Validation of Trypanosoma cruzi inactivation techniques for laboratory use.

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.

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

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 Ki 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.

Short-course combination treatment for experimental chronic 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 Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 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.

RES-Seq-a barcoded library of drug-resistant Leishmania donovani allowing rapid assessment of cross-resistance and relative fitness.

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions (MoAs) are required to consolidate progress toward control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritization of anti-leishmanial compounds acting via novel MoA. We have amassed a large collection of Leishmania donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterized. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC50 for 3 days) and applied to screening of a complex library with VL preclinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies.IMPORTANCEVisceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000-30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

Identification of a Proteasome-Targeting Arylsulfonamide with Potential for the Treatment of Chagas' Disease.

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.

The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.

Identification of 6-amino-1H-pyrazolo[3,4-d]pyrimidines with in vivo efficacy against visceral leishmaniasis.

Visceral leishmaniasis (VL) affects millions of people across the world, largely in developing nations. It is fatal if left untreated and the current treatments are inadequate. As such, there is an urgent need for new, improved medicines. In this paper, we describe the identification of a 6-amino-N-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine scaffold and its optimization to give compounds which showed efficacy when orally dosed in a mouse model of VL.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas' disease drug discovery.

Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments.

Chemical Validation of Methionyl-tRNA Synthetase as a Druggable Target in Leishmania donovani.

Methionyl-tRNA synthetase (MetRS) has been chemically validated as a drug target in the kinetoplastid parasite Trypanosoma brucei. In the present study, we investigate the validity of this target in the related trypanosomatid Leishmania donovani. Following development of a robust high-throughput compatible biochemical assay, a compound screen identified DDD806905 as a highly potent inhibitor of LdMetRS (Ki of 18 nM). Crystallography revealed this compound binds to the methionine pocket of MetRS with enzymatic studies confirming DDD806905 displays competitive inhibition with respect to methionine and mixed inhibition with respect to ATP binding. DDD806905 showed activity, albeit with different levels of potency, in various Leishmania cell-based viability assays, with on-target activity observed in both Leishmania promastigote cell assays and a Leishmania tarentolae in vitro translation assay. Unfortunately, this compound failed to show efficacy in an animal model of leishmaniasis. We investigated the potential causes for the discrepancies in activity observed in different Leishmania cell assays and the lack of efficacy in the animal model and found that high protein binding as well as sequestration of this dibasic compound into acidic compartments may play a role. Despite medicinal chemistry efforts to address the dibasic nature of DDD806905 and analogues, no progress could be achieved with the current chemical series. Although DDD806905 is not a developable antileishmanial compound, MetRS remains an attractive antileishmanial drug target.

Discovery and Optimization of 5-Amino-1,2,3-triazole-4-carboxamide Series against Trypanosoma cruzi.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is the most common cause of cardiac-related deaths in endemic regions of Latin America. There is an urgent need for new safer treatments because current standard therapeutic options, benznidazole and nifurtimox, have significant side effects and are only effective in the acute phase of the infection with limited efficacy in the chronic phase. Phenotypic high content screening against the intracellular parasite in infected VERO cells was used to identify a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC). Optimization of the ATC series gave improvements in potency, aqueous solubility, and metabolic stability, which combined to give significant improvements in oral exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which demonstrated significant suppression of parasite burden in a mouse model of Chagas' disease.

The relationship of endotoxaemia to peripheral and central nervous system inflammatory responses in Human African Trypanosomiasis.

Endotoxaemia has been described in cases of Human African trypanosomiasis (HAT), but it is unclear if this phenomenon influences inflammatory pathology either in the periphery or central nervous system (CNS). We studied endotoxin concentrations in the plasma and cerebrospinal fluid (CSF) of Trypanosoma brucei rhodesiense patients using the chromogenic Limulus Amoebocyte lysate assay. The relationship of endotoxin concentration to the presentation of gross signs of inflammation and the inflammatory/counter-inflammatory cytokine profile of the relevant compartments were analysed. We demonstrate that HAT patients exhibit parasitaemia-independent plasma endotoxaemia, and that this is associated with splenomegaly and lymphadenopathy. Endotoxin concentrations normalize rapidly after treatment. There was no evidence of endotoxin release in the CNS. A rapid normalization of endotoxin levels after treatment and lack of association with parasitaemia suggest that gut leakage is the main source of endotoxin in the circulation. Low CSF endotoxin concentrations and a lack of any association with neuroinflammatory markers or neurological sequelae suggest that endotoxin does not play a role in the pathogenesis of the disease in the CNS.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.

African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.

Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response.

BACKGROUND: Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance. METHODOLOGY/PRINCIPAL FINDINGS: This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-ß levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF. CONCLUSIONS: Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value.

The orthologue of Sjögren's syndrome nuclear autoantigen 1 (SSNA1) in Trypanosoma brucei is an immunogenic self-assembling molecule.

Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.