Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxido-reductase activity

Garavaglia, Patricia Andrea; Cannata, Joaquı́n J.B.; Ruiz, Andrés M.; Maugeri, Dante; Durán, Rosario; Galleano, Mónica; García, Gabriela Andrea

doi:10.1016/j.molbiopara.2010.05.019

Cited by 20 publications

(35 citation statements)

References 44 publications

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“…Expression and purification of recombinant TcAKR (recTcAKR) were performed as previously described from Escherichia coli (M15) transformed with the plasmid pQE30-TcAKR (19). Native TcAKR was purified from the soluble fraction of CL Brener clone epimastigotes by Cibacron Blue-Sepharose CL-6B resin (Pharmacia Biotech) eluted with 0.7 mM NADPH (19).…”

Section: Methodsmentioning

confidence: 99%

“…The TcAKR gene was excised from the recombinant plasmid pGEMTcAKR (19) with NotI and introduced into the inducible expression vector pTcIndex (23) excised with the same restriction enzyme to form pTcIndex-TcAKR. This plasmid was introduced by electroporation into Adriana epimastigotes previously transformed with plasmid pLEW13.…”

Section: Methodsmentioning

confidence: 99%

“…The generation of free radicals is concomitant with o-NQ reduction, suggesting that TcAKR participates in their metabolism (19,20,21). Trochine et al recently reported that TcAKR interacts with an immobilized derivative of Bz, suggesting that it also participates in the Bz mode of action (22).…”

mentioning

confidence: 99%

“…We have previously described a novel NADPH-dependent aldo-keto reductase from T. cruzi, TcAKR, which reduces commonly used AKR substrates and -naphthoquinones (-NQ) with trypanocidal effects, such as ␤-lapachone (19). The generation of free radicals is concomitant with o-NQ reduction, suggesting that TcAKR participates in their metabolism (19,20,21).…”

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confidence: 99%

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Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism

Garavaglia

Laverrière

Cannata

et al. 2016

Antimicrob Agents Chemother

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b Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC 50 ) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH-and NADH-dependent reductases.C hagas' disease, caused by the protozoan Trypanosoma cruzi, affects 11 million people in Latin America (1). The parasite is usually transmitted to humans and other mammals by triatomine bugs. However, transmission can also be oral (through contaminated food), congenital, or transfusional or by organ transplantation, even in countries where the disease is not endemic (2). The course of infection includes an acute phase followed by an indeterminate phase without symptoms, which lasts throughout life in most infected people. Approximately 30% of patients can develop a chronic phase characterized by cardiac and/or digestive pathologies that can lead to death (3).Benznidazole (Bz) and, to a lesser extent, nifurtimox (Nx) are the drugs currently used for treatment of T. cruzi infection (4). Although the mechanism of action of these nitroheterocyclic compounds is not clearly understood, it is known that, to exert their trypanocidal effects, they need to be activated through the reduction of their nitro group (5). Several studies have proposed that an NADH-dependent trypanosomal type I nitroreductase (NTR I) is a key enzyme which catalyzes Bz and Nx activation in vivo (6,7,8,9). However, evidence indicates that although Bz is reduced to its anion radical, redox cycling may not be relevant for its mode of action, as ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism

Garavaglia

Laverrière

Cannata

et al. 2016

Antimicrob Agents Chemother

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“…However, the mechanism(s) by which such agents mediate their antiparasitic activity is unclear. Based on in vitro enzyme studies and the belief that protozoan parasites have a limited enzymatic capacity to metabolize reactive oxygen species, it had been postulated that some quinones may function by inhibiting key essential enzymes such as trypanothione reductase or lipoamide dehydrogenase, while others are thought to induce oxidative stress within the parasite (18,19,21,29,37,51). However, no demonstration of a functional link between the antiparasitic activity of any quinonebased agent and a proposed protein target within the pathogen has been reported and it is now well established that trypanosomatids possess a series of novel enzymatic oxidative defense pathways.…”

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confidence: 99%

Targeting the Substrate Preference of a Type I Nitroreductase To Develop Antitrypanosomal Quinone-Based Prodrugs

Hall

Meredith

Wilkinson

2012

Antimicrob Agents Chemother

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Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, having K cat /K m values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound.

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Biochemicals

Ncibi¹

2017

A Sustainable Bioeconomy

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Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxido-reductase activity

Cited by 20 publications

References 44 publications

Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism

Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism

Targeting the Substrate Preference of a Type I Nitroreductase To Develop Antitrypanosomal Quinone-Based Prodrugs

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