qHTS validation assay of beta-arrestin-biased ligands of beta2-adrenergic receptor
The concept of biased agonism  is an emerging concept of molecular pharmacology which proposes that seven transmembrane receptors (7TMRs) or G protein-coupled receptors (GPCRs), exist in multiple signaling conformations and complexes and that ligands (either agonists or antagonists) can induce signaling biased responses for distinct signal transduction cascades. ..more
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: Fast-track/HHSN271700800025C
PI: Bryan Roth and Bob Lefkowitz
The concept of biased agonism  is an emerging concept of molecular pharmacology which proposes that seven transmembrane receptors (7TMRs) or G protein-coupled receptors (GPCRs), exist in multiple signaling conformations and complexes and that ligands (either agonists or antagonists) can induce signaling biased responses for distinct signal transduction cascades.
Recently, this concept has been highlighted by beta-arrestin-biased signaling through 7TMRs . beta-arrestins have been known to interact with agonist-occupied 7TMRs and thus to sterically inhibit coupling of the receptor to G proteins, leading to termination of signaling. Furthermore, accumulating evidence over the last decade has drawn attention to a novel function of beta-arrestins as signal transducers that scaffold various signaling molecules upon activation of 7TMRs. In particular, beta-arrestins have been shown to mediate activation of several signaling pathways upon stimulation of 7TM receptors even in the absence of G protein activation . This beta-arrestin-biased signaling presumably leads to distinct physiological outcomes from those by G protein activation.
Additionally, the active receptor conformations which prefer on this beta-arrestin signaling are likely different from those activating G protein signaling
One of the systems in which biased agonism has been thoroughly elucidated is beta2-adrenergic receptors (beta2-ARs), where the 'antagonist' ligand carvedilol has been demonstrated to selectively activate beta-arrestin-mediated signaling pathways .
The goal of this screening project will be to discover and optimize novel beta-arrestin-biased ligands for beta2-AR signaling. A luminescent cell-based assay for beta2-AR-mediated agonism, known as the Tango assay , has been validated in which ligand binding of a recombinant beta2-AR and subsequent recruitment of a recombinant beta-arrestin lead to the release of the transcriptional activator tTA, which upregulates transcription of a tTA-responsive luciferase gene. Compounds with beta2-AR agonist activity lead to an increase in luciferase expression, and upon addition of a luciferase detection reagent, demonstrate an increase in luminescence. Compounds were screened as a concentration-titration series that ranged from 46 microM to 0.6 nM.
NCGC Assay Protocol Summary:
Tango cells were dispensed into white, solid, tissue-culture-treated 1536-well plates at 1500 cells/well in DMEM, containing 1% fetal bovine serum (FBS). Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. Plates were incubated at 37 degrees C, 5% CO2 for 18 hr. Then 2.5 uL luminescent substrate mix (in-house prepared: 150 mM Tris-Cl, pH 7.6, 3 mM MgCl2, 1% Triton X-100, 5 mM DTT, 0.5 mM CoA, 0.15 mM ATP, 0.44 mM D-luciferin, final concentrations) was added to each well. Plates were then incubated for 15 minutes at room temperature, and subsequently measured on a ViewLux plate reader for luminescent signal using a clear filter with a 10 second exposure and 2x binning. The %Activity was determined from the corrected luminescence values. Isoproterenol, a well-characterized beta2-AR full agonist, and metaproterenol, a less potent beta2-AR agonist, were included both in 16-pt titration and at full response concentration to normalize %Activity of identified agonists; DMSO-pinned controls were used to normalize 0%Activity (zero agonism), while EC100 isoproterenol and/or EC100 metaproterenol were used to normalize 100%Activity (full agonism).
Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active agonists showed concentration-dependent increases in luminescence, concordant with an increase in bound beta2-AR and luciferase expression. Inactive compounds showed no effect on luminescence signal.
Keywords: biased agonism, beta2-AR, beta-arrestin, GPCR, 7TMR, Tango, luminescence, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)