In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

A screening pipeline for antiparasitic agents targeting cryptosporidium inosine monophosphate dehydrogenase.

BACKGROUND: The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have developed a pipeline of tools to accelerate the in vivo screening of inhibitors of C. parvum IMPDH. We have genetically engineered the related parasite Toxoplasma gondii to serve as a model of C. parvum infection as the first screen. This assay provides crucial target validation and a large signal window that is currently not possible in assays involving C. parvum. To further develop compounds that pass this first filter, we established a fluorescence-based assay of host cell proliferation, and a C. parvum growth assay that utilizes automated high-content imaging analysis for enhanced throughput. CONCLUSIONS AND SIGNIFICANCE: We have used these assays to evaluate C. parvum IMPDH inhibitors emerging from our ongoing medicinal chemistry effort and have identified a subset of 1,2,3-triazole ethers that exhibit excellent in vivo selectivity in the T. gondii model and improved anti-cryptosporidial activity.