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
BioActive Compounds: 4
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.
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.
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: 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
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).
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)