thioredoxin peroxidase [Schistosoma mansoni] [gi:4325211]
Conserved Domains     Related Protein Structures

Compounds:   All: 63786   Active: 40   Inactive: 63392   Inconclusive: 364
Substances:   All: 64651   Active: 44   Inactive: 64241   Inconclusive: 366
Taxonomy: 1 Link

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.

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

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

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Project Category: MLSCN