Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy

Abstract

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

Keywords: Cryptosporidiosis treatment; bumped kinase inhibitors.; calcium-dependent protein kinase 1.

Figure 1.

Criteria for continuing or ceasing additional investigation of bumped-kinase inhibitor (BKIs). A, The colors reflect distinct stages of evaluating BKIs for their suitability as a therapy for cryptosporidiosis. Criteria for advancement (ie, continuing additional investigation) are listed for each assay. B, Summary of the progression of each individual BKI. Colored blocks are filled in when the BKI satisfies the criteria for advancement and left blank when the BKI fails to meet the criteria and was eliminated from additional investigation. Abbreviations: CC₅₀, 50% cytotoxicity concentration; C_max, maximum concentration; CpCDPK1, Cryptosporidium parvum calcium-dependent protein kinase 1; C. parvum, Cryptosporidium parvum; EC₅₀, 50% effective concentration; IC₅₀, 50% inhibitory concentration; IFN-γ, interferon γ; KO, knockout. ^aIncluded as a control.

Figure 2.

Efficacy of bumped-kinase inhibitor (BKIs) in mouse models of Cryptosporidium parvum infection. A–C, Efficacies against C. parvum excretion in adult interferon γ knockout mice, based on nanoluciferase activity log RLU plotted on y-axis (A–C), and efficacy of BKI 1534 in SCID/beige mice, using reverse transcription–polymerase chain reaction to quantify infection (D). A, BKIs were delivered orally for 5 days, beginning 3 days after infection. In vivo inhibition by BKIs 1266, 1294, 1534, and 1550. BKI 1266, with limited inhibition of the C. parvum calcium-dependent protein kinase 1 target enzyme, was included as a negative control. BKIs 1294 and 1534 significantly reduced parasite shedding as compared to controls. The C. parvum infectious dose was 10000 oocysts. B and C, Significant initial reduction of parasite burden by BKIs, even in the case of higher fecal luciferase levels, which reflect higher parasites load. Parasite excretion rebounds when control animals show a higher infectious burden (B). BKIs 1294 (100 mg/kg once daily), 1369 (100 mg/kg once daily or 50 mg/kg twice daily), and 1649 (10 mg/kg once daily) efficacies (uninfected control mice had the highest recorded levels; C). BKIs substantially reduced Cryptosporidium proliferation, as shown by lower oocyst counts. A once-daily BKI 1369 dose of 100 mg/kg significantly reduced the infection burden (by 100000-fold), relative to that in untreated controls. BKI 1649 was administered at lower doses than other compounds owing to its high oral exposure, but it still significantly reduced infection relative to controls. The C. parvum infectious dose was 1000 oocysts. D, BKI 1534 delivered orally at a dose of 60 mg/kg for 5 days cleared infection with no recurrence. Assays were performed at the University of Georgia (Athens; A), the University of Washington (Seattle; B and C), and the University of Texas Medical Branch (Galveston; D). For assays with results in panels A and B, the percentage inhibition of infection, relative to that in controls, after treatment (day 10) was calculated for each compound as follows: 1294, 99.5%; 1534, 99.4%; and 1550, 59.7% (A); and 1294, 99.9%; 1318, 37.6%; 1369, 99.9%; and 1649, 98.9% (B). There were 3 mice in each experimental group.

Figure 3.

Efficacy in adult interferon γ knockout mice with dosing starting on day 6 after infection. In all mice, the nanoluciferase-expressing Cryptosporidium parvum infectious dose was 1000 oocysts. A, Efficacy plots of bumped-kinase inhibitors (BKIs) 1294, 1369, 1534, and 1649 used for treatment of highly established infection when administered orally at 60 mg/kg once daily for 5 days. BKI 1649 was administered at 6 mg/kg once daily. All BKIs showed significant decreases in infection. B, Efficacy of subcutaneous versus oral dosing of mice with 1294 at 60 mg/kg, starting on day 6 after infection, for 5 days. BKI 1294 showed a significantly greater reduction in oocyst excretion when delivered orally as compared to an equivalent dose delivered subcutaneously (P < .05). This is probably due to a fecal concentration of orally administered 1294 that was higher than the 50% effective concentration (EC₅₀) early in the treatment period and to the longer presence of the orally administered 1294 in the lumen relative to the subcutaneously administered dose, for which the level after a single dose was below the EC₅₀. C, Efficacy of subcutaneous versus oral dosing with 1534 at 60 mg/kg, starting on day 6 after infection, for 5 days. No significant difference was seen in parasite excretion between the groups receiving the BKI doses.

Figure 4.

Comparison of the in vitro Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) 50% inhibitory concentration (IC₅₀) to the C. parvum 50% effective concentration (EC₅₀). A, Graphed cellular C. parvum EC₅₀ versus CpCDPK1 IC₅₀ from Table 1. Bumped-kinase inhibitor 1243 was excluded because of its lack of an R1 group. Values show a statistically significant correlation (r₁₆ = 0.93; P < .0001). B, Values graphed on logarithmic scale to better show the separation of IC₅₀ values <0.1 mM.