Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug clas

High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity

Trypanothione reductase (TR), an enzyme that buffers oxidative stress in trypanosomatid parasites, was screened against commercial libraries containing approximately 134,500 compounds. After secondary screening, four chemotypes were identified as screening positives with selectivity for TR over human glutathione reductase. Thirteen compounds from these four chemotypes were purchased, and their in vitro activity against TR and Trypanosoma brucei are described

Chemical, genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome

Pyridoxal-5′-phosphate (vitamin B(6)) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B(6)de novo and rely on uptake of B(6) vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg(−1) and a K(m)(app) with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress▿ §

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man5GlcNAc2 to Man9GlcNAc2 glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway

Synthesis and Evaluation of Indatraline-Based Inhibitors for Trypanothione Reductase

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition

Generation of cell lines resistant to Oxaborole-1.

<p><b>A.</b> Stepwise generation of resistance to oxaborole. <b>B.</b> Representative drug sensitivity plots. The parental cloned line had an EC₅₀ value of 64.4 ± 1.8 nM and the resistant clones 1, 2 and 3 had EC₅₀ values of 530 ± 21, 390 ± 17 and 340 ± 27, nM, respectively. Data are the weighted mean of three independent determinations.</p

Structure of oxaboroles and affinity chromatography resins.

<p>Structure of oxaboroles and affinity chromatography resins.</p

Copy number variations (CNVs) in the drug-resistant and -revertant parasite lines identified by whole genome sequencing.

<p>The read coverage plots show the relative read depth across selected chromosomal regions in the different parasite lines. (<b>A, B</b>) The panels depict the relative read coverage across the core regions of chromosomes 1 and 4, analysed in windows of 12 kb width, by plotting the ratio of the median read depth observed in a given window over the median read depth of all chromosomes for that parasite line. A relative read depth of 1.5 therefore represents a 50% copy number increase of the respective chromosome compared to the rest of the genome, which indicates an absolute copy number increase from two to three in the diploid parasite genome. (<b>C-E</b>) These panels depict the relative read coverage across selected chromosomal regions, analysed in windows of 600 bp width, by plotting the log₂-based ratio of the median read depth observed in a given window over the median read depth of the entire core region of that chromosome for that parasite line. A log₂-based relative read depth of 1 therefore represents a 100% copy number increase of the respective region compared to the rest of that chromosome, whereas a log₂-based relative read depth of -1 indicates a 50% reduction in read depth which is likely due to the loss of one of the two alleles in that region (hemizygosity). In both cases of allele loss, the central area with 50% diminished read depth is flanked by two regions that are approximately 99% identical to one another (D, E). The location of genes along chromosomal regions is indicated below the graphs (C-E). Note the variable but highly reproducible (across the different parasite lines) nature of the read coverage which is in part caused by differences in sequence read mapping efficiency across the genome especially in regions of repetitive sequence.</p