A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.
A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.
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DOI：
10.1128/msphere.00539-21
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发表时间：
2021-08-25
期刊：
影响因子：
4.8
通讯作者：
Krysan DJ
中科院分区：
文献类型：
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作者：
Beattie SR;Krysan DJ
侵袭性霉菌感染的治疗受到缺乏有效对抗这些致命感染的足够药物选择的限制。使用传统的抗真菌生长测定法进行霉菌的高通量筛选是有问题的，并且极大地限制了我们识别新的霉菌活性剂的能力。在这里，我们提出了一个高通量筛选平台，用于与烟曲霉，最常见的病原体侵入性霉菌感染，发现新的霉菌活性抗真菌药。该试验通过释放胞质酶腺苷酸激酶来检测细胞溶解，因此不依赖于生物量或代谢的变化来检测抗真菌活性。特异性检测细胞裂解的能力是该测定的独特方面，其允许鉴定破坏真菌细胞完整性的分子，例如细胞壁活性分子。我们还发现萌发的A.烟曲霉分生孢子释放低水平的腺苷酸激酶，并且该背景的减少使我们能够鉴定抑制分生孢子萌发的分子，从而扩大了发现新的抗真菌化合物的潜力。在这里，我们描述了这种检测方法的验证和概念验证的试点筛选，确定了一种新的抗真菌化合物，PIK-75，破坏细胞壁的完整性。该筛选试验为高通量筛选A.用于鉴定抗霉菌药物。由霉菌引起的真菌感染在人类真菌感染中死亡率最高。这些毁灭性的感染很难治疗，可用的抗真菌药物往往无效。因此，鉴定新的具有霉菌活性的抗真菌药物至关重要。用霉菌进行药物筛选具有挑战性，并且可用于直接用这些生物体鉴定新的抗真菌化合物的测定方法有限。在这里，我们提出了一种适用于高通量筛选与常见的霉菌病原体的测定。这种检测方法具有令人兴奋的未来潜力，用于鉴定治疗这些致命感染的新药。
Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.