title: 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.
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: April C
authors.initials: AC
authors.lastname: Joice
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Harris
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Kahney
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: Heidi C
authors.initials: HC
authors.lastname: Dodson
authors.affiliation: Department of Biological Sciences, Chicago State University, Chicago, IL 60628, United States.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Maselli
authors.affiliation: Department of Chemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Whitehead
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.
authors.forename: James C
authors.initials: JC
authors.lastname: Morris
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