Analysis of the Physicochemical Properties of Anti-Schistosomal Compounds to Identify Next-Generation Leads.

To investigate the physicochemical properties of anti-schistosomal compounds reported between 2008 and 2023, a simple but extensive literature scrutiny was conducted. Keywords were searched in Chemical Abstracts Service (CAS) SciFinder and primary medicinal chemistry and pharmacology literature to locate publications with compounds displaying ex vivo and/or in vivo anti-schistosomal activity. A total of 57 repurposed U.S. Food and Drug Administration (FDA)-approved drugs, hits and their derivatives were manually extracted, curated and compared to known anti-schistosomal oral drugs in view of establishing trends of calculated critical molecular properties. From this analysis, it was determined that more than 65% of the compounds display cLogD7.4 > 3 values, whereas oxamniquine, metrifonate and praziquantel (PZQ), previous and currently used oral anti-schistosomal drugs, possess lower cLogD7.4 values (≤2.5). Furthermore, the lipophilicity associated with PZQ corresponds to a highly permeable and sparingly soluble compound, characteristics that favor drug absorption and compound penetration in the parasite. These physicochemical properties together with PZQ's anti-schistosomal activity make PZQ an essential medicine for the treatment of schistosomiasis and demonstrate the importance of finding the right balance among potency (e.g., EC50 < 5 and 0.5 µM), cell permeability (e.g., Papp > 2 × 106 cm/s) and kinetic aqueous solubility (e.g., >10 µM) to provide high-quality hits and/or leads for the discovery of new oral anti-schistosomal therapeutics.

The Anthelmintic Activity of Praziquantel Analogs Correlates with Structure-Activity Relationships at TRPMPZQ Orthologs.

The anthelmintic drug praziquantel remains a key clinical therapy for treating various diseases caused by parasitic flatworms. The parasite target of praziquantel has remained undefined despite longstanding usage in the clinic, although a candidate ion channel target, named TRPMPZQ, has recently been identified. Intriguingly, certain praziquantel derivatives show different activities against different parasites: for example, some praziquantel analogs are considerably more active against cestodes than against schistosomes. Here we interrogate whether the different activities of praziquantel analogs against different parasites are also reflected by unique structure-activity relationships at the TRPMPZQ channels found in these different organisms. To do this, several praziquantel analogs were synthesized and functionally profiled against schistosome and cestode TRPMPZQ channels. Data demonstrate that structure-activity relationships are closely mirrored between parasites and their TRPMPZQ orthologs, providing further support for TRPMPZQ as the therapeutically relevant target of praziquantel.

Propensity of selecting mutant parasites for the antimalarial drug cabamiquine.

We report an analysis of the propensity of the antimalarial agent cabamiquine, a Plasmodium-specific eukaryotic elongation factor 2 inhibitor, to select for resistant Plasmodium falciparum parasites. Through in vitro studies of laboratory strains and clinical isolates, a humanized mouse model, and volunteer infection studies, we identified resistance-associated mutations at 11 amino acid positions. Of these, six (55%) were present in more than one infection model, indicating translatability across models. Mathematical modelling suggested that resistant mutants were likely pre-existent at the time of drug exposure across studies. Here, we estimated a wide range of frequencies of resistant mutants across the different infection models, much of which can be attributed to stochastic differences resulting from experimental design choices. Structural modelling implicates binding of cabamiquine to a shallow mRNA binding site adjacent to two of the most frequently identified resistance mutations.

Genetic profiles of Schistosoma haematobium parasites from Malian transmission hotspot areas.

BACKGROUND: Although schistosomiasis is a public health issue in Mali, little is known about the parasite genetic profile. The purpose of this study was to analyze the genetic profile of the schistosomes of Schistosoma haematobium group in school-aged children in various sites in Mali. METHODS: Urine samples were collected from 7 to 21 November 2021 and subjected to a filtration method for the presence S. haematobium eggs. The study took place in two schistosomiasis endemic villages (Fangouné Bamanan and Diakalèl), qualified as hotspots according to the World Health Organization (WHO) definition. Molecular genotyping on both Cox1 and ITS2/18S was used for eggs' taxonomic assignation. RESULTS: A total of 970 miracidia were individually collected from 63 school-aged children and stored on Whatman FTA cards for molecular analysis. After genotyping 42.0% (353/840) and 58.0% (487/840) of miracidia revealed Schistosoma bovis and S. haematobium Cox1 profiles, respectively; 95.7 (885/925) and 4.3% (40/925) revealed S. haematobium and S. haematobium/S. curassoni profiles for ITS/18S genes, respectively. There was a significant difference in the Cox1 and ITS2/18S profile distribution according to the village (P < 0.0001). Overall, 45.6% (360/789) were hybrids, of which 72.0% (322/447) were from Diakalèl. Three hybrids' profiles (Sb/Sc_ShxSc with 2.3%; Sb/Sc_ShxSh with 40.5%; Sh_ShxSc with 2.8%) and one pure profile (Sh_ShxSh with 54.4%) were identified. CONCLUSION: Our findings show, for the first time to our knowledge, high prevalence of hybrid schistosomes in Mali. More studies are needed on population genetics of schistosomes at the human and animal interface to evaluate the parasite's gene flow and its consequences on epidemiology of the disease as well as the transmission to humans.

Anti-infectives Developed as Racemic Drugs in the 21st Century: Norm or Exception?

This viewpoint outlines the case for developing new chemical entities (NCEs) as racemates in infectious diseases and where both enantiomers and racemate retain similar on- and off-target activities as well as similar PK profiles. There are not major regulatory impediments for the development of a racemic drug, and minimizing the manufacturing costs becomes a particularly important objective when bringing an anti-infective therapeutic to the marketplace in the endemic settings of infectious diseases.

Causal chemoprophylactic activity of cabamiquine against Plasmodium falciparum in a controlled human malaria infection: a randomised, double-blind, placebo-controlled study in the Netherlands.

BACKGROUND: Cabamiquine is a novel antimalarial that inhibits Plasmodium falciparum translation elongation factor 2. We investigated the causal chemoprophylactic activity and dose-exposure-response relationship of single oral doses of cabamiquine following the direct venous inoculation (DVI) of P falciparum sporozoites in malaria-naive, healthy volunteers. METHODS: This was a phase 1b, randomised, double-blind, placebo-controlled, adaptive, dose-finding, single-centre study performed in Leiden, Netherlands. Malaria-naive, healthy adults aged 18-45 years were divided into five cohorts and randomly assigned (3:1) to receive cabamiquine or placebo. Randomisation was done by an independent statistician using codes in a permuted block schedule with a block size of four. Participants, investigators, and study personnel were masked to treatment allocation. A single, oral dose regimen of cabamiquine (200, 100, 80, 60, or 30 mg) or matching placebo was administered either at 2 h (early liver-stage) or 96 h (late liver-stage) after DVI. The primary endpoints based on a per-protocol analysis set were the number of participants who developed parasitaemia within 28 days of DVI, time to parasitaemia, number of participants with documented parasite blood-stage growth, clinical symptoms of malaria, and exposure-efficacy modelling. The impact of cabamiquine on liver stages was evaluated indirectly by the appearance of parasitaemia in the blood. The Clopper-Pearson CI (nominal 95%) was used to express the protection rate. The secondary outcomes were safety and tolerability, assessed in those who had received DVI and were administered one dose of the study intervention. The trial was prospectively registered on ClinicalTrials.gov (NCT04250363). FINDINGS: Between Feb 17, 2020 and April 29, 2021, 39 healthy participants were enrolled (early liver-stage: 30 mg [n=3], 60 mg [n=6], 80 mg [n=6], 100 mg [n=3], 200 mg [n=3], pooled placebo [n=6]; late liver-stage: 60 mg [n=3], 100 mg [n=3], 200 mg [n=3], pooled placebo [n=3]). A dose-dependent causal chemoprophylactic effect was observed, with four (67%) of six participants in the 60 mg, five (83%) of six participants in the 80 mg, and all three participants in the 100 and 200 mg cabamiquine dose groups protected from parasitaemia up to study day 28, whereas all participants in the pooled placebo and 30 mg cabamiquine dose group developed parasitaemia. A single, oral dose of 100 mg cabamiquine or higher provided 100% protection against parasitaemia when administered during early or late liver-stage malaria. The median time to parasitaemia in those with early liver-stage malaria was prolonged to 15, 22, and 24 days for the 30, 60, and 80 mg dose of cabamiquine, respectively, compared with 10 days for the pooled placebo. All participants with positive parasitaemia showed documented blood-stage parasite growth, apart from one participant in the pooled placebo group and one participant in the 30 mg cabamiquine group. Most participants did not exhibit any malaria symptoms in both the early and late liver-stage groups, and those reported were mild in severity. A positive dose-exposure-efficacy relationship was established across exposure metrics. The median maximum concentration time was 1-6 h, with a secondary peak observed between 6 h and 12 h in all cabamiquine dose groups (early liver-stage). All cabamiquine doses were safe and well tolerated. Overall, 26 (96%) of 27 participants in the early liver-stage group and ten (83·3%) of 12 participants in the late liver-stage group reported at least one treatment-emergent adverse event (TEAE) with cabamiquine or placebo. Most TEAEs were of mild severity, transient, and resolved without sequelae. The most frequently reported cabamiquine-related TEAE was headache. No dose-related trends were observed in the incidence, severity, or causality of TEAEs. INTERPRETATION: The results from this study show that cabamiquine has a dose-dependent causal chemoprophylactic activity. Together with previously demonstrated activity against the blood stages combined with a half-life of more than 150 h, these results indicate that cabamiquine could be developed as a single-dose monthly regimen for malaria prevention. FUNDING: The healthcare business of Merck KGaA, Darmstadt, Germany.

Metabolism of (R)-Praziquantel versus the Activation of a Parasite Transient Receptor Potential Melastatin Ion Channel.

Praziquantel (PZQ) is an essential anthelmintic drug recently established to be an activator of a Transient Receptor Potential Melastatin (TRPMPZQ ) ion channel in trematode worms. Bioinformatic, mutagenesis and drug metabolism work indicate that the cyclohexyl ring of PZQ is a key pharmacophore for activation of trematode TRPMPZQ , as well as serving as the primary site of oxidative metabolism which results in PZQ being a short-lived drug. Based on our recent findings, the hydrophobic cleft in schistosome TRPMPZQ defined by three hydrophobic residues surrounding the cyclohexyl ring has little tolerance for polarity. Here we evaluate the in vitro and in vivo activities of PZQ analogues with improved metabolic stability relative to the challenge of maintaining activity on the channel. Finally, an estimation of the respective contribution to the overall activity of both the parent and the main metabolite of PZQ in humans is reported.

Perspective on Schistosomiasis Drug Discovery: Highlights from a Schistosomiasis Drug Discovery Workshop at Wellcome Collection, London, September 2022.

In September 2022, the Drug Discovery Unit at the University of Dundee, UK, organised an international meeting at the Wellcome Collection in London to explore the current clinical situation and challenges associated with treating schistosomiasis. The aim of this meeting was to discuss the need for new treatments in view of the clinical situation and to ascertain what the key requirements would be for any potential new anti-schistosomals. This information will be essential to inform ongoing drug discovery efforts for schistosomiasis. We also discussed the potential drug discovery pathway and associated criteria for progressing compounds to the clinic. To date, praziquantel (PZQ) is the only drug available to treat all species causing schistosomiasis, but it is often unable to completely clear parasites from an infected patient, partially due to its inactivity against juvenile worms. PZQ-mediated mass drug administration campaigns conducted in endemic areas (e.g., sub-Saharan Africa, where schistosomiasis is primarily prevalent) have contributed to reducing the burden of disease but will not eliminate the disease as a public health problem. The potential for Schistosoma to develop resistance towards PZQ, as the sole treatment available, could become a concern. Consequently, new anthelmintic medications are urgently needed, and this Perspective aims to capture some of the learnings from our discussions on the key criteria for new treatments.

Modulation of Host-Parasite Interactions with Small Molecules Targeting Schistosoma mansoni microRNAs.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from Schistosoma mansoni, has been shown to influence the fate of host T-cells through manipulation of the NF-κB pathway. We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated manipulation of the host immune system. We used a fragment-based screening approach by means of nuclear magnetic resonance (NMR) to identify binders to the precursor of the parasite sma-microRNA-10 present in their extracellular vesicles. The small fragments identified were used to select larger molecules. These molecules were shown to counteract the inhibition of NF-κB activity by sma-microRNA-10 in cell-based assays.

Praziquantel Reduces Maternal Mortality and Offspring Morbidity by Enhancing Anti-Helminthic Immune Responses.

Alongside the wide distribution throughout sub Saharan Africa of schistosomiasis, the morbidity associated with this chronic parasitic disease in endemic regions is often coupled with infection-driven immunomodulatory processes which modify inflammatory responses. Early life parasite exposure is theorized to drive immune tolerance towards cognate infection as well as bystander immune responses, beginning with in utero exposure to maternal infection. Considering that 40 million women of childbearing-age are at risk of infection worldwide, treatment with Praziquantel during pregnancy as currently recommended by WHO could have significant impact on disease outcomes in these populations. Here, we describe the effects of anthelminthic treatment on parasite-induced changes to fetomaternal cross talk in a murine model of maternal schistosomiasis. Praziquantel administration immediately prior to mating lead to clear re-awakening of maternal anti-parasite immune responses, with persistent maternal immune activation that included enhanced anti-schistosome cytokine responses. Clearance of parasites also improved capacity of dams to endure the additional pressure of pregnancy during infection. Maternal treatment also drove lasting functional alterations to immune system development of exposed offspring. Prenatal anthelminthic treatment skewed offspring immune responses towards parasite clearance and reduced morbidity during cognate infection. Maternal treatment also restored offspring protective IgE antibody responses directed against schistosome antigens, which were otherwise suppressed following exposure to untreated maternal infection. This was further associated with enhanced anti-schistosome cytokine responses from treatment-exposed offspring during infection. In the absence of cognate infection, exposed offspring further demonstrated imprinting across cellular populations. We provide further evidence that maternal treatment can restore a more normalized immune profile to such offspring exposed in utero to parasite infection, particularly in B cell populations, which may underlie improved responsiveness to cognate infection, and support the WHO recommendation of anthelminthic treatment during pregnancy.

Translation of liver stage activity of M5717, a Plasmodium elongation factor 2 inhibitor: from bench to bedside.

BACKGROUND: Targeting the asymptomatic liver stage of Plasmodium infection through chemoprevention could become a key intervention to reduce malaria-associated incidence and mortality. METHODS: M5717, a Plasmodium elongation factor 2 inhibitor, was assessed in vitro and in vivo with readily accessible Plasmodium berghei parasites. In an animal refinement, reduction, replacement approach, the in vitro IC99 value was used to feed a Population Pharmacokinetics modelling and simulation approach to determine meaningful effective doses for a subsequent Plasmodium sporozoite-induced volunteer infection study. RESULTS: Doses of 100 and 200 mg would provide exposures exceeding IC99 in 96 and 100% of the simulated population, respectively. CONCLUSIONS: This approach has the potential to accelerate the search for new anti-malarials, to reduce the number of healthy volunteers needed in a clinical study and decrease and refine the animal use in the preclinical phase.

Pre-erythrocytic Activity of M5717 in Monotherapy and Combination in Preclinical Plasmodium Infection Models.

Combination therapies have emerged to mitigate Plasmodium drug resistance, which has hampered the fight against malaria. M5717 is a potent multistage antiplasmodial drug under clinical development, which inhibits parasite protein synthesis. The combination of M5717 with pyronaridine, an inhibitor of hemozoin formation, displays potent activity against blood stage Plasmodium infection. However, the impact of this therapy on liver infection by Plasmodium remains unknown. Here, we employed a recently described 3D culture-based hepatic infection platform to evaluate the activity of the M5717-pyronaridine combination against hepatic infection by P. berghei. This effect was further confirmed in vivo by employing the C57BL/6J rodent Plasmodium infection model. Collectively, our data demonstrate that pyronaridine potentiates the activity of M5717 against P. berghei hepatic development. These preclinical results contribute to the validation of pyronaridine as a suitable partner drug for M5717, supporting the clinical evaluation of this novel antiplasmodial combination therapy.

Detection of Mycolactone by Thin Layer Chromatography.

By means of thin layer chromatography coupled to a fluorescence enhancer, a highly sensitive and operationally simple method to detect the mycolactones stemming from the human pathogen Mycobacterium ulcerans was developed and applied to various sample sources.

Praziquantel Treatment of Schistosoma mansoni Infected Mice Renders Them Less Susceptible to Reinfection.

Beyond transient control of the infection, additional benefits of mass drug administration of praziquantel in endemic communities have been suggested in communities but not mechanistically investigated experimentally. The present study sought to evaluate the additional and hitherto unreported benefits of repeated mass drug administration of praziquantel. We used a tractable mouse model of Schistosoma mansoni infection to assess the effects of repeated infection-treatment cycles on the host susceptibility to reinfection. Parasitaemia was assessed by quantification of Schistosoma egg burden in liver tissues and morbidity was followed up by histological observation of liver lesions by microscopy and using biochemical measurement of liver transaminases. Immune responses were further determined by serum probing of schistosoma-specific antibodies, cytokines and quantification of liver cellular and soluble mediator responses by flow cytometry and ELISA, respectively. At similar ages and comparable gender distribution, groups of mice undergoing higher number of infections treatment cycles over a longer period, remained susceptible to reinfection by the parasite, as judged by the presence of eggs and the associated increasing pathology in the liver tissues. However, notably, there was a clear and significantly higher propensity to lower egg burden upon reinfection when compared to counterparts undergoing a lower number of infection-treatment cycles. This relative reduction of susceptibility to infection was paralleled by a more robust humoral response against parasite antigens, elevated serum IL-4 and liver cytokines. Of note, praziquantel treatment of infected mice left them at a higher baseline of serum IL-4, IgE and liver cytokines but lower CD4+ T cell -derived cytokines when compared to infected non-treated mice supporting an immunological treatment-induced advantage of previously infected mice over naïve mice and infected/not treated mice. Notably, repeated infection-treatment cycles did not preclude the infection-driven aggravation of collagen deposition in the livers over time and was corroborated by a more robust local production of inflammatory cytokines in the most exposed livers. Taken together, our data reveal that treatment of S. mansoni-infected hosts with praziquantel rewires the immune system to a conformation less permissive to subsequent reinfection in mice. Provided the data are translatable from mouse to human, our findings may provide mechanistic support to the potential benefits of more frequent MDAs in high transmission areas to allow rapid acquisition of protective immunity against reinfection.

Mechanism of praziquantel action at a parasitic flatworm ion channel.

Praziquantel (PZQ) is an essential medicine for treating parasitic flatworm infections such as schistosomiasis, which afflicts over 250 million people. However, PZQ is not universally effective, lacking activity against liver flukes of the Fasciola genus. The reason for this insensitivity is unclear, as the mechanism of PZQ action is unknown. Here, we use ligand- and target-based methods to demonstrate that PZQ activates a transient receptor potential melastatin ion channel (TRPMPZQ) in schistosomes by engaging a hydrophobic ligand binding pocket within the voltage sensor­like domain of the channel to cause calcium entry and worm paralysis. PZQ activates TRPMPZQ homologs in other PZQ-sensitive flukes, but not Fasciola hepatica. However, a single amino acid change in the F. hepatica TRPMPZQ binding pocket, to mimic schistosome TRPMPZQ, confers PZQ sensitivity. After decades of clinical use, the molecular basis of PZQ action at a druggable TRP channel is resolved.

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium.

BACKGROUND: Anthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay. METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3-6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug. CONCLUSION: With this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).

X-SPEC: a 70†eV to 15†keV undulator beamline for X-ray and electron spectroscopies.

X-SPEC is a high-flux spectroscopy beamline at the KIT (Karlsruhe Institute of Technology) Synchrotron for electron and X-ray spectroscopy featuring a wide photon energy range. The beamline is equipped with a permanent magnet undulator with two magnetic structures of different period lengths, a focusing variable-line-space plane-grating monochromator, a double-crystal monochromator and three Kirkpatrick-Baez mirror pairs. By selectively moving these elements in or out of the beam, X-SPEC is capable of covering an energy range from 70 eV up to 15 keV. The flux of the beamline is maximized by optimizing the magnetic design of the undulator, minimizing the number of optical elements and optimizing their parameters. The beam can be focused into two experimental stations while maintaining the same spot position throughout the entire energy range. The first experimental station is optimized for measuring solid samples under ultra-high-vacuum conditions, while the second experimental station allows in situ and operando studies under ambient conditions. Measurement techniques include X-ray absorption spectroscopy (XAS), extended X-ray absorption fine structure (EXAFS), photoelectron spectroscopy (PES) and hard X-ray PES (HAXPES), as well as X-ray emission spectroscopy (XES) and resonant inelastic X-ray scattering (RIXS).

Alternatives to Praziquantel for the Prevention and Control of Schistosomiasis.

Schistosomiasis, a neglected tropical disease provoked by infection with parasitic blood flukes of the genus Schistosoma, affects almost 240 million people worldwide, and more than 700 million people live in endemic areas. However, 40 years after the approval of praziquantel as an anthelmintic drug, the pipeline is nearly empty, and no other therapeutic alternative has reached the market. In its roadmap to "eliminate Schistosomiasis as a public health problem by 2030", the World Health Organization calls for the development of new therapeutic interventions. A viewpoint on the learnings from praziquantel research as well as shaping the next generation of drugs is shared.