Compounds
KDU731 was synthesized as described earlier24. KDU731 was found to induce micronuclei in vitro in human peripheral blood lymphocytes and also in in vivo micronucleus assay in rat peripheral blood. The genotoxicity liability of KDU731 is mitigated by substituting cyanopyridine with chlorobenzene moiety while retaining potency and selectivity. All the soft-drug analogues described in this report were made with chlorobenzene/trifluoro-oxy-benzene substitution. Synthesis of EDI048, its metabolites and all other soft-drug candidates described in chemical synthesis along with analytical data is described below and also in a related patent31. Chemical structures were drawn using ChemDraw Professional v.22.0.0.22 (PerkinElmer).
KDU731 3–(4-carbamoylphenyl)-N-(5-cyanopyridin-2-yl)-N-methylpyrazolo [1,5-a]pyridine-5-carboxamide
KDU731 is a yellow-colour solid. 1H NMR (400 MHz, DMSO-d6): δ 8.87 (dd, J = 2.3, 0.8 Hz, 1H), 8.72 (dd, J = 7.2, 0.9 Hz, 1H), 8.53 (s, 1H), 8.26 (dd, J = 8.6, 2.3 Hz, 1H), 8.01 (s, 1H), 7.99–7.94 (m, 3H), 7.66–7.59 (m, 3H), 7.37 (s, 1H), 6.86 (dd, J = 7.2, 1.8 Hz, 1H), 3.54 (s, 3H); ESILC/MS: m/z 398 [M + H]+. J is the coupling constant and is a measure of the interaction between spin-coupled protons.
Compound 1 (ethyl N-(4-chlorophenyl)-N-(3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carbonyl)glycinate)
Ethyl (4-chlorophenyl)glycinate (1.3 g, 5.9 mmol) was added to a solution of 3-bromopyrazolo[1,5-a]pyridine-5-carbonyl chloride (1.4 g, 5.4 mmol) in dichloromethane (DCM) (5 ml), followed by N,N-diisopropyl-N-ethylamine (1.9 ml, 10.8 mmol). The mixture was stirred overnight and concentrated under vacuum onto silica gel, followed by purification on additional silica gel with a 0% to 100% ethyl acetate (EtOAc) in heptane ramped to isolate ethyl N-(3-bromopyrazolo[1,5-a]pyridine-5-carbonyl)-N-(4-chlorophenyl)glycinate (1.0 g, 2.29 mmol, 42.4% yield) as a solid. This solid was dissolved in 1,4-dioxane (10 ml) and water (2.0 ml) in a microwave vial. N-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (492 mg, 2.75 mmol), K2HPO4 (1.5 g, 6.9 mmol) and PdCl2(dppf)·CH2Cl2 (340 mg, 0.46 mmol) were added and the solution degassed with N2. The mixture was heated to 100 °C for 10 min, then filtered, concentrated and purified by supercritical fluid chromatography (SFC) to yield the title compound as a solid (27.6 mg, 2.4% yield). LC–MS (m/z): 491.3 [M + 1]+, retention time (RT) = 0.79 min. 1H NMR (500 MHz, DMSO-d6): δ 8.69 (d, J = 7.2 Hz, 1H), 8.47 (d, J = 13.6 Hz, 2H), 7.92 (d, J = 4.4 Hz, 2H), 7.74 (s, 1H), 7.50–7.42 (m, 4H), 7.37 (t, J = 4.9 Hz, 2H), 6.87–6.82 (m, 1H), 4.64 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 2.82 (d, J = 4.5 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H).
Compound 2 (N-(4-chlorophenyl)-N-(3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carbonyl)glycine)
Ethyl N-(4-chlorophenyl)-N-(3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carbonyl)glycinate (250 mg, 0.509 mmol) and LiOH (12 mg, 0.509 mmol) were dissolved in 3 ml 1:1:1 methanol (MeOH):1,4-dioxane:water and stirred overnight. Product was precipitated with 6 N hydrochloric acid (HCl), and the solid was filtered and washed with water. The material was purified by SFC to yield the title compound as a solid (80 mg, 33.6%). LC–MS (m/z): 463.1 [M + 1]+, RT = 1.389 min. 1H NMR (400 MHz, DMSO-d6): δ 8.67 (dd, J = 7.2, 0.9 Hz, 1H), 8.46 (d, J = 6.6 Hz, 2H), 7.95–7.86 (m, 2H), 7.72 (d, J = 1.6 Hz, 1H), 7.49–7.40 (m, 4H), 7.39–7.30 (m, 2H), 6.83 (dd, J = 7.2, 1.8 Hz, 1H), 4.54 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H).
Compound 3 (2-(dimethylamino)-2-oxoethyl 3-(N-(4-chlorophenyl)-3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamido)propanoate)
3-(N-(4-Chlorophenyl)-3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamido)propanoic acid (100 mg, 0.21 mmol) was added to a solution of 2-bromo-N,N-dimethylacetamide (30 mg) in dimethylformamide (2 ml). K2CO3 (87 mg) was added and the mixture stirred for 12 h. The mixture was cooled to 0 °C and poured into ice water. The aqueous layer was extracted with EtOAc and the combined organic layers dried over Na2SO4, filtered and concentrated. The material was purified by reverse phase HPLC (Zorbax C18, H2O/CAN, 18 ml min−1) to provide the title compound as a solid (25 mg, 21%). LC–MS (m/z): 561.75 [M + 1]+, RT = 1.42 min. 1H NMR (400 MHz, DMSO-d6): δ 8.65 (d, J = 7.2 Hz, 1H), 8.50–8.45 (m, 2H), 7.96–7.89 (m, 2H), 7.81 (s, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.45 (s, 4H), 6.88 (dd, J = 7.3, 1.8 Hz, 1H), 4.75 (s, 2H), 4.12 (t, J = 7.3 Hz, 2H), 2.90 (s, 3H), 2.83 (d, J = 4.4 Hz, 3H), 2.77 (s, 3H), 2.74 (t, J = 7.4 Hz, 2H).
Compound 4 ((R)-3-(4-(methylcarbamoyl)phenyl)-N-((5-oxotetrahydrofuran-2-yl)methyl)-N-(4-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamide)
To a suspension of (R)-3-bromo-N-((5-oxotetrahydrofuran-2-yl)methyl)-N-(4-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamide (65 mg, 0.13 mmol) in tetrahydrofuran (THF) (2 ml) was added (4-(methylcarbamoyl)phenyl)boronic acid (58 mg), triethylamine (Et3N) (55 µl) and H2O (1 ml), and the mixture was purged with N2. PdCI2(dtbpf) (8.5 mg) was added. The mixture was stirred at 100 °C for 2 h, then filtered, concentrated and subjected to SFC (2-ethylpyridine column, CO2/MeOH, 80 ml min−1, 2.18 min) to provide the title compound (27 mg, 37%) as a solid. LC–MS (m/z): 553.3 [M + 1]+, RT = 0.97 min. 1H NMR (500 MHz, DMSO-d6): δ 8.66 (d, J = 7.2 Hz, 1H), 8.46 (d, J = 5.9 Hz, 2H), 7.94–7.89 (m, 2H), 7.76 (s, 1H), 7.55–7.47 (m, 4H), 7.38 (d, J = 8.4 Hz, 2H), 6.86 (dd, J = 7.2, 1.8 Hz, 1H), 4.83–4.77 (m, 1H), 4.19 (dd, J = 14.6, 3.9 Hz, 1H), 4.11 (dd, J = 14.6, 8.0 Hz, 1H), 2.82 (d, J = 4.4 Hz, 1H), 2.56–2.49 (m, 2H), 2.34–2.24 (m, 1H), 1.94 (dq, J = 12.7, 9.1 Hz, 1H).
Compound 5 (methyl 2-(3-(4-carbamoylphenyl)-N-methylpyrazolo[1,5-a]pyridine-5-carboxamido)-5-chlorobenzoate)
Methyl 2-(3-bromo-N-methylpyrazolo(1,5-a)pyridine-5-carboxamido)-5-chlorobenzoate (0.79 g, 1.9 mmol), (4-carbamoylphenyl)boronic acid (0.46 g), PdCI2(dtbpf) (0.27 g) and K3PO4 (1.2 g) were taken up in dioxane (15 ml) and water (3.1 ml) in a microwave vial. The vial was purged with N2 for 10 min and then heated in a microwave at 100 °C for 10 min. The mixture was filtered, concentrated and purified by HPLC (amino column C3 20–25, CO2/MeOH, 80 ml min−1) to yield the title compound (650 mg, 1.4 mmol, 75% yield) as a yellow solid. LC–MS (m/z): 463.2 [M + 1]+, RT = 0.76 min. 1H NMR (400 MHz, chloroform-d): δ 8.69 (d, J = 1.8 Hz, 1H), 8.51 (d, J = 7.2 Hz, 1H), 8.27 (s, 1H), 8.12 (d, J = 8.2 Hz, 2H), 8.02 (s, 1H), 7.84 15 (dd, J = 8.6, 2.1 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.38 (s, 1H), 6.78 (d, J = 7.2 Hz, 1H), 3.96 (s, 3H), 3.64 (s, 3H).
EDI048 (methyl 2-chloro-5-(N-methyl-3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamido)benzoate)
To a suspension of methyl 5-(3-bromo-N-methylpyrazolo[1,5-a]pyridine-5-carboxamido)-2-chlorobenzoate (14.4 g, 34.1 mmol) in THF (360 ml) was added (4-(methylcarbamoyl)phenyl)boronic acid (8.6 g), Et3N (14.3 ml) and H2O (67 ml). The mixture was degassed and purged with N2 three times. PdCI2(dtbpf) (222 mg) was added. The mixture was stirred at 53 °C for 3 h, diluted with EtOAc and water, and then filtered. The layers were separated, the organic layer dried over Na2SO4, filtered and some of the volatiles removed. The resulting slurry was filtered to collect the solids. The solids were dissolved in ethanol and EtOAc, stirred with Pd-scavenging resin, filtered and fully concentrated. The solid was dissolved in hot EtOAc (150 ml), slowly cooled and collected to provide the title compound (10 g, 21 mmol, 62% yield) as a yellow solid. LC–MS (m/z): 477.0 [M + H]+, RT = 0.75 min (LC–MS source data provided). 1H NMR (400 MHz, chloroform-d): δ 8.33–8.25 (m, 1H), 8.06 (s, 1H), 7.80–7.75 (m, 2H), 7.71 (d, J = 2.80 Hz, 1H), 7.52 (d, J = 15 0.80 Hz, 1H), 7.32 (d, J = 8.80 Hz, 1H), 7.24–7.17 (m, 3H), 7.04 (dd, J = 2.80, 8.40 Hz, 1H), 6.83 (dd, J = 2.00, 7.20 Hz, 1H), 6.39 (br s, 1H), 3.92–3.82 (m, 3H), 3.51–3.40 (m, 3H), 3.00 (d, J = 5.20 Hz, 3H).
Compound 6 (2-chloro-5-(N-methyl-3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamido)benzoic acid)
t-Butyl 2-chloro-5-(N-methyl-3-(4-(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyridine-5-carboxamido)benzoate (130 mg, 0.25 mmol) was taken up in DCM (5.0 ml). The solution was cooled to 0 °C and trifluoroacetic acid (5.0 ml) was added. The mixture was stirred at r.t. for 6 h and the volatiles were removed under vacuum. The resulting solid was washed with diethyl ether and purified by HPLC (Kinetex EVO, 150 mm × 21.2 mm, 20 ml min−1; A = 0.1% trifluoroacetic acid in water, B = acetonitrile, 20–30% B over 2 min, 30–44% B over 7 min) to provide the title compound (25 mg, 0.055 mmol, 22%) as a solid. LC–MS (m/z): 463.15 [M + H]+, RT = 0.63 min. 1H NMR (400 MHz, DMSO-d6): δ 8.68 (d, J = 7.2 Hz, 1H), 8.52–8.40 (m, 2H), 7.95–7.82 (m, 4H), 7.57 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 3.2 Hz, 2H), 6.87 (d, J = 7.2 Hz, 1H), 3.42 (s, 3H), 2.81 (d, J = 4.4 Hz, 3H).
Compound 7 (4-(5-((4-chloro-3-(methoxycarbonyl)phenyl)(methyl)carbamoyl)pyrazolo[1,5-a]pyridin-3-yl)benzoic acid)
(4-(tert-Butoxycarbonyl)phenyl)boronic acid (74 mg, 0.33 mmol) was dissolved in THF (3 ml), H2O (560 µl) and triethylamine (100 µl). Methyl 5-(3-bromo-N-methylpyrazolo[1,5-a]pyridine-5-carboxamido)-2-chlorobenzoate (100 mg, 0.24 mmol) was added and the solution purged with N2. PdCl2(dtbpf) (7.7 mg) was added. The mixture was stirred at 72 °C for 1 h, when the reaction was judged complete by LC–MS. The mixture was cooled to r.t. and diluted with EtOAc and water. The organic layer was washed with brine, dried with Na2SO4, concentrated and purified by flash chromatography (0–100% EA/Hep) to give methyl 5-(3-(4-(tert-butoxycarbonyl)phenyl)-N-methylpyrazolo[1,5-a]pyridine-5-carboxamido)-2-chlorobenzoate (112 mg, 0.215 mmol, 91% yield). This solid was diluted in DCM, and 4.0 M HCl in dioxane (538 µl) was added. Reaction was completed by LC–MS in 2 h. The volatiles were removed under vacuum to dryness and purified by preparative HPLC. The material was isolated and the resulting solid was collected and dried under air to yield the title compound (86 mg, 0.18 mmol, 84% yield). LC–MS (m/z): 464.3 [M + 1]+, RT = 0.82 min. 1H NMR (500 MHz, DMSO-d6): δ 12.93 (s, 1H), 8.72 (d, J = 7.1 Hz, 1H), 8.50 (s, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.96 (d, J = 2.6 Hz, 1H), 7.84 (s, 1H), 7.59–7.51 (m, 3H), 7.48 (d, J = 8.6 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 3.84 (s, 3H), 3.43 (s, 3H).
HsPI(4)K specific inhibitor (MI14)
The HsPI(4)K specific inhibitor (MI14) used in this study for enzymatic assays was synthesized as described earlier48.
Cell and parasites
Human ileocaecal colorectal adenocarcinoma cells (HCT-8) were purchased from American Type Culture Collection (ATCC) (CCL-34). Cells were cultured at 37 °C with 5% CO2 in a humidified tissue culture incubator using complete media consisting of RMPI-1640 medium (Gibco, A1049101) supplemented with either 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, 12306C), 120 U ml−1 penicillin and 120 µg ml−1 streptomycin (Gibco, 15140-122). For cytopathic effect (CPE) assays, RPMI media were supplemented with 10% heat-inactivated horse serum (Thermo Fisher, 26050), 1× MEM nonessential amino acids (Thermo Fisher, 11140), 10 mM HEPES (Thermo Fisher, 15630), 100 U ml−1 penicillin and 100 U ml−1 streptomycin at 37 °C in a humidified incubator with 5% CO2. HCT-8 cells were only used for experiments between passage number P7 and P30. Cells were counted using a ChemoMetec NucleoCounter with NucleoView software v.1.2.0.0. C. parvum Iowa isolate oocysts were purchased from Bunch Grass Farm (Deary, Idaho) and the C. hominis TU502 isolate purchased from Dr Saul Tzipori (Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts). C. parvum Iowa nanoluciferase-expressing oocysts were a kind gift from D. Boris Striepen from the University of Pennsylvania49 and routinely passaged in interferon gamma knockout (IFNγ KO) or NOD SCID gamma (NSG) mouse models50.
In vitro activity against Cryptosporidium species
The inhibition of the asexual life cycle in the standard (48 h) assays was determined using the CPE assay51. Oocysts were artificially excysted using 10 mM HCl in 1X Hank’s balanced salt solution (HBSS) for 10 min at 37 °C using a shaker at 1,000 r.p.m., followed by exposure to pre-warmed, pre-gassed 2 mM sodium taurocholate in parasite infection medium (PIM, 1:1 formulation of Leibovitz’s L-15 medium and UltraCULTURE medium) for 5 min at 37 °C. HCT-8 cells were infected in a T-175 flask with oocysts triggered for excystation at a multiplicity of infection (MOI) of 3 for C. parvum and MOI of 4 for C. hominis. At 3 h post infection, the infected HCT-8 cells were dissociated via TrypLE (Gibco, 12604021) treatment for 30 min at 37 °C in 5% CO2. Cryptosporidium-infected cells were then pelleted, resuspended in HCT-8 culture media and seeded at a cell density of 2.75 × 104 cells per well in 30 µl per well. For seeding, we used a MultiDrop reagent dispenser (Thermo Fisher) and standard tube dispensing cassette at a high speed setting, onto a black polystyrene 384-well plate (Greiner, 781091). The 384-well plate was previously dry spotted with 60 nl per well of compounds (diluted in DMSO) in a ten-point dose response with 3-fold compound dilution or with controls (DMSO or KDU731 at a final concentration of 2 µM) using an Echo Acoustic liquid handler (BioTek Instruments). Assay plates were incubated at 37 °C in a humidified incubator with 5% CO2 for 48 h, and cells were then lysed with 20 µl r.t. CellTitre-Glo 2.0 (Promega, G9241) and incubated for 30 min at r.t. in the dark. After 30 min, luminescence was measured using the luminometer (CLARIOstar with software v.5.40 R2, BMG Labtech) at 0.1 s per well and 3,500 gain. Raw data files were exported and results were expressed as percent stimulation, where 100% stimulation was equal to the median of the active control wells and 0% stimulation was equal to the median of the DMSO-treated negative control wells. Cell viability curves were analysed using DAVID Helios software v.3.01.00.360 (ref. 52).
Cell-based mode of action, washout and time-kill assays
The invasion and DNA replication assay using fluorescence microscopy, live imaging and transmission electron microscopy were performed as described earlier35. Washout and time-kill assays were adapted from previous studies35,53 and performed using nanoluciferase-expressing parasites. For all assays, oocysts were primed for excystation by treatment with 10 mM HCl in water for 10 min at 37 °C, followed by exposure to 2 mM sodium taurocholate in Dulbecco’s phosphate-buffered saline with calcium and magnesium for 10 min at 16 °C. Primed oocysts were pelleted at 14,000 g for 4.5 min at 4 °C and resuspended in warm complete media for infection. Complete media consisted of RMPI-1640 medium (Gibco, A1049101) supplemented with either 10% heat-inactivated FBS (Sigma-Aldrich, 12306C), 120 U ml−1 penicillin and 120 µg ml−1 streptomycin (Gibco, 15140-122).
Host cell invasion was assayed by allowing C. parvum to invade host cell monolayers in the presence of compound and enumerating parasites and host cells after just 3 h (that is, before completion of a parasite division cycle). Wiskostatin, a known inhibitor of Neural Wiskott–Aldrich Syndrome protein, was used as an active control as host cell actin remodelling is required for Cryptosporidium invasion and parasitophorous vacuole formation35. HCT-8 cells were seeded at 25 µl per well in 384-well black polystyrene assay plates (Corning, 353962) such that the wells were 95–100% confluent at the time of infection. At 1 h before infection, DMSO control, wiskostatin and test compounds were added at twice the indicated concentrations incubated at 37 °C in a humidified 5% CO2 incubator for 1 h. At 1 h post compound addition, 50,000 primed oocysts at 25 µl per well were used to infect each well such that the final concentration of compounds was the same as the indicated concentrations. Assay plates were then incubated for 3 h in a humidified incubator at 37 °C under 5% CO2. For all the following steps, 1X phosphate-buffered saline (PBS) was used (prepared by diluting 10X PBS (Thermo Fisher, 70011-044) 10-fold with distilled water). At 3 h post infection, assay plates were washed 3 times with 1X PBS with 0.1% Tween 20, fixed with 4% paraformaldehyde (PFA) in 1X PBS for 15 min at r.t. and permeabilized with 0.25% Triton X-100 in 1X PBS for 10 min at 37 °C. Following permeabilization of the monolayer, HCT-8 cells were washed 3 times with 1X PBS with 0.1% Tween 20 and blocked with 4% bovine serum albumin (BSA) in 1X PBS for 2 h at 37 °C or 4 °C overnight. Parasitophorous vacuoles were stained with 1.33 µg ml−1 of fluorescein-labelled Vicia villosa lectin (VVL) (Vector laboratories, FL-1231) diluted in 1% BSA in 1X PBS for 1 h at 37 °C, followed by nuclei staining with Hoechst 33258 (AnaSpec, AS-83219) at a final concentration of 0.09 mM diluted in water for 15 min at 37 °C. Assay plates were then washed 5 times in 1X PBS with 0.1% Tween 20, and thereafter were ready for imaging. A Nikon Eclipse Ti2 epifluorescence microscope with a motorized stage and a Nikon DS-Qi2 camera with a wide field of view was used for imaging. NIS-Elements Advanced Research (AR) software v.5.02.01 (Nikon), incorporating the Nikon Perfect Focus System, was used to automatically focus and collect images from each well with a CFI Plan Apo Lambda ×20, NA 0.75 objective. For image analysis, NIS-Elements AR Analysis software v.5.20.02 and ImageJ v.1.53t were also used.
The DNA synthesis assay measures replication of the parasites inside intestinal cells. HCT-8 cells were seeded in 384-well polystyrene glass-bottomed plates (Cellvis, P384-1.5-N) pre-treated with 20 µl per well of freshly dissolved 40 µg ml−1 fibronectin in 1X PBS (prepared as described under the invasion assay method above) for 2 h at r.t. or at 4 °C overnight. Cells were seeded at 25 µl per well at a range of concentrations between 5,000 to 25,000 cells per well such that the cells were 90–100% confluent at the time of infection. Growth media were removed and cells were infected with 5 × 104 oocysts artificially triggered for excystation as described above. Assay plates were incubated for 3 h at 37 °C in 5% CO2 before the addition of different concentrations of EDI048 or controls. DMSO was used as a neutral control, and the lysyl-rRNA synthetase inhibitor cladosporin26 was used as an active control. Assay plates were incubated at 37 °C in 5% CO2 until ~9 h post infection. At ~9 h post infection, 10 µM 5-ethynyl-2’deoxyuridine (EdU) was added to all wells and incubated at 37 °C in 5% CO2 for 2 h. At 11 h post infection, assay plates were washed twice with 1X PBS, fixed with 4% PFA in 1X PBS for 15 min at r.t. and left in 2% PFA in 1X PBS fixative at 4 °C overnight. In the morning, fixative was removed and wells were washed twice with 3% BSA in 1X PBS. Cells were then permeabilized with 0.5% Triton X-100 for 20 min at r.t. and washed again twice with 3% BSA in 1X PBS. The Click-ITTM EdU Alexa Fluor 647 imaging kit was then used for EdU staining (Invitrogen, 10340). Briefly, this involved making a Click-iT reaction cocktail and incubating for 30 min at r.t., protected from light. Post-EdU staining, wells were again washed with 3% BSA in 1X PBS, and parasitophorous vacuole and nuclei were stained as described above. Wells were then washed 3 times with 1X PBS and imaged. Imaging was as described above for the invasion assay, except that a CFI S Plan Fluor ELWD ×40, NA 0.6 objective was used.
Live imaging of C. parvum was performed to assay the life stage effect of PI(4)K inhibitors. Fibronectin-coated glass-bottomed 96-well plates were infected with 150,000 primed oocysts per well. Assay plates were incubated for 3 h (h) at 37 °C under 5% CO2. At 3 h post infection, wells were washed 3 times with 2 ml of warm complete media and then incubated with 0.5 µM KDU731 (approximate C. parvum CPE assay EC90) or DMSO. Thereafter, live microscopy on the Nikon Eclipse Ti2 epifluorescence microscope was set up within the live humidified chamber at 37 °C under 5% CO2. Cells were imaged at 20 min intervals with a CFI S Plan Fluor ELWD 40XC, NA 0.6 objective. All images were strung together to make a video from 6 h to 21 h post infection.
TEM was performed to visualize the effect of compounds on meronts. HCT-8 cells were grown to >90% confluence in polystyrene 12-well cell culture plates (Corning, cCLS3513), and complete media were removed before infection. HCT-8 cells were infected with 100,000 primed oocysts per well. At 3 h post infection, wells were washed 6 times with 2 ml of warm complete media and then incubated with 0.5 µM (approximate C. parvum CPE assay EC90) KDU731 or DMSO in complete media for 10.5 h at 37 °C under 5% CO2. At 10.5 h post infection, Cryptosporidium-infected cells were de-adhered via 0.25% Trypsin–2.21 mM EDTA treatment for 5 min at 37 °C. Infected cells were pelleted at 150 g for 5 min and resuspended in 1 ml half-strength Karnovsky’s fixative: 1% PFA, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Infected cells were fixed for 1 h at 4 °C and then pelleted as before. Fixed cells were thereafter washed 3 times in total with 0.1 M sodium cacodylate buffer wash reagent for 5 min at r.t. The washed and fixed samples were shipped to the University of Vermont Microscopy Imaging Centre for further treatment and generation of TEM images. Briefly, this involved crosslinking and dehydration of samples, followed by semi-thin sectioning (60–80 nm) and mounting onto a mesh copper grid for imaging with a JEM 1400 transmission electron microscope (JEOL).
C. parvum Iowa isolate oocysts expressing nanoluciferase (Nluc) under control of the enolase promoter were a kind gift from Dr Boris Striepen from the University of Pennsylvania. C. parvum was passaged in mice following published methods50. Briefly, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from The Jackson Laboratory and orally infected with 104 Nluc-expressing oocysts, and fresh faeces were collected within 2 h of excretion for several weeks post infection. Luminescence readings were taken for a fraction of the faeces to quantify the Nluc signal using the Nano-Glo Luciferase Assay System kit (Promega, N1130) and the CLARIOstar microplate reader with software v.5.40 R2 (BMG LABTECH). Oocysts were purified from faeces by homogenization, filtration and centrifugation, followed by sucrose and caesium chloride gradients. Purified oocysts were confirmed for expression of nanoluciferase by phase contrast microscopy and by quantifying luminescence reading, and were confirmed for viability by microscopic evaluation of excystation. Purified oocysts were stored at 4 °C and used within 2 months from the date of shedding.
Time-kill assays were used to assess whether EDI048 is parasiticidal or parasitistatic for C. parvum. HCT-8 cells were seeded in 384-well white-bottomed plates (Greiner, 781207) such that the wells were ~90% confluent at the time of infection. HCT-8 cells were then infected with oocysts primed for excystation immediately after resuspending oocysts in warm complete media. Oocysts (400 per well) were added and ~24 h after infection, compounds were added at indicated concentrations ranging from 20 µM to 1 nM. Time of compound addition (~24 h post infection) was considered time 0. At timepoints 0, 4, 8, 24, 48 and 72 h, luminescence (relative luminescence units, RLU) was measured using the Nano-Glo Luciferase Assay System kit (Promega, N1130) following manufacturer instructions and readings taken using the CLARIOstar microplate reader with software v.5.40 R2. For each timepoint, luminescence from each well was normalized to the average luminescence of the DMSO control wells to calculate percent (%) RLU relative to DMSO control. The RLU values as well as the DMSO control-normalized % RLU for each timepoint and each concentration of EDI048 and nitazoxanide were plotted using GraphPad Prism v.8.1.2.
Washout assays were performed to evaluate the effect of compounds on parasite growth stages. HCT-8 cells were seeded in 384-well white-bottomed plates (Greiner, 781207), and upon achieving ~90% confluence were infected with 400 per well of total Nluc-expressing oocysts primed for excystation immediately after resuspending oocysts in warm complete media. Compounds were added and washed at the indicated concentrations and timepoints. For each wash, media were removed and cells were washed at least two times with 100 µl per well of complete media. RLU was measured at indicated times using the Nano-Glo Luciferase Assay System kit (Promega, N1130) following manufacturer instructions and readings taken using the CLARIOstar microplate reader with software v.5.40 R2. The RLU values taken soon after infection were considered day 0 RLU and were used to normalize RLU values at each timepoint. Data were plotted using GraphPad Prism v.8.1.2.
Cytotoxicity assay
Cytotoxicity against HepG2 ATCC CRL-10741 was determined as previously described24. Briefly, cells were seeded into 384-well plates at 400 cells per well, incubated at 37 °C for 24 h and exposed to 3-fold serially diluted compounds for 96 h. Cell viability was monitored using Cell Counting Kit-8.
Expression and purification of CpPI(4)K and HsPI(4)K recombinant proteins for enzyme assays and structural studies
Baculovirus cloning, expression and purification of the full-length mutant C. parvum PI(4)K constructs were performed as previously described for wild-type enzyme24. Briefly, the full-length coding sequence of C. parvum PI(4)K (cgd8_4500) with the mutations described (Y705A, Y907A or both) was codon optimized for baculovirus expression, synthesized and cloned into pFastBac-HTb (Invitrogen) in frame with an amino (N)-terminal polyhistidine tag using the BamHI and HindIII restriction sites. Primers for mutagenesis were designed using NEBaseChanger (https://nebasechanger.neb.com/) from New England BioLabs and were ordered from Elim Biopharm. Mutagenesis was performed using the Q5 Mutagenesis kit (New England BioLabs, E0552S). Recombinant pFastBac-HTb-C. parvum PI(4)K bacmid clones were generated by site-specific transposition in Escherichia coli DH10Bac (Thermo Fisher). Bacmid isolation, transfection and selection of the recombinant viruses were performed according to manufacturer protocol (Bac-to-Bac System, Thermo Fisher, 10359-016). SF9 cells, cultured in ESF921 protein-free medium (Expression Systems, 96-001-01), were transfected with liposomes and recombinant baculovirusat 4:1 ratio and incubated at 27 °C for 4–7 days. For protein expression, Sf21 cells were infected with 3%, Passage2, virus at a cell density of 1.5 × 106 cells per ml. Cells were collected at 48 h post infection by centrifugation at 1,000 × g for 30 min at 4 °C and resuspended in cell lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM dithiothreitol (DTT), 20 mM imidazole, 0.01% Triton X-100 and 1X complete protease inhibitor cocktail without ethylenediamine tetra-acetic acid (EDTA, Roche)). The cell suspension was lysed by sonication and the clarified supernatant was loaded onto a 1 ml HisTrap affinity column (GE Healthcare) pre-equilibrated with buffer A (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 20 mM imidazole and 1X complete protease inhibitor cocktail without EDTA). The column was washed with buffer A containing 45 mM imidazole and the bound protein was eluted with buffer A with 90 mM imidazole. Fractions containing mutant C. parvum PI(4)K were identified by size-exclusion chromatography, pooled, concentrated using Amicon Ultra-15, purified by a gel-filtration column (Hi-Load 26/60 Superdex 200, GE Healthcare) and equilibrated with 20 mM Tris pH 7.5, 300 mM NaCl, 1 mM DTT and 1X protease inhibitor cocktail without EDTA. Aliquots were flash frozen in liquid nitrogen and immediately stored at −80 °C.
Full-length wild-type Human PI(4)Kb isoform 2 was codon optimized for expression in Spodoptera frugiperda. The gene was split into two gene tiles with overlap and cloned into a linearized pFastBac1 vector at BamH1 and Xho1 sites.
Truncated HsPI(4)K proteins for X-ray crystallography and enzyme assays were all based on the construct described earlier54 in which HsPI(4)K isoform 2 (1–801) was truncated at the N (1–121) and C (785–801) termini, deleted internally at 249–287 and 408–507 and mutated at S294A. The sequence was codon optimized for expression in E. coli and cloned into pET24 at Nco1 and BamH1 sites with an N-terminal hexa-His tag followed by a TEV protease sequence. For the enzyme activity assays, a variant of the HsPI(4)K X-ray crystallography construct that retains C-terminal residues 785–801 was cloned into pET28a containing an N-terminal hexa-His, MBP, TEV sequence. It has been previously shown that the C termini of HsPI(4)K are necessary for the enzyme activity34,54. The HsCpPI(4)K chimaeric construct included the site-directed mutations, L374Y and P597Y. Both these tyrosine residues are highly conserved across all apicomplexan PI(4)K enzymes and are expected to have a profound effect on the binding pocket surface and allow Pi-stacking with aromatic scaffolds seen in the structures of other PIKK members55. For probing enzyme activity of the HsCpPI(4)K chimaera, the dynamic C-terminal region (C-tail, residues 785–801) of HsPI(4)K was retained as described above.
All four plasmids (HsPI(4)K and HsCpPI(4)K for both X-ray crystallography and enzyme assays) were transformed into the BL21 Star expression strain, grown in 2YT media (+antibiotic) in an incubator/shaker at 37 °C and 250 r.p.m., induced with isopropyl β-d-1-thiogalactopyranoside at 1 optical density (OD), then incubated overnight at 18 °C. Cells were collected by centrifugation (15 min at 5,000 × g) and cell pellets stored at −70 °C until purification. All purification buffers had 20 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol and 1 mM Tris(2-carboxyethyl)phosphine in common, and all column fractions were analysed by gel electrophoresis and intact mass MS before pooling. Cells were lysed with a microfluidizer in buffer containing a commercial protease inhibitor cocktail and universal nucleases. HsPI(4)K proteins for X-ray crystallography were purified by a 3-step protocol: (1) HisTrap (5 ml) capture and 0–250 mM imidazole elution, (2) simultaneous overnight TEV cleavage and imidazole dialysis of the pooled PI(4)K fractions, then passage over a second HisTrap column to capture His-tagged TEV and impurities, followed by (3) final purification of XRC protein in a SEC column (Superdex 200). The pooled fractions were concentrated by centrifugation to ~10 mg ml−1 and flash frozen into liquid nitrogen for storage. Proteins for enzyme activity were purified by a variation of this 3-step protocol in that a no-cleavage step was performed due to the stabilizing effect of the MBP solubility tag. Assay proteins were purified by HisTrap capture and elution, followed by two rounds of SEC chromatography. E. coli expressed HsPI(4)K and HsCpPI(4)K enzymes showed comparable biochemical activity. Recombinant HsRab11 protein used for XRC studies was expressed and purified as described earlier34.
For PI(4)K enzyme assays, all solutions were prepared in deionized water. Triton X-100 (X100-500ML) and manganese(II) chloride tetrahydrate (M3634-100G) were purchased from Sigma-Aldrich. Tris-HCl solutions were prepared in-house as 1 M stocks with appropriate pH. Octylglucoside (OG) powder was used to prepare a 3% solution in 10 mM Tris pH 7.0. OG was used to solubilize 10 mg bovine liver l-α-phosphatidylinositol (PI; Avanti Polar Lipids, 84002P) to a final concentration of 30 mM PI. After addition of 3% OG into the glass vial, it was sealed with parafilm and allowed to sit on ice for 30 min. The solution was then vortexed until all PI was fully solubilized. ADP-Glo reagent, kinase detection reagent, Ultra Pure ATP and adenosine diphosphate (ADP) were purchased from Promega as an ADP-Glo kit (V9101). Luminescence measurements were carried out in white 384-well polypropylene plates (Greiner, 781207). Compound-mediated inhibition of PI(4)K variants was assessed in a total reaction volume of 10 μl. First, 5 µl of PI(4)K enzyme at twice the final concentration (6 nM enzyme for full-length enzymes or 10 nM for truncated constructs) in 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% Triton X-100 and 5 mM MnCl2 was dispensed into assay-ready plates dry spotted with compound (dissolved in DMSO) via Echo liquid handler (Labcyte). The enzyme was allowed to incubate with compound for 5 min at r.t. The reaction was started with the addition of substrate at twice the final concentration (20 μM PI, 6 μM ATP, 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% Triton X-100, 5 mM MnCl2). The final ATP concentration of 3 μM is below the ATP KM. No notable differences in KM were observed for mutant PI(4)K constructs relative to their corresponding wild-type enzymes. The reaction was allowed to proceed for 50 min at r.t. Subsequent steps to quench and detect were followed as described in the ADP-Glo kit manual. The reaction was quenched by the addition of 10 μl of ADP-Glo reagent and allowed to incubate at r.t. for at least 40 min. After this incubation, 20 μl of kinase detection reagent was added and allowed to incubate at r.t. for 30 min, after which the plate was read using a BMG Labtech CLARIOstar plate reader (with software v.5.40 R2) using a gain of 3,600, 0.2 s measurement interval and focal height of 10 mm. IC50 values were determined using in-plate duplicate 10-point 3-fold serial dilutions of each compound covering a concentration range of 20 μM to 1 nM. The neutral control (NC; DMSO) and the active control (AC; 10 μM KDU731) were present in columns 1, 12, 13 and 24 of each plate. The active control was also present in dose response on each plate. Data were converted to % activity, with 0% representing no inhibition (comparable to DMSO) and −100% representing full inhibition (comparable to the AC) based on the equation −100 × ((x − NC) / (AC − NC)), where x is the measured value. DAVID Helios software v.3.01.00.360 (ref. 52) was used to further analyse the data. The normalized values were plotted against compound concentration, and the IC50 was calculated using four-parameter nonlinear regression. The reported IC50 values were calculated as µM concentrations.
Crystallization and structure determination of HsCpPI(4)K/HsRab11a with EDI048
Crystallization
The protein used for X-ray crystallography was HsPI(4)KIIIβ with truncated dynamic regions34 and containing two mutations (P597Y and L374Y) in the ATP-binding pocket to mimic Cryptosporidium enzymes ligand-binding pocket. This truncated HsPI(4)KIIIβ::P597Y: L374Y enzyme is referred to as ‘HsCpPI(4)K’. HsCpPI(4)K/Rab11a complex was crystallized by vapour diffusion methods by incubating 10 mg ml−1 HsCpPI(4)K protein with 12 mg ml−1 HsRab11a in an ~1:1 molar ratio for 30 min at a final concentration of ~5–6 mg ml−1. Hanging crystallization drops were set up containing 1 µl complex and 1 µl well solution in 15-well crystallization plates. The well solution was composed of 500 mM ammonium sulfate, 88 mM sodium citrate, 5% glycerol and 875 mM lithium sulfate. The plates were then incubated for crystallization at 18–25 °C for 2–3 days. Crystals were collected for compound soaking in cryo-soak solutions. For soaking, equal volumes of 100% glycerol and 1 M HEPES pH 7.5 were mixed with sufficient mass of EDI048 to result in a final concentration of 100 mM. Protein crystallization mixture (20 µl) was dispensed and mixed with 5 µl of the compound solution to make a cryo-soaking solution. Cryo-soaking solution (10 µl) was dispensed into sitting drop wells in a 24-well plate, and HsCpPI(4)K/HsRab11a crystals were applied for soaking for ~24 h. Soaked crystals were collected and flash frozen in liquid nitrogen using a cryoprotectant composed of 25% ethylene glycol and 75% cryo-soak.
Data collection
A full data set (useable data to 2.9 Å) was collected at APS on the MCAT beamline. The data collection was performed at 100 K and wavelength = 1.0000 Å. The crystal used was of the space group P212121, with average unit cell lengths of a = 48.57 Å, b = 105.22 Å, c = 186.41 Å; α = β = γ = 90°. The raw data were integrated, indexed and scaled using the Autoproc suite from Global Phasing.
Refinement
Phaser (Phenix Consortium) as implemented in the CCP4 Suite of programs was used for the molecular replacement. An in-house structure of Hs-Cp-PI(4)K/HsRab11a was used as the search model. Refinement and manual rebuilding were performed in iterative cycles using Phenix Refine (Phenix Consortium) and Coot software v.0.9.8.92 (ccp4)56. Partial fitting of the protein was performed before refinement of the inhibitor. Idealized atomic coordinates for the inhibitor were generated with Molecular Operating Environment (MOE) v.2022.02 (Chemical Computing Group) and fit into an Fo−Fc difference electron density map contoured at the 3σ level using Coot. Structural parameters for EDI048 were generated by grade/mogul (Global Phasing, CCDC). The inhibitor was then refined along with the rest of the structure until the refinement converged. Solvent molecules were located and automatically assigned by Coot and Phenix.refine. Solvent that had good geometries (that is, trajectory and distances consistent with potential hydrogen bonds) were modelled and checked for consistent 2Fo−Fc difference density contoured at the 1σ level in subsequent cycles. Ramachandran statistics used were: Outliers: 0%; Allowed: 4%; Favoured: 96%. Relevant data and refinement statistics are presented in Extended Data Table 3. The PDB (Protein Data Bank) accession code for the HsCpPI(4)K/HsRab11a-EDI048 co-crystal structure is 8VOF. A ligand-interaction plot was generated using MOE v.2022.02 (Chemical Computing Group). A simulated annealing omit map (mFo−DFc) was calculated as additional confirmation of the ligand-binding mode (Phenix Consortium).
Solubility and permeability assays
Solubility was measured using a high-throughput equilibrium solubility assay employing a miniaturized shake-flask approach and streamlined HPLC analysis as described earlier57. In vitro permeability parallel artificial membrane assays were carried out using standard protocol58. Passive permeability along with P-glycoprotein-mediated efflux was measured in MDR-transfected MDCK cells as described earlier59.
In vitro liver microsomes and hepatocytes metabolic stability assessment
The metabolic stability in liver microsomes and hepatocytes was determined using the compound depletion approach and quantified by LC–MS/MS. The assay measures the rate and extent of metabolism as determined by the disappearance of the parent compound, which allows the determination of in vitro half-life (t1/2), intrinsic clearance (Clint), prediction of metabolic clearance (CL) and hepatic extraction ratio in various species60,61.
In vitro stability in intestinal S9 fraction, in vitro plasma protein binding and plasma stability
Stability assays in plasma and intestinal S9 fraction were performed as previously described62. Briefly, the metabolic reaction was initiated by addition of compounds to the matrices. Incubation was conducted in a shaking water bath at 37 °C and timepoint measurements were sequentially taken. Samples were quenched with ice-cold acetonitrile and analysed by LC–MS/MS. The t1/2 was calculated from the rate of compound depletion. In vitro plasma protein binding was determined using the rapid equilibrium dialysis method63.
Cytochrome P450 analysis
EDI048 was subjected to CYP450 inhibition analysis using 3 different isoforms following standard procedure64 and assessed for time-dependent inhibition using CYP3A4 (ref. 65). CYP induction assay was carried out using PXR reporter gene assay. A PXR binding-based hepatoma cell assay was employed to measure CYP3A4 transactivation. This assay uses a human hepatoma cell line DPX-2 overexpressing the human PXR and a CYP3A4 promoter-luciferase reporter gene66,67.
Cardiotoxicity and phototoxicity
Cardiotoxicity risk was measured as previously described67. Briefly, for hERG binding assay, concentration–response curves were typically generated as 6-point dose response from 0.37 to 30 µM using hERG-expressing HEK293 cells; for hNav1.5 and hCav1.2 patch clamp assays, as 7-point dose response from 0.1 to 50 µM in HEK293 cells expressing respective ion channels. The effect of the compound on hERG potassium channel current was evaluated (4-point dose response of 1–30 µM) at near-physiological temperature in stably transfected mammalian cells that express the hERG gene. Phototoxicity assay was performed following the OECD Guideline for Testing of Chemicals 432: ‘in vitro 3T3 NRU phototoxicity test’68. Briefly, in vitro photo safety evaluation was conducted in 3T3 fibroblast cells (seeded into 96-well microtitre plates treated with a range of concentration of compound) using neutral red uptake assay, tested up to the limit of solubility in HBSS in accordance with current regulatory guidelines.
Mini-ames, TK6 micronucleus assay and in vitro safety profiling assays
Mini-ames genotoxicity risk was measured as previously described69. Briefly, mutagenic potential of the compound was evaluated by its effects on one or more histidine-auxotrophic Salmonella typhimurium strains TA98 and TA100 in the absence or presence of rat liver metabolizing S9 system. The in vitro micronucleus test in TK6 cells was performed as previously described70. All assays for binding to proteins known to bear potential safety liabilities in humans were high-throughput competitive binding assays using specific radiolabelled ligands67.
In vitro human lymphocyte (HuLy) micronucleus assay
In vitro HuLy micronucleus assay was performed using pooled blood (two donors), in accordance with Labcorp standard operating procedures, the United Kingdom Good Laboratory Practice Monitoring Authority, Good Laboratory Practice Regulations 1999 and in compliance with the OECD guidelines71. The clastogenic and aneugenic potential of EDI048 was evaluated by analysing the frequency of micronuclei formation in cultured human peripheral blood lymphocytes treated in the absence and presence of a rat liver metabolizing system (S9). Briefly, 50–500 µg ml−1 of EDI048 was incubated for either 3 h or 24 h with 21 h or 24 h recovery period, respectively, with and without S9. Cells were centrifuged and resuspended in a minimal amount of fresh fixative, and slide analysis was carried out using fluorescence microscopy.
In vivo rat micronucleus and Comet assays
On days 3 and 15, blood samples for peripheral blood micronucleus assay were collected in randomized order from 2 weeks rat toxicology study animals and analysed by flow cytometer as described in ref. 72. Dosing details are described in ‘Rat toxicology study’. For in vivo Comet assay with EDI048, groups of 6 male Wistar rats were dosed at 200, 600 and 2,000 mg kg−1 d−1 for 2 days. Liver and duodenum tissue were sampled at ~Tmax following the second dose, and Comet assays were performed as described previously73.
In vitro identification of metabolites in hepatocytes
The metabolic disposition of EDI048 was assessed following in vitro incubation with mouse, rat, dog and human hepatocytes after 2 h of incubation. Briefly, 10 µM concentration of test compound was added directly to wells containing hepatocytes. Time zero samples were immediately quenched with an equal volume of acetonitrile and mixed well. Two-hour samples were incubated at 37 °C in a humidified CO2 incubator (5% CO2 and 90% relative humidity) for 2 h on an orbital shaker at 400 r.p.m. Following this, hepatocyte incubations were quenched with acetonitrile, centrifuged (~6,000 g for 10 min at ~20 °C) and the supernatant analysed using LC–UV/MS with accurate mass detection.
In vivo pharmacokinetic analysis
In vivo pharmacokinetic studies were conducted using non-randomized C57BL/6 male mice (n = 3, 8–10 weeks old), Wister male Sprague Dawley rats (n = 3, 8–10 weeks old) and male Beagle dogs (n = 3, 12–14 weeks old). Neonatal calf pharmacokinetic studies were performed as part of the efficacy study on day 1 of treatment. All procedures involving animals were reviewed and approved by the respective Institutional Animal Care And Use Committees (IACUC). Mice PK was performed according to the IACUC regulations of Charles River Laboratories (No. 2100230). Mice were housed in Innovive disposable micro-isolator cages with bedding. Rat PK was performed according to the IACUC regulations of Novartis Institute for BioMedical Research (No. 2100231). The jugular vein catheter/portal vein catheter rats were housed individually in Allentown reusable cages. Dog PK was performed according to the IACUC regulations of Charles River Laboratories (No. 2100232). All animals were group housed in pens with contact bedding. Calf PK was carried out in compliance with the Virginia Tech Institutional Animal Care and Use Committee (No. 00271) and housing conditions are elaborated in ‘Neonatal calf efficacy study’. No statistical methods were used to predetermine sample size. Sample size was determined on the basis of the minimum number of animals required for good data distribution and statistics. Although animals were selected randomly for each group, animal dosing was not blinded, as compounds need to be formulated in vehicle at different concentrations. However, for the sample collection and analysis, analysts were blinded or followed an unbiased approach using an automated platform. EDI048 was formulated in a suspension formulation for oral dosing (methylcellulose:Tween 80:water at 0.5:0.1:99.4 w/w for mouse, rats and dogs; methylcellulose:Tween 80:water at 0.5:0.5:99 w/w for calves) and solution formulation for intravenous dosing (NMP:4% BSA in PBS 10/90 v/v for mouse; NMP:PEG200 10/90 v/v for rats and dogs).
The blood samples for pharmacokinetic studies were collected between 0 and 24 h post dose. Blood samples were collected into 2 ml tubes containing 6 mg Na2EDTA and 3 mg sodium fluoride (BD Hemogard) and kept on ice until centrifugation. These samples were centrifuged within 30 min of collection for 10 min in a refrigerated centrifuge (set to maintain at 4 °C) at ~2,000 g. The resultant plasma was collected into fresh tubes placed on ice until transferred to a freezer, set to maintain −60 to −80 °C. The concentrations of compounds at various timepoints were determined by high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Plasma samples from pharmacokinetic studies were extracted with acetonitrile:methanol (50:50) containing CHIR073911 as internal standard, using a 4:1 extractant:plasma ratio. Analyte quantitation was performed by LC–MS/MS. Liquid chromatography was performed using a Waters ultra-performance liquid chromatography (UPLC) system with the Waters C18 SB column (2.1 × 50 mm) at an oven temperature of 50 °C, coupled with an API6500 triple quadruple mass spectrometer (Sciex Applied Biosystems). Instrument control and data acquisition were performed using Applied Biosystems software Analyst v.1.6.2. Pharmacokinetic parameters were determined by non-compartmental analysis using Phoenix WinNonLin v.8.3 (Certara).
Rat toxicology study
The rat toxicology study including analyses was conducted at designated test sites in compliance with the principles of Good Laboratory Practice Standards. All procedures involving animals were reviewed and approved by the IACUC of Covance Laboratories (No. 1970604). EDI048 was formulated in a suspension formulation (methylcellulose:Tween 80:water at 0.5:0.5:99 w/w) and orally administered twice daily to 10 male and 10 female Wistar rats at a daily oral dose of 50, 250 or 1,000 mg kg−1 body weight d−1 for 2 weeks. The number of animals in the toxicity protocols is considered to be the minimum necessary for statistical, regulatory and scientific reasons. For rat toxicology studies, 10 animals per sex per group was considered the minimum number that would account for the expected variability among these animals. Rats were obtained from Envigo and subjected to 5 days of quarantine and acclimatization before the study began. Rats were ~9–10 weeks at initiation of dosing and were pair or triple housed, by group and sex, in solid bottom cages with cellulose-based contact bedding. An enrichment device as well as a hut, tunnel or house were provided in each cage at all times. All animals were subjected to daily clinical observation, and body weight and food consumption were determined appropriately for all animals enrolled in the study. Clinical laboratory evaluations (haematology and clinical chemistry) were performed at the scheduled necropsy on day 15. Organs were examined for gross pathology and weighed before fixation and preparation for histology. Samples from organs and tissues prepared from animals assigned to control and high-dose groups were examined microscopically. Specifically, the adrenal glands, aorta (thoracic), bone marrow (femur, including joint, sternal), brain, caecum, colon, duodenum, epididymides, oesophagus, eyes, harderian glands, heart, ileum, jejunum, kidneys, lacrimal glands, liver, lymph nodes (mandibular, mesenteric), mammary gland, nerve (optic, sciatic), ovaries, pancreas, Peyer’s patches, pituitary, prostate, rectum, salivary glands (mandibular, parotid, sublingual), seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, cervix, vagina and gross lesions were examined for histopathological changes. Blood samples were collected on days 1 and 14 predose and at ~0.5, 1, 3 and 8 h post the first daily dose. These samples were processed to generate plasma, and concentrations were analysed for EDI048, and compounds 6 and 7 as described above.
Dog toxicology study
The dog toxicology study including analyses was conducted at designated test sites in compliance with the principles of Good Laboratory Practice Standards. All procedures involving animals were reviewed and approved by the IACUC of Covance Laboratories (No. 1970605). EDI048 was formulated in a suspension formulation (methylcellulose:Tween 80:water at 0.5:0.5:99) and orally administered twice daily to 3 male and 3 female beagle dogs at a daily oral dose of 15, 50, 150 or 1,000 mg kg−1 body weight d−1 for 2 weeks. The number of animals in the toxicity protocols is considered to be the minimum necessary for statistical, regulatory and scientific reasons. For dog toxicology studies, 3 animals per sex per group was considered the minimum number to account for the expected variability among these animals. Dogs were obtained from Marshall BioResources and subjected to at least 1 week of quarantine and acclimatization before the study began. Dogs were prepubertal/pubertal (~10–11 months old at dose initiation) and were housed in pairs or triplets, by group and sex, in elevated stainless-steel cages. All animals were subjected to daily clinical observation and body weight and food consumption were determined appropriately for all animals enrolled in the study. Clinical laboratory evaluations (haematology and clinical chemistry) were performed on day 7 and at the scheduled necropsy on day 15. Organs were examined for gross pathology and weighed before fixation and preparation for histology. All tissue samples described above in the rat toxicology section were collected and examined microscopically. Blood samples were collected from all animals on days 1 and 14 predose and at ~0.5, 1, 3 and 8 h post the first daily dose. These samples were processed to generate plasma, and concentrations were analysed for EDI048, and compounds 6 and 7 as described above.
Mouse efficacy study
All mouse studies described in this section were reviewed and approved by the IACUC of the Novartis Institute for Biomedical Research (animal use protocol no. 2017-055). Female C57BL/6 IFN-γ-knockout mice (B6.129S7-Ifngtm1Ts/J, Jackson Laboratories) aged 6–8 weeks were selected randomly for each group (n = 3) and infected with 10,000 C. parvum oocysts (Iowa isolate purified from experimentally challenged neonatal calves obtained from Bunch Grass Farms; oocysts used in these experiments were within 3–4 months from shedding). Sample size was determined on the basis of the minimum number of animals, technical and biological replicates required for good data distribution and statistics. For all these studies, n = 3 animals per dose group was used. EDI048 was formulated in 0.5% w/v methylcellulose and 0.5% w/v polysorbate in water and administered to mice for 5 days by oral gavage on day 3 post infection. Control mice were given only vehicle. Animal dosing was not blinded, as compounds need to be formulated in vehicle at different concentrations; however, for the sample collection and analysis, analysts were blinded or followed an unbiased approach using an automated platform. Faecal samples were collected daily from days 3 to 8 post infection. Measurements were performed on faecal material collected from cage-wide (pooled) collections. To measure oocyst load by qPCR, DNA was isolated from 50–100 mg of pooled faecal samples using the Quick-DNA faecal/soil microbe prep kit (Zymo Research). Samples were shaken for 20 min (25 °C, 1,450 r.p.m.) on an Eppendorf ThermoMixer F1.5 and subjected to 4–5 rounds of freeze–thaw cycles using liquid nitrogen. qPCR was performed using sample DNA extracted from faeces of infected mice along with DNA standards prepared by spiking varying numbers of oocysts into uninfected faeces. PCR primers JVAF and JAVR and probe 5’FAM labelled JVAR74 were used with the following cycling parameters: denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s. Each 10 μl PCR reaction contained 5 μl of SSoAdvanced universal probes supermix (Bio-Rad), 0.075 μM of each primer, 0.125 μM probe and 2 μl DNA. A standard calibration curve was prepared to estimate the number of oocysts per gram faeces. Oocyst dilutions (100 µl) ranging from 1 × 102 to 1 × 106 were added to each tube containing ~50 mg of faecal samples. DNA isolation and qPCR were performed as described above. PBS (100 µl) was used as a negative control. A calibration curve was freshly prepared and analysed with every set of study samples. Based on the observed cycle threshold (Ct) values corresponding to the lowest oocyst concentration, the limit of detection ranged between 80 and 280 oocysts per 50 g faeces (corresponding Ct values were 35 and above). To determine EDI048 and compound 6 concentrations, blood samples were collected on day 1 at various timepoints between 0 and 24 h post dose, processed and analysed as described above.
Neonatal calf efficacy study
Calf enrollment
All calves used in this study were cared for in compliance with the Virginia Tech IACUC. Sample size was calculated assuming that 85% of treated calves stop shedding oocysts by the end of the study observation period as compared with 15% of control calves. Assuming a type I error risk of 5% and a type II error risk of 80%, 7 calves were needed in each group of the Cryptosporidium-infected group. Fifteen Holstein–Friesian breed bull and heifer calves (Bos taurus taurus) were purchased from a local commercial dairy and enrolled into the study at birth. Study personnel attended the births to ensure that calves were delivered aseptically and that exposure to pathogens was limited. All calves enrolled were randomized to treatment with EDI048 (n = 7), positive infection control (n = 7) and negative infection control (n = 1) at birth. The perineum of the dam was thoroughly cleaned with povidone–iodine scrub and calves were delivered onto single-use plastic sheets to prevent exposure to environmental pathogens. Calves with abnormal physical examination findings and those weighing less than 30 kg at birth were excluded. Enrolled calves received 4 l ≥50 g IgG/L Land O’Lakes colostrum replacer (Purina Mills) and a 3 ml subcutaneous injection of vitamin E and selenium (BoSe, Merck). Calves were then transported from the commercial dairy farm to Cornell University (College of Veterinary Medicine, Ithaca, New York) in a dedicated trailer bedded with sterile straw.
Calf management and oocyst challenge
At Cornell University, calves were housed in individual box stalls in a Biosafety Level 2 facility. Shatter-proof mirrors were provided for enrichment. Within the first 48 h of birth, blood samples were collected and evaluated for adequate passive transfer of colostral immunity. Calves were offered a commercial 20% protein/20% fat non-medicated milk replacer (Land O’Lakes) every 12 h via nipple bucket. At each feeding, calves were fed an average of 7.6 g dry matter kg−1 birth weight for the duration of the study. Water was provided ad libitum. All calves randomized to the EDI048 treatment or positive control group were experimentally challenged within the first 48 h of life with 5 × 107 C. parvum oocysts (Iowa II strain, Bunch Grass Farm) through the rigid portion of an oroesophageal feeding tube. Oocysts were within 1 month of isolation, cleaned in 0.6% sodium hypochlorite for 1 min and then washed four times with PBS. The negative control calf was sham challenged to maintain blinding of study personnel, and study personnel were blinded during drug dosing, clinical scoring and qPCR analysis of faecal samples. At the end of the study, calves treated with the experimental compound were humanely euthanized using American Veterinary Medical Association approved methods to prevent accidental introduction into the food chain, following federal regulations. Calves that did not receive experimental compound were offered for adoption.
Administration of EDI048 and biological sampling
To facilitate collection of blood for plasma pharmacokinetic analysis, a long-term intravenous catheter (MILACATH, MILA) was aseptically placed in the jugular vein of each calf within the first 48–72 h of life. Calves were sedated with 0.1 ml intravenous xylazine (20 mg ml−1) (Akorn Animal Health). Sedation was reversed with 0.1 ml intramuscular atipamezole (5 mg ml−1) (Zoetis). After oral oocyst challenge, a faecal sample was collected directly from the rectum of each calf every 24 h. A complete physical examination was performed every 12 h, and clinical data including appetite, mentation, faecal consistency and hydration status were recorded. Clinical data were evaluated on a scale of 1 (normal) to 3 (severe) in accordance with previously described methods75,76.
EDI048 was prepared as 5 mg ml−1 suspension formulation in 0.5% w/v methylcellulose and 0.5% w/v Tween 80 in water, and EDI048 treatment was initiated when a calf began shedding oocysts in the stool and had a faecal consistency score of 3. Calves were induced to suckle and EDI048 was then given orally via an oral dosing syringe. Calves were treated every 12 h for 7 days at a dose of 10 mg kg−1 birth weight at least 2 h after feeding. Pharmacokinetic sampling was conducted on day 1 of treatment. Blood was drawn before and at multiple timepoints (between 0 and 12 h) after EDI048 administration. To determine the concentration of EDI048 and QPL621, blood samples were processed and analysed as described above. A faecal sample was collected at 1 h and 12 h post administration. On day 3 of life, a faecal sample was tested for Escherichia coli K99 and on day 7 for Salmonella, rotavirus and coronavirus.
Faecal oocyst enumeration
Oocysts counts were interpolated by qPCR at the Cornell Animal Health Diagnostic Center using serial dilutions of commercially purified C. parvum oocysts (Waterborne). Total nucleic acid was extracted from supernatants of 200 mg of faecal sample, oocyst suspension or negative control homogenized in 400 µl of PBS using a magnetic-bead-based automated procedure (AM1840, Applied Biosytems). An exogenous control (MS2 phage) was added to the lysis buffer to control for PCR inhibition77. qPCR for Cryptosporidium spp. 18S rRNA was performed on an Applied Biosystems 7500-FAST platform using commercial master mix (ToughMix, Quantabio) and oligonucleotides as previously described78. This count was standardized by the faecal dry weight percentage. A 5–10 g portion of each original faecal sample was dried at 108 °C for a minimum of 24 h (Squaroid Vaccuum Oven, Labline) and weighed75.
Data analysis
log normalized oocyst shedding per gram of faecal dry matter data over time was determined to have a normal distribution by Shapiro–Wilk, Anderson–Darline, D’Agostino and Pearson, and Kolmogorov–Smirnov tests, and were analysed using Student’s t-test (two-tailed, paired). LOD to detect parasites in the faecal samples by qPCR assay was determined to be 50 oocysts per gram faeces. Any samples that were undetected or below the lower limit of LOD that is, LOD/2 were represented as 25 oocysts per gram faeces. The clinical scores, that is, severe diarrhea (score = 3), moderate-to-severe dehydration (scores 2 or 3), mentation (scores 2 or 3) and appetite (scores of 2 or 3) were calculated for each calf, and statistical differences between vehicle control and EDI048-treated calves were analysed using the Student’s t-test (unpaired, two-tailed). Data were analysed using GraphPad Prism v.9.1.2 and v.9.5.1. In this study, infected calves showed diarrhoeal symptoms as measured by faecal consistency scores, the primary symptom for cryptosporidiosis. However, the neonatal calves did not develop severe impact on mentation, dehydration or appetite scores. Thus, the clinical effect of EDI048 compared to untreated calves was evaluated on faecal consistency scores only (Fig. 3c and Extended Data Fig. 5). The calf efficacy source data used for the analysis shown in Fig. 3 and Extended Data Fig. 5 are included as Microsoft Excel files (v.2405) in the Supplementary Information.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.