Bioluminescence Resonance Energy Transfer as a Screening Assay: Focus on Partial and Inverse Agonism

Abstract

The reported data for compound screening with the bioluminescence resonance energy transfer (BRET 2 ) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven β2 adrenergic receptor (β2-AR) agonists were tested for full or partial agonism in an improved version of the receptor/β-arrestin2 BRET 2 assay and in 2 cyclic adenosine monophosphate (cAMP) assays (column cAMP assay and ALPHAscreen™ cAMP assay). Tested in the highly sensitive ALPHAscreen™ cAMP assay, all selected agonists behaved as full agonists, using isoproterenol as a reference compound. In the less sensitive column cAMP assay, ephedrine and dopamine had a clear partial response. For the BRET 2 assay, a highly graded picture was obtained. Moreover, β2-AR antagonists were tested for inverse agonism. Pronounced inverse agonism was detected in the ALPHAscreen™ cAMP assay. Only marginal inverse agonistic responses were seen for alprenolol and pindolol in the column cAMP assay, and no inverse agonism was seen in the BRET 2 assay. For the β2-AR, the BRET 2 assay may be superior for secondary screening of agonists where a separation of full and partial agonists is needed and the ALPHAscreen™ cAMP assay may be preferred for primary screening of agonists where all receptor activating compounds are desired. (Journal of Biomolecular Screening 2007:41-49)

Quantification of Ligand Bias for Clinically Relevant β2-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

“…Hypertension, angina, IHD, heart failure (Helfand et al, 2007;Baker et al, 2011) Bucindolol 8.92 (Ponicke et al, 2002Baker, 2010) ↑ 9.40 (Baker, 2010) Nonselective bAR antagonist Heart failure (Pauwels et al, 1991) .5.00 (Galandrin and Bouvier, 2006) Carvedilol 9.02-9.40 (Ponicke et al, 2002;Hoffmann et al, 2004;Baker, 2005) ↑ 9.10 (Baker et al, 2003) a 1 AR and bAR antagonist Hypertension, IHD, heart failure, post-MI (Helfand et al, 2007;Ram, 2010;Baker et al, 2011) .5.00 (Galandrin and Bouvier, 2006) (2)-Epinephrine 5.99-6.54 (Frielle et al, 1988;Del Carmine et al, 2002;Hoffmann et al, 2004;Baker, 2010) ↑ 7.74-8.22 (Liapakis et al, 2004;Elster et al, 2007;Baker, 2010) Nonselective endogenous aAR and bAR agonist Allergic reactions, asthma, cardiac failure, hemorrhage, shock (McLeanTooke et al, 2003)…”

“…Hypertension, heart failure (Ram, 2010;Baker et al, 2011) (2)-Norepinephrine 4.19-5.41 (Frielle et al, 1988;Hoffmann et al, 2004;Del Carmine et al, 2002;Baker, 2010) ↑5.61-7.46 (Liapakis et al, 2004;Elster et al, 2007;Baker, 2010) Nonselective endogenous aAR and bAR agonist Severe hypotension (Ferguson-Myrthil, 2012) Pindolol 8.32-9.32 (Pauwels et al, 1988;Del Carmine et al, 2002;Hoffmann et al, 2004;Baker, 2010) ↑ 8.96 (Baker et al, 2003) Nonselective bAR antagonist Hypertension (Helfand et al, 2007) ↓ 7.44 (Chidiac et al, 1994) (6)-Propranolol 9.08-9.37 (Del Carmine et al, 2002;Ponicke et al, 2002;Hoffmann et al, 2004;Baker, 2005) ↓ 8.10-9.17 (Baker et al, 2003;Galandrin and Bouvier, 2006) Noncardioselective bAR antagonist Hypertension, angina, atrial arrhythmia, portal hypertension, anxiety, tremor, thyrotoxicosis, migraine …”

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

“…BRET-The protocol for BRET measurements was developed for the Mithras LB 940 plate reader (Berthold Technologies) as described previously (15,16). Briefly, 48 h after transfection, the cells were harvested and washed twice in PBS.…”

“…For the bioluminescence resonance energy transfer (BRET) assay, the B2AR was tagged C-terminally with Renilla luciferase and cloned into the pcDNA3.1ϩ vector. ␤-arrestin2 was cloned into pcDNA3.1ϩ vector and tagged N-terminally with green fluorescent protein (GFP) as described previously (15,16).…”

Beyond Standard Molecular Dynamics: Investigating the Molecular Mechanisms of G Protein-Coupled Receptors with Enhanced Molecular Dynamics Methods

“…One, close to the inactive, s ~0.2, was marginally (~1 kcal/mol) more stable than the second state, at s ~0.6. This type of free energy profile illustrates possible reasons for some studies having found alprenolol to behave in different ways in different assays: most notably, as an inverse agonist (Elster et al 2007; Hopkinson et al 2000) or as a partial agonist (Callaerts-Vegh et al 2004). …”