Sequence: ATCDPMEK (4-(CYTIDINE 5'-PHOSPHO)-2-C-METHYL-D-ERITHRITOL KINASE); 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase [Arabidopsis thaliana]
Description ..   

Gene:CDPMEK     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Probe	Comment
1379	Counterscreen for Luciferase (Kinase-Glo TM) Inhibition	confirmatory
1868	qHTS Assay for Inhibitors of Human Galactokinase (GALK)	confirmatory
2114	qHTS Assay for Inhibitors of Human Galactokinase (GALK): Summary	summary	1
2547	Secondary Assay for Inhibitors of Human Galactokinase (GALK): HEK-293 Cell Viability Assay	confirmatory
493188	Confirmation Assay for Inhibitors of Human Galactokinase (GALK): SAR round 2	confirmatory

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
The GHMP kinase family is a unique class of important enzymes that plays an essential role in many metabolic processes. It contains more than 170 protein members among which are the galactokinase (GALK) and the CMK enzymes. GALK plays a crucial role in galactose processing and is a potential therapeutic target for Classic Galactosemia (OMIM: 230400) [1]. CMK is involved for the non-mevalonate synthesis of isoprenoids precursors and natural products like sterols or ubiquinones which are important to many biological functions [2]. Although the sequence identity between GALK and CMK is low, the three-dimensional structures and active sites are highly similar [3]. Therefore to determine the validity and selectivity of an active compound observed in the GALK qHTS screen (AID: 1868), a bioluminescent counter-screen targeting bacterial CMK was developed. In addition, because the mevalonate independent synthesis of isoprenoids are more prevalent in opportunistic pathogens such as bacteria [4], CMK is a potential novel therapeutic target where selective inhibitors could be used to determine potential antibacterial effects.
The CMK (provided by Kent Lai) was assayed using ATP and 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME; Echelon, cat# I-M052) as substrates. 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 [5] luciferase and D-luciferin which generates a bioluminescence signal from the remaining ATP. Kinase reactions run with and without the enzyme were use as negative and positive controls together with treatment of Protein Tyrosine Phosphatase CD45 inhibitor (EMD Bioscience cat# 540215) which is observed to weakly inhibit the CMK.

NCGC Assay Protocol Summary:
Three uL/well of substrate solution (15 uM ATP, 30 uM CDP-ME, 20 mM HEPES pH8.0, 5 mM MgCl2, 40 mM KCl, 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 and control solutions (23 nL) were transferred to the assay plate using the Kalypsys 1536-pin tool. One uL/well enzyme solution (0.25 ug/mL CDP-Me kinase, 20 mM HEPES pH8.0, 5 mM MgCl2, 40 mM KCl, 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. After 1 min, luminescence was detected with the ViewLux plate reader (Perkin Elmer, Waltham, MA) using a 1 sec exposure time and 2x binning settings.

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