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.
