A Fluorescence-Based High-Throughput Screening Assay to Identify Growth Inhibitors of the Pathogenic Fungus Aspergillus fumigatus
Due to the advancements in modern medicine that have resulted in an increased number of immunocompromised individuals, the incidences and the associated mortality of invasive aspergillosis have continued to rise over the past three decades despite appropriate treatment. As a result, invasive aspergillosis has emerged as a leading cause of infection-related mortality in immunocompromised individuals. Utilizing the resazurin to resorufin conversion fluorescence readout to monitor cell viability, herein, we outline a high-throughput screening method amenable to profiling a large pharmaceutical library against the clinically relevant but less frequently screened fungal pathogen Aspergillus fumigatus. This enables the user to conduct high-throughput screening using a disease-relevant fungal growth assay and identify novel antifungal chemotypes as drug leads.
Key wordsAspergillus fumigatus Fungus Antifungal Resazurin High-throughput screening (HTS)
We would like to thank the Aspergillus fumigatus inhibitor project team including Neil Ryder, Kathryn Thompson, Roger Fujimoto, Joseph Drumm, and Dominic Hoepfner for scientific input and advice during the screen. We would like to thank Heidi Faw for safety considerations regarding Aspergillus handling. We would like to thank Alejandra Raimondi for initial assay development work. We would like to thank Melissa Grippo for compound purity testing by LC-MS.
- 4.Morgan J, Wannemuehler K, Marr K, Hadley S, Kontoyiannis D, Walsh T, Fridkin S, Pappas P, Warnock D (2005) Incidence of invasive aspergillosis following hematopoietic stem cell and solid organ transplantation: interim results of a prospective multicenter surveillance program. Med Mycol 43(1):S49–S58CrossRefGoogle Scholar
- 8.Brakhage J (2006) Molecular determinates of virulence in Aspegillus fumigatus. In: Heitman J (ed) Molecular principles of fungal pathogenesis. ASM Press, Washington, DCGoogle Scholar
- 9.Latgé J (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12(2):310–350Google Scholar
- 10.Herbrecht R, Denning D, Patterson T, Bennett J, Greene R, Oestmann J, Kern W, Marr K, Ribaud P, Lortholary O, Sylvester R, Rubin R, Wingard J, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar P, Hodges M, Schlamm H, Troke P, Bd P (2002) Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 347(6):408–415CrossRefGoogle Scholar
- 12.Pesch K, Simmert U (1929) Combined assays for lactose and galactose by enzymatic reactions. Milchw Forsch 8:551Google Scholar
- 17.Monteiro M, Mdl C, Cantizani J, Moreno C, Tormo J, Mellado E, Lucas JD, Asensio F, Valiante V, Brakhage A, Latgé J, Genilloud O, Vicente F (2012) A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin. J Biomol Screen 17(4):542–549CrossRefGoogle Scholar