Adenosine kinase of T. b. rhodesiense identified as the putative target of 4-[5-(4-phenoxyphenyl)-2 H-pyrazol-3-yl]morpholine using chemical proteomics

Abstract

Background: Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H- pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC50 of 1.0 μM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT. Methodology/ Principal Findings: In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition. Conclusions/Significance: The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides. © 2009 Kuettel et al.