Skip to main content

High-Content Screening for Cryptosporidium Drug Discovery

  • Protocol
  • First Online:
Cryptosporidium

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2052))

Abstract

High-content screening (HCS) is a cell-based type of phenotypic screening that combines multiple simultaneous readouts with a high level of throughput. A particular benefit of this form of screening for drug discovery is the ability to perform the interrogation in a biologically relevant system. This approach has greatly advanced the field of drug discovery for cryptosporidiosis, a diarrheal disease caused by protozoan parasites of Cryptosporidium spp. These parasites are obligate intracellular parasites and cannot be cultured in vitro without the support of a host cell, limiting the options for potential assay readout. Here we describe an established 384- or 1536-well format high-content imaging (HCI) assay of Cryptosporidium-infected HCT-8 human ileocecal adenocarcinoma cells. This HCS assay is a powerful tool to assess large numbers of compounds to power drug discovery, as well as to phenotypically characterize known Cryptosporidium-active compounds.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acacio S, Biswas K, O’Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM (2013) 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(9888):209–222. https://doi.org/10.1016/S0140-6736(13)60844-2

    Article  PubMed  Google Scholar 

  2. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, McCormick BJ, McGrath M, Olortegui MP, Samie A, Shakoor S, Mondal D, Lima IF, Hariraju D, Rayamajhi BB, Qureshi S, Kabir F, Yori PP, Mufamadi B, Amour C, Carreon JD, Richard SA, Lang D, Bessong P, Mduma E, Ahmed T, Lima AA, Mason CJ, Zaidi AK, Bhutta ZA, Kosek M, Guerrant RL, Gottlieb M, Miller M, Kang G, Houpt ER (2015) Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health 3(9):e564–e575. https://doi.org/10.1016/s2214-109x(15)00151-5

    Article  PubMed  PubMed Central  Google Scholar 

  3. Arrowood MJ (2002) In vitro cultivation of Cryptosporidium species. Clin Microbiol Rev 15(3):390–400

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hijjawi NS, Meloni BP, Morgan UM, Thompson RC (2001) Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. Int J Parasitol 31(10):1048–1055

    Article  CAS  PubMed  Google Scholar 

  5. Upton SJ, Tilley M, Nesterenko MV, Brillhart DB (1994) A simple and reliable method of producing in vitro infections of Cryptosporidium parvum (Apicomplexa). FEMS Microbiol Lett 118(1–2):45–49

    Article  CAS  PubMed  Google Scholar 

  6. Bessoff K, Sateriale A, Lee KK, Huston CD (2013) Drug repurposing screen reveals FDA-approved inhibitors of human HMG-CoA reductase and isoprenoid synthesis that block Cryptosporidium parvum growth. Antimicrob Agents Chemother 57(4):1804–1814. https://doi.org/10.1128/aac.02460-12

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Murphy RC, Ojo KK, Larson ET, Castellanos-Gonzalez A, Perera BG, Keyloun KR, Kim JE, Bhandari JG, Muller NR, Verlinde CL, White AC Jr, Merritt EA, Van Voorhis WC, Maly DJ (2010) Discovery of potent and selective inhibitors of calcium-dependent protein kinase 1 (CDPK1) from C. parvum and T. gondii. ACS Med Chem Lett 1(7):331–335. https://doi.org/10.1021/ml100096t

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Sharling L, Liu X, Gollapalli DR, Maurya SK, Hedstrom L, Striepen B (2010) A screening pipeline for antiparasitic agents targeting Cryptosporidium inosine monophosphate dehydrogenase. PLoS Negl Trop Dis 4(8):e794. https://doi.org/10.1371/journal.pntd.0000794

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Umejiego NN, Gollapalli D, Sharling L, Volftsun A, Lu J, Benjamin NN, Stroupe AH, Riera TV, Striepen B, Hedstrom L (2008) Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against cryptosporidiosis. Chem Biol 15(1):70–77. https://doi.org/10.1016/j.chembiol.2007.12.010

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Gorla SK, McNair NN, Yang G, Gao S, Hu M, Jala VR, Haribabu B, Striepen B, Cuny GD, Mead JR, Hedstrom L (2014) Validation of IMP dehydrogenase inhibitors in a mouse model of cryptosporidiosis. Antimicrob Agents Chemother 58(3):1603–1614. https://doi.org/10.1128/aac.02075-13

    Article  PubMed  PubMed Central  Google Scholar 

  11. Bessoff K, Spangenberg T, Foderaro JE, Jumani RS, Ward GE, Huston CD (2014) Identification of Cryptosporidium parvum active chemical series by repurposing the open access malaria box. Antimicrob Agents Chemother 58(5):2731–2739. https://doi.org/10.1128/aac.02641-13

    Article  PubMed  PubMed Central  Google Scholar 

  12. Manjunatha UH, Vinayak S, Zambriski JA, Chao AT, Sy T, Noble CG, Bonamy GMC, Kondreddi RR, Zou B, Gedeck P, Brooks CF, Herbert GT, Sateriale A, Tandel J, Noh S, Lakshminarayana SB, Lim SH, Goodman LB, Bodenreider C, Feng G, Zhang L, Blasco F, Wagner J, Leong FJ, Striepen B, Diagana TT (2017) A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis. Nature 546(7658):376–380. https://doi.org/10.1038/nature22337

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Chen JJ, Rateb ME, Love MS, Xu Z, Yang D, Zhu X, Huang Y, Zhao LX, Jiang Y, Duan Y, McNamara CW, Shen B (2018) Herbicidins from Streptomyces sp. CB01388 showing anti-Cryptosporidium activity. J Nat Prod 81(4):791–797. https://doi.org/10.1021/acs.jnatprod.7b00850

    Article  PubMed  CAS  Google Scholar 

  14. Love MS, Beasley FC, Jumani RS, Wright TM, Chatterjee AK, Huston CD, Schultz PG, McNamara CW (2017) A high-throughput phenotypic screen identifies clofazimine as a potential treatment for cryptosporidiosis. PLoS Negl Trop Dis 11(2):e0005373. https://doi.org/10.1371/journal.pntd.0005373

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Giuliano KA, Haskins JR, Taylor DL (2003) Advances in high content screening for drug discovery. Assay Drug Dev Technol 1(4):565–577. https://doi.org/10.1089/154065803322302826

    Article  PubMed  CAS  Google Scholar 

  16. Ang ML, Pethe K (2016) Contribution of high-content imaging technologies to the development of anti-infective drugs. Cytometry A 89(8):755–760. https://doi.org/10.1002/cyto.a.22885

    Article  PubMed  PubMed Central  Google Scholar 

  17. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73. https://doi.org/10.1177/108705719900400206

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Case W. McNamara .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Love, M.S., McNamara, C.W. (2020). High-Content Screening for Cryptosporidium Drug Discovery. In: Mead, J., Arrowood, M. (eds) Cryptosporidium. Methods in Molecular Biology, vol 2052. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9748-0_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9748-0_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9747-3

  • Online ISBN: 978-1-4939-9748-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics