Comparative Characterization of CpCDPK1 and CpCDPK9, Two Potential Drug Targets Against Cryptosporidiosis

Abstract

As the invasion, egress, and growth of Cryptosporidium spp. are regulated by the calcium ion, calcium-dependent protein kinases (CDPKs) are considered potential drug targets against these pathogens. In this study, we expressed CpCDPK1 of Cryptosporidium parvum encoded by the cgd3_920 gene and CpCDPK9 encoded by the the cgd7_1260 gene in Escherichia coli, and we conducted some comparative studies with quantitative PCR, immunofluorescence staining, and in vitro neutralization assays. By immunofluorescence microscopy, CpCDPK1 was expressed over the entirety of the sporozoites, while CpCDPK9 was mainly expressed in the apical region. The expression of the cgd3_920 gene was the highest at 12 h of the in vitro culture, whereas the expression of the cgd7_1260 gene peaked between 2 h and 6 h. Polyclonal antibodies against these two CpCDPK proteins had similar neutralization efficiency on C. parvum growth, reaching approximately 40%. Of the 50 candidate compounds from the molecular docking of CpCDPK1, 10 had significant in vitro anti-cryptosporidial effects, but only one inhibited enzyme activity. For CpCDPK9, five of the forty-five candidate compounds showed significant in vitro anti-cryptosporidial effects. Results obtained from this study suggest that CpCDPK1 and CpCDPK9 might function differently in C. parvum infection.

An update on Cryptosporidium biology and therapeutic avenues

“…In the life cycle of apicomplexan parasites, the regulation of Ca 2+ binding by calcium-dependent protein kinases (CDPKs) is necessary for parasite secretion, motility, and growth (Etzold et al 2014 ). Therefore, CDPKs are thought to be potential therapeutic targets against the parasites (Su et al 2022 ). After understanding the crystal structures of CDPKs having a gatekeeper glycine residue, specific bumped-kinase inhibitors (BKIs) were developed that inhibit CDPK1 functions through the hydrophobic pocket that opens up next to the glycine residue (Huang et al 2017 ; Hulverson, et al 2017a , b ; Van Voorhis et al 2021 ).…”

Characterization of Calcium-Dependent Protein Kinase 2A, a Potential Drug Target Against Cryptosporidiosis

“…In recent years, several bumped kinase inhibitors of Cp CDPK1 have become the leading candidates for the development of novel treatment of cryptosporidiosis ( Van Voorhis et al, 2021 ). Previously, using a similar approach, we also identified a Cp CDPK1 inhibitor (F083-0116) with inhibitory activities on the enzyme and C. parvum growth ( Su et al, 2022 ). Thus, Cp CDPK2A might be another valid target for the development of treatment of cryptosporidiosis.…”

“…Data from the present study suggest that Cp CDPK2A is another CDPK that is expressed in multiple life cycle stages of C. parvum , including sporozoites, trophozoites, type 1 meronts, and macrogamonts. However, data obtained from studies of Cp CDPK1, Cp CDPK2A, Cp CDPK3, Cp CDPK4, Cp CDPK5, Cp CDPK6, and Cp CDPK9 indicate that these CDPKs are all expressed in sporozoites and asexual stages of C. parvum , although the relative levels of them could be different ( Zhang et al, 2020 , 2021 ; Su et al, 2022 ). Their subcellular locations, however, appear to differ among Cp CDPKs.…”

“…Inhibition of Cp CDPK1 expression had caused a reduction in parasite load in vitro ( Castellanos-Gonzalez et al, 2016 ; Choudhary et al, 2020 ). Similarly, Cp CDPK1, Cp CDPK4, Cp CDPK6, and Cp CDPK9 have all been shown to participate in the invasion of host cells by C. parvum ( Zhang et al, 2021 ; Su et al, 2022 ). In contrast, Cp CDPK3 and Cp CDPK5 might play some roles mostly during the growth of the parasite ( Zhang et al, 2020 ).…”

Investigation of the prophylactic and therapeutic effectiveness of oral thyme extract in rats experimentally infected with cryptosporidium parvum

“…In animals, azithromycin, nitazoxanide, paromomycin, halofuginone lactate, tylosin, and decoquinate are some active substances that may be partially effective (Shahiduzzaman and Daugschies 2012 ; Yasa Duru et al 2013 ; Yagci et al 2017 ). Considering the limited efficacy of the agents used for both humans and animals, their failure in eradication, and their toxic effects, there is an urgent need for new, effective, and safe anticryptosporidial agents (Nguyen-Ho-Bao et al 2022 ; Su et al 2022 ).…”