  • High throughput –can be used for compound profiling in HTS formats

Fig. Binding of fluorescently tagged propranolol to transiently expressed HiBit-β2-AR

HEK-293 cells were transiently transfected with HiBit-β2-AR, grown overnight on scaffolds, then treated with various concentrations of fluorescently tagged propranolol in the absence or presence of 30μM unmodified propranolol. After a 90-minute incubation, HiBit detection reagent was added and cells were incubated for 5 minutes prior to BRET measurement. Data demonstrates specific binding of the fluorescently tagged propranolol to β2-AR that can be competed with an excess of unmodified propranolol (n=3+/-S.D.)

Fig. Competition binding of compounds to β-AR expressed in ‘fresh cells in 2-D’, ‘fresh cells in 3-D’ and cryo-preserved cells in 3-D on scaffolds.

HEK-293 cells were transiently transfected on scaffolds then either cryo-preserved or maintained in culture in 3-D on scaffolds or 2-D in TC treated 96 well white plates. Upon defrosting and a further 24 hrs in culture, all groups were treated with fluorescently tagged propranolol (2nM=EC80) together with various concentrations of competing compounds. After a 90-minute incubation period, cells were incubated with HiBit detection reagent for 5 minutes before BRET was measured.

Dta demonstrates competitive binding of known β2-AR compounds through the displacement of fluorescently tagged propranolol. In addition, cells in 3-D on scaffolds give responses similar to those in 2-D. Furthermore, when recovered from cryo-preservation, cells on scaffolds exhibited very similar IC50 values to those obtained using ‘2-D fresh’, ‘3-D fresh cells’.

Fig. Association rate of fluorescently tagged propranolol to β2-AR.

HEK-293 cells expressing HiBit-β2-AR on 3-D scaffolds were incubated with HiBit detection reagent for 15 minutes, then treated with various concentrations of fluorescently tagged propranolol. Immediately following the addition, BRET was measured every minute for over 10 minutes. Data demonstrates the rapid association rate of fluorescently tagged propranolol for β2-AR (n=3+/-S.D.)

Fig. Rate of displacement of fluorescence tracer from β2-AR by competing compounds. Transiently transfected HEK-293 cells on 3-D scaffolds were incubated with HiBit detection reagent for 15 minutes, then treated simultaneously with both fluorescently tagged propranolol at 2nM (EC80) together with competing compounds at 10 times their IC50 concentration for each compound. Immediately following additions, BRET was measured every minute for 15 minutes. Data demonstrates the rate of displacement of the tracer by each of the compounds and suggests that Salmeterol binds more slowly and Pindolol binds more rapidly to β2-AR than the other compounds (n=3+/-S.D.)

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