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
BioActive Compounds: 11
Depositor Specified Assays
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.
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)
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
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)