Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): Phenol-HRP redox assay counterscreen for probe SAR
Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132) ..more
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 MH085689-01
Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132)
NCGC Assay Overview:
One mM dithiothreitol (DTT) was used as a reducing agent to maintain the galactokinase (GALK) enzyme in its active form in the GALK qHTS screen (AID: 1868). However, it has been shown that in the presence of DTT a number of compounds also undergo redox recyling, generating a significant amount of H2O2 that can oxidize crucial cysteine residues in the enzyme, leading to and a false-positive result .
To evaluate and flag redox recycling compounds for compounds re-acquired from the qHTS screen, an endpoint colorimetric assay developed by Johnston et al.  was adopted and optimized for a 1536-well plate format. In brief, the assay detects the presence of H2O2 by following the change in absorbance at 600 nm due to oxidation of phenol red (Sigma, cat# 114537) catalyzed by horseradish peroxidase (HRP; Invitrogen). A stop reagent, 1N NaOH was added prior to reading the absorbance. DMSO and different concentrations of hydrogen peroxide (Sigma, cat# 216763) in the GALK buffer were used as negative and positive controls respectively.
NCGC Assay Protocol Summary:
Two uL/well of GALK buffer (20mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was dispensed into a 1536-well assay plates (Greiner, black clear bottom plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound and control solutions (23 nL each) were transferred to the assay plate in their respective wells using Kalypsys pin tool equipped with a 1536-pin tool head and incubated at room temperature for 30 min. Presence of H2O2 produced by redox cycling compounds was detected by adding 2 uL/well phenol red - HRP detection reagent (100 uL/ml phenol red in 1x Hanks Balanced Salt Solution, 25 U/mL HRP final concentrations). After 15 min of room temperature incubation, the reaction was stopped by adding 2 uL/well 1N NaOH and incubated for an additional 1 hr at room temperature. The absorbance at 600 nm was measured using the ViewLux plate reader (Perkin Elmer, Waltham, MA) with 6000 light energy, 2 sec exposure, and 2x binning instrument settings.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)