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.

Keywords: Elongation factor 1Bγ; Giardia; NBDHEX; Thioredoxin reductase.

Graphical abstract

Fig. 1

Proteomic identification of NBDHEX targets. A) Representative SDS-PAGE of soluble proteins (50 μg) from Giardia trophozoites treated or not with NBDHEX under UV light (right) and after Coomassie staining (left). Molecular size markers are indicated on the left. B) and C) Representative MS/MS spectra of gTrxR peptide 133–148 (B) and gEF1Bγ peptide 30–42 (C) acquired after trypsin digestion of fluorescent bands 1 and 6. NBDHEX indicates cysteines (Cys137 in gTrxR and Cys34 in gEF1Bγ) with mass increase of 265 Da and IAM indicates iodoacetamide-alkylated cysteines.

Fig. 2

NBDHEX effect on target protein electrophoretic mobility and intracellular localization. A) Representative Western blot analysis of soluble proteins from Giardia trophozoites incubated or not with NBDHEX. The low molecular weight bands of α-gTUB (arrow) and gEF1Bγ (triangle) are indicated. Asterisks indicate the molecular size corresponding to monomeric (*), dimeric (**) and multimeric (***) gEF1Bγ. The circle indicates the gOCT band shifted at higher molecular size. The square bracket indicates a gTrxR bands pair. The panel corresponding to the Western blot with anti-gEF1Bγ is a combination of two exposure times B) CLSM analysis of Giardia trophozoites incubated with (panels d, e, f, h, i, m and n) or without (panels a, b, c, g and l) NBDHEX. NBDHEX (green) was directly visualized using the laser light at λem 488 nm. Parasites were stained with the indicated mouse antibodies and anti-mouse AlexaFluor 647-conjugated (red). Nuclei were stained with DAPI (blue). Pn: perinuclear area; mb: median body; ifr: intracellular flagellar roots. Displayed micrographs correspond to a single z-stack. T, transmission light acquisition. Scale bar 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

NBDHEX binds to recombinant His-tagged gTrxR and HIS-gEF1Bγ expressed in bacteria. Recombinant proteins, expressed in E. coli and affinity purified after treatment of bacteria with NBDHEX (50 μM) or ethanol for 2 h, were separated on 4–12% PAGE, visualized on UV-transilluminator (UV), stained with Coomassie or blotted and probed with mAb anti-HIS (αHIS). The double bands of gEF1Bγ are indicated by the “less than” symbol (<).

Fig. 4

NBDHEX binding to and inhibitory effect on HIS-gTrxR A) Analysis of HIS-gTrxR intrinsic fluorescence quenching after incubation was reacted with different amounts of NBDHEX. Experimental points were fitted to a bi-phasic binding curve. B) Inhibition of HIS-gTrxR thioreductase activity by NBDHEX. DTNB thioreductase activity was followed at 412 nm where the enzymatic product (TNB⁻) absorbs. Inhibition data were obtained incubating HIS-gTrxR, NADPH and DTNB with increasing amounts of NBDHEX as detailed in Methods.

Fig. 5

Effect of NBDHEX on NADPH oxidase activity of gTrxR and NADPH-dependent gTrxR activity on NBDHEX. A) Oxidation of NADPH by gTrxR in presence or absence of saturating NBDHEX (60 μM). B) NBDHEX-dependent oxidation of NADPH by gTrxR at pH 7.4. Emiactivation is at 16 μM. C) UV–visible spectrum of NBDHEX recorded either in the presence of NADPH alone (dark green line) after the addition of HIS-gTrxR at t = 0 (mustard line) or after 80 min of incubation (orange line). Maximal absorption wavelength (nm) is indicated (arrow). D) Fluorescence spectra of NBDHEX (excitation at 430 nm) incubated with HIS-gTrxR at t = 0 (solid line) or after 80 min (dashed line) in the presence of NADPH. The change in color of the reaction is shown in the insert. E) SDS-PAGE of HIS-gTrxR after 80 min incubation with or without NBDHEX, in the presence or not of NADPH. Subsequent treatment of the reaction mixture with sodium dithionite (Na₂S₂O₄) is also indicated. Gel was photographed under UV light (UV, right panel) before staining with Coomassie blue (Coomassie, left panel). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

NBDHEX binding to gEF1By. A) UV–visible spectrum of HIS-gEF1Bγ recorded at 25 °C. Peak maxima are reported. Spectrum is representative of three independent experiments. B) Binding of NBDHEX to HIS-EF1Bγ. Quenching of intrinsic HIS-EF1Bγ fluorescence caused by NBDHEX binding was monitored at 340 nm (excitation at 280 nm). C) Changes in NBDHEX fluorescence emission spectra due to HIS-EF1Bγ binding. NBDHEX in the absence of HIS-gEF1Bγ (gray line) and in the presence of increasing HIS-gEF1Bγ amounts: blue, green, red and dashed line. Excitation wavelength was at 432 nm. Vertical lines indicate ∼530 and ∼520 nm, while the arrow indicates the “blue-shift” of NBDHEX fluorescence. D) SDS-PAGE of HIS-EF1Bγ after 80 min incubation with or without NBDHEX. Subsequent treatment of the reaction mixture with sodium dithionite (Na₂S₂O₄) is also indicated. Gel was photographed under UV light (UV, right panel) before staining with Coomassie blue (Coomassie, left panel). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)