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

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.

SUBMITTER: Khan SM

PROVIDER: S-EPMC8761912 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.ResultsThe C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀ values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.ConclusionsIdentification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

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

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