A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis

Abstract

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

Figure 1. Pyrazolopyridine analogues exhibit potent anti-Cryptosporidium activity.

a, C. parvum phenotypic screening against a focused library of 6,220 compounds (percentage inhibition at 5 μM). Hits with inhibition >3 s.d. are shown in red. b, Structure of the pyrazolopyridine lead KDU731. c, In vitro activity of KDU731 (red), MMV3900048 (maroon), and nitazoxanide (NTZ, black) against C. parvum (solid line) and C. hominis (dashed line). d, Correlation of growth inhibition (EC₅₀) of selected compounds between C. parvum (Cp) and P. falciparum (Pf). Pyrazolopyridine analogues are shown in red, imidazopyrazines in blue, quinoxaline in green, and diaryl-aminopyridine in maroon. e, Correlation of growth inhibition (EC₅₀) between C. parvum and C. hominis (Ch) for selected compounds. f, Inhibition of phosphatidylinositide kinase activity of purified enzyme by KDU731, KDU691, MMV390048, and BQR695 (means ± s.e.m. with at least three biological replicates) in the presence of 3 μM ATP. g, Correlation between inhibition of PI kinase activity of purified CpPI(4)K enzyme (IC₅₀) and growth inhibition (EC₅₀) of C. parvum with selected compounds (colours as in d). h, CpPI(4)K activity across a range of ATP concentrations in the presence of 1.56–50 nM KDU731. Data shown in c and f represent mean ± s.e.m., n = 3 biological replicates, representative data shown. Experiments shown in d, e, and g were repeated at least twice and geometric mean EC₅₀ values are plotted; experiment in h was repeated twice (biological replicates) and one representative assay is shown. (Error bars are from the technical replicate, n = 2.) PowerPoint slide

Figure 2. KDU731 has potent activity against Cryptosporidium in immunocompromised IFN-γ knockout mice.

a, Mice were infected with 10,000 C. parvum UGA1 Nluc or UGA2 Fluc oocysts. Parasite load in the faeces was determined by measuring faecal Nluc activity and parasite tissue load was quantified by whole-animal imaging of Fluc activity. Different 7-day treatment courses are indicated in red. Faecal luciferase measurements of individual mice with treatment initiated after 3 (b) or 11 (c) days of infection (red; vehicle control shown in black), n = 5 and n = 9 mice respectively, representative of two biological replicates for b and c. d, Histology of the ileum of infected mice (shown in c) after 1 week of KDU731 treatment compared with vehicle-treated control on day 18 (n = 3 biological replicates, representative images shown here). Note numerous intracellular parasite stages (white arrowheads) and extracellular oocysts (black arrowhead) in the control, absent in the treated mice. Vehicle-treated animals showed disorganized columnar epithelial cells and loss of brush border compared with KDU731-treated mice. White box indicates section shown at higher magnification to the right. Mice (n = 5) infected with UGA2 FLuc were treated on day 7 with 10 mg per kg (body weight) of KDU731 (red, e and g), 100 mg per kg (body weight) nitazoxanide (red, f; also see Extended Data Fig. 6), or vehicle (black, e and f) for 1 week. Animals were monitored by whole-animal imaging and a radiance scale and quantification of total flux in photons per second is shown. *Animals shown on the baseline were below the level of detection. PowerPoint slide Source data

Figure 3. Therapeutic efficacy of KDU731 in neonatal calf clinical model of cryptosporidiosis.

a, Within 48 h of birth, calves were challenged with 5 × 10⁷ C. parvum oocysts. Faecal oocyst shedding was enumerated by qPCR and calves were clinically evaluated every 12 h. Oral treatment with KDU731 (5 mg per kg (body weight) every 12 h for 7 days) was initiated when calves showed severe diarrhoea (faecal consistency score (FCS) = 3) and oocysts in their faeces. KDU731-treated calves shed significantly fewer oocysts in their stool (b), had significantly fewer days of severe diarrhoea (c), and were significantly less dehydrated (d) than untreated calves. e, Day 1 and day 7 plasma pharmacokinetic profile of KDU731. Data shown here (b–e) are from infected calves that were treated with vehicle (Veh.) (n = 6) or KDU731 (n = 7); n represents the number of calves. In vitro EC₉₀ is shown as dotted line. Error bars, s.e.m. Data shown here (c, d) as a ‘box and whiskers’ plot; the box extends from the 25th to 75th percentiles, and whiskers with minimum to maximum showing all data points. Data in b and e were determined to display non-Gaussian distribution and were log-transformed before statistical analysis using t-tests with two-tailed *P < 0.05, **0.01, ***0.001, and ****0.0001; data in c and d were determined to be normally distributed and analysed using t-tests. PowerPoint slide Source data

Extended Data Figure 1. Structures of the pyrazolopyridines and other known PI(4) kinase inhibitors.

Compounds described in Table 1. Important structural determinants required for anti-Cryptosporidium activity in pyrazolopyridines are shown in blue.

Extended Data Figure 2. Anti-Cryptosporidium activity does not correlate with mammalian cell toxicity.

Correlation of C. parvum cytopathic effect versus HepG2 cytotoxicity assay for selected pyrazolopyridine and imidazopyrazine analogues along with BQR695 and MMV390048. Data shown here are geometric mean EC₅₀ values, with at least two biological replicates.

Extended Data Figure 3. Recombinant C. parvum cgd8_4500 shows phosphatidylinositide kinase activity.

C. parvum cgd8_4500 was expressed in insect cells using a Baculovirus system and recombinant enzyme was purified. A Michaelis–Menten plot of phosphatidylinositide kinase reaction with 3 nM CpPI(4)K enzyme at varying ATP concentrations is shown. Data shown here are a representative graph of two independent biological replicates.

Extended Data Figure 4. KDU731 inhibits C. parvum Nluc parasites in vitro and in vivo.

a, EC₅₀ determination of KDU731 against UGA1 Nluc transgenic parasites grown in HCT-8 cultures using luciferase activity as read out. Representative data are shown, three technical replicates. b, Mice (n = 5) were infected with 10,000 UGA1 Nluc oocysts and treated orally 3 days after infection with 1, 5, or 10 mg per kg (body weight) KDU731 for 1 week. Faecal oocyst load was determined by measuring parasite luciferase activity (b) or parasite DNA by qPCR (c) in faeces pooled from entire cage of five mice (20 mg faeces for Nluc and 100 mg for PCR assay). b, c, Means for three technical replicates are shown. Error bars, s.d. Pooled Nluc experiments for vehicle and 10 mg per kg (body weight) dose were repeated in three biological replicates and a representative result is shown. Source data

Extended Data Figure 5. Parasite intestinal load measured by qPCR correlates with faecal shedding and tissue luminescence.

a, Mice (n = 4) were infected with 50,000 UGA2 FLuc oocysts and imaged after 1 week. Mice were killed and the small intestine was resected and imaged (representative image shown). Infection of the intestine ranged in intensity from heavy in the ileum to more moderate in the jejunum and caecum (see radiance scale bar for comparison). Intestines were cut into 12 segments and the luminescence of each segment was recorded. b, qPCR analysis of intestinal segments was performed in triplicate and plotted against the respective luminescence measurements. Regression analysis found robust correlation of tissue luminescence and PCR for parasite DNA, with r² = 0.8. c, Mice were infected with 10,000 UGA2 FLuc oocysts, and 7 days after infection animals were treated daily for a week with vehicle or 10 mg per kg (body weight) KDU731. Whole-animal imaging during the treatment period is shown in Fig. 2g. Faecal oocyst load was determined by measuring parasite DNA by qPCR in faeces pooled from a cage of five mice. Error bars, s.d. Source data

Extended Data Figure 6. Nitazoxanide does not reduce intestinal parasite load in IFN-γ knockout mice.

Mice (n = 5) were infected with 10,000 UGA2 FLuc oocysts, and 7 days after infection animals were treated daily for a week with 100 mg per kg (body weight) nitazoxanide or vehicle. Mice were monitored by whole-animal imaging. Radiance scale shows total flux in photons per second.

Extended Data Figure 7. Effect of KDU731 on severity of diarrhoea and dehydration in the neonatal calf model of cryptosporidiosis.

Severity of diarrhoea and dehydration in individual calves challenged with 5 × 10⁷ C. parvum oocysts. Infected calves were treated with vehicle (n = 6) or with KDU731 (n = 7); n represents the number of calves. Every 12 h, calves were stimulated to defecate, faecal consistency was evaluated, and hydration status was assessed. Faecal consistency and hydration scores were assigned according to the study rubric (see Supplementary Information). The schematic representation shows the faecal consistency (a) and hydration scores (b) throughout the drug treatment period. Faecal consistency and hydration began to improve within 48 h of initiating treatment with KDU731. Source data