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BioAssay: AID 624147

qHTS of Trypanosoma Brucei Inhibitors: LOPAC Validation

Members 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 more ..
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 Tested Compounds
 Tested Compounds
All(1265)
 
 
Active(7)
 
 
Inactive(1219)
 
 
Inconclusive(39)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(7)
 
 
Inactive(1234)
 
 
Inconclusive(39)
 
 
AID: 624147
Data Source: NCGC (TbPyK001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-05-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 7
Depositor Specified Assays
AIDNameTypeComment
624155qHTS of Trypanosoma Brucei Inhibitors: Summarysummary
Description:
Members 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. These diseases cause severe public health and economic burdens on populations that are often already caught in a tragic cycle of poverty, poor nutrition and disease. Collectively, parasites of the trypanosomatid family infect 30 million people worldwide (~500 million live in endemic areas), and account for ~130K deaths annually. Existing treatments have developed little in the past 50 years, and suffer from toxicity, poor efficacy and resistance; the goal of this project is to develop lead drugs that will be suitable for entry into pre-clinical trials.

This project will build on previous work that has demonstrated carbohydrate metabolism to be a strong drug target in trypanosomatids. {Verlinde, 2001} Blood-stream or infective stages of T. brucei are entirely dependent on glycolysis as a source of ATP, because 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. {Engel, 1987} This project aims to discover and optimize selective inhibitors of the glycolytic enzyme pyruvate kinase (PYK) from Trypanosoma brucei, the parasite that causes sleeping sickness. TbPYK has been validated as a drug target by RNAi where RNAi knockdown of PYK in T. brucei was found to be lethal to cultured parasites, with a 50% reduction in glycolytic flux sufficient to kill the cells.{Albert, 2005} Detailed structural information for PYK is available. Trypanosomatid PYKs show significant differences from their human counterparts including only 47-50% sequence identity and different allosteric and activation properties.{Callens, 1992} To this end, a qHTS was done against the MLSMR to discover inhibitors.

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

Grant: MH095452
PI Name: Malcolm Walkinshaw, University of Edinburgh
Protocol
2 uL of TbPYK/substrate buffer (columns 1-3, 5-48) are dispensed into Greiner white solid bottom 1536-well assay plates; column 4 receives only TEA buffer as 0x TbPYK controls. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Following addition of compound, 2 uL of ADP (130 uM final concentration, all columns) is added to initiate the reaction. The plates are then incubated at room temperature for 45 minutes. Kinase-Glo Plus reagent is added (3 uL) 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 50 mM TEA (pH 7.2), 100 mM KCl, 10 mM MgCl2, 0.05% BSA and 0.01% Tween-20.

All screening operations will be performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staeubli, Duncan, SC). Library plates will be screened starting from the lowest and proceeding to the highest concentration. Vehicle-only plates, with DMSO being pin-transferred to columns 5-48, will be inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data will be corrected, normalized, and concentration-effect relationships will be derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).
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
Show more
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.00329 uM (0.00329484μM**)% Activity at given concentration.Float%
16Activity at 0.00590 uM (0.0059μM**)% Activity at given concentration.Float%
17Activity at 0.015 uM (0.0147μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
19Activity at 0.029 uM (0.0295μM**)% Activity at given concentration.Float%
20Activity at 0.074 uM (0.0737μM**)% Activity at given concentration.Float%
21Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
22Activity at 0.147 uM (0.147μM**)% Activity at given concentration.Float%
23Activity at 0.369 uM (0.369μM**)% Activity at given concentration.Float%
24Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
25Activity at 0.737 uM (0.737μM**)% Activity at given concentration.Float%
26Activity at 1.840 uM (1.84μM**)% Activity at given concentration.Float%
27Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
28Activity at 3.690 uM (3.69μM**)% Activity at given concentration.Float%
29Activity at 9.516 uM (9.51569μM**)% Activity at given concentration.Float%
30Activity at 18.40 uM (18.4μM**)% Activity at given concentration.Float%
31Activity at 47.58 uM (47.5785μM**)% Activity at given concentration.Float%
32Activity at 92.20 uM (92.2μM**)% Activity at given concentration.Float%
33Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH095452

Data Table (Concise)
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