Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) In Vitro by Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture

Abstract

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration.

FIG 1

Structures of the compounds used in this study. Compound 21 is the original PP inhibitor, previously referred to as PP1 (49). Compound 22 is an unrelated trisubstituted pyrrole that inhibits PKG (44). MW, molecular weight.

FIG 2

Comparison of the activities of TgCDPK1 and CpCDPK1 enzyme inhibitors and inhibition of C. parvum growth. (A) Comparison of the sensitivities of the CpCDPK1 and TgCDPK1 enzymes to inhibitors in vitro. Linear regression is shown; dashed lines indicate 95% confidence intervals. (B) Plot of IC₅₀s of individual compounds for the CpCDPK1 and TgCDPK1 enzymes. Linear regression is shown; dashed lines indicate 95% confidence intervals. (C) Sensitivity of CpCDPK1 enzyme activity (IC₅₀) versus growth (EC₅₀) of C. parvum in culture to inhibitors. (D) Plot of IC₅₀s of individual compounds for CpCPKD1 enzyme activity versus EC₅₀s against C. parvum growth. IC₅₀s and EC₅₀s are from Table 1.

FIG 3

Comparison of the effects of compounds on invasion and growth of C. parvum in HCT-8 cells in vitro. Compounds were tested in C. parvum growth or invasion assays by using RT-qPCR assays to quantify parasite numbers as described in Materials and Methods. Invasion was monitored by pretreatment (15 min) of isolated sporozoites, followed by challenge of monolayers and removal of the compound (Cmpd) at 4 h. Growth was monitored by inoculation of isolated sporozoites, followed by the addition of compounds at 4 h postinfection and continual culture in the compounds. Relative expression values represent the values of experimental samples (plus drug) normalized to those of control samples (no drug) (ratio of gene expression at each drug concentration/control). Triplicate PCR assays were performed, and relative expression values are presented as means ± standard deviations (n = 3). Curve fitting was performed by nonlinear regression with Prism (GraphPad). EC₅₀s are provided in Table 1.

FIG 4

Effects of selected compounds on C. parvum growth as determined by IFA assays. (A) Dose response of inhibition of growth was monitored by IFA assay detection of intracellular C. parvum forms as described in Materials and Methods. FFU values represent means ± standard deviations (n = 3). Curve fitting was performed by nonlinear regression with Prism (GraphPad). (B) Representative images of IFA staining of control and compound (Cmpd) 1- and 21-treated cultures. The upper set of images depicts representative single-well montage photomicrographs (comprising nine fields) of a 96-well plate as described in Materials and Methods. The lower images are each enlargements representing one of the nine image frames shown in the corresponding upper images. EC₅₀s are provided in Table 1. Scale bars, 200 μm.

FIG 5

Time dependence of the sensitivity of C. parvum growth to compounds. (A) Sensitivity of C. parvum growth to compound (Cmpd) 1. (B) Sensitivity of C. parvum growth to compound 21. Open circles, growth in the absence of inhibitors; black triangles, inhibitors added at 4 h postinfection (dashed arrow) representing a positive control demonstrating that compounds completely blocked C. parvum growth during the first 24 h; filled circles, inhibitors added at 24 h postinfection (solid arrow) and then replaced every 24 h thereafter (see Materials and Methods for additional details). Relative expression is defined as normalized expression (see Materials and Methods) at the time points indicated with and without the drug divided by the normalized expression at the initial 4-h time point (representing initial sporozoite invasion but no growth) as described in Materials and Methods. Values represent means ± standard deviations (n = 3).