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BioAssay: AID 485352

HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 2

b-galactosidase (b-gal), a hydrolase enzyme that catalyzes the hydrolysis of b-galactosides to monosaccharides is utilized in many different screening technologies involving enzyme reaction coupling and reporter assays, for example DiscoverX b-Arrestin GPCR assays such as the APJ Agonist or Antagonist. ..more
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 Tested Compounds
 Tested Compounds
All(385)
 
 
Active(11)
 
 
Inactive(374)
 
 
 Tested Substances
 Tested Substances
All(385)
 
 
Active(11)
 
 
Inactive(374)
 
 
AID: 485352
Data Source: Burnham Center for Chemical Genomics (SBCCG-A440-beta-GAL-Assay-2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-05
Modify Date: 2011-07-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
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AIDNameTypeComment
1966HTS Dose response counterscreen for assays utilizing the enzyme, b-galactosidaseConfirmatorydepositor-specified cross reference: Set 1.
2569Summary assay for small molecule antagonists of the APJ receptorSummarydepositor-specified cross reference: Summary AID.
2521uHTS identification of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
2766Single concentration confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
2784Dose Response confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
463109SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
463214Dose Response screen for antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptorConfirmatorysame project related to Summary assay
488803SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay - Set 2Confirmatorysame project related to Summary assay
488810SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptorConfirmatorysame project related to Summary assay
488992SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay - Set 3Confirmatorysame project related to Summary assay
488994SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor - Set 2Confirmatorysame project related to Summary assay
492984SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor - Set 3Confirmatorysame project related to Summary assay
492986SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay - Set 4Confirmatorysame project related to Summary assay
504436SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay - Set 5Confirmatorysame project related to Summary assay
504438SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor - Set 4Confirmatorysame project related to Summary assay
504455SAR analysis of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay - Set 6Confirmatorysame project related to Summary assay
504457SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor - Set 5Confirmatorysame project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1X01DA026208-01
Assay Provider: Dr. Lawrance Barak , Duke University, Durham, NC

b-galactosidase (b-gal), a hydrolase enzyme that catalyzes the hydrolysis of b-galactosides to monosaccharides is utilized in many different screening technologies involving enzyme reaction coupling and reporter assays, for example DiscoverX b-Arrestin GPCR assays such as the APJ Agonist or Antagonist.
This assay was developed and performed as a counterscreen for screening assays that utilize b-gal and a reaction that it catalyzes. By detecting inhibitors and activators of this enzyme, it is possible to attribute activity not to the primary assay in question, but rather to interaction with the method of detection.

References

Fowler et al. (1970). "The amino acid sequence of b-galactosidase". J. Biol. Chem. 245 (19): 5032. http://www.jbc.org/cgi/reprint/245/19/5032.
Matthews B (2005). "The structure of E. coli beta-galactosidase". C R Biol 328 (6): 549 V56.

Zhao, X et al. (2008). A homogeneous enzyme fragment complementation-based b-Arrestin translocation assay for high-throughput screening of G-protein-Coupled receptors. J Biomol Screen. 13(8):737-747.

McGuinness, D, et al. (2009) Characterizing Cannabinoid CB2 Receptor Ligands using DiscoveRx PathHunter Assay. J Biol Chem. 284(18):12328-12338 (ProLink Cloning Vector and HEK 293 EA-Arrestin Parental Cell Line)
Protocol
Assay materials:

1) Beta-Galactosidase from Sigma-Aldrich (Cat# G4155)
2) Assay Medium: Opti-MEM Medium supplemented with 1% hiFBS, 1X Pen/Strep/Glu, 125 ug/mL Hygromycin (1/2 recommended), 250 ug/mL Geneticin (1/2 recommended)
3) DiscoverX b-Arrestin Detection Reagents: Galacton Star, Emerald II, and Cell Assay Buffer
Dose Response protocol:
1) Add 5.0 uL of b-gal diluted in assay media to 0.03 U/mL to all wells (0.02 U/mL final assay concentration) of a 1536-well plate with the exception of the positive control wells where 5.0 uL of assay media alone is added.
2) Spin plates at 500 rpm for 1 min.
3) Prepare Detection Reagent Solution from DiscoveRx (1 part Galacton Star: 5 parts Emerald II and 19 parts Cell Assay Buffer).
4) Using a Labcyte Echo, DMSO and test compounds are transferred to assay wells. DMSO only is transferred to positive and negative control wells, while varying volumes of test compounds are transferred to test compound wells to achieve the desired test concentrations. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
5) Spin plates at 500 rpm for 1 min.
6) Add 2.5 uL of Detection Reagent Solution to all wells of assay plate and immediately spin plates at 500 rpm for 1 min.
7) After 15 minutes, read assay plates on a Perkin Elmer Envision using a luminescence protocol
Comment
Compounds with EC50 < 40 uM are considered "active" in this assay. Due to the counter-screen goal of the assay, activity in this assay is an indicator of compounds being potential artifacts in the primary assays based on b-galactosidase detection. This interference needs to be assessed for each primary assay individually by comparing the results of the two assays. In general, activity comparable in potency in the counterscreen relative to the primary screen is a strong indication that the compound is a false positive of the primary assay, merely interfering with the b-galactosidase detection.

To simplify the distinction between the actives of the primary screen and of the confirmatory dose response, a tiered activity scoring system was devised to take into consideration compound efficacy and potential interference with the assay. The outline of the scoring system utilized for the screen is as follows:

1) First tier (0-40 range) is reserved for primary screening data and therefore is not applicable in this assay.

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The score is calculated as:
Score = 44 + 6*(pEC50 - 3),
where pEC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_QualifierThis qualifier is to be used with the next TID, EC50. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
2EC50*EC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(EC50)Standard Error of EC50 valueFloat
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
6% Activity at 40 uM_first_point (40μM**)% Activity at a given concentrationFloat%
7% Activity at 20 uM_first_point (20μM**)% Activity at a given concentrationFloat%
8% Activity at 10 uM_first_point (10μM**)% Activity at a given concentrationFloat%
9% Activity at 5 uM_first_point (5μM**)% Activity at a given concentrationFloat%
10% Activity at 2.5 uM_first_point (2.5μM**)% Activity at a given concentrationFloat%
11% Activity at 1.25 uM_first_point (1.25μM**)% Activity at a given concentrationFloat%
12Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
13% Activity at 40 uM_second_point (40μM**)% Activity at a given concentrationFloat%
14% Activity at 20 uM_second_point (20μM**)% Activity at a given concentrationFloat%
15% Activity at 10 uM_second_point (10μM**)% Activity at a given concentrationFloat%
16% Activity at 5 uM_second_point (5μM**)% Activity at a given concentrationFloat%
17% Activity at 2.5 uM_second_point (2.5μM**)% Activity at a given concentrationFloat%
18% Activity at 1.25 uM_second_point (1.25μM**)% Activity at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1X01DA026208-01

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