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BioAssay: AID 492951

Human Glck Counter Screen Assay

The purpose of this assay is to identify compounds that are nonselective or possess potentially undesirable cross reactivity, due to inhibition of the related enzyme, hGlck. This assay employs recombinant hGlck in place of the TbHK1 in the coupled assay. Ideally, IC50s for hGLk will be >10 microM. Please note that while we prefer selective inhibitors of TbHK1, inhibition of the human enzyme does more ..
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 Tested Compounds
 Tested Compounds
All(192)
 
 
Active(77)
 
 
Inactive(115)
 
 
 Tested Substances
 Tested Substances
All(192)
 
 
Active(77)
 
 
Inactive(115)
 
 
AID: 492951
Data Source: Molecular Libraries Program, Specialized Chemistry Center, University of Kansas (KUA10005)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-12-02
Modify Date: 2011-02-14

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 77
Related Experiments
AIDNameTypeProbeComment
1430HTS assay for inhibitors of Trypanosoma brucei hexokinase 1Screening depositor-specified cross reference
1632HTS assay for inhibitors of Trypanosoma brucei hexokinase 1: IC50 determinationsConfirmatory depositor-specified cross reference
2230Confirmation assay for inhibitors of Trypanosoma brucei hexokinase 1-Analogue-first seriesConfirmatory depositor-specified cross reference
2516G6DPH counterscreen for TbHK1 inhibitors - primary screen of DPI cherry picked compoundsOther depositor-specified cross reference
2560Rescreen of TbHK1 primary actives - DPI cherry picked compoundsOther depositor-specified cross reference
2579G6DPH counterscreen for TbHK1 inhibitors - Analogues seriesConfirmatory depositor-specified cross reference
2600Identification of Inhibitors of Trypanosoma Brucei Hexokinases - summary assaySummary1 depositor-specified cross reference
449725IMR-90 (cell viability counter screen)Confirmatory depositor-specified cross reference
Description:
The purpose of this assay is to identify compounds that are nonselective or possess potentially undesirable cross reactivity, due to inhibition of the related enzyme, hGlck. This assay employs recombinant hGlck in place of the TbHK1 in the coupled assay. Ideally, IC50s for hGLk will be >10 microM. Please note that while we prefer selective inhibitors of TbHK1, inhibition of the human enzyme does not indicate removal from consideration, as the trypanosome is exclusively reliant on TbHK1 for metabolism, while host organisms (including humans) have a varied metabolism that may allow them to tolerate inhibition of equivalent enzymes. Compounds determined to inhibit hGlck will be addressed on a case by case basis.
Protocol
Assays are performed in triplicate using purified recombinant human HK 4 (human glucokinase, hGlk, GenBank accession no. BC001890) in a coupled enzyme HK assay in a 96-well format. Briefly, test and control compounds are added to a 96 well microtiter plate containing reaction reagents (0.1M TEA, pH 7.9 containing 1.0 mM ATP, 3.3 mM MgCl2, glucose 6-phosphate dehydrogenase (1 unit/assay, EMD Biosciences, Inc, Sand Diego, CA) and 0.75 mM NAD+) and hGlk, for a final test compound concentration of 10 microM. This mixture is incubated 10 min at RT, with the reaction initiated by addition of 20 mM glucose. The coupling enzyme reduces NAD+ to NADH during the oxidation of glucose-6-P to 6-phosphogluconic acid, a reaction that is monitored spectrophotometrically at absorbance OD340 on a GENios spectrophotometer (Tecan Group Ltd., Switzerland). Negative (vehicle) controls containing 1% DMSO and positive controls containing 10 M ebselen are typically included on experimental plates.
Comment
Compounds with % inhibition > 10 were considered active. For active compounds - activity score range is 100-12, for inactive 11-0. The PubChem activity score was calculated by normalizing the data so the most active compound recieved an activity score of 100.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1% hGlck Inhibition at 10 microMPercent inhibition of hGlck at 10 micromolar.Float%
2Standard Error in % hGlck Inhibition at 10 microMStandard error in Percent inhibition of hGlck at 10 micromolar.Float
Additional Information
Grant Number: 1 R03 MH082340-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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