Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Abstract

BackgroundHuman 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 FindingsIn 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/SignificanceThe 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.

TTAS a new stilbene derivative that induces apoptosis in Leishmania infantum

“…The loss of mitochondrial membrane potential suggests that the TTAS-induced apoptosis is mediated by the mitochondrial apoptotic pathway. To discover the main molecular target/s of TTAS in Leishmania parasites we took advantage of a chemical proteomic approach that was recently successfully applied by some of us (Kuettel et al, 2009). Thus the amino-stilbene derivative TTAS, chosen based on the presented activity data, was immobilized and the corresponding affinity matrix was used to find out potential targets.…”

The Pharmaceutical Biochemistry Group: Where Pharmaceutical Chemistry Meets Biology and Drug Delivery

“…The chemical proteomics approach, based on affinity chromatography techniques, has been applied successfully for the identification of adenosine kinase (AK) as the target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine in Trypanosoma brucei rhodesiense. [57,58] Chemical validation using recombinant protein indeed showed specific and tight interaction. Interestingly, amino acid that is crucial for the conformational transition and DFG flip of c-Src by combining mutagenesis, X-ray crystallography, activity measurements, isothermal titration calorimetry and full atomistic very long molecular dynamics including free energy calculations performed in collaboration with Dr. Gervasio.…”

Affinity purification in target identification: the specificity challenge

“…First, a ''negative matrix'' can be generated by immobilizing the positive ligand at a position that would abolish its biological activity (Ding et al 2003;Kuettel et al 2009). Second, the method of selective elution uses free, unbound small molecule to elute specific targets ( Fig.…”