From hit to target : identification and characterization of the 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine target in 'Trypanosoma brucei rhodesiense'

The aim was to identify and validate the target(s) of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine (compound 1, IC ₅₀ of 1 µM on "Trypanosoma brucei rhodesiense") by the use of a chimical proteomics approach. Out of the total parasite cell lysate, "T.b. rhodesiense" adenosine kinase (TbrAK) was isolated and identified based on the interaction with an immobilized derivative of compound 1. The "ak" gene was cloned and recombinant protein was purified to homogeneity for chemical validation. TbrAK was confirmed to be the intracellular target of compound 1 by thermal protein stability assays and isothermal titration calorimetry. Activity measurements revealed that compound 1 was a strong concentration dependent activator of TbrAK enzymatic activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK was due to the abolishment of the intrinsic substrate inhibition. X-Ray crystallographic studies resulted in the resolution of the first 3D-structures of TbrAK-compound 1 and TbrAK-diadenosine pentaphosphate