Abstract
Cryptosporidium parvum 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. C. parvum lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, C. parvum 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 C. parvum, both in vitro and in vivo. Herein, we developed an in vitro 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 C. parvum using in vitro assays, and an in vivo mouse infection model. We identified six CpPyK inhibitors that blocked in vitro growth and proliferation of C. parvum 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 C. parvum and demonstrated their efficacy against the parasite in vitro and in vivo. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.
Highlights
Intracellular eukaryotic protozoan parasites belonging to the genus Cryptosporidium are members of the phylum Apicomplexa
The 1,581 bp long open reading frame of the C. parvum pyruvate kinase (CpPyK) gene translates into a 526 amino acid protein with an estimated molecular weight of 56.4 kDa and an isoelectric point of 6.78.2 We sequenced the cloned CpPyK coding sequence amplified from C. parvum cDNA and observed a 100% homology when aligned with the nucleotide sequence reported in genome databases (CryptoDB gene ID: cgd1_2040; GenBank accession number: XM_628040)
Using the ATP detection-based CpPyK enzymatic assay (Figure 1A), we found that recombinant CpPyK (rCpPyK) protein depicted concentration- and pH-dependent catalytic activity with optimal activity at 6 ng/μl and pH 7.5, respectively
Summary
Intracellular eukaryotic protozoan parasites belonging to the genus Cryptosporidium are members of the phylum Apicomplexa. Out of the currently documented 42 species of Cryptosporidium (Zahedi and Ryan, 2020), Cryptosporidium parvum is recognized as the major zoonotic species responsible for diarrheal infections in animals and humans worldwide Among humans in developing countries, Cryptosporidium is the main cause of linear growth faltering and the second leading cause of moderate-to-severe diarrhea in infants (0–11 months of age) and a major cause of mortality and stunted growth in the second year of life (Kotloff et al, 2013; Nasrin et al, 2021). While C. parvum has been recognized as an important etiological agent of diarrhea for over four decades (Navin and Juranek, 1984), neither fully effective therapeutic drugs nor prophylactic vaccines are currently available. There is an urgent need for the development of efficacious anti-cryptosporidial drugs
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