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BioAssay: AID 651728

Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset2

Assay Provider: Jonathan Jarvik, Carnegie Mellon University Screening Center/ PI: UNMCMD/ Larry Sklar ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(4)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(4)
 
 
Inactive(4)
 
 
AID: 651728
Data Source: NMMLSC (UNMCMD_BA_DR_COUNTER_CCR5_POWDERSET2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-10-30
Modify Date: 2012-12-06

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: CCR5 [Homo sapiens]
Description ..   

Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 4
Related Experiments
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AIDNameTypeComment
651701Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactionsSummarydepositor-specified cross reference: Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactions
504448Summary of HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor InternalizationSummarysame project related to Summary assay
588775Dose response for HTS for Beta-2AR agonists via FAP method from Powderset1Confirmatorysame project related to Summary assay
623882Dose response for HTS for Beta-2AR agonists via FAP method from Powderset2Confirmatorysame project related to Summary assay
623947Dose response for HTS for Beta-2AR agonists via FAP method from Powderset3Confirmatorysame project related to Summary assay
651694Cytotoxicity Dose Response with AM2.2-beta2AR cells for PowderSet01Confirmatorysame project related to Summary assay
651698Counter screen for HTS for Beta-2AR agonists with FAP-tagged mouse CCR5 with Powderset1Confirmatorysame project related to Summary assay
651729Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset2Confirmatorysame project related to Summary assay
651853Counter screen for HTS for Beta-2AR agonists with FAP-tagged human GPR32 with Powderset3Confirmatorysame project related to Summary assay
651855Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset3Confirmatorysame project related to Summary assay
651870Cytotoxicity Dose Response with AM2.2-beta2AR cells for PowderSet4Confirmatorysame project related to Summary assay
651872Dose response for HTS for Beta-2AR agonists via FAP method from Powderset4Confirmatorysame project related to Summary assay
651873Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset4Confirmatorysame project related to Summary assay
651875Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset4Confirmatorysame project related to Summary assay
651912Dose response for HTS for Beta-2AR agonists via FAP method from Powderset5Confirmatorysame project related to Summary assay
651913Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset5Confirmatorysame project related to Summary assay
651914Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset5Confirmatorysame project related to Summary assay
651936Evaluation of reversibility of compound binding to AM2.2-beta2AR cells with Powder Set 3and4Othersame project related to Summary assay
651938Fluorogen/soluble FAP binding competition assayOthersame project related to Summary assay
Description:
University of New Mexico Assay Overview:
Assay Support: R03 MH093192-01
Project Title: HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor Internalization
Assay Provider: Jonathan Jarvik, Carnegie Mellon University Screening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist: Yang Wu
Chemistry Center/ PI: Vanderbilt Specialty Chemistry Center/Craig Lindsley Chemistry Center Lead: Shaun Stauffer
Assay Implementation: Yang Wu, Phillip Tapia

Assay Background and Significance:

G protein-coupled receptors represent the largest family of proteins in the human genome with an estimated number of approximately 800. Because of their central involvement in almost every aspect of human physiology, they also represent the largest target for medical intervention [Lin and Civelli, Annu Med 36 (2004), 204-14]. Today, GPCRs represent the target of approximately 30-40% of all drugs on the market. Indeed, of the 50 top-selling drugs in the United States in 2007, 18 target GPCRs, with combined sales of approximately 25 billion dollars.

Of about 800 GPCRs, 500 are chemosensory, representing the chemokine/chemoattractant GPCRs, and the olfactory and gustatory GPCRs. Although the former have been thoroughly characterized for the most part, members of the latter, particularly the olfactory receptors which may include the important category of pheromones, have had only a small number of ligands identified. The remaining GPCRs, approximately 360, constitute the transmitter GPCRs. Of these, approximately 100 receptors still have no known ligand. Such receptors with no known physiological ligand are referred to as orphan receptors and arose from cloning strategies based on limited homology within almost all GPCRs (predominantly during the 1990's) as well as the sequencing of the human genome (in 2000) [Chung et al, Br J Pharmacol 153 Suppl 1 (2008), S339-46].

The search for endogenous ligands for orphan GPCRs has been challenging. This process has given rise to the field of reverse pharmacology, which uses orphan GPCRs to identify novel ligands, which together often lead to the characterization of new physiological paradigms. Over the last 20 years, this approach has led to the deorphanization of about 300 GPCRs. Many of the ligands were already known and their biology characterized, but their receptor was unknown. But in some of these instances, this process also led to the identification of novel transmitters [Civelli et al., Pharmacol Ther 110 (2006), 525-32].

During the 1990's, approximately 10 GPCRs were deorphanized per year. However, very few have been deorphanized since 2004. In addition, no novel transmitters have been identified since that time [Chung et al, Br J Pharmacol 153 Suppl 1 (2008), S339-46]. Given these facts, the question arises as to whether the remaining orphan GPCRs will be readily deorphanized. One major issue is that the pool of known transmitters for which no receptor is known has been essentially depleted. Since all the possible transmitters have now been matched to GPCRs, the current orphan GPCRs can only bind unknown ligands, for which the identification is also slow and resource intense. This is particularly true given that the concentrations of many transmitters in vivo is exceedingly low, making their identification difficult. It is also possible that the expression of some ligands may be developmentally or environmentally regulated.

Almost all current reverse pharmacological/screening approaches rely on monitoring second messenger levels such as calcium mobilization, cAMP production and transcriptional activation. Thus, successful screening requires knowledge of the pathway for a given receptor, in particular the G protein to which the receptor couples. As the number of heterotrimeric G protein combinations is large, this is not a trivial task.

An alternate approach to measuring signal transduction is the monitoring receptor internalization. Virtually all known GPCRs undergo activation-dependent internalization as a mechanism to reduce cell surface receptor numbers. Internalization does not require G protein coupling. Instead, the activity of one of four G protein receptor kinases (GRKs) is required. In most instances, the binding of an accessory protein, arrestin, is also required. One screening approach that has been developed is the recruitment of GFP-tagged arrestin to either the plasma membrane or intracellular endosomes.

The beta-Arrestin clustering assay, developed by Norak Technologies, requires high resolution imaging, well spread, adherent cells, and extensive image analysis to determine the response to a treatment. Other methods, such a receptor desensitization measurements on non-permeabilized cells rely on measurement of subtle changes in intensity. As described below, the CMU TCNP is developing sensors for GPCR responses that are readily compatible with HTS flow cytometry and multiplexing when the GPCRs are expressed in suspension cell lines.
Protocol
This is a counter screen for the beta-2AR screens. This assay measures binding of fluorescent FAP-tagged humanCCR5 in the presence of test compounds to assess if compound interferes with FAP binding.

1. Spin down MG13-hCCR5 cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5x10^6 cells/milliL.
2. Add 5microL serum free RPMI to Columns 2-24 of the assay plate by Nanoquot
3. Add 5microL of freshly prepared Rantes in RPMI full media to Column 1 of all the plates as PCntrls by Microflow
4. Add 100nL of library compounds to assay plates by FX or NX
5. Add 3microL of cells to Columns 1 - 22 of the assay plates by Nanoquot.
6. Shake the plates and put them in 37 deg C incubator for 90mins.
7. Add 3microL 210nM MG2p to assay plates by Microflow or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.

Calculations:

Median Channel fluorescence are calculated from flow cytometric data by HyperView (IntelliCyt, Albuquerque, NM). Then these values for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with EC50 less than 10 microM are labeled active and the PubChem_Score is calculated based on EC50 by the following equation:
PubChem Score = 100 * (1 - EC50/10 microM)
Keywords: beta2AR, UNMCMD
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_QUALIFIERQualifier for the value of EC50String
2LogEC50Log of the EC50 estimateFloat
3 EC50_MICROM*Effective concentration of half maximal event count as estimated by curve fitFloatμM
4EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
5EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
6BOTTOMResponse value at the bottom plateauFloat
7TOPResponse value at the top plateauFloat
8HILLSLOPEHill slope estimate for the fitted dose response curveFloat
9STD_BOTTOMStandard error for the response value at the bottom plateauFloat
10STD_TOPStandard error for the response value at the top plateauFloat
11STD_HILLSLOPEStandard error for the hillslopeFloat
12PERCENT_SPANSpan between MCF values at top test concentration and bottwom test concentrationFloat
13RSQRCorrelation coefficient for the fitted dose response curveFloat
14N_POINTSNumber of data points for each dose response curveInteger
15RAW_MCF_0.000_MICROM (0μM**)Average MCF (Median Channel Fluorescence) measured with no test compound, n=2Float
16RAW_MCF_0.003_MICROM (0.003μM**)Average MCF (Median Channel Fluorescence) measured with 0.003 micromolar concentration of test compound, n=2Float
17RAW_MCF_0.010_MICROM (0.01μM**)Average MCF (Median Channel Fluorescence) measured with 0.01 micromolar concentration of test compound, n=2Float
18RAW_MCF_0.031_MICROM (0.03μM**)Average MCF (Median Channel Fluorescence) measured with 0.03 micromolar concentration of test compound, n=2Float
19RAW_MCF_0.098_MICROM (0.1μM**)Average MCF (Median Channel Fluorescence) measured with 0.1 micromolar concentration of test compound, n=2Float
20RAW_MCF_0.310_MICROM (0.31μM**)Average MCF (Median Channel Fluorescence) measured with 0.31 micromolar concentration of test compound, n=2Float
21RAW_MCF_0.985_MICROM (0.99μM**)Average MCF (Median Channel Fluorescence) measured with 0.99 micromolar concentration of test compound, n=2Float
22RAW_MCF_3.128_MICROM (3.13μM**)Average MCF (Median Channel Fluorescence) measured with 3.13 micromolar concentration of test compound, n=2Float
23RAW_MCF_9.927_MICROM (9.93μM**)Average MCF (Median Channel Fluorescence) measured with 9.93 micromolar concentration of test compound, n=2Float
24RAW_MCF_100.0_MICROM (100μM**)Average MCF (Median Channel Fluorescence) measured with 100 micromolar concentration of test compound, n=2Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH093192-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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