Host cells with transient overexpression of MDR1 as a novel in vitro model for evaluating on-target effect for activity against the epicellular Cryptosporidium parasite.

OBJECTIVES: To rapidly generate host cells with resistance to multiple compounds for differentiating drug action on parasite target or the host cell target (i.e. on-target or off-target effect) against the zoonotic enteric parasite Cryptosporidium parvum. METHODS: Transient overexpression of a multidrug resistance protein 1 (MDR1) gene in host cells (HCT-8 cell line) was explored to increase drug tolerance of the host cells to selected anti-cryptosporidial leads. In vitro cytotoxicity and anti-cryptosporidial efficacy of selected compounds were evaluated on the parasite grown in WT parental and transiently transfected HCT-8 cells. The approach was based on the theory that, for an epicellular parasite receiving consistent exposure to compounds in culture medium, overexpressing MDR1 in HCT-8 cells would increase drug tolerance of host cells to selected compounds but would not affect the anti-cryptosporidial efficacy if the compounds acted solely on the parasite target and the drug action on host cell target played no role on the antiparasitic efficacy. RESULTS: Six known anti-cryptosporidial leads were tested. Transient overexpression of MDR1 increased drug tolerance of HCT-8 cells on paclitaxel, doxorubicin HCl and vincristine sulphate (2.11- to 2.27-fold increase), but not on cyclosporin A, daunorubicin HCl and nitazoxanide. Increased drug tolerance in host cells had no effect on antiparasitic efficacy of paclitaxel, but affected that of doxorubicin HCl. CONCLUSIONS: Data confirmed that, at efficacious concentrations, paclitaxel acted mainly on the parasite target, while doxorubicin might act on both parasite and host cell targets. This model can be employed for studying the action of additional anti-cryptosporidial leads, and adapted to studying drug action in other epicellular pathogens. The limitation of the model is that the anti-cryptosporidial leads/hits need to be MDR1 substrates.

Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture.

The widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro.

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

High-Content Screening for Cryptosporidium Drug Discovery.

High-content screening (HCS) is a cell-based type of phenotypic screening that combines multiple simultaneous readouts with a high level of throughput. A particular benefit of this form of screening for drug discovery is the ability to perform the interrogation in a biologically relevant system. This approach has greatly advanced the field of drug discovery for cryptosporidiosis, a diarrheal disease caused by protozoan parasites of Cryptosporidium spp. These parasites are obligate intracellular parasites and cannot be cultured in vitro without the support of a host cell, limiting the options for potential assay readout. Here we describe an established 384- or 1536-well format high-content imaging (HCI) assay of Cryptosporidium-infected HCT-8 human ileocecal adenocarcinoma cells. This HCS assay is a powerful tool to assess large numbers of compounds to power drug discovery, as well as to phenotypically characterize known Cryptosporidium-active compounds.

High-Throughput Screening of Drugs Against the Growth of Cryptosporidium parvum In Vitro by qRT-PCR.

An effective method to quantify the parasite loads is the key to the evaluation of anti-cryptosporidial drug efficacy in vitro. However, high-throughput screening (HTS) of drugs against Cryptosporidium parvum in vitro was impractical by the labor-intensive traditional assays. Here we describe a simplified quantitative RT-PCR assay suitable for HTS of compounds and for evaluating drug efficacy against the growth of C. parvum in vitro.

In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds.

Hollow fiber technology is a powerful tool for the culture of difficult-to-grow cells. Cryptosporidium parvum has a multistage sexual and asexual life cycle that has proved difficult to culture by conventional in vitro culture methods. Here, we describe a method utilizing a hollow fiber bioreactor for the continuous in vitro growth of C. parvum that produces sexual and asexual stages. The method enables the evaluation of potential therapeutic compounds under conditions that mirror the dynamic conditions found in the gut facilitating preliminary pharmacokinetic and pharmacodynamic data to be obtained.

Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.

The Action of the Hexokinase Inhibitor 2-deoxy-d-glucose on Cryptosporidium parvum and the Discovery of Activities against the Parasite Hexokinase from Marketed Drugs.

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2-deoxy-d-glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG-6-phosphate (2DG6P), the anti-cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose-6-phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti-cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC50  = 0.53, 1.77, 8.1 and 165 µM, respectively). The anti-CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase.

Anti-protozoal activity of extracts from chicory (Cichorium intybus) against Cryptosporidium parvum in cell culture.

Cryptosporidium spp. are responsible for severe public health problems and livestock production losses. Treatment options are limited to only one drug available for human and bovine cryptosporidiosis, respectively, and both drugs exhibit only partial efficacy. Sesquiterpene lactones (SL) are plant bioactive compounds that function as a defence mechanism against herbivores. SL have demonstrated anti-parasitic properties against a range of parasitic taxa but knowledge about their anti-Cryptosporidium efficacy is limited. The effect of SL-rich leaf and root extracts from chicory (Cichorium intybus cv. Spadona) was investigated using human colon adenocarcinoma (HCT-8) cells infected with Cryptosporidium parvum. C. parvum oocysts were inoculated onto the cell monolayer and i) incubated for 4 hours with extracts (leaf and root extracts 300, 150, 75, 37.5, 18.75 and 9.375 µg/mL) in triplicates followed by incubation in bioactive free media (sporozoite invasion assays) or ii) incubated for 4 hours in bioactive free media followed by 48-hours incubation with extracts (growth inhibition assays). Extract toxicity on HCT-8 cells was assessed via water-soluble tetrazolium (WST)-1 assay prior to quantifying parasitic growth via immunofluorescence. Both extracts demonstrated dose-dependent inhibition in the growth inhibition assays (p = < 0.0001 for both extracts) but not in the invasion assays. Anti-parasitic activity did not appear to be solely related to SL content, with the extract with lower SL content (leaf) exhibiting higher inhibition at 300 µg/ml. However, given the limited treatment options available for Cryptosporidium spp., our study encourages further investigation into the use of chicory extracts to identify novel active compound(s) inhibiting these protozoa.

Efficacy of chitosan, a natural polysaccharide, against Cryptosporidium parvum in vitro and in vivo in neonatal mice.

Cryptosporidiosis is a zoonotic disease caused by species in the genus Cryptosporidium. In young ruminants, Cryptosporidium parvum causes economically significant disease with mild to severe clinical signs and occasional death. The typical clinical course in animals aged 1-3 weeks old is acute diarrhoea. Currently there are no available treatments that are fully effective against cryptosporidiosis in either humans or animals. Therefore there is a critical need for the development of new therapeutic agents. We adapted two in vitro culture systems (HCT-8 and Caco-2 cell lines) for C. parvum infection to investigate the "anticryptosporidial" activity of two chitosans; Chitosan NAG and Chitosan Mix. Chitosan-a naturally-occurring polysaccharide compound-has been found to be active against a variety of diseases, possessing both antimicrobial and anticancer properties. We investigated both chitosan's toxicity and effects on C. parvum in the two in vitro models. To evaluate chitosan's effects on oocyst shedding in vivo, CD-1 neonate mice were orally inoculated with C. parvum oocysts (Iowa strain), treated with chitosan, and compared to infected non-treated animals. Paromomycin, a classical drug used in veterinary medicine, was used as a reference compound. Immunofluorescence techniques were used to analyse the parasites. Our results showed significant reductions in Cryptosporidium oocyst viability (>95%) after oocyst pre-incubation with either paromomycin (P < 0.001), Chitosan Mix or Chitosan NAG (P < 0.001), for 24 h at 37 °C. Additionally, paromomycin, Chitosan Mix, and Chitosan NAG significantly inhibited C. parvum multiplication in HCT-8 and Caco-2 cell lines (P < 0.005). These effects were dose-dependent. In in vivo studies, treatment with both chitosans (Chitosan NAG, Chitosan Mix) or paromomycin sulfate significantly reduced parasite shedding in infected treated newborn mice (-56%, -34.5% and -58%, respectively). In conclusion, these findings provide the first in vitro and in vivo evidence of the anticryptosporidial activities of this natural polysaccharide.

Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia and Cryptosporidium parvum.

There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed ≥ 75% inhibition of parasite growth in the initial screen (16 µM), with fifteen showing ≥ 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with ≥ 75% parasite growth inhibition at 10 µM, with six showing ≥ 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.

Herbicidins from Streptomyces sp. CB01388 Showing Anti- Cryptosporidium Activity.

A high-content imaging assay was used to screen the fraction collection of the Natural Product Library at The Scripps Research Institute for inhibitors of Cryptosporidium parvum. A chemical investigation of one strain, Streptomyces sp. CB01388, resulted in the isolation of six herbicidins (1-6), one of which is new (herbicidin L, 1). Five of the six herbicidins (1-3, 5, 6) showed moderate inhibitory activity against C. parvum, with 1 and 6 comparable to the FDA-approved drug nitazoxanide, and 2-6 showed no toxicity to the host HCT-8 cells and human HEK293T and HepG2 cells. These findings highlight the herbicidin scaffold for anti- Cryptosporidium drug development.

Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/µg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 µM; Ki = 36.33 µM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 µM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 µM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target.

Development of a Cytopathic Effect-Based Phenotypic Screening Assay against Cryptosporidium.

Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum ( Cp) and Cryptosporidium hominis ( Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti- Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials.

Effect of oregano essential oil and carvacrol on Cryptosporidium parvum infectivity in HCT-8 cells.

Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1×106) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1×104) in two modalities: 1) 4h co-culture with bioactive (0-250µg/mL) followed by washing and incubation (48h, 37°C, 5% CO2) in bioactive-free media; and 2) 4h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0-250µg/mL) during 48-h incubation. Cell viability was tested using Live/Dead™ assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125µg/mL and 60µg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6±10.4% and 45.8±4.1% at 60 and 30µg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.

Improvement of in vitro evaluation of chemical disinfectants for efficacy on Cryptosporidium parvum oocysts.

Cryptosporidium parvum has been suggested as a suitable target for in vitro efficacy testing of disinfectants. To improve validity of a method based on exposure of HCT-8 monolayers to C. parvum oocysts we here critically evaluate and we propose certain procedural steps needed for the validation of disinfectants. Within a range of 0.02% to 0.4%, sodium taurocholate at 0.2% stimulated infection most efficiently while preserving host cell integrity. The course of invasion was monitored for periods of 30-240min post infection (p.i.). FACS analysis revealed that the proportion of sporozoites liberated from oocysts in the presence of 0.2% sodium taurocholate increased within 120min of incubation but remained constant thereafter. Maximum invasion of cells measured by qPCR was reached 180min p.i. and therefore set as invasion endpoint. As monolayers harvested 24h or 48h p.i. did not differ in the quantity of parasite hsp70 gene copies, DNA extraction can be performed as early as 24h p.i. Incubation of oocysts with 20% H2O2 for 2h resulted in inactivation of more than 99.5% both at room temperature and 10°C and appeared thus suitable as positive chemical treatment control. Four washing procedures considered to remove potentially toxic residual disinfectant from oocyst suspensions were tested. An application of a combination of DMSO (Dimethylsulfoxid), Tween20 and WSH (water of standardized hardness) appeared most efficient without deleterious effect of disinfectant residuals on the cell monolayer viability when oocysts accordingly washed were applied. In conclusion, for standardized in vitro evaluation of chemical disinfectants in C. parvum infected HTC-8 monolayers. (i) excystation medium should contain 0.2 % sodium taurocholate. (ii) excystation medium should be replaced by growth medium after 180 min. (iii) monolayers should be harvested 24 h p.i. for DNA preparation. (iv) ocysts exposed to 20 % H2O2 should be included as positive controls. (v) disinfected oocysts should be washed with DMSO/Tween20/WSH before they are transferred to monolayers.

A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis.

Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.

In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum.

Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250-500 µg mL(-1), IC50 = 361 (279-438) µg mL(-1), IC90 = 467 (398-615) µg mL(-1)). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds.

Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite.

The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM).