Pharmacological validation of Trypanosoma brucei phosphodiesterases as novel drug targets.

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets.

The exopolyphosphatase TbrPPX1 of Trypanosoma brucei.

BACKGROUND: Exopolyphosphatases and pyrophosphatases play important but still incompletely understood roles in energy metabolism, and also in other aspects of cell biology such as osmoregulation or signal transduction. Earlier work has suggested that a human exopolyphosphatase, Prune, might exhibit cyclic nucleotide phosphodiesterase activity. RESULTS: The kinetoplastida, a large order of unicellular eukaryotes that contains many important pathogens such as Trypanosoma brucei (human sleeping sickness), Trypanosoma cruzi (Chagas disease) or Leishmania ssp (several clinically dinstinct leishmaniases) all contain several exo- and pyrophosphatases. The current study provides a systematic classification of these enzymes, which now allows to situate the information that is already available on some of these enzymes. It then analyses the exopolyphosphatase TbrPPX1 of T. brucei in detail, using RNA interference and genetic knockouts in an attempt to define its function, and immunofluorescence microscopy to study its subcellular localization.TbrPPX1 is an exopolyphosphatase that does hydrolyze pentasodium triphosphate, but not organic triphosphates such as ATP, pyrophosphate or long-chain polyphosphates. Finally, the study investigates the potential cyclic nucleotide phosphodiesterase activity of TbrPPX1. CONCLUSIONS: All kinetoplastid genomes that are currently available contain genes for an exopolyphosphatase and two classes of pyrophosphatases, one associated with the acidocalcisomes and one cytoplasmic. TbrPPX1 represents the T. brucei exopolyphosphatase. It is located throughout the cytoplasm, and its genetic ablation does not produce a dramatic phenotype. Importantly, TbrPPX1 does not exhibit any cyclic nucleotide specific phosphodiesterase activity, which definitively eliminates it as an additional player in cAMP signalling of the kinetoplastida.

The N terminus of phosphodiesterase TbrPDEB1 of Trypanosoma brucei contains the signal for integration into the flagellar skeleton.

The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.