Putative role of the aldo-keto reductase from Trypanosoma cruzi (TcAKR) in benznidazole metabolism

GARAVAGLIA, PA; LAVERRIÈRE, M; CANNATA, JB; GARCIA, GA

Buenos Aires

Encuentro; XXVII Reunión Anual de la Soc. Argentina de Protozoología.; 2015

Sociedad Argentina de Protozoologia

Benznidazole (Bz), the drug used for of Chagas? disease treatment caused by Trypanosoma cruzi, needs to be reductivelyactivated to exert its effect. The NADH-dependent trypanosomal type I nitroreductase (NTR I) plays a key role in Bzactivation, however, several studies indicate that other enzymes are involved. In this study, we evaluated whether the aldoketo reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz assubstrate. TcAKR purified from epimastigotes by Cibacron Blue affinity chromatography showed reductase activity towardsBz using NADPH, but not NADH, as co-factor, with a specific activity of 73.11± 3.65 nmol of NADPH/min/mg of protein. Tounderstand the role of TcAKR in Bz susceptibility, we genetically engineered epimastigotes for tetracycline-inducible overexpression of TcAKR. TcAKR-over-expressing parasites showed increased NADPH-dependent-Bz reductase activity(7.73±0.25 vs. 13.27±1.91 nmol NADPH/min/mg of protein) and higher resistance to this drug (IC50 9.90±1.30 uM vs. 17.4± 1.25 uM), suggesting that this enzyme may be involved in Bz detoxification. We also studied TcAKR expression and its newenzymatic activity in two T. cruzi strains with differential susceptibility to Bz, CL and Nicaragua, with IC50 values of 10 and20.5 uM, respectively. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected thatthe more resistant strain, Nicaragua, had higher TcAKR levels than CL. However, the results were the opposite. Incorrelation with TcAKR expression, CL showed 5.7-fold higher NADPH-Bz reduction than Nicaragua. In addition, NADHdependent Bz reductase activity, characteristic of the NTRI, was also higher in CL than in Nicaragua, indicating anassociation between this enzyme and Bz susceptibility. We conclude that TcAKR activity is not a determinant of Bzresistance in wild type strains and may be overcome by other enzymes involved in Bz activation