Manu De Rycker

Trypanosoma cruzi , the causative agent of Chagas disease, exhibits a complex life cycle with multiple hosts, stages and differentiation steps. We present a cell atlas for the T. cruzi life cycle, based on single cell transcriptomes for over 31,000 cells and population-based transcriptomics. The atlas reveals many life cycle associated genes and can be utilised to accurately annotate life cycle stages. It provides detailed insights into cell heterogeneity, including cell-specific repertoires of surface antigens in trypomastigotes, with key implications for immune responses. Enabled by single-cell resolution, we define the transcriptomic changes that occur across the epimastigote to metacyclic trypomastigote differentiation axis. Furthermore, we provide comprehensive UTR annotation, identifying previously unannotated transcripts as well as revealing alternative polyadenylation and an unanticipated complexity of reverse strand and antisense transcripts. This T. cruzi atlas provides a comprehensive resource and unlocks a range of new avenues for research on this important human pathogen.

Assessing and understanding the impacts of all possible mutations at a drug binding site remain challenging. Here we use multiplex oligo targeting for mutational profiling, and computational modelling, to decode efficacy and resistance space at the otherwise native binding site for an anti-trypanosomal proteasome inhibitor. We saturation-edit twenty codons in the Trypanosoma brucei proteasome and subject the resulting libraries to stepwise drug selection and codon variant scoring, yielding dose-response profiles for >100 resistance-conferring mutants. Codon variant scores are predictive of relative resistance observed using a bespoke set of mutants, while fitness profiling reveals otherwise extensive constraints on mutational fitness and resistance space. The resistance profile is predictive of routes to spontaneous drug resistance observed within ‘accessible’, single nucleotide mutational space, while in silico predictions are closely aligned with impacts on drug resistance observed in cellulo. Thus, multiplex oligo targeting facilitates assessment of all possible mutations at a drug binding site.

The drug discovery pipeline for neglected kinetoplastid diseases remains sparse. In particular, the field of leishmaniasis drug discovery has had limited success in translating potential drug candidates into viable therapies. Here, we describe the development of two lead compounds, BZ-1 and BZ1-I, which have potent in vitro anti-leishmanial activity against Leishmania donovani DD8 intracellular amastigotes (0.59 ± 0.13 µM and 0.40 ± 0.38 µM) with corresponding selectivity ( > 33.89 and > 49.12) for differentiated THP-1 cells (Human monocytic cell line), respectively. Further characterization and biological profiling identified that in addition to the activity against L. donovani DD8 (Old World - Indian strain), compounds were active against intracellular parasites from other species and strains of the Old and New World, namely L. donovani (Old World - Sudanese strain) and L. infantum chagasi (New World-South American strain). In vivo evaluation using the hamster model illustrated that the activity observed in vitro was translated in vivo, with outstanding results. Our data suggests that these compounds represent a promising starting point for developing a novel lead series for future anti-leishmanial therapeutics.

Two related series of benzodiazepinediones were synthesised, profiled for their suitability as novel treatments for Chagas disease, and progressed to a T. cruzi washout assay.

Trypanosoma cruzi , the causative agent of Chagas disease, exhibits a complex life cycle with multiple hosts, stages and differentiation steps. We present a complete cell atlas for the T. cruzi life cycle, based on single cell transcriptomes for over 31,000 cells and population-based transcriptomics. The atlas reveals many life cycle associated genes and can be utilised to accurately annotate life cycle stages. It provides detailed insights into cell heterogeneity, including cell-specific repertoires of surface antigens in trypomastigotes, with key implications for immune responses. Enabled by single-cell resolution, we define the transcriptomic changes that occur across the epimastigote to metacyclic trypomastigote differentiation axis. Furthermore, we provide comprehensive UTR annotation, identifying previously unannotated transcripts as well as revealing alternative poly-adenylation and an unanticipated complexity of reverse strand and antisense transcripts. This T. cruzi atlas provides a comprehensive resource and unlocks a range of new avenues for research on this important human pathogen.

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas’ disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, T. cruzi is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe T. cruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three T. cruzi. inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4–6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all T. cruzi CLBrener Luc life cycle stages and Silvio x10/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated T. cruzi Silvio x10/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated T. cruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, T. cruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated T. cruzi inactivation methods which can be applied to other related ACDP Hazard Group 2–3 kinetoplastid parasites.

Background: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. Methodology/Principal findings: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC₅₀ < 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with K_i 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. Conclusions/Significance: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi , affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Q _i active site of T. cruzi cytochrome b, part of the cytochrome bc ₁ complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.