Journal article

Publication year: 2003

Pages: 13319-13330

Journal: Biochemistry

Volume number: 42

Issue number: 45

ISSN: 0006-2960

DOI Link: https://doi.org/10.1021/bi0353629

Publisher: American Chemical Society

Abstract: 
Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading, to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M-r electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.

Harvard Citation style: Davioud-Charvet, E., McLeish, M., Veine, D., Giegel, D., Arscott, L., Andricopulo, A., et al. (2003) Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity, Biochemistry, 42(45), pp. 13319-13330. https://doi.org/10.1021/bi0353629

APA Citation style: Davioud-Charvet, E., McLeish, M., Veine, D., Giegel, D., Arscott, L., Andricopulo, A., Becker, K., Müller, S., Schirmer, R., Williams, C., & Kenyon, G. (2003). Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity. Biochemistry. 42(45), 13319-13330. https://doi.org/10.1021/bi0353629