Bookmark and Share
BioAssay: AID 448

Schistosoma Mansoni Peroxiredoxins (Prx2)

Schistosoma mansoni, a causative agent of schistosomiasis, resides in the bloodstream of their host up to 30 years without being eliminated by the host immune attack. One proposed survival mechanism is the production of an antioxidant "firewall" that neutralizes the oxidative assault of the host's immune attack. Schistosoma mansoni peroxiredoxins (Prx) are important parasite antioxidant proteins more ..
_
   
 Tested Compounds
 Tested Compounds
All(63781)
 
 
Active(38)
 
 
Inactive(63389)
 
 
Inconclusive(364)
 
 
 Tested Substances
 Tested Substances
All(64651)
 
 
Active(44)
 
 
Inactive(64241)
 
 
Inconclusive(366)
 
 
AID: 448
Data Source: NCGC (prx2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-07-14
Modify Date: 2008-01-04

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 38
Depositor Specified Assays
AIDNameTypeComment
1011Confirmation Concentration-Response Assay for Inhibitors of the Schistosoma mansoni Redox Cascadeconfirmatory
485363Probe Development Summary for Inhibitors of Schistosoma Mansoni Peroxiredoxinssummary
485364qHTS Assay for the Inhibitors of Schistosoma Mansoni Peroxiredoxinsconfirmatory
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
David Williams [Illinois State University]

NCGC Assay Overview:
Schistosoma mansoni, a causative agent of schistosomiasis, resides in the bloodstream of their host up to 30 years without being eliminated by the host immune attack. One proposed survival mechanism is the production of an antioxidant "firewall" that neutralizes the oxidative assault of the host's immune attack. Schistosoma mansoni peroxiredoxins (Prx) are important parasite antioxidant proteins that play a crucial role in redox balance mechanisms. Data strongly suggest the possible use of Prx as novel drug targets. First, the proteins are essential for the parasite survival. Second, the proteins exhibit sufficient biochemical and structural differences from host Prx proteins. Third, the protein(s) is amenable for study at the molecular level and can be produced in bacteria in large quantities, in soluble and active form. Inhibition of Prx activity was screened in a coupled-enzyme format with reducing equivalents being transferred from NADPH to glutathione intermediate via thioredoxin/glutathione reductase (TGR)-catalyzed reaction, then from thioredoxin to hydrogen peroxide via Prx-catalyzed electron transfer. A decrease in the fluorescence intensity of NADPH was used to measure the enzyme activity. Purified S. mansoni TGR and Prx were provided by Professor David L. Williams, Illinois State University.
Protocol
NCGC Assay Protocol Summary:

Buffer:
100 mM phosphate buffer, pH 7.4, 10 mM EDTA, 0.01% Tween-20.

Reagents / Controls:
100 uM NADPH in columns 3 and 4 as negative control (fully inhibited, zero % activity).

100 uM NADPH/25 nM TGR/700 uM GSH/50 nM Prx2 mixture in columns 1, 2, 5-48. Titration of control potassium antimonyl tartrate (PAT, from 1 mM, then 1:5) pin-transferred to lower half of column 2. Column 1 and upper half of column 2 are neutral (100% activity).

Assay Steps:
Three uL of reagents were dispensed to 1536-well Greiner black plates. Compounds and controls (20 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then a 1 uL aliquot of 400 uM NADPH/700 uM GSH was added, immediately followed by a 1uL aliquot of 2.5 mM H2O2 (to start the reaction). The plate was transferred to ViewLux (Perkin-Elmer) High-throughput CCD imager where kinetic measurements (every 30 sec, for a total of 8 min) of NADPH fluorescence were taken using 365 nm excitation/450 nm emission filter set. During dispense, enzyme-containing reagent bottles were kept submerged into 4 deg C water bath to minimize degradation.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, David Williams, Peroxyredoxin, Schistosoma mansoni, prx2, prx, , 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

2. Within each curve class, compounds are ranked by potency
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity DirectionIndicates direction of activity: inactive, decreasing, increasing.String
2Activity QualifierIndicates if AC50 is "=" to value or if it is infinite activity (i.e, inactive), ">".String
3Qualified AC50Qualified AC50; can be an IC50 or EC50.Float
4Log of AC50Log of AC50.Float
5Hill CoefficientHill slope of fitted curve.Float
6Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
7Data TypeNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String
8Compound QCNCGC comment on compound QC: 'DPI QC', 'NCGC Purified'String
9Data Analysis QCNCGC comment on confidence of data: 'Curve Verfied', etcString
10NCGC CommentAnnotation/notes on a particular compound's data: e.g, 'cytotoxic', 'fluorescent artifact'String
11Curve Fit ModelCurve fitting model used.String
12Hill S0S0 of Hill EquationFloat
13Hill SinfSinf of Hill EquationFloat
14Hill dSdS of Hill EquationFloat
15Log AC50 Std ErrorStandard error for Log AC50.Float
16Excluded PointsIndividual points excluded from curve fitting.String
17Number of PointsNumber of valid points used for fitting.Integer
18Activity at 0.732nM% Activity at given concentration.Float%
19Activity at 1.636nM% Activity at given concentration.Float%
20Activity at 3.658nM% Activity at given concentration.Float%
21Activity at 8.18nM% Activity at given concentration.Float%
22Activity at 18.291nM% Activity at given concentration.Float%
23Activity at 0.041uM% Activity at given concentration.Float%
24Activity at 0.091uM% Activity at given concentration.Float%
25Activity at 0.204uM% Activity at given concentration.Float%
26Activity at 0.457uM% Activity at given concentration.Float%
27Activity at 1.022uM% Activity at given concentration.Float%
28Activity at 2.286uM% Activity at given concentration.Float%
29Activity at 5.111uM% Activity at given concentration.Float%
30Activity at 11.429uM% Activity at given concentration.Float%
31Activity at 25.556uM% Activity at given concentration.Float%
32Activity at 0.057mM% Activity at given concentration.Float%
33Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'MLSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
PageFrom: