title: 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 (BRET2) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven beta2 adrenergic receptor (beta2-AR) agonists were tested for full or partial agonism in an improved version of the receptor/beta-arrestin2 BRET2 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 BRET2 assay, a highly graded picture was obtained. Moreover, beta2-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 BRET2 assay. For the beta2-AR, the BRET2 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.
authors.affiliation: 7TM Pharma Fremtidsvej 3, Hørsholm Denmark. le@7tm.com
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Elster
authors.forename: Christian
authors.initials: C
authors.lastname: Elling
authors.forename: Anders
authors.initials: A
authors.lastname: Heding
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists / Q000032:analysis* / Q000494:pharmacology*; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000420:Albuterol / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000494:pharmacology; D004735:Energy Transfer; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D000068299:Salmeterol Xinafoate; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D000068299:Salmeterol Xinafoate; D000242:Cyclic AMP; D007545:Isoproterenol; D000420:Albuterol