Cryptosporidium parvum Pyruvate Kinase Inhibitors With in vivo Anti-cryptosporidial Efficacy

Abstract

<i>Cryptosporidium parvum</i> is a highly prevalent protozoan parasite that causes a diarrheal disease in humans and animals worldwide. Thus far, the moderately effective nitazoxanide is the only drug approved by the United States Food and Drug Administration for treating cryptosporidiosis in immunocompetent humans. However, no effective drug exists for the severe disease seen in young children, immunocompromised individuals and neonatal livestock. <i>C. parvum</i> lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, <i>C. parvum</i> possesses two unique enzymes, the bacterial-type lactate dehydrogenase (CpLDH) and the plant-like pyruvate kinase (CpPyK), that catalyze two sequential steps for generation of essential metabolic energy. We have previously reported that inhibitors of CpLDH are effective against <i>C. parvum</i>, both <i>in vitro</i> and <i>in vivo</i>. Herein, we developed an <i>in vitro</i> assay for the enzymatic activity of recombinant CpPyK protein and used it to screen a chemical compound library for inhibitors of CpPyK's activity. The identified inhibitors were tested (at non-toxic concentrations) for efficacy against <i>C. parvum</i> using <i>in vitro</i> assays, and an <i>in vivo</i> mouse infection model. We identified six CpPyK inhibitors that blocked <i>in vitro</i> growth and proliferation of <i>C. parvum</i> at low micromolar concentrations (EC₅₀ values ranging from 10.29 to 86.01 μM) that were non-toxic to host cells. Among those six compounds, two (NSC252172 and NSC234945) were found to be highly efficacious against cryptosporidiosis in immunocompromised mice at a dose of 10 mg/kg body weight, with very significant reduction in parasite load and amelioration of intestinal pathologies. Together, these findings have unveiled inhibitors for an essential molecular target in <i>C. parvum</i> and demonstrated their efficacy against the parasite <i>in vitro</i> and <i>in vivo</i>. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.