ABSTRACT
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.
Subject(s)
Antiprotozoal Agents/isolation & purification , Cryptosporidium parvum/drug effects , Cryptosporidium parvum/growth & development , Drug Evaluation, Preclinical/methods , Epithelial Cells/parasitology , Antiprotozoal Agents/pharmacology , Cell Line , HumansABSTRACT
Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Cryptosporidiosis/drug therapy , Cryptosporidiosis/parasitology , Cryptosporidium/drug effects , Cryptosporidium/enzymology , Pyrazoles/pharmacology , Pyridines/pharmacology , Animals , Animals, Newborn , Cattle , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Immunocompromised Host , Interferon-gamma/deficiency , Interferon-gamma/genetics , Male , Mice , Mice, Knockout , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyridines/chemistry , Pyridines/pharmacokinetics , Rats , Rats, WistarABSTRACT
The apicomplexan parasite Cryptosporidium is the second most important diarrheal pathogen causing life-threatening diarrhea in children, which is also associated with long-term growth faltering and cognitive deficiency. Cryptosporidiosis is a parasitic disease of public health concern caused by Cryptosporidium parvum and Cryptosporidium hominis. Currently, nitazoxanide is the only approved treatment for cryptosporidium infections. Unfortunately, it has limited efficacy in the most vulnerable patients, thus there is an urgent need for a safe and efficacious cryptosporidiosis drug. In this work, we present our current perspectives on the target product profile for novel cryptosporidiosis therapies and the perceived challenges and possible mitigation plans at different stages in the cryptosporidiosis drug discovery process.
Subject(s)
Antiprotozoal Agents/pharmacology , Cryptosporidiosis/parasitology , Cryptosporidium/drug effects , Animals , Cryptosporidiosis/diagnosis , Cryptosporidiosis/drug therapy , Cryptosporidium/genetics , Cryptosporidium/growth & development , Cryptosporidium/physiology , Drug Discovery , HumansABSTRACT
Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ß-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease.
