A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions
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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.
KeywordsCryptococcus neoformans Cryptococcal infections Cryptococcal meningitis High-throughput screening ATP content assay
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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