A New Approach to Drug Discovery: High-Throughput Screening of Microbial Natural Extracts against Aspergillus fumigatus Using Resazurin
A New Approach to Drug Discovery: High-Throughput Screening of Microbial Natural Extracts against Aspergillus fumigatus Using Resazurin
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DOI：
10.1177/1087057111433459
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发表时间：
2012-04-01
影响因子：
--
通讯作者：
Vicente, Francisca
中科院分区：
文献类型：
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作者：
Candida Monteiro, Maria;de la Cruz, Mercedes;Vicente, Francisca
天然产物是药物发现的取之不尽的源泉。然而，初步筛选试验的验证和选择对于确保选择具有相关药理作用和值得跟进的提取物或分子至关重要。该分析必须快速、简单、易于实施，并产生快速结果，最好是以低成本进行。在这项工作中，我们开发并验证了一种使用Resazurin活性染料的比色微量滴度分析方法。优化并建立了利用天然提取物进行烟曲霉菌高通量筛选(HTS)的条件。每孔加入提取物+含有孢子的改良RPMI1640型培养基和0.002%的瑞珠灵。孵育24~30h后进行荧光读数。在测定不同抗真菌药物对烟曲霉菌的最低抑菌浓度时，重天青与阿拉马蓝一样适用，并能有效地分析杀菌和抑菌化合物。对两株烟曲霉菌株进行了12000种微生物提取物的高温高温杀菌试验，其中2.7%的提取物具有抗真菌活性。我们小组首次使用这种方法筛选了一系列天然提取物，以鉴定对医学上重要的人类病原体烟曲霉菌具有抗真菌活性的化合物。
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.