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.

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References

  1. 1.

    et al. Global, regional, and national causes of under-5 mortality in 2000–15: an updated systematic analysis with implications for the Sustainable Development Goals. Lancet 388, 3027–3035 (2016)

  2. 2.

    et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382, 209–222 (2013)

  3. 3.

    . et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob. Health 3, e564–e575 (2015)

  4. 4.

    et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect. Dis. 15, 85–94 (2015)

  5. 5.

    et al. High dose prolonged treatment with nitazoxanide is not effective for cryptosporidiosis in HIV positive Zambian children: a randomised controlled trial. BMC Infect. Dis. 9, 195 (2009)

  6. 6.

    et al. Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet 360, 1375–1380 (2002)

  7. 7.

    Parasitic infections: time to tackle cryptosporidiosis. Nature 503, 189–191 (2013)

  8. 8.

    et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477–480 (2015)

  9. 9.

    et al. Spiroindolones, a potent compound class for the treatment of malaria. Science 329, 1175–1180 (2010)

  10. 10.

    et al. Gift from nature: cyclomarin A kills mycobacteria and malaria parasites by distinct modes of action. ChemBioChem 16, 2433–2436 (2015)

  11. 11.

    et al. Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334, 1372–1377 (2011)

  12. 12.

    et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature 504, 248–253 (2013)

  13. 13.

    et al. Lead optimization of imidazopyrazines: a new class of antimalarial with activity on Plasmodium liver stages. ACS Med. Chem. Lett. 5, 947–950 (2014)

  14. 14.

    et al. Preparation of pyrazolopyridine compounds for the treatment of parasitic diseases. US patent WO 2014078802 A1 (2014)

  15. 15.

    Molecular epidemiology of cryptosporidiosis: an update. Exp. Parasitol. 124, 80–89 (2010)

  16. 16.

    et al. PI4 kinase is a prophylactic but not radical curative target in Plasmodium vivax-type malaria parasites. Antimicrob. Agents Chemother. 60, 2858–2863 (2016)

  17. 17.

    et al. Identification of Plasmodium PI4 kinase as target of MMV390048 by chemoproteomics. Malar. J. 13 (Suppl. 1), P38 (2014)

  18. 18.

    et al. A proposed target product profile and developmental cascade for new cryptosporidiosis treatments. PLoS Negl. Trop. Dis. 9, e0003987 (2015)

  19. 19.

    ., ., & Cryptosporidiosis drug discovery: opportunities and challenges. ACS Infect. Dis. 2, 530–537 (2016)

  20. 20.

    et al. Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal. Biochem. 396, 290–297 (2010)

  21. 21.

    , , , & Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrob. Agents Chemother. 42, 1959–1965 (1998)

  22. 22.

    et al. Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves. Parasitol. Res. 112, 1247–1254 (2013)

  23. 23.

    et al. Cryptosporidium parvum: determination of ID50 and the dose-response relationship in experimentally challenged dairy calves. Vet. Parasitol. 197, 104–112 (2013)

  24. 24.

    et al. A comparison of fecal percent dry matter and number of Cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves. J. Parasitol. 97, 349–351 (2011)

  25. 25.

    et al. 3,5-Diaryl-2-aminopyridines as a novel class of orally active antimalarials demonstrating single dose cure in mice and clinical candidate potential. J. Med. Chem. 55, 3479–3487 (2012)

  26. 26.

    et al. Cryptosporidium infection of human intestinal epithelial cells increases expression of osteoprotegerin: a novel mechanism for evasion of host defenses. J. Infect. Dis. 197, 916–923 (2008)

  27. 27.

    , , & Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth. Antimicrob. Agents Chemother. 57, 1804–1814 (2013)

  28. 28.

    et al. A screening pipeline for antiparasitic agents targeting cryptosporidium inosine monophosphate dehydrogenase. PLoS Negl. Trop. Dis. 4, e794 (2010)

  29. 29.

    et al. Direct inhibitors of InhA are active against Mycobacterium tuberculosis. Sci. Transl. Med. 7, 269ra3 (2015)

  30. 30.

    et al. High-throughput in vitro profiling assays: lessons learnt from experiences at Novartis. Expert Opin. Drug Metab. Toxicol. 2, 823–833 (2006)

  31. 31.

    Artificial membrane assays to assess permeability. Curr. Drug Metab. 9, 886–892 (2008)

  32. 32.

    , & An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl Acad. Sci. USA 70, 782–786 (1973)

  33. 33.

    , , , & Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays. J. Med. Microbiol. 57, 1099–1105 (2008)

  34. 34.

    , & Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J. Clin. Microbiol. 43, 4551–4557 (2005)

  35. 35.

    , , , & Correlation between diarrhea severity and oocyst count via quantitative PCR or fluorescence microscopy in experimental cryptosporidiosis in calves. Am. J. Trop. Med. Hyg. 92, 45–49 (2015)

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Acknowledgements

We thank S. Tzipori and D. Girouard for C. hominis oocysts; B. Nare for screening; B. H. Lee and J. Selva for high-content imaging data; M. Weaver for rat toxicology studies; I. Mueller for monkey pharmacokinetics; B. Yeung, O. Simon, J. Roland, V. Bollu, A. Chatterjee, A. Nagle, R. Moreau, and P. K. Mishra for compound synthesis; other Novartis Institutes for Biomedical Research (NIBR) colleagues for profiling; J. Burrows and K. Chibale for MMV390048; and M. Meissner for a plasmid carrying the red-shifted Fluc gene. This work was supported in part by the NIBR, the Wellcome Trust (Pathfinder 107678/Z/15/Z to B.S. and U.H.M.), and the National Institutes of Health (NIH R01AI112427 to B.S.). Inhibitors of the Plasmodium PI4K were discovered with the support of translational grants (WT078285 and WT096157) from the Wellcome Trust and funding from the Medicines for Malaria Venture (M.M.V.). B.S. is a Georgia Research Alliance Distinguished Investigator and A.S. is supported by NIH Fellowship F32AI124518. We thank our colleagues from Novartis Institute for Tropical Diseases, University of Georgia, Athens, Washington State University’s Office of the Campus Veterinarian, Animal Resource Unit, and the Office of Research Support and Operations and R. Anderson, 5D Dairy Farm, for their support. We are also grateful to the animal science and veterinary students at Washington State University for their participation in data collection and care of the research calves.

Author information

Author notes

    • Ujjini H. Manjunatha
    • , Sumiti Vinayak
    •  & Jennifer A. Zambriski

    These authors contributed equally to this work.

Affiliations

  1. Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670, Singapore

    • Ujjini H. Manjunatha
    • , Alexander T. Chao
    • , Christian G. Noble
    • , Ghislain M. C. Bonamy
    • , Ravinder R. Kondreddi
    • , Bin Zou
    • , Peter Gedeck
    • , Suresh B. Lakshminarayana
    • , Siau H. Lim
    • , Christophe Bodenreider
    • , Gu Feng
    • , Francesca Blasco
    • , Juergen Wagner
    • , F. Joel Leong
    •  & Thierry T. Diagana
  2. Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia 30602, USA

    • Sumiti Vinayak
    • , Carrie F. Brooks
    • , Gillian T. Herbert
    • , Adam Sateriale
    •  & Boris Striepen
  3. Washington State University, College of Veterinary Medicine, Paul G. Allen School for Global Animal Health, Pullman, Washington 99164, USA

    • Jennifer A. Zambriski
    • , Tracy Sy
    •  & Susan Noh
  4. Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA

    • Jayesh Tandel
    •  & Boris Striepen
  5. USDA-Agricultural Research Service, Animal Disease Research Unit, Washington State University, Pullman, Washington 99164, USA

    • Susan Noh
  6. Washington State University, Department of Veterinary Microbiology and Pathology, Washington Animal Disease Diagnostic Laboratory, Pullman, Washington, USA

    • Susan Noh
  7. Cornell University, College of Veterinary Medicine, Department of Population Medicine and Diagnostic Sciences, Ithaca, New York 14853, USA

    • Laura B. Goodman
  8. China Novartis Institutes for Biomedical Research, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, China

    • Lijun Zhang

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Contributions

U.H.M., S.V., J.A.Z., B.S., and T.T.D. conceived and designed the study; B.S. wrote grant applications with contributions from U.H.M.; U.H.M., A.T.C., and G.M.C.B. developed C. parvum screening assays; C.G.N. and S.H.L. developed enzyme assays; C.B. analysed P. falciparum EC50 data; U.H.M., P.G., and T.T.D. assembled the screening library; R.R.K. and B.Z. performed compound synthesis; U.H.M., B.Z., and J.W. analysed the structure–activity relationship; S.B.L. and F.B. analysed in vivo pharmacokinetics data; L.Z. optimized formulation; U.H.M., G.F., F.J.L., and T.T.D. analysed in vivo efficacy and toxicology results; S.V., A.S., and B.S. designed mouse models based on transgenic parasites; S.V., A.S., and J.T. constructed transgenic parasites; S.V., A.S., C.F.B. and G.T.H. validated mouse models; G.T.H., S.V., and C.F.B. tested compounds; J.A.Z. developed the calf model and analysed calf data; T.L.S. executed the calf model; S.N. conducted anatomic pathology reviews for efficacy and toxicity; L.B.G. developed and executed calf stool analytics; and B.S., S.V., U.H.M., and T.T.D. wrote the manuscript with contributions from J.A.Z., A.T.C., C.G.N., and S.B.L.

Competing interests

R.R.K. and B.Z. are named as inventors on a pyrazolopyridine patent application related to this work (WO 20140788002 A1). U.H.M. and T.T.D. are named as inventors on a pending cryptosporidiosis patent application related to this work. All Novartis Institute for Tropical Diseases-affiliated authors are employees of Novartis and some own shares in Novartis.

Corresponding authors

Correspondence to Jennifer A. Zambriski or Boris Striepen or Thierry T. Diagana.

Reviewer Information Nature thanks J. S. Doggett, N. S. Gray and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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    This file contains further details on assembly and screening of NITD parasite box and calf efficacy study and Supplementary Table 1.

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https://doi.org/10.1038/nature22337

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