title: A new approach to drug discovery: high-throughput screening of microbial natural extracts against Aspergillus fumigatus using resazurin.
abstract: Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatus were optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatus and effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatus strains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus.
authors.affiliation: Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Armilla, Granada, Spain. mcandida.monteiro@medinaandalucia.es
authors.forename: Maria Cândida
authors.initials: MC
authors.lastname: Monteiro
authors.forename: Mercedes
authors.initials: M
authors.lastname: de la Cruz
authors.forename: Juan
authors.initials: J
authors.lastname: Cantizani
authors.forename: Catalina
authors.initials: C
authors.lastname: Moreno
authors.forename: José R
authors.initials: JR
authors.lastname: Tormo
authors.forename: Emilia
authors.initials: E
authors.lastname: Mellado
authors.forename: J Ramón
authors.initials: JR
authors.lastname: De Lucas
authors.forename: Francisco
authors.initials: F
authors.lastname: Asensio
authors.forename: Vito
authors.initials: V
authors.lastname: Valiante
authors.forename: Axel A
authors.initials: AA
authors.lastname: Brakhage
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Latgé
authors.forename: Olga
authors.initials: O
authors.lastname: Genilloud
authors.forename: Francisca
authors.initials: F
authors.lastname: Vicente
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects*; D045424:Complex Mixtures / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D005658:Fungi / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010078:Oxazines; D014966:Xanthenes
chemical_list: D000935:Antifungal Agents; D045424:Complex Mixtures; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin