The adenosine analog tubercidin inhibits glycolysis in Trypanosoma brucei as revealed by an RNA interference library.
We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.
Drew
Mark E
ME
Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.
Morris
James C
JC
Wang
Zefeng
Z
Wells
Lance
L
Sanchez
Marco
M
Landfear
Scott M
SM
Englund
Paul T
PT
D000222:Adaptation, Physiological / Q000235:genetics; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D004163:Diphosphoglyceric Acids / Q000032:analysis; D004351:Drug Resistance; D006019:Glycolysis / Q000187:drug effects*; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000378:metabolism; D014372:Tubercidin / Q000494:pharmacology*
D000981:Antiprotozoal Agents; D004163:Diphosphoglyceric Acids; D034741:RNA, Small Interfering; C015891:glycerate 1,3-biphosphate; D006593:Hexokinase; D010735:Phosphoglycerate Kinase; D014372:Tubercidin