Cryptosporidium PI(4)K inhibitor EDI048 is a gut-restricted parasiticidal agent to treat paediatric enteric cryptosporidiosis

Abstract

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis.

Fig. 1. Oral soft drug strategy to engineer GI stability and limit systemic exposure to treat cryptosporidiosis.

a, Schematic representation of C57BL/6 IFN-γ knockout mouse efficacy model. Infected animals were treated with vehicle (grey) or KDU731 orally (p.o.) at 10 mg kg⁻¹ (red) or intravenously (i.v.) at 1 mg kg⁻¹ (blue) with 3 mice per group. Parasite shedding per gram faeces was measured by qPCR; data are mean ± s.d. of 3 replicates. Statistical analysis was done using one-way analysis of variance (ANOVA). ^NSP = 0.5797; ****P < 0.0001. KDU731 systemic exposure in mice was measured in uninfected mice (mean data from n = 3 mice for oral and n = 1 for i.v. dosing are shown). b, Overview of an oral soft drug strategy to maximize intestinal exposure and minimize systemic exposure to limit off-target toxicity. c, Chemical structures of compounds described in this report to summarize structure–activity relationship for soft drug strategy by introducing metabolically labile spots shown in blue. Two soft drug candidates 1 and EDI048 along with their respective primary metabolites are shown in dashed boxes. d,e, In vivo efficacy (d) and systemic exposure (e) of the various analogues. Experimental design is as described in a. All animals were orally treated once daily with vehicle or 10 mg kg⁻¹ for all compounds except nitazoxanide (NTZ) (100 mg kg⁻¹) for 5 days. log₁₀ oocyst reduction per gram faeces compared to vehicle control (d) and AUC (e) are mean ± s.d. of 3 replicates. AUC is the area under the curve from time 0 to the last timepoint measured from the mouse efficacy studies. The PK parameters are for active metabolite Tizoxanide following NTZ dosing at 100 mg kg⁻¹. f, In vivo dose–response study with EDI048. On day 3 post infection, mice were treated once daily with vehicle (black) or indicated doses of EDI048 for 5 days, 3 mice per group. The PK was measured on day 1 post treatment and PK parameters are shown in Extended Data Table 2. Mean ± s.d. of oocysts shed per gram faeces (n = 3) are shown. Dashed line indicates the qPCR assay LOD. Statistical analyses were performed comparing untreated versus EDI048-treated groups (n = 3 each) on respective days using multiple unpaired parametric t-test (Holm–Šídák approach). Source data

Fig. 2. Structural insights into the ligand binding pocket of EDI048 and its mechanism of anti-Cryptosporidium activity in the enterocyte infection model.

a, Concentration–response curves for CpPI(4)K wild type (solid lines) and CpPI(4)K::Y705A:Y907A double mutant (dashed lines) with EDI048 (blue) or KDU731 (orange). Data are mean ± s.d. of at least 2 replicates. b, X-ray co-crystal structure of HsCpPI(4)K-HsRab11 complex with EDI048, highlighting the hinge H-bond between V598-NH and the pyrazolopyridine core (C atoms, orange; N atoms, blue), Pi-stacking of Y597 (lavender) with the pyarzolopyridine core (C atoms, orange), Pi-stacking between Y374 (lavender) with the chlorophenyl moiety of EDI048 (C atoms, orange), and H-bonding between K549 and carbonyl of the phenyl-amide moiety. c, EDI048 does not affect DNA synthesis. Cryptosporidium-infected cells were treated with compounds at 3 h post infection and 10 µM of EdU was added at 9 h post infection and 2 h later washed, fixed, stained and imaged with incorporated EdU representing replicating parasites (magenta), nuclei (blue) and parasitophorous vacuoles (green). White arrows indicate representative Cryptosporidium parasites. Scale bar, 5 µm. d, EDI048 inhibits formation of functional merozoites and blocks egress. Cryptosporidium-infected cells were treated with compounds at 3 h post infection followed by time-lapse imaging (also see Extended Data Fig. 4c and Supplementary Videos 1, 2 and 3 for time-lapse videos). Scale bar, 5 µm. Images in c and d are representative of 2 independent experiments, each with 2 technical replicates. e, Effect and reversibility of CpPI(4)K inhibition on early asexual stages of Cryptosporidium growth. Left: cells infected with nanoluciferase-expressing Cryptosporidium were treated with compounds as indicated in blocks of 4 h post infection, followed by drug washout, and then allowed to continue growing until 72 h post infection. Right: RLU normalized to time 0 are plotted, and data are mean ± s.d. of 4 technical replicates and representative of 2 biological replicates. NTZ, nitazoxanide. f, Time-kill curve with EDI048 compared to nitazoxanide. Cells were infected with nanoluciferase-expressing parasites and at 24 h post infection treated with indicated concentrations of EDI048 or NTZ (also see Supplementary Fig. 1 for complete dose–response data). Data are mean ± s.d. of at least 2 replicates. Source data

Fig. 3. Therapeutic efficacy in clinical neonatal calf model of cryptosporidiosis and in vivo safety of EDI048.

a, Schematic representation of a cryptosporidiosis calf model monitoring parasitological and clinical efficacy of EDI048. Calves were orally challenged with 5 × 10⁷ C. parvum oocysts within 48 h of birth. Clinical parameters were assessed every 12 h and parasites shed in faeces were quantified daily by qPCR. Upon onset of diarrhea and detection of oocysts in faeces, calves were treated twice daily with 10 mg kg⁻¹ (body weight) of EDI048 for 7 days. b,c, Compared with vehicle control (open circles), EDI048 (filled circles)-treated calves shed significantly fewer oocysts in faeces (b) and had improved clinical scores of diarrhea (faecal consistency scores) (c). Parasitological and clinical time-course data shown are mean ± s.e.m. of 7 calves tested per group. For AUC analysis, each circle represents an individual calf over 7 days and lines are mean ± s.e.m. of n = 7 calves per group. Statistical analyses were performed using two-tailed unpaired parametric t-tests; **P = 0.0062, ****P < 0.0001. d,e, Dose–exposure–response relationship for EDI048 (d) and compound 6 (e). Mouse exposure at 1, 3 and 10 mg kg⁻¹ d⁻¹ (blue, AUC_last), calf exposure at 10 mg kg⁻¹ twice a day (BID) (red, AUC_0–12), rat exposures at 50, 250 and 1,000 mg kg⁻¹ d⁻¹ (green, AUC_0–8) and dog exposures at 15, 50, 250 and 1,000 mg kg⁻¹ d⁻¹ (purple, AUC_0–8) are shown. Overall, efficacy exposures in green and safety exposures in pink zone. 1,000 mg kg⁻¹ d⁻¹ is the NOAEL dose in both rat and dog toxicity study. Exposure multiples for EDI048 is 70× comparing between 1,000 mg kg⁻¹ d⁻¹ NOAEL exposure (day 14) and 10 mg kg⁻¹ d⁻¹ mouse efficacious exposure. f, Schematic representation of Cryptosporidium life cycle and site of action of EDI048 in the GI tract. Orally delivered EDI048 is absorbed by the intestinal cells at the site of infection. Herein, EDI048 demonstrates parasiticidal activity on the intracellular Cryptosporidium parasites by inhibiting membrane biogenesis. The absorbed EDI048 is metabolized in the liver, limiting systemic exposure and thus increasing safety margins. Source data

Extended Data Fig. 1. Schematic representation of cross species metabolism for EDI048 in primary hepatocytes.

EDI048 was metabolized via ester hydrolysis to Compound 6 in all species, with amide hydrolysis to Compound 7 observed in dogs. Further, Compound 6 undergoes N-demethylation to Compound 8 or 9, and/or glucuronidation to Compound 10. No unique human specific metabolites were observed using hepatocytes in vitro.

Extended Data Fig. 2. Multiple sequence alignment of Cryptosporidium PI(4)K homologues highlighting key residues interacting with EDI048.

a, Multiple sequence alignment of CpPI(4)K, ChPI(4)K, PfPI(4)K and HsPI(4)K, highlighting key residues interacting with EDI048 along with catalytic lysine (K858). The critical residues identified to be interacting with EDI048 (Cryptosporidium Y705, Y907 and I908) are highlighted in blue box. b, Multiple sequence alignment of PI(4)K from various Cryptosporidium species. CpPI(4)K sequences were obtained from CryptoDB are labeled with their species name followed by a hyphen and the CryptoDB gene identifier. Highlighted are the two key tyrosine residues (Y705 and Y907) that interact with EDI048 and the conserved Lysine (K858) in C. parvum cgd8_4500.

Extended Data Fig. 3. Highly conserved tyrosine residues in Cryptosporidium PI(4)K (Y705 and Y907) enable inhibition of Cryptosporidium over human PI(4)K.

a, Characterization of single and double mutations in CpPI(4)K at Y705A and Y907A. Fold shift in IC50 of KDU731 and EDI048 against the CpPI(4)K single and double mutants over wild-type enzyme are plotted, the data is the mean of two independent experiments. (b, c), Introducing two-point mutations P597Y & L374Y (corresponding to Y907 and Y705 in CpPI(4)K) in the HsPI(4)K that is, HsCpPI(4)K significantly shifted the IC50s of CpPI(4)K inhibitor analogs (solid circles) to be much more like that of CpPI(4)K. HsPI(4)K specific inhibitor shown in solid triangle. Data are mean of at least 2 replicates. Schematic diagrams of protein-EDI048 interaction plot using Molecular Operating Environment (MOE) (d) and the simulated annealing OMIT mFo-DFc electron density map for EDI048 (e) are shown. Source data

Extended Data Fig. 4. EDI048 does not affect C. parvum sporozoites invasion, parasitophorous vacuole formation or growth but arrests C. parvum at meront stage.

a, Cartoon of life stages investigated in the invasion assay along with schematic of the assay. HCT-8 cells were pre-treated with 2x concentration of compounds for 1 hour (h). Primed C. parvum Iowa strain oocysts were then added and allowed to excyst and invade in presence of 1x compound concentration. At 3 h post-infection parasites that did not invade were washed off and then cells stained for parasitophorous vacuoles using FITC conjuated Vicia villosa lectin (green) and nuclei (blue). The active control wiskostatin (11.3 µM), an inhibitor of N-WASp and its activation by cdc42, prevented sporozoite invasion and formation of trophozoites, whereas 20 µM of EDI048 and KDU731 were inactive (b). c, Graphical representation of stages visualized by time-lapse microscopy and schematic representation of the experimental methodology. HCT-8 cells were infected by oocysts for 3 hours (h) and then washed and treated with 1 µM EDI048 or 0.5 µM KDU731. Time-lapse microscopy images were taken every 20 minutes from 4 h post-infection, that is, 1 h after compound addition (time 0 h in the videos) up to 24 h post-infection and are shown in Supplementary Videos 1 (DMSO), 2 (EDI048), and 3 (KDU731). d, Experimental overview to determine the effect of KDU731 on meronts by transmission electron microscopy (TEM). HCT-8 cells were infected with C. parvum oocysts induced for excystation and 0.5 µM of KDU731 was added at 3 h post-infection and parasite morphology analyzed by TEM at 10.5 h post-infection (e). Scale bars: 5 µm (b and Supplementary Videos 1 to 3) and 500 nm (e). Images and videos are representative of at least 2 independent experiments.

Extended Data Fig. 5. EDI048 improves clinical diarrhea symptoms in experimentally challenged calves.

a, EDI048 reduces overall frequency of severe diarrhea and moderate-to-severe diarrhea. There were 7 calves per group, and each square (untreated) and circle (EDI048 treated) represents frequency of diarrhea for an individual calf over 7 days. p values were determined using unpaired, two-tailed t test. Data shown 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, and the center is median. b. Microbiological and clinical end-points measured 7 days post-cessation of drug treatment, details as described in Fig. 3b,c. Source data

Extended Data Fig. 6. Dose Cmax response relationship for EDI048 and Compound 6.

Dose and Cmax relationship for EDI048 (a) and Compound 6 (b) in mouse Cmax at 1, 3 and 10 mg/kg/day (Blue). Calf Cmax at 10 mg/kg BID (Red), Rat Cmax at 50, 250 and 1000 mg/kg/day (Green) and dog Cmax at 15, 50, 250 and 1000 mg/kg/day (Purple). Overall, efficacy Cmax in green zone and safety Cmax in pink zone. 1000 mg/kg/day is the NOAEL dose in both rat and dog tox study. Cmax multiples for EDI048 is 42× comparing between 1000 mg/kg/day NOAEL Cmax (Day 14) and 10 mg/kg/day mouse efficacious Cmax. Source data