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 Cys 
535'-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.