Synthesis of 5,5 '-dithiobis(2-nitrobenzamides) as alternative substrates for trypanothione reductase and thioredoxin reductase: A microtiter colorimetric assay for inhibitor screening

Citation
E. Davioud-charvet et al., Synthesis of 5,5 '-dithiobis(2-nitrobenzamides) as alternative substrates for trypanothione reductase and thioredoxin reductase: A microtiter colorimetric assay for inhibitor screening, ANALYT BIOC, 268(1), 1999, pp. 1-8
Citations number
38
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
268
Issue
1
Year of publication
1999
Pages
1 - 8
Database
ISI
SICI code
0003-2697(19990301)268:1<1:SO5'AA>2.0.ZU;2-9
Abstract
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 inhib ition of a putative TrxR from Plasmodium falciparum (PfTxR) might prove eff ective against malaria. The natural substrates of the first two enzymes are very expensive and difficult to obtain; in the case of PfTrxR, the physiol ogical substrate has not yet been identified. We have therefore synthesized and tested three different 5,5'-dithiobis(2-nitrobenzamides) as alternativ e 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 conver ted to their corresponding chromophoric thiolates; however, they have much lower K-m 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 scre ening. (C) 1999 Academic Press.