title: Structural basis for inactivation of Giardia lamblia carbamate kinase by disulfiram.
abstract: Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850.
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850.
authors.forename: Liudmila
authors.initials: L
authors.lastname: Kulakova
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850.
authors.forename: Kap
authors.initials: K
authors.lastname: Lim
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Catherine Z
authors.initials: CZ
authors.lastname: Chen
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850; National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
authors.forename: Illarion V
authors.initials: IV
authors.lastname: Turko
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850; Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742. Electronic address: osnat@umd.edu.
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000981:Antiprotozoal Agents / Q000737:chemistry; D020134:Catalytic Domain; D049109:Cell Proliferation; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004221:Disulfiram / Q000737:chemistry*; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005260:Female; D016829:Giardia lamblia / Q000201:enzymology*; D005873:Giardiasis / Q000188:drug therapy* / Q000201:enzymology; D013058:Mass Spectrometry; D008795:Metronidazole / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053058:Trophozoites / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D008795:Metronidazole; D000255:Adenosine Triphosphate; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D014357:Trypsin; D003545:Cysteine; D004221:Disulfiram
keywords: Animal Models; Carbamate Kinase; Crystallography; Disulfiram; Drug Discovery; Enzyme Inhibitors; Enzyme Structure; Giardia lamblia; Parasite