Listing of strains, their history and TbrPDEB2 genotype.

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

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

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

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

Discovery of Novel <i>Trypanosoma brucei</i> Phosphodiesterase B1 Inhibitors by Virtual Screening against the Unliganded TbrPDEB1 Crystal Structure

<i>Trypanosoma brucei</i> cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the unliganded TbrPDEB1 and its use for the in silico screening for new TbrPDEB1 inhibitors with novel scaffolds. The TbrPDEB1 crystal structure shows the characteristic folds of human PDE enzymes but also contains the parasite-specific P-pocket found in the structures of <i>Leishmania major</i> PDEB1 and <i>Trypanosoma cruzi</i> PDEC. The unliganded TbrPDEB1 X-ray structure was subjected to a structure-based in silico screening approach that combines molecular docking simulations with a protein–ligand interaction fingerprint (IFP) scoring method. This approach identified six novel TbrPDEB1 inhibitors with IC₅₀ values of 10–80 μM, which may be further optimized as potential selective TbrPDEB inhibitors

Catechol Pyrazolinones as Trypanocidals: Fragment-Based Design, Synthesis, and Pharmacological Evaluation of Nanomolar Inhibitors of Trypanosomal Phosphodiesterase B1

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of <i>Trypanosoma brucei</i>, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC₅₀ values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (<b>20b</b>): <i>T. brucei rhodesiense</i> IC₅₀ = 60 nM, <i>T. brucei brucei</i> IC₅₀ = 520 nM, <i>T. cruzi</i> = 7.6 μM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of <b>20b</b> was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target