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BioAssay: AID 540360

Confirmatory Assay to Find Inhibitors of T. brucei phosphofructokinase: probe SAR

Various species of the protozoan family Trypanosomatidae are responsible for a range of serious human diseases in tropical and subtropical areas of the world. The subspecies Trypanosoma brucei is one of three known to cause sleeping sickness in sub-Saharan Africa, significantly contributing to the millions of people worldwide who are infected by these parasites and endangering hundreds of millions more. Many of the disorders caused by trypanosomatids are fatal if left untreated, but most currently used drugs are inefficient and toxic. ..more
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 Tested Compounds
 Tested Compounds
All(40)
 
 
Active(29)
 
 
Inactive(5)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(40)
 
 
Active(29)
 
 
Inactive(5)
 
 
Inconclusive(6)
 
 
AID: 540360
Data Source: NCGC (PFK100)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-08-17
Hold-until Date: 2012-08-05
Modify Date: 2012-08-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 29
Depositor Specified Assays
AIDNameTypeComment
485367qHTS Assay to Find Inhibitors of T. brucei phosphofructokinaseconfirmatoryqHTS assay
488768qHTS Assay to Find Inhibitors of T. brucei Phosphofructokinase: Summarysummarysummary assay
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

Grant: 1 R03 MH092153-01
PI Name: Malcolm Walkinshaw, University of Edinburgh

NCGC Assay Overview:

Various species of the protozoan family Trypanosomatidae are responsible for a range of serious human diseases in tropical and subtropical areas of the world. The subspecies Trypanosoma brucei is one of three known to cause sleeping sickness in sub-Saharan Africa, significantly contributing to the millions of people worldwide who are infected by these parasites and endangering hundreds of millions more. Many of the disorders caused by trypanosomatids are fatal if left untreated, but most currently used drugs are inefficient and toxic.
Of many possible drug targets in trypanosomatid parasites, the carbohydrate metabolism pathway is seen as potentially one of the most selective, as T. brucei, when in the bloodstream of its mammalian host, is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply [1]. Oxidative metabolism involving the mitochondrial tricarboxylic acid cycle and oxidative phosphorylation are repressed in these parasites, and recent RNA interference (RNAi) experiments have shown that even partial depletion of certain individual glycolytic enzymes can lead to the death of cultured parasites [2]. One such glycolytic enzyme, phosphofructokinase (PFK), catalyzes the formation of fructose-1,6-bisphosphate (F1,6BP) and ADP from fructose-6-phosphate (F6P) and ATP, and in many metabolic circumstances makes an important contribution to the control of flux through the glycolytic pathway. As a specific glycolytic target, PFK is particularly attractive as it catalyzes the first irreversible step in glycolysis, and structural and kinetic studies have shown very substantial and essential differences from corresponding host enzymes [3], allowing for the discovery of parasite-selective inhibitors.
The goal of this screen is to find small-molecule inhibitors of T. brucei PFK using a biochemical assay that measures PFK activity as a function of ADP production. A luminescence-based assay for T. brucei PFK activity has been developed which uses luciferase to detect production of ADP. Traditional luciferase-based nucleotide detection reagents rely on ATP production as a direct substrate for the luciferase reaction; however, PFK catalyzes the transfer of a phosphate from ATP to F6P, resulting in the net depletion of ATP. To directly measure the ADP product of the PFK reaction, we used the ADP-Glo detection kit (Promega), which utilizes two separate reactions to 1) deplete all remaining uncatalyzed ATP and 2) convert all remaining ADP to ATP, which is then detected through a traditional luciferase-coupled reaction. Compounds were screened as a concentration-titration series that ranged from 57 uM to 0.3 nM.
Protocol
NCGC Assay Protocol Summary:

NCGC Assay Protocol Summary:

Substrate buffer was dispensed into white, solid 1536-well plates at 3uL/well in 0.1M triethanolamine (TEA) buffer, pH 8.0, containing final concentrations of 5 mM MgCl2, 0.01% Tween20, 0.5 mM fructose-6-phosphate (F6P) and 0.1mM ATP. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL T. brucei PFK (0.1M TEA, 0.01% Tween20, 0.1% bovine serum albumin (BSA) and 1.25nM PFK, final concentrations) was then dispensed, and plates were incubated at room temperature for 45 min. The luminescent detection reagent, ADP-Glo (Promega), was then added in two steps: the first reagent, ADP-Glo (which converts remaining uncatalyzed ATP), was added at 2.5uL/well and incubated for ten minutes at room temperature, and the second component, Kinase Detection reagent (which converts ADP to ATP as a substrate for luciferase-based luminescence), was added immediately after at 5uL/well, followed by one final ten minute room temperature incubation. The plates were measured on a ViewLux plate reader for luminescent signal using a clear filter with a one second exposure. The %Activity was determined from the corrected luminescence values. As no specific T. brucei PFK inhibitors have been identified in the literature, 1x (1.25nM) and 0x PFK enzyme controls (untreated) were included to normalize %Activity of identified inhibitors; 0x enzyme values corresponded to 100%Activity (full inhibition), while 1x PFK enzyme values were used to normalize 0%Activity (no inhibition).

Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent decreases in luminescence, concordant with a decrease in PFK activity and ADP production. Inversely, active activators showed a concentration-dependent increase in luminescence signal above 1x PFK control levels, suggestive of an increase in ADP production. Inactive compounds showed no effect on luminescence signal.

Keywords: T. brucei, phosphofructokinase, PFK, glycolysis, luminescence, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
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.
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 0.0003244253 uM (0.000324425μM**)% Activity at given concentration.Float%
15Activity at 0.0009732759 uM (0.000973276μM**)% Activity at given concentration.Float%
16Activity at 0.00292 uM (0.00291983μM**)% Activity at given concentration.Float%
17Activity at 0.00876 uM (0.00875954μM**)% Activity at given concentration.Float%
18Activity at 0.026 uM (0.0262786μM**)% Activity at given concentration.Float%
19Activity at 0.079 uM (0.0788357μM**)% Activity at given concentration.Float%
20Activity at 0.237 uM (0.236507μM**)% Activity at given concentration.Float%
21Activity at 0.710 uM (0.709522μM**)% Activity at given concentration.Float%
22Activity at 2.129 uM (2.12857μM**)% Activity at given concentration.Float%
23Activity at 6.386 uM (6.3857μM**)% Activity at given concentration.Float%
24Activity at 19.16 uM (19.1571μM**)% Activity at given concentration.Float%
25Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
26Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH092153-01

Data Table (Concise)
Classification
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