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BioAssay: AID 485364

qHTS Assay for the Inhibitors of Schistosoma Mansoni Peroxiredoxins

Schistosomiasis is a major neglected tropical disease that currently affects over 200 million. It is caused by different species of flatworms, such as Schistosoma mansoni. Over the last century, drugs to treat the disease have evolved from potassium antimonyl tartrate, artemisinin, oxamniquine, to praziquantel, with praziquantel remaining the universally-used single drug of choice over the past more ..
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 Tested Compounds
 Tested Compounds
All(356652)
 
 
Active(10735)
 
 
Inactive(331528)
 
 
Inconclusive(14556)
 
 
 Tested Substances
 Tested Substances
All(359841)
 
 
Active(10784)
 
 
Inactive(334477)
 
 
Inconclusive(14580)
 
 
AID: 485364
Data Source: NCGC (TGR5001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-06

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 10735
Related Experiments
AIDNameTypeComment
448Schistosoma Mansoni Peroxiredoxins (Prx2)Confirmatorydepositor-specified cross reference: Schistosoma Mansoni Peroxiredoxins (Prx2) and thioredoxin glutathione reductase (TGR) coupled assay
1011Confirmation Concentration-Response Assay for Inhibitors of the Schistosoma mansoni Redox CascadeConfirmatorydepositor-specified cross reference: Confirmation Concentration-Response Assay for Inhibitors of the Schistosoma mansoni Redox Cascade
485363Probe Development Summary for Inhibitors of Schistosoma Mansoni PeroxiredoxinsSummarydepositor-specified cross reference
Description:
Schistosomiasis is a major neglected tropical disease that currently affects over 200 million. It is caused by different species of flatworms, such as Schistosoma mansoni. Over the last century, drugs to treat the disease have evolved from potassium antimonyl tartrate, artemisinin, oxamniquine, to praziquantel, with praziquantel remaining the universally-used single drug of choice over the past three decades. However, transmission rates have changed little with praziquantel, and there is evidence for the development of drug resistant parasites. Because there is currently no suitable alternative therapy available, there is an urgent need to identify new targets and drugs for schistosomiasis treatment. Thioredoxin glutathione reductase (TGR), one uniquely positioned S. mansoni enzyme, has been identified as a major component of the worms' distinct and compressed antioxidant "firewall". TGR is a multifunctional enzyme that catalyzes the interconversion between reduced and oxidized forms of both glutathione (GSH) and thioredoxins, thus making it an attractive new antiparasitic target.

Recently, a quantitative high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR, ~71K at the time of the screen in 2007) was performed using an assay that targeted both TGR and peroxiredoxin2 (Prx2, an H2O2-reducing enzymatic component of the S. mansoni redox "firewall") by following the decrease of NADPH fluorescence. The screen led to the identification of furoxan (4-phenyl-3-furoxancarbonitrile, PubChem CID 1756) (oxadiazole-2-oxide class) that acted as a TGR inhibitor and later was shown to possess potent ex vivo worm killing ability at modest concentrations (10 uM) and against all developmental stages of worm. Additional mechanistic link between exogenous NO donation and parasitic injury for the oxadiazole-2-oxide class of compounds was also expanded and better defined. Although the screen addressed both TGR and Prx2, the assay required post-screen target deconvolution for further characterization of the actives. In addition, the fluorescence assay was also subject to fluorescence interference from compound library members.

We have developed a 1536-well-based kinetic HTS assay to address TGR alone. The assay followed the reduction of 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) (Ellman's reagent) by NADPH and measured the increase in absorbance at 412 nm of the reaction product, TNB. Furoxan inhibition that was observed in the screening assay was recapitulated in this miniaturized absorbance assay; furthermore, concentration-response curves of furoxan were almost identical upon separate testing, with IC50s falling within the 4.0-7.9 uM range. This assay format can be used to screen the current MLSMR collection (with >300K compounds available) to find novel TGR inhibitors. Furoxan is a good choice to serve as a positive control in the primary HTS assay.
Protocol
Three microliters of reagents (100 uM NADPH and 20 nM TGR or 100 uM NADPH as no-enzyme control) were dispensed into 1,536-well black clear-bottomed plates. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The plates were then be incubated for 15 min at room temperature, and 1 uL aliquot of 500 uM NADPH were added, immediately followed by a 1 uL aliquot of 15 mM DTNB to start the reaction. The plate was transferred to a ViewLux high-throughput CCD imager (Perkin-Elmer, Wellesley, MA) where kinetic measurements of the TNB absorbance were acquired using a 405 excitation filter. Throughout the screen, reagent bottles and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a "double-dipping" step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity overtime. Screening data were 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). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.
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 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
15Activity at 0.091 uM (0.0914507μM**)% Activity at given concentration.Float%
16Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
17Activity at 2.290 uM (2.29μM**)% Activity at given concentration.Float%
18Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
19Activity at 22.90 uM (22.9μM**)% Activity at given concentration.Float%
20Activity at 57.10 uM (57.1μM**)% Activity at given concentration.Float%
21Activity at 114.0 uM (114μM**)% Activity at given concentration.Float%
22Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH076449-01

Data Table (Concise)
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