Trichuris muris egg-hatching assay for anthelminthic drug discovery and characterization.

Trichuriasis is a neglected tropical disease widely distributed among tropical and sub-tropical areas and associated with poverty and lack of access to safe drinking water, sanitation and hygiene. Existing drugs have limited efficacy and face a constant risk of developing resistance, necessitating the search for alternative treatments. However, drug discovery efforts are sparse and little research has been performed on anthelminthic effects on embryonated eggs, the infectious life stage of Trichuris spp. We examined bacterial species dependent egg hatching of the murine model parasite Trichuris muris and identified Escherichia coli, Pseudomonas aeruginosa and Enterobacter hormaechei effective as hatching inducers, resulting in hatching yields of 50-70%. Streptococcus salivarius, reported to be associated with reduced drug efficacy of ivermectin-albendazole coadministration in Trichuris trichiura infected patients, did not promote egg hatching in vitro. We optimized hatching conditions using E. coli grown in luria broth or brain-heart infusion media to reach consistently high hatching yields to provide a sensitive, robust and simple egg-hatching assay. Oxantel pamoate demonstrated the strongest potency in preventing hatching, with an EC50 value of 2-4 µM after 24 h, while pyrantel pamoate, levamisole and tribendimidine exhibited only moderate to weak inhibitory effects. Conversely, all tested benzimidazoles and macrolide anthelminthics as well as emodepside failed to prevent hatching (EC50 > 100 µM). Our study demonstrates that egg-hatching assays complement larval and adult stage drug sensitivity assays, to expand knowledge about effects of current anthelminthics on Trichuris spp. Further, the developed T. muris egg-hatching assay provides a simple and cheap screening tool that could potentially lead to the discovery of novel anthelminthic compounds.

Development of a hookworm egg hatching assay to determine the ovicidal effects of anthelminthics.

BACKGROUND: Few anthelminthics are currently available, manifesting the urgent need for new treatment options. In vitro profiling of current anthelminthics against larval and adult stage helminths displayed varying effects on closely related worm species and between life stages of the same species. Conversely, limited research has been performed on the egg stage of human hookworms, and the effects of investigational compounds on the egg stage are not routinely assessed. METHODS: We profiled the development and hatching of Heligmosomoides polygyrus, Ancylostoma duodenale and Necator americanus eggs isolated from rodent faeces in liquid media with various nutrient levels, osmolar concentrations, and acidities in dependence on incubation temperature and light exposure. Incubation conditions were optimised to allow the study of drug effect on immature and embryonated eggs. We analysed concentration-effect relationships of commercially available anthelminthics over 72 h. RESULTS: Rapid embryonation and hatching were observed at room temperature with and without light exposure without nutrient supplementation in a wide range of acidities. Hookworms hatched optimally at room temperature in PBS achieving > 75% hatching over 34 h. Developmental delays were seen when eggs were stored at 4 °C with no effect on viability. Similar delays were also seen with increased osmolar concentrations resulting in decreased viability. Benzimidazole anthelminthics effectively reduced the viability and prevented hatching of hookworm eggs, with albendazole and thiabendazole eliciting particularly potent effects at EC50 values below 1 µM. Macrolide anthelminthics as well as emodepside, oxantel pamoate, and pyrantel pamoate were inactive while monepantel, levamisole, and tribendimidine displayed varied potencies among the hookworm species. CONCLUSION: The presented egg-hatching assay will complement ongoing anthelminthic drug discovery and allow a full characterisation of drug activity against all life stages. In the development and application of the egg-hatching assay, good accordance was observed between the three hookworm species evaluated. Marketed anthelminthics show differences of drug action compared to larval and adult stages highlighting the importance of profiling drug activity against all life stages.

In Vitro and In Vivo Antischistosomal Activity Profiling and Pharmacokinetics of Ozonide Carboxylic Acids.

Praziquantel, the only drug in clinical use for the treatment and control of schistosomiasis, is inactive against developing infections. Ozonides are synthetic peroxide derivatives inspired by the naturally occurring artemisinin and show particularly promising activity against juvenile schistosomes. We conducted an in-depth characterization of the in vitro and in vivo antischistosomal activity and pharmacokinetics of lead ozonide carboxylic acid OZ418 and four of its active analogs. In vitro, the ozonides featured rapid and consistent activity against schistosomula and adult schistosomes at double-digit micromolar EC50 values. Potency did not vary considerably between Schistosoma spp. The zwitterionic OZ740 and OZ772 were more active in vivo compared to their non-amphoteric carboxylic acids OZ418 and OZ748, despite their much lower systemic plasma exposure (AUC). The most active compound in vivo was ethyl ester OZ780, which was rapidly transformed to its parent zwitterion OZ740 and achieved ED50 values of 35 ± 2.4 and 29 ± 2.4 mg/kg against adult and juvenile Schistosoma mansoni, respectively. Ozonide carboxylic acids represent promising candidates for further optimization and development due to their good efficacy against both life stages together with their broad activity range against all relevant parasite species.

Improving translational power in antischistosomal drug discovery.

Schistosomiasis is a poverty-associated tropical disease caused by blood dwelling trematodes that threaten approximately 10% of the world population. Praziquantel, the sole drug currently available for treatment, is insufficient to eliminate the disease and the clinical drug development pipeline is empty. Here, we review the characteristics of the patent Schistosoma mansoni mouse model used for in vivo antischistosomal drug discovery, highlighting differences in the experimental set-up across research groups and their potential influence on experimental results. We explore the pharmacokinetic/pharmacodynamic relationship of selected drug candidates, showcasing opportunities to improve the drug profile to accelerate the transition from the early drug discovery phase to new clinical candidates.

Real-time and automated monitoring of antischistosomal drug activity profiles for screening of compound libraries.

Schistosomiasis is a neglected tropical disease that affects over 200 million people annually. As the antischistosomal drug pipeline is currently empty, repurposing of compound libraries has become a source for accelerating drug development, which demands the implementation of high-throughput and efficient screening strategies. Here, we present a parallelized impedance-based platform for continuous and automated viability evaluation of Schistosoma mansoni schistosomula in 128 microwells during 72 h to identify antischistosomal hits in vitro. By initially screening 57 repurposed compounds against larvae, five drugs are identified, which reduce parasite viability by more than 70%. The activity profiles of the selected drugs are then investigated via real-time dose-response monitoring, and four compounds reveal high potency and rapid action, which renders them suitable candidates for follow-up tests against adult parasites. The study shows that our device is a reliable tool for real-time drug screening analysis of libraries to identify new promising therapeutics against schistosomiasis.

Discovery of novel antischistosomal scaffolds from the open access Pandemic Response Box.

BACKGROUND: Treatment and control of schistosomiasis rely on a single drug, praziquantel. New orally active antischistosomals featuring novel molecular scaffolds are urgently needed to prevent the emergence of resistance. METHODS: We screened 400 drug-like compounds contained in the open-access Pandemic Response Box (PRB) against newly transformed schistosomula (NTS) at a concentration of 10 µM scoring death, changes in motility, and morphological alterations. Compounds displaying an activity ≥66% at 72 h underwent testing against adult Schistosoma mansoni in vitro. Fast-acting (≥66% at 24 h), nontoxic drugs focusing on late-stage and approved drugs were investigated in the patent S. mansoni mouse model. RESULTS: We identified 26 hits active against NTS, of which 17 elicited ≥66% activity against adult S. mansoni following 24 h of drug exposure. The highest activity against adult S. mansoni was observed with MMV1581558 (EC50 value of 0.18 ± 0.01 µM) and nitazoxanide (0.47 ± 0.07 µM). Of the five compounds tested in vivo, MMV1581558 and the approved drug ozanimod reduced average worm burden versus controls by 42 % and 36 %, respectively, after a single oral dose of 200 mg/kg bodyweight in mice harboring a chronic S. mansoni infection. CONCLUSION: MMV1581558 discovered from screening the PRB represents a novel antischistosomal scaffold with high in vitro antischistosomal activity amenable to chemical modification for drug development.

Parallelized Impedance-Based Platform for Continuous Dose-Response Characterization of Antischistosomal Drugs.

Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma, which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance-based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose-response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug-induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose-response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance-based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.

In vitro qualitative and quantitative CT assessment of iodinated aerosol nasal deposition using a 3D-printed nasal replica.

Computed tomography can provide high-resolution details on nasal anatomy being potentially useful for the assessment of nasal spray deposition. The purpose of this technical note was to present a method based on CT imaging to assess qualitatively and quantitatively the in vitro spray deposition patterns within the sinonasal cavities of a nasal replica obtained by three-dimensional (3D) printing, using iodinated contrast agent labelled solutions with high spatial and temporal resolution. Using a third generation dual-source CT scanner in single energy mode, scans of a nasal replica were acquired following application of iodinated contrast agent labelled aerosols with an iodine concentration of 92.5 mgl/mL. Two software programmes were then utilised (Osirix MD v.9.0, Pixmeo, Geneva, Switzerland; 3mensio, Pie Medical Imaging, Bilthoven, Netherlands) to generate three-dimensional reconstructions of the scans, thus enabling the rapid detection and visualisation of administered single droplets and their voxel-by-voxel localisation. Using this approach, we achieved recovery rates between 84-98% and 89-109% (depending on the software programme) of the total applied aerosol volume.

A DNA-Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition.

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a Kd value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.

"Cap-and-Catch" Purification for Enhancing the Quality of Libraries of DNA Conjugates.

The potential of DNA-encoded combinatorial libraries (DECLs) as tools for hit discovery crucially relies on the availability of methods for their synthesis at acceptable purity and quality. Incomplete reactions in the presence of DNA can noticeably affect the purity of DECLs and methods to selectively remove unreacted oligonucleotide-based starting products would likely enhance the quality of DECL screening results. We describe an approach to selectively remove unreacted oligonucleotide starting products from reaction mixtures and demonstrate its applicability in the context of acylation of amino-modified DNA. Following an amide bond forming reaction, we treat unreacted amino-modified DNAs with biotinylating reagents and isolate the corresponding biotinylated oligonucleotides from the reaction mixture by affinity capture on streptavidin-coated sepharose. This approach, which yields the desired DNA-conjugate at enhanced purity, can be applied both to reactions performed in solution and to procedures in which DNA is immobilized on an anion exchange solid support.