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