Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition

Abstract

Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbHK1 and inhibitor following incubation did not temper inhibition suggesting conjugate formation. Using mass spectrometry to analyze EbSe-based modifications revealed that two Cys residues (C327 and C369) were oxidized after treatment. Site-directed mutagenesis of C327 led to enzyme inactivation indicating that C327 was essential for catalysis. C369 was not essential, suggesting that EbSe inhibition of TbHK1 was the consequence of modification of C327 via thiol oxidation. Additionally, neither EbSe treatment nor mutation of the nine TbHK1 Cys residues appreciably altered enzyme quaternary structure.

Keywords: BSF, bloodstream form; EbS, 2-phenyl-12-benzisothiazol-3(2H)-one; EbSe, ebselen (2-phenyl-12-benzisoselenazol-3(2H)-one); Ebselen; G6-P, glucose-6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GK, glycerol kinase; Gly3P, glycerol-3-phosphate; HK, hexokinase; Hexokinase; Inhibitors; PF, procyclic form; TbHK, T. brucei hexokinase; Trypanosoma brucei; rTbHK1, recombinant Trypanosoma brucei hexokinase 1.

Graphical abstract

Fig. 1

EbSe, a known Cys-reactive compound, inhibits TbHK1 activity. (A) Structures of ebselen (EbSe) and ebsulfur (EbS), SID 17387000. (B) EbS and EbSe inhibition are irreversible by dilution. TbHK1 (32 ng) was incubated with EbSe or EbS in the assay for 15 min. Alternatively, inhibitor was incubated with enzyme prior to addition of other assay components, which yielded a 200-fold dilution of enzyme and inhibitor. (C) DTT can block but not reverse TbHK1 inhibition by EbSe. TbHK1 (32 ng) was incubated with EbS or EbSe (hatched bars) followed by the addition of DTT (100 mM) prior to assay. Experiments were performed in triplicate and standard deviation is indicated.

Fig. 2

Two Cys residues on the large lobe of TbHK1 are modified in EbSe treated samples. The predicted distribution of TbHK1 Cys residues, based on modeling to the yeast structure, with the nine Cys residues and the catalytic base (D214) included to indicate the active site (Chambers et al., 2008c). The ^* indicates the Cys found by ESI–MS/MS to be oxidized in both untreated and EbSe-treated TbHK1, while the ^** indicates the two oxidized Cys residues observed only in peptides from treated samples.

Fig. 3

Relative hexamer, monomer, and free Cys abundance for TbHK1 and variants. (A.) Relative hexamer and monomer abundance of TbHK1 and Cys variants. Relative hexamer abundance (hatched bars) was determined by densitometry of appropriately sized bands visualized by silver staining of a 4% native gel followed by comparison to TbHK1 (lane 1). Relative monomer abundance (black bars) was determined by western blot analysis of the appropriate region of a 4% native gel using an anti-RGSH₆ antibody and comparison to TbHK1. (B.) MPB modification of the monomer was determined by incubating variants with MPB (50 μM) for 30 min and monitoring the appropriate region of the gel by western blotting using an anti-biotin antibody. Experiments were performed in triplicate and standard deviation is indicated, with representative data included above each bar graph.

Fig. 4

Negative stain TEM analysis of globular TbHK1 hexamers. Samples of WT TbHK1 (upper) and C445A variant TbHK1 (lower) were analyzed using a JEOL JEM 1200 EX transmission electron microscope after negative staining. Inset contains enlargement of four representative protein particles. Scale bar = 100 nm.

Fig. 5

Potential scheme for EbSe-based oxidation of Cys residues in TbHK1.