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 ..
BioActive Compounds: 10735
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.
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)