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 target

Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei

Cyclic nucleotide specific phosphodiesterases are important regulators of cyclic nucleotide signalling in eukaryotes. In many organisms, including humans and trypanosomes, some of these enzymes contain specific domains (GAF domains) that bind cyclic nucleotides, and that are involved in the regulation of the catalytic domain. In the parasitic protozoon that causes human sleeping sickness, Trypanosoma brucei, two closely related phosphodiesterases each contain two such GAF domains, GAF-A and GAF-B. Their genes are tandemly located on chromosome 9, spaced by only a few thousand nucleotides. We here show that a gene conversion event has exchanged the region that codes for the GAF-A domain of the downstream gene by the closely similar corresponding sequence of the upstream gene. This domain exchange has no effect on intracellular localization of the two enzymes. The gene conversion event has occurred in one particular strain of trypanosomes (Lister427) and is found in all its derivatives, but not in any other strain or isolate. The presence or absence of this gene conversion represents a useful analytical marker for the stringent discrimination of Lister427 derivatives from other trypanosome strains

Restriction enzyme analysis of TbrPDEB2a and TbrPDEB2b.

<p>Panel A: Restriction map of the TbrPDEB2 locus on chromosome 9. HindIII¹⁾: site present only in the converted allele TbrPDEB2b; BlnI²⁾: site present only in non-converted allele TbrPDEB2a. Box underneath horizontal line: Open reading frame of TbrPDEB2. A,B: GAF domains, CAT: catalytic domain. Hybridization probe: detects both alleles, TbrPDEB2a and TbrPDEB2b; corresponds to nucleotides 158–518 of TbrPDEB2. Panel B: Southern blot analysis of singly or doubly digested genomic DNA establishes the presence of both alleles in the genome of strain Lister427. Circles: Fragments derived from allele TbrPDEB2a; asterisks: fragments derived from allele TbrPDEB2b; no tag: fragments common to both. Sizes of molecular weight markers are indicated to the right.</p

Strain discrimination based on the presence or absence of gene conversion in TbrPDEB2.

<p>A: upper panel: amplification with primer pair TbrB2-for and TbrGAFA2-rev (specific for the wild-type allele); lower panel: amplification with primer pair TbrB2-for and TbrGAFA1-rev (specific for the converted allele). Templates were genomic DNAs of the following strains: 1: procyclic 427; 2: BS221; 3: 427var3; 4: 927; 5: AnTat1.1; 6: STIB247; 7: GVR 35; 8: STIB345; 9: STIB900 (<i>T. b. rhodesiense</i>). B: Duplex PCR for the simultaneous detection of TbrPDEB1 (as an internal control) and the converted allele TbrPDEB2b, using the three primers TbrB1-for, TbrB2-for and TbrGAFA1-rev. Templates were genomic DNAs of strains 427 (1) and STIB247 (2). The 597 bp fragment from TbrPDEB1 corresponds to two gene equivalents, while the 299 bp fragment from TbrPDEB2b corresponds to one gene equivalent.</p

Subcellular localization of TbrPDEB2 is not altered by gene conversion.

<p>Immunoflourescence analysis of c-Myc tagged TbrPDEB2 alleles. Panel A: STIB247 wt (negative control); panel B: STIB247, homozygous for wild-type TbrPDEB2a, one gene copy C-terminally tagged with c-Myc; panel C: Lister 427, heterozygous; the gene-converted allele TbrPDEB2b is tagged with c-Myc. Upper row: Alexa Fluor 488 and DAPI fluorescence; bottom row: phase contrast microscopy. Bars represent 10 um.</p

Immunofluorescence of Triton-X100 extracted cytoskeletons of procyclic trypanosomes.

<p>Triton-resistant, c-Myc tagged TbrPDEB2a and B2b (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000455#pntd-0000455-g006" target="_blank">Fig. 6</a>) both remain associated with the flagellum. Panel A: STIB247 wt; panel B: STIB427 with tagged TbrPDEB2a; panel C: 427 with tagged TbrPDEB2b.</p

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.ISSN:1471-218

Restriction enzyme analysis of the wild-type and converted TbrPDEB2 open reading frames.

<p>These were amplified from genomic DNA of strain 427var3 (containing one converted allele) and strain 927 (no gene conversion). Panel A: restriction digests. Panel B: expected fragment sizes correspond exactly to those obtained experimentally.</p

Listing of strains, their history and TbrPDEB2 genotype.

<p>Listing of strains, their history and TbrPDEB2 genotype.</p