AID	Name	Type	Comment
448	Schistosoma Mansoni Peroxiredoxins (Prx2)	confirmatory
485363	Probe Development Summary for Inhibitors of Schistosoma Mansoni Peroxiredoxins	summary
485364	qHTS Assay for the Inhibitors of Schistosoma Mansoni Peroxiredoxins	confirmatory

NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: MH076449-01
Assay Provider: Prof David Williams, Illinois State University

This is a confirmation assay for PubChem BioAssay AID 448. 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.

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-2, tgr, qHTS, NCGC

Compounds are ranked (PUBCHEM_ACTIVITY_SCORE) by potency using ceiling of -10*Fit_LogAC50 value. All inactive compounds had a score of 0.