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BioAssay: AID 504712

HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 3

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. ..more
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 Tested Compounds
 Tested Compounds
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Active(4)
 
 
Inactive(279)
 
 
 Tested Substances
 Tested Substances
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Active(4)
 
 
Inactive(279)
 
 
AID: 504712
Data Source: Burnham Center for Chemical Genomics (SBCCG-A641-CCR6-beta-GAL-Assay-3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-04-29

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
493098uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assayscreening
493121Summary assay for selective small molecule antagonists of the CCR6 receptorsummarySummary AID.
493159Summary assay for small molecule antagonists of the CCR6 receptorsummarySummary AID.
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.

This assay was developed and performed as a counterscreen for the primary assay originally identified as "uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assay", AID 493098. 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: Hams F-12 supplemented with 2.5% hiFBS, 1X Pen/Strep/Glu,
3) DiscoverX b-Arrestin Detection Reagents: Galacton Star, Emerald II, and Cell Assay Buffer

Dose-Response Protocol:
I. Enzyme addition
1. Using Biotek dispenser, dispense 6.0 ul of assay media to columns 1-4 and 6.0 uL of galactosidase enyme in assay buffer to columns 5-48 of a Corning, white 1536-well plate. Final concentration of enzyme is 0.05 units/ml. This concentration gave ~5% enzyme turnover in 15min.

II. Compound Addition:
2. Using LabCyte Echo 555, transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4 and 45-48, respectively. Using a dose response protocol, transfer compounds from 10mM and 0.312 mM Echo qualified plates into assay plate columns 5 - 45. (Final concentrations range 66 uM to 0.128 uM, 10 doses, with 0.66% DMSO.)
3. Centrifuge plates at 500 rpm for 1 min.

II. Set up of enzyme assay:
4. Using Biotek dispenser, dispense 3 ul Detection mix to all wells.
5. Spin the plates at 1000 rpm for 1 min. Incubate at room temperature for 15 mins.
6. Read plates using a Perkin Elmer Envision using a luminescence protocol (0.1s/well).
Comment
Compounds with IC50 < 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*(pIC50 - 3),
where pIC50 is a negative log(10) of the IC50 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
1IC50_QualifierThis qualifier is to be used with the next TID, . If the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50*IC50 value determined using a sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of the IC50 valueFloatμM
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 66.6 uM_first_point (66.6μM**)% inhibition at the test concentrationFloat%
7% Activity at 33.3 uM_first_point (33.3μM**)% inhibition at the test concentrationFloat%
8% Activity at 16.65 uM_first_point (16.65μM**)% inhibition at the test concentrationFloat%
9% Activity at 8.325 uM_first_point (8.325μM**)% inhibition at the test concentrationFloat%
10% Activity at 4.1625 uM_first_point (4.1625μM**)% inhibition at the test concentrationFloat%
11% Activity at 2.08125 uM_first_point (2.08125μM**)% inhibition at the test concentrationFloat%
12% Activity at 1.040625 uM_first_point (1.04062μM**)% inhibition at the test concentrationFloat%
13% Activity at 0.5203125 uM_first_point (0.520312μM**)% inhibition at the test concentrationFloat%
14% Activity at 0.2601562 uM_first_point (0.260156μM**)% inhibition at the test concentrationFloat%
15% Activity at 0.1300781 uM_first_point (0.130078μM**)% inhibition at the test concentrationFloat%

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

Data Table (Concise)
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