ACS DIVISION OF BIOLOGICAL CHEMISTRY

February 20th, 2014 by Galkin, A., Kulakova, L., Lim, K., Chen, C. Z., Zheng, W., Turko, I. V., Herzberg, O.

Carbamate kinase from Giardia lamblia (glCK1) 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 glCK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here we examine the structural basis for glCK 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 glCK soaked with disulfiram revealed that the compound thiocarbamoylated Cys242, a residue located at the edge of the active site. The modified Cys242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr245 side chain against the adenine moiety, an interaction seen in the structure of glCK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys242 in solution. The G. lamblia viability studies in the metronidazole-resistant strain and the glCK 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 glCK is an attractive drug target for development of novel antigiardial therapies, and that disulfiram, a FDA approved drug, is a promising candidate for drug repurposing.