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BioAssay: AID 504547

qHTS Validation Assay to Find Inhibitors of Phosphoglycerate Kinase

throughout the tropical and subtropical world. Sleeping sickness, caused by two subspecies of Trypanosoma brucei, is endemic to areas of sub-Saharan Africa, while Chagas disease, a manifestation of Trypanosoma cruzi infection, is prevalent throughout Central and South America. In addition, many species of Leishmania are responsible for a range of cutaneous and visceral diseases, causing ulcers, more ..
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 Tested Compounds
 Tested Compounds
All(1266)
 
 
Active(19)
 
 
Inactive(1212)
 
 
Inconclusive(35)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(19)
 
 
Inactive(1226)
 
 
Inconclusive(35)
 
 
AID: 504547
Data Source: NCGC (PGK001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-23

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: phosphoglycerate kinase [Trypanosoma brucei]
Description ..   
Protein Family: Phosphoglycerate_kinase

Gene:PGKC     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 19
Related Experiments
AIDNameTypeComment
504632Inhibitors of Phosphoglycerate Kinase: SummarySummarydepositor-specified cross reference
602233qHTS Assay to Find Inhibitors of Phosphoglycerate KinaseConfirmatorydepositor-specified cross reference
686980qHTS for inhibitors of Phosphogylcerate Kinase: Hit Validation ScreenConfirmatorydepositor-specified cross reference
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH093212-01
PI Name: Malcolm Walkinshaw, University of Edinburgh

NCGC Assay Overview:

Several species of the Trypanosomatidae family of parasitic protozoans cause a spectrum of diseases
throughout the tropical and subtropical world. Sleeping sickness, caused by two subspecies of Trypanosoma brucei, is endemic to areas of sub-Saharan Africa, while Chagas disease, a manifestation of Trypanosoma cruzi infection, is prevalent throughout Central and South America. In addition, many species of Leishmania are responsible for a range of cutaneous and visceral diseases, causing ulcers, destructive lesions and kalaazar, a lethal infection of the viscera. Collectively, these organisms infect 30 million people worldwide (~500 million live in endemic areas), and account for ~130,000 deaths annually. Given their debilitating and prevalent nature, trypanosomatid parasites impose a heavy medical, economic and social burden on entire populations, primarily in the developing world. The problem is further exacerbated by the fact that existing treatments have progressed little in the past 50 years, and current drugs show high toxicity and poor efficacy.

This project will build on previous work that has demonstrated carbohydrate metabolism to be a strong drug target in trypanosomatids [1]. Blood-stream stages of T. brucei are entirely dependent on glycolysis as a source of ATP, and the tricarboxylic acid cycle and oxidative phosphorylation are both repressed. Glycolysis in axenically cultured T. cruzi, representing the intracellular pathogenic stage, is also essential for these parasites [2]. Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner, with the first seven of the ten enzymes sequestered inside organelles called glycosomes [3], with the consequence that many enzymes differ significantly from their host counterparts. Previous studies have shown that the glycolytic enzyme, phosphoglycerate kinase (PGK), is a viable target for drugs to kill these parasites. Known inhibitors of TbPGK (glycosomal isoenzyme) include the small molecules suramin [4], Ib1 [5], a bisubstrate analog [6], as well as a series of adenosine derivatives [7]; several such compounds were demonstrated to effectively stunt the growth of T. brucei and T. cruzi parasites in culture [1].
Protocol
NCGC Assay Protocol Summary:
A stepwise description of the 1536-well assay is shown in Table 2. Briefly, 3ul of substrate/PGK buffer (columns 1,2,5-48) are dispensed into Greiner white solid bottom 1536-well assay plates; columns 3-4 receive only TEA buffer as 0x PGK controls. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10nl slotted pins, V&P Scientific, San Diego, CA). Following addition of compound, 1ul of GAPDH (100nM final concentration, all columns) is added to initiate the reaction (this upstream coupling enzyme is necessary for in-well production of PGK substrate, 1,3-bisphosphoglycerate (1,3-BPG), which is unstable and not commercially available). The plates are then incubated at room temperature for 60 minutes. Kinase-Glo Plus reagent is added (2ul) and plates incubated for 10 minutes, before being transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein single end-point measurements of luminescence are acquired using a clear filter. All reagents are diluted in an assay buffer consisting of 100mM TEA (pH 7.6), 5mM MgCl2 and 0.01% Tween-20.
Comment
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". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00368 uM (0.00368μM**)% Activity at given concentration.Float%
15Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
16Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
17Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
18Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
19Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
20Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
21Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH093212-01

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