A series of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives, prepared by two synthetic routes, were in vitro assayed against three Trypanosoma strains, Leishmania donovani, and Plasmodium falciparum K1. Seven out of 17 compounds showed moderate to very good activity against blood stage T. b. rhodesiense, with 10 and 17 exhibiting highest potency (IC50 of 1.0 and 1.1 microM, respectively). Interestingly, the beta-diketone precursors 1-3 had good antitrypanosomal activity toward the insect stage, with IC50 values of 1.0-3.4 microM. Among different compounds with moderate activity against T. cruzi, compound 17 showed the lowest IC50 value of 9.5 microM; thus, the series seemed to act selectively toward the different Trypanosoma parasites. Eight compounds were moderately active against L. donovani, with 2, 3, and 12 being the most promising ones (IC50 values of 2.3-5.2 microM), whereas compound 14 was the only derivative with good activity against P. falciparum (IC50 of 3.7 microM).

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.

The enzymes phosphomannomutase (PMM), phospho-N-acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N-acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose-phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.