A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis

Abstract

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

Keywords: Calcium-dependant Protein kinase; CpCDPK-1; Cryptosporidium parvum; antiparasitic; bumped kinase inhibitor; cryptosporidiosis.

Figure 1.

In vitro anti-Cryptosporidium activity of 1294. A, Chemical structure of 1294. B, Five initial studies demonstrated consistent submicromolar efficacy of 1294. The 2 independent experiments shown in the figure used a wider range of concentrations to quantify the half maximal effective concentration of 1294 against Cryptosporidium parvum in HCT-8 cells. Mean values±SD (indicated with vertical bars) are shown.

Figure 2.

The effects of 1294 treatment in a severe combined immunodeficiency–beige mouse model of Cryptosporidium infection. Median oocyst shedding by mice treated with 1294 (dotted line) or placebo (solid line) is shown for each time point. Findings of stool specimen assays for each mouse are represented with the same color and shape through the experiment. Mice treated with 1294 (triangles [n = 7]; treatment regimen, 1 dose of 100 mg/kg by oral gavage once daily on days 4–14 after infection) or placebo (circles [n = 7]; treatment regimen, vehicle only by oral gavage once daily on days 4–14 after infection) are indicated, and horizontal bars represent median values. The sky-blue box indicates the treatment period, stool specimens from day 4 collected before initiation of dosing. The number of parasites shed in per 25 mg of stool among treated and untreated groups was quantified by real-time polymerase chain reaction and normalized to the number of oocysts, using a standard curve. Differences between groups were compared at each time point, and P values were calculated by Kruskal-Wallis analysis. Only 1 of seven 1294-treated mice had detectable parasites on day 33 after treatment, yet 6 of 7 placebo-treated mice had parasites after treatment. Abbreviation: ND, none detected.

Figure 3.

Intestinal damage in the cryptosporidiosis severe combined immunodeficiency (SCID)–beige mouse model. A–C, Microscopic analysis of representative hematoxylin-eosin–stained sections of intestine from 1294-treated and infected (A), vehicle-treated and infected (B), and uninfected (C) SCID-beige mice. Note that findings for 1294-treated and infected mice (A) and uninfected controls (C) are essentially identical, but intestines of vehicle-treated and infected mice had profoundly blunted villi, epithelial loss, damaged villi, and villous inflammation (B).

Figure 4.

Determination of apoptotic damage in intestinal tissue. Intestinal sections were prepared and double-stained for apoptotic cells by the TUNEL technique (green) and counterstained with DAPI (blue). A–D, Representative terminal small intestinal areas of uninfected controls (A), 1294-treated and infected mice (B), and placebo (ie, vehicle)–treated and infected controls (C and D). E, An apoptotic score was determined for each section, and the score was normalized to the rate of TUNEL-positive cells found in untreated matched mouse intestines (apoptosis score, 1.0). Data are mean ± SD. *P < .05, by the Student t test.