Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

Abstract

Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.

Structural characterization of Giardia duodenalis thioredoxin reductase ( g TrxR) and computational analysis of its interaction with NBDHEX

“…Experimental evidences suggest that NBDHEX can tightly bind to gTrxR in its oxidized fluorescent form, also in the absence of NADPH, likely by cysteine attack at two different drug positions (C4 and C6) of the bicyclic system. We also observed that the binding and enzyme inhibition follows a biphasic behavior [6]. Adducts between the nitro-reduced form of the drug and, mainly, the catalytic Cys137 and Cys140 of gTrxR could be detected by mass spectrometry analysis but only when NADPH was present [6].…”

“…The full-length coding sequence of TrxR from the isolate WB-C6 of Giardia duodenalis (gTrxR, GL50803_9827) was cloned and expressed as N-terminal 6XHIS tagged protein in E. coli M15 strain as previously reported [6]. HIS-gTrxR was affinity purified on native condition on Ni-NTA Agarose (Qiagen, Germany) and eluted with 250 mM imidazole according to the manufacturer.…”

“…We then engaged in the investigation of NBDHEX targets in order to better clarify its mode of action. We showed that NBDHEX administration to G. duodenalis trophozoites results in a significant reduction of the parasite FAD-dependent glycerol-3-phosphate dehydrogenase (gG3PD) [5] activity and that this compound also tightly binds thioredoxin reductase (gTrxR) [6]. Upon gTrxR binding, NBDHEX concomitantly inhibits the NADPH-dependent disulphide reductase activity and promote NADPH oxidase activity of the enzyme in a dose-dependent manner [6].…”

“…We showed that NBDHEX administration to G. duodenalis trophozoites results in a significant reduction of the parasite FAD-dependent glycerol-3-phosphate dehydrogenase (gG3PD) [5] activity and that this compound also tightly binds thioredoxin reductase (gTrxR) [6]. Upon gTrxR binding, NBDHEX concomitantly inhibits the NADPH-dependent disulphide reductase activity and promote NADPH oxidase activity of the enzyme in a dose-dependent manner [6]. Administration of NBDHEX to parasite cells and in vitro to recombinant gTrxR in presence of NADPH, led to the formation of covalent adducts with the catalytic cysteines (Cys137 and Cys140) and nitro-reduced form of the drug.…”

“…Administration of NBDHEX to parasite cells and in vitro to recombinant gTrxR in presence of NADPH, led to the formation of covalent adducts with the catalytic cysteines (Cys137 and Cys140) and nitro-reduced form of the drug. Moreover, the NBDHEX is modified by gTrxR in vitro, likely by nitroreduction [6]. Interaction of NBDHEX with gTrxR is highly interesting due to the specific redox metabolism of Giardia.…”