Development of a high throughput-compatible assay for screening filamentous fungi

项目总结 开发具有霉菌活性的抗真菌药物的一个主要障碍是有限的高效 用模具进行生产能力筛选。烟曲霉和镰刀菌。是两种常见的霉菌 免疫受损患者的侵袭性感染。尽管目前的抗真菌治疗仅限于三种 作为一种独特的药物，这些感染的死亡率非常高。除了严重的 霉菌对免疫低下患者健康构成威胁，霉菌导致免疫能力感染 个人，特别是以角膜感染或真菌性角膜炎的形式。真菌性角膜炎是导致 全世界范围内的单眼性失明，只有大约一半的由曲霉菌或 镰刀菌对治疗有反应。全球真菌感染的现状，结合新出现的 对当前抗真菌药物的抗药性，需要新型的霉菌活性抗真菌药物来对抗这些生命- 有感染危险。 这项建议旨在通过将腺苷酸激酶检测应用于丝状菌来填补该领域的技术空白。 真菌进行高通量筛选。腺苷酸激酶的检测已经被验证并成功。 用于酵母和细菌的高通量筛选，并具有使其成为 非常适合应用于模具。我们已经证明，这种方法可以可靠地检测到烟曲霉菌的裂解。 优化了适合高通量筛选的条件。在这项提案中，我们的目标是使这项测试 然后用曲霉和含有多个小分子的镰刀菌进行筛选。 天然产品库。除了筛选野生型烟曲霉菌外，我们还将进行一项重新用途的 使用一株具有常见伏立康唑耐药机制的菌株筛选FDA批准的药物。在这 通过这种方式，我们希望确定具有霉菌活性的抗真菌药物的新类别，以及将被重新用途和 加速用于治疗真菌感染的临床应用。

PROJECT SUMMARY A major obstacle in the development of mold-active antifungals is limited methodologies for high throughput screening with molds. Aspergillus fumigatus and Fusarium spp. are two common molds that cause invasive infections in immune-compromised patients. Despite current antifungal treatments, limited to only three unique classes of drugs, mortality rates of these infections are devastatingly high. In addition to the serious health threat molds pose to immune-compromised patients, molds cause infections in immune-competent individuals, particularly in the form of corneal infections, or fungal keratitis. Fungal keratitis is a leading cause of monocular blindness worldwide and only about half of patients with corneal infections caused by Aspergillus or Fusarium respond to treatment. The current state of fungal infections worldwide, combined with emerging resistance to current antifungal drugs, calls for novel classes of mold-active antifungals to combat these life- threatening infections. This proposal aims to fill a technical gap in the field by applying the adenylate kinase assay to filamentous fungi to perform high throughput screening. The adenylate kinase assay has been validated and successfully used for high throughput screening with yeast and bacteria and possesses several qualities that make it an excellent candidate for application to molds. We have shown that this assay reliably detects lysis of A. fumigatus and optimized conditions suitable for high throughput screening. In this proposal, our goal is to adapt this assay to Fusarium oxysporum, then perform screens with Aspergillus and Fusarium with multiple small molecule and natural product libraries. In addition to screening against wild type A. fumigatus, we will perform a repurposing screen of FDA-approved drugs using a strain harboring a common voriconazole resistance mechanism. In this way, we hope to identify novel classes of mold-active antifungal drugs, as well as drugs to be repurposed and expedited to clinical use to treat fungal infections.