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Analytical and Bioanalytical Chemistry

, Volume 405, Issue 21, pp 6823–6829 | Cite as

A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions

  • Seameen J. Dehdashti
  • Jennifer Abbott
  • Dac-Trung Nguyen
  • John C. McKew
  • Peter R. Williamson
  • Wei Zheng
Research Paper

Abstract

Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1 % and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3′-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.

Keywords

Cryptococcus neoformans Cryptococcal infections Cryptococcal meningitis High-throughput screening ATP content assay 

Notes

Acknowledgments

This work was supported by the Intramural Research Programs of the Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences (NCATS), and National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). The authors thank Paul Shinn and compound management team at NCATS for their assistance.

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Seameen J. Dehdashti
    • 1
  • Jennifer Abbott
    • 2
  • Dac-Trung Nguyen
    • 1
  • John C. McKew
    • 1
  • Peter R. Williamson
    • 2
    • 3
  • Wei Zheng
    • 1
  1. 1.National Center for Advancing Translational SciencesNational Institute of HealthBethesdaUSA
  2. 2.Translational Mycology Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious DiseasesNational Institutes of HealthBethesdaUSA
  3. 3.Section of Infectious Diseases, Department of MedicineUniversity of Illinois at ChicagoChicagoUSA

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