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:
Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called Classic Galactosemia (OMIM 230400). Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. Therefore, finding inhibitors of GALK is a potential novel therapy for this inherent metabolic disease and is the long term goal of this project.
GALK (provided by the assay submitter) was assayed using ATP and D-(+)-Galactose (Sigma-Aldrich cat# G0750) as substrates [1]. Promega Kinase-Glo Plus (cat# V3774) technology was used to detect the residual ATP following kinetic reaction. Briefly, the Kinase-Glo Plus contains Ultra-Glo [2] luciferase and D-luciferin which generates a bioluminescence signal from the remaining ATP. ATP-galactose solution (35 uM ATP, 100 uM galactose, 20mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) and Protein Tyrosine Phosphatase CD45 inhibitor (EMD Bioscience cat# 540215) which was shown to inhibit GALK, were used as a positive controls[1].

NCGC Assay Protocol Summary:

Three uL/well of ATP-buffer solution (35 uM ATP, 20mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was dispensed into 1536-well, assay plates (Greiner, solid white medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using the Kalypsys 1536-pin tool. One uL/well GALK-galactose solution (5 nM GALK, 100 uM galactose, 20 mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was then added using the FRD yielding a total kinase reaction volume of 4 uL/ well. After 1 hour of room temperature incubation, 4 uL Kinase-Glo Plus reagent was added for a final assay volume of 8 uL/ well. Luminescence was detected with the ViewLux plate reader (Perkin Elmer, Waltham, MA) after 10 min incubation using a 1 sec exposure time and 2x binning.

Compound Ranking:

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.
3. Compounds that interfere with the Ultra-Glo luciferase could interfere with this assay. PubChem AID: 1379 can be used as counter-screen for this [2]

Grant Number: R03 MH085689-01