Synthesis of 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates for trypanothione reductase and thioredoxin reductase: a microtiter colorimetric assay for inhibitor screening.
Trypanothione reductases (TR; EC 1.6.4.8) and thioredoxin reductases (TrxR; EC 1.6.4.5.) are enzymes central to cellular thiol metabolism. Trypanosoma cruzi TR (TcTR) is therefore considered as a potential candidate for drug design against trypanosomiasis. Inhibition of human TrxR (hTrxR) is likely to be beneficial in psoriasis, cancer, and autoimmune diseases, while inhibition of a putative TrxR from Plasmodium falciparum (PfTrxR) might prove effective against malaria. The natural substrates of the first two enzymes are very expensive and difficult to obtain; in the case of PfTrxR, the physiological substrate has not yet been identified. We have therefore synthesized and tested three different 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates of the above enzymes. As with 5, 5'-dithiobis(2-nitrobenzoate) (DTNB), which can be reduced by TRs and TrxRs, the new compounds are converted to their corresponding chromophoric thiolates; however, they have much lower Km values and are therefore less likely to interfere with inhibitor testing. Using the new substrates, a novel enzyme assay has been developed which is identical for all three enzymes, can be performed in a microtiter plate, and is amenable to automation. Thus, the assay provides a versatile and inexpensive tool for kinetic studies and high-throughput inhibitor screening.
Institut de Biologie de Lille-Institut Pasteur de Lille, URA CNRS 1309, 1 rue du Professeur Calmette, Lille Cedex, 59021, France. elisabeth.davioud@pasteur-lille.fr
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Davioud-Charvet
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Becker
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Landry
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Gromer
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D000818:Animals; D001549:Benzamides / Q000138:chemical synthesis*; D014355:Chagas Disease / Q000188:drug therapy; D003124:Colorimetry / Q000379:methods*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology
D001549:Benzamides; D004791:Enzyme Inhibitors; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D013880:Thioredoxin-Disulfide Reductase