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.

5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum

“…In this study, AC-BKIs were added before host cell invasion. Nine out of 15 compounds had EC 50 s of Յ1 M, which were lower than those of PP-BKIs, which are uniformly of low concentration (14). Compounds within each group exhibited similar C. parvum EC 50 s. AC-BKIs in group 1 were all quite potent, with EC 50 s ranging from 0.41 to 1.0 M, with a Ͻ3-fold inhibition difference.…”

“…New AC-BKIs (8,11,14,17) were synthesized and characterized following previously reported procedures (8,12,13) (see Text S1 in the supplemental material). Methods for solubility testing, inhibition of CpCDPK1 and C. parvum cellular growth, drug pharmacokinetic and fecal concentration analysis, plasma protein binding, and determination of efficacy in neonatal mice were performed as previously described (9 Fig.…”

“…be the best predictor of efficacy in neonatal mice, as low exposure does not eliminate efficacy (r ϭ Ϫ0.18). The poor correlation may be due to differences in drug exposure in adult and neonatal mice (14). AC-BKIs 4 and 16 were very active against C. parvum in vitro, but AC-BKI 4 demonstrated lower in vivo efficacy than AC-BKI 16.…”

“…AC-BKI 4 had higher systemic levels and much lower stool levels than AC-BKI 16 ( Table 2). The emerging consensus, based on a recent study (14), is that higher fecal levels of AC-BKI 16 may be a good proxy for elevated intraepithelial concentrations in the gastrointestinal tract, the site of drug action against C. parvum, whereas low fecal levels of AC-BKI 4 may indicate lower intraepithelial levels and low in vivo efficacy. Nevertheless, a number of AC-BKIs exhibited efficacy in neonatal mice, which is a good predictor of efficacy in the calf model, and remain promising for the treatment of cryptosporidiosis in animals and humans (16).…”

Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis

“…These BKIs have excellent activity against C. parvum and T. gondii parasites in vitro and in vivo. A recently reported series of pyrazolo[2,3- d ]pyrmidine (PP) BKIs are effective in experimental therapeutic treatments for cryptosporidiosis (Hulverson et al, 2017). Since that publication, additional data make it clear that one compound, BKI 1369, is emerging as a pre-clinical lead for cryptosporidiosis therapy.…”