Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

Indole metabolites generated by microbiota inhibit Cryptosporidium growth.

Funkhouser-Jones et al. recently identified gut metabolites that affected Cryptosporidium growth. A key focus, indole, was shown to inhibit the parasite in vivo and in vitro by decreasing the host mitochondria function and the membrane potential of parasite mitosomes. These findings help clarify the role microflora and metabolites play in host resistance.

Anticryptosporidium Efficacy of Olea europaea and Ficus carica Leaves Extract in Immunocompromised Mice Associated with Biochemical Characters and Antioxidative System.

Cryptosporidiosis is caused by an opportunistic protozoan parasite (Cryptosporidium parvum and C. hominis) known as a parasite of humans, especially children and immunocompromised patients. The current study was designed to evaluate the therapeutic efficacy of a mixture of fig and olive leaf extracts as an alternative medicinal plant. Parasitological examination for oocysts in the stool and histopathological alterations in the small intestines were examined. Additionally, biochemical analyses of liver and kidney functions in addition to antioxidant parameters such as superoxide dismutase (SOD), glutathione peroxidase (GSH) and catalase (CAT) in the plasma were evaluated. Our results showed that marked reduction in oocysts shedding and amelioration in intestinal histopathological changes and hepatic or renal functions were detected in all treated groups compared to the control infected group. Additionally, the treated groups with tested extracts at ratios 1:3 and 1:5 showed a significant decrease in the number of oocysts compared to the other treated groups. Results exhibited a significant increase in the plasma SOD, CAT and GSH levels in treated groups compared to the infected control one. This study suggested that a mixture of fig and olive leaf extracts is a convenient promising therapeutic agent for Cryptosporidiosis.

Optimization of the Urea Linker of Triazolopyridazine MMV665917 Results in a New Anticryptosporidial Lead with Improved Potency and Predicted hERG Safety Margin.

Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.

Use-case scenarios for an anti-Cryptosporidium therapeutic.

Cryptosporidium is a widely distributed enteric parasite that has an increasingly appreciated pathogenic role, particularly in pediatric diarrhea. While cryptosporidiosis has likely affected humanity for millennia, its recent "emergence" is largely the result of discoveries made through major epidemiologic studies in the past decade. There is no vaccine, and the only approved medicine, nitazoxanide, has been shown to have efficacy limitations in several patient groups known to be at elevated risk of disease. In order to help frontline health workers, policymakers, and other stakeholders translate our current understanding of cryptosporidiosis into actionable guidance to address the disease, we sought to assess salient issues relating to clinical management of cryptosporidiosis drawing from a review of the literature and our own field-based practice. This exercise is meant to help inform health system strategies for improving access to current treatments, to highlight recent achievements and outstanding knowledge and clinical practice gaps, and to help guide research activities for new anti-Cryptosporidium therapies.

Phenotypic screening techniques for Cryptosporidium drug discovery.

Introduction: Two landmark epidemiological studies identified Cryptosporidium spp. as a significant cause of diarrheal disease in pediatric populations in resource-limited countries. Notably, nitazoxanide is the only approved drug for treatment of cryptosporidiosis but shows limited efficacy. As a result, many drug discovery efforts have commenced to find improved treatments. The unique biology of Cryptosporidium presents challenges for traditional drug discovery methods, which has inspired new assay platforms to study parasite biology and drug screening. Areas covered: The authors review historical advancements in phenotypic-based assays and techniques for Cryptosporidium drug discovery, as well as recent advances that will define future drug discovery. The reliance on phenotypic-based screens and repositioning of phenotypic hits from other pathogens has quickly created a robust pipeline of potential cryptosporidiosis therapeutics. The latest advances involve new in vitro culture methods for oocyst generation, continuous culturing capabilities, and more physiologically relevant assays for testing compounds. Expert opinion: Previous phenotypic screening techniques have laid the groundwork for recent cryptosporidiosis drug discovery efforts. The resulting improved methodologies characterize compound activity, identify, and validate drug targets, and prioritize new compounds for drug development. The most recent improvements in phenotypic assays are poised to help advance compounds into clinical development.

Supply and demand-heme synthesis, salvage and utilization by Apicomplexa.

The Apicomplexa phylum groups important human and animal pathogens that cause severe diseases, encompassing malaria, toxoplasmosis, and cryptosporidiosis. In common with most organisms, apicomplexans rely on heme as cofactor for several enzymes, including cytochromes of the electron transport chain. This heme derives from de novo synthesis and/or the development of uptake mechanisms to scavenge heme from their host. Recent studies have revealed that heme synthesis is essential for Toxoplasma gondii tachyzoites, as well as for the mosquito and liver stages of Plasmodium spp. In contrast, the erythrocytic stages of the malaria parasites rely on scavenging heme from the host red blood cell. The unusual heme synthesis pathway in Apicomplexa spans three cellular compartments and comprises enzymes of distinct ancestral origin, providing promising drug targets. Remarkably given the requirement for heme, T. gondii can tolerate the loss of several heme synthesis enzymes at a high fitness cost, while the ferrochelatase is essential for survival. These findings indicate that T. gondii is capable of salvaging heme precursors from its host. Furthermore, heme is implicated in the activation of the key antimalarial drug artemisinin. Recent findings established that a reduction in heme availability corresponds to decreased sensitivity to artemisinin in T. gondii and Plasmodium falciparum, providing insights into the possible development of combination therapies to tackle apicomplexan parasites. This review describes the microeconomics of heme in Apicomplexa, from supply, either from de novo synthesis or scavenging, to demand by metabolic pathways, including the electron transport chain.

Recent advances in genetic manipulation of Cryptosporidium.

Cryptosporidium is a leading cause of diarrhea-associated morbidity and mortality in young children. Currently, there is no fully effective drug to treat cryptosporidiosis and a complete lack of vaccine to prevent disease. For a long time, progress in the field of Cryptosporidium research has been hindered due to unavailability of methods to propagate the parasite, lack of efficient animal infection models and most importantly, the absence of technology to genetically manipulate the parasite. The recent advent of molecular genetics has been transformative for Cryptosporidium research, and is facilitating our fundamental understanding of parasite biology, and accelerating the pace of drug discovery. This review summarizes recent advancements in genetic manipulation and its applications for studying parasite gene function, host-parasite interactions and discovery of anti-cryptosporidial drugs.

Aetiology of acute diarrhoea and antimicrobial usage among children aged under five years at health centres in Bahir Dar, Ethiopia.

The aetiology of diarrhoea with Cryptosporidium species and pathogenic bacteria has not been identified in the Ethiopian primary healthcare system. Thus, antibiotic consumption for unconfirmed pathogenic diarrhoea is not infrequent. A total of 344 children aged <5 years with diarrhoea were included in the present study to determine protozoal or bacterial aetiology and antibiotic use. Overall, 47.1% of children with diarrhoea had either protozoan or bacterial pathogens. The prevalence of Cryptosporidium species was 12.8% while bacterial pathogens were 13.1%. Based on direct microscopic stool examination, 77.6% (267/344) of children with diarrhoea were treated with antimicrobials. Of these, 16.7%-51.4% of prescribed antimicrobials were appropriate. None of the children with Cryptosporidium diarrhoea were treated with the appropriate antimicrobial. Enteric bacterial pathogens showed a resistance of 51.6% to cotrimoxazole. Empirical treatments are not appropriate for the majority of child cases of diarrhoea.

Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates.

The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches.

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.

Evaluation of the effects of anthelmintic administration on the fecal microbiome of healthy dogs with and without subclinical Giardia spp. and Cryptosporidium canis infections.

BACKGROUND: The gastrointestinal microbiome plays an important role in host health and there is increasing concern regarding the deleterious effects of pharmaceuticals on the fecal microbiome. The effect of anthelmintic therapy on the fecal microbiome in dogs has not yet been evaluated. The purpose of this study was to evaluate the effect of anthelmintic administration on the fecal microbiome of dogs with and without subclinical Giardia species and Cryptosporidium canis infections. METHODOLOGY/PRINCIPAL FINDINGS: Part 1: 6 healthy adult research beagles with subclinical giardiasis and cryptosporidiosis were administered a commercially available preparation of febantel combined with pyrantel and praziquantel (FPP) orally daily for three days. Part 2: 19 healthy staff-owned dogs without giardiasis or cryptosporidiosis were divided into a treatment group (n = 9) that was administered fenbendazole orally daily for five days and an untreated control group (n = 10). For both parts of the study, feces were collected at multiple time points before and after anthelmintic (FPP or fenbendazole) administration. Fecal DNA was extracted for Illumina sequencing of the bacterial 16S rRNA gene and qPCR assays. Neither FPP nor fenbendazole treatment caused a significant change in alpha or beta diversity or the relative abundance of bacterial species. Upon univariate statistical analysis neither FPP or fenbendazole caused minimal changes in the fecal microbiota. CONCLUSION: FPP administration was associated with minimal alterations of the fecal microbiome of healthy research beagles with subclinical giardiasis and cryptosporidiosis. Fenbendazole administration was associated with minimal alterations of the fecal microbiome of healthy staff owned dogs.

P-Glycoprotein-Mediated Efflux Reduces the In Vivo Efficacy of a Therapeutic Targeting the Gastrointestinal Parasite Cryptosporidium.

Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy.

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.

Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater.

Wastewater from different sources is contaminated by protozoan parasites including Cryptosporidium and Giardia. Many protozoan parasites are becoming resistant to chemical treatment. The challenge of finding alternatives is presented to researchers by exploring other methods of eliminating protozoan parasites from wastewater. The aim of this study was to assess the speciation and the viability of Cryptosporidium and Giardia in environmental samples with the specific objective of evaluating if effluent chlorination and UV affect the viability. Different doses of chlorine with different exposure times were experimented with both distilled water and waste water spiked with (oo)cysts derived from environmental samples. UV irradiation at different doses was also experimented using the same spiked samples. Two methods of quantification and detection, namely, microscopy and flow cytometry, were used in the experiment. Two vital dyes, Syto-9+PI and DAPI+PI, were the used for staining the collected wastewater samples. It was found that the (oo)cysts responded to chlorination and UV treatments with Giardia responding better than Cryptosporidium. Giardia responded very well to UV irradiations with almost 0 percent remaining viable after a low dose of UV. Cryptosporidium was found to be resistant to chlorination even at high doses but responded well to high UV doses. DAPI+PI dye gave a lower mean percentage viability values than Syto-9+PI. Flow cytometry gave higher mean percentage than microscopy from the results. It is concluded that UV is a promising alternative to Chlorine in removing Cryptosporidium and Giardia from waste water. Appropriate treatment method for wastewater is necessary to minimize water resources pollution when wastewater is released into water systems.

A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis.

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.

Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research.

The intestinal apicomplexan parasite Cryptosporidium is a major cause of diarrheal disease in humans worldwide. However, treatment options are severely limited. The search for novel interventions is imperative, yet there are several challenges to drug development, including intractability of the parasite and limited technical tools to study it. This review addresses recent, exciting breakthroughs in this field, including novel cell culture models, strategies for genetic manipulation, transcriptomics, and promising new drug candidates. These advances will stimulate the ongoing quest to understand Cryptosporidium and the pathogenesis of cryptosporidiosis and to develop new approaches to combat this disease.