BioActive Compounds: 40
Related BioAssays:
Protein Target:
thioredoxin peroxidase [Schistosoma mansoni] [gi: 4325211] Conserved Domains Related Protein Structures
Links:
Compounds:
All: 63786
Active: 40
Inactive: 63392
Inconclusive: 364
Substances:
All: 64651
Active: 44
Inactive: 64241
Inconclusive: 366
Taxonomy: 1 Link
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:
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TID | Name | Description | Type | Unit | | Outcome | The BioAssay activity outcome | String | |
| Score | The BioAssay activity ranking score | Integer | |
1 | Activity Direction | Indicates direction of activity: inactive, decreasing, increasing. | String | |
2 | Activity Qualifier | Indicates if AC50 is "=" to value or if it is infinite activity (i.e, inactive), ">". | String | |
3 | Qualified AC50 | Qualified AC50; can be an IC50 or EC50. | Float | |
4 | Log of AC50 | Log of AC50. | Float | |
5 | Hill Coefficient | Hill slope of fitted curve. | Float | |
6 | Curve R2 | R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit. | Float | |
7 | Data Type | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String | |
8 | Compound QC | NCGC comment on compound QC: 'DPI QC', 'NCGC Purified' | String | |
9 | Data Analysis QC | NCGC comment on confidence of data: 'Curve Verfied', etc | String | |
10 | NCGC Comment | Annotation/notes on a particular compound's data: e.g, 'cytotoxic', 'fluorescent artifact' | String | |
11 | Curve Fit Model | Curve fitting model used. | String | |
12 | Hill S0 | S0 of Hill Equation | Float | |
13 | Hill Sinf | Sinf of Hill Equation | Float | |
14 | Hill dS | dS of Hill Equation | Float | |
15 | Log AC50 Std Error | Standard error for Log AC50. | Float | |
16 | Excluded Points | Individual points excluded from curve fitting. | String | |
17 | Number of Points | Number of valid points used for fitting. | Integer | |
18 | Activity at 0.732nM | % Activity at given concentration. | Float | % |
19 | Activity at 1.636nM | % Activity at given concentration. | Float | % |
20 | Activity at 3.658nM | % Activity at given concentration. | Float | % |
21 | Activity at 8.18nM | % Activity at given concentration. | Float | % |
22 | Activity at 18.291nM | % Activity at given concentration. | Float | % |
23 | Activity at 0.041uM | % Activity at given concentration. | Float | % |
24 | Activity at 0.091uM | % Activity at given concentration. | Float | % |
25 | Activity at 0.204uM | % Activity at given concentration. | Float | % |
26 | Activity at 0.457uM | % Activity at given concentration. | Float | % |
27 | Activity at 1.022uM | % Activity at given concentration. | Float | % |
28 | Activity at 2.286uM | % Activity at given concentration. | Float | % |
29 | Activity at 5.111uM | % Activity at given concentration. | Float | % |
30 | Activity at 11.429uM | % Activity at given concentration. | Float | % |
31 | Activity at 25.556uM | % Activity at given concentration. | Float | % |
32 | Activity at 0.057mM | % Activity at given concentration. | Float | % |
33 | Compound Type | NCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'MLSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization' | String | |
Additional Information:
Project Category: MLSCN
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