Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi

Abstract

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH) ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH) metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH) even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity

“…The PCR products were ligated into the pTREXn vector by standard methodologies. Construction of plasmids for γECS and TryS overexpression has been previously reported [29]. Genes' nucleotide sequences and in-frame plasmid constructs were verified by automated Sanger DNA sequencing.…”

“…Epimastigotes overexpressing γECS and TryS were created as reported elsewhere [29]; the same protocol was used to obtain clones overexpressing TryR and TXN1. Briefly, 3 × 10 8 epimastigotes resuspended in 350 μL of cold non-supplemented LIT medium were transfected with 100 μg of cesium chloride-purified plasmid DNA by electroporation with a BTX ECM 830 electroporator (Harvard Apparatus; Holiston, MA, USA) at 300 V for 70 ms in 2 mm gap BTX electroporation cuvettes.…”

“…Control (Wt or mock) and different stable OE-epimastigote clones were cultured to the late logarithmic phase, harvested by centrifugation at 4250 x g and washed with PBS. The soluble-enriched fractions were prepared as described before [29] by lysing the parasites with three cycles of freezing/thawing and the lysate was centrifuged to collect the soluble fraction and immediately used for activity determination.…”

“…Activities of γECS and TryS in the OE clones and GS in all parasites were determined as described before [29] by coupling the ADP production via pyruvate kinase/lactate dehydrogenase (PyK/LDH) to NADH oxidation and the change in absorbance was monitored in real time under initial velocity conditions at 340 nm and 37 °C in a diode array spectrophotometer (Agilent, Santa Clara, CA, USA). In all assays it was made sure that the activity was linear with respect to the amount of soluble cell protein.…”

“…Aliquots of 90 μL were transferred to 1.5 mL tubes and incubated at 37 °C for 0, 15, 30 or 60 min. After each time, the reaction was stopped by adding perchloric acid (PCA) at 3% v/v final concentration and T(SH) 2 was determined by HPLC as described in section 2.7 [29].…”