Ajoene Is an Inhibitor and Subversive Substrate of Human Glutathione Reductase and Trypanosoma cruzi Trypanothione Reductase:  Crystallographic, Kinetic, and Spectroscopic Studies

Abstract

Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.

S-glutathionylation: relevance in diabetes and potential role as a biomarker

“…The enzyme has been identified as one of the target molecules of the widely applied antitumor drug bis-chloroethylnitrosourea (Frischer and Ahmad, 1977), and a target of ajoene, a compound present in the garlic extract. Its inactivation may result in a 400-fold increased activity of NADPH oxidase (NOX), generating superoxide (Gallwitz et al, 1999). As it is the case with many antioxidant enzymes, Nrf2 is a key regulator of GR expression (Harvey et al, 2009).…”

Natural Products Active Against African Trypanosomes: A Step Towards New Drugs

“…82 They showed that ajoene 118, another garlic-derived natural product, affects the antioxidant thiol metabolism of T. cruzi, leading to increased oxidative stress by inhibition of the trypanothione reductase. 82 Manumycin A 119, an antibiotic produced by Streptomyces microorganisms, is an inhibitor of Ras farnesyltransferase, which is of interest for antitumor therapy. Compound 119 is potently active in vitro against the growth of both bloodstream and procyclic forms of T. b. brucei (IC 50 = 1.5 and 0.4 µM respectively).…”

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

“…Using either rational or empiric approaches, different classes of compounds, including peptides and peptoid, substituted polyamines, quinacrine and acridine analogues, 2-aminodiphenylsulfides, phenothiazine derivatives, substituted piperazines and nitrofuran derivatives, were studied for their inhibitory potential (see reviews by Werbovetz 2000, Augustyns et al 2001, Schmidt & KrauthSiegel 2002. Investigation of natural products, although not so extensive, disclosed interesting inhibitors such as ajoene from garlic (Gallwitz et al 1999), bisbenzylisoquinoline alkaloids (Fournet et al 1998(Fournet et al , 2000, and polyamines such as lunarine (Bond et al 1999) and kukoamine (Ponasik et al 1995).…”

“…Using either rational or empiric approaches, different classes of compounds, including peptides and peptoid, substituted polyamines, quinacrine and acridine analogues, 2-aminodiphenylsulfides, phenothiazine derivatives, substituted piperazines and nitrofuran derivatives, were studied for their inhibitory potential (see reviews by Werbovetz 2000, Augustyns et al 2001, Schmidt & KrauthSiegel 2002. Investigation of natural products, although not so extensive, disclosed interesting inhibitors such as ajoene from garlic (Gallwitz et al 1999), bisbenzylisoquinoline alkaloids (Fournet et al 1998(Fournet et al , 2000, and polyamines such as lunarine (Bond et al 1999) and kukoamine (Ponasik et al 1995).After the initial work of Henderson and co-workers (1988) naphthoquinone derivatives were further investigated for their action on TR (Jockers-Scherubl et al 1989, Salmon-Chemin et al 2000. In a previous work we reported the in vitro effect of several naphthothiophenquinone derivatives against epimastigote and trypomastigote forms of T. cruzi and the inhibition of the recombinant enzyme trypanothione reductase (Zani et al 1997).…”