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

项目摘要 霉菌活性抗真菌剂开发中的主要障碍是用于高霉菌活性抗真菌剂的有限方法。 通过模具筛选。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，仅限于三个 由于药物种类独特，这些感染的死亡率非常高。除了严重的 霉菌对免疫功能低下的患者构成健康威胁，霉菌会导致免疫功能正常的患者感染， 个体，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角膜炎的主要原因。 全世界只有大约一半的单眼失明患者患有由曲霉菌引起的角膜感染， 镰刀菌对治疗有反应。全球真菌感染的现状，加上新出现的 目前抗真菌药物的耐药性，呼吁新型的霉菌活性抗真菌药，以打击这些生命， 威胁感染。 该提案旨在填补该领域的技术空白，将腺苷酸激酶测定应用于丝状菌 真菌进行高通量筛选。腺苷酸激酶测定法已得到验证， 用于酵母和细菌的高通量筛选，具有使其成为 适用于模具的优秀候选材料。我们已经表明，该测定法可靠地检测A.烟曲霉 并优化了适合高通量筛选的条件。在本提案中，我们的目标是调整该测定法， 对尖孢镰刀菌，然后用多个小分子进行曲霉和镰刀菌筛选， 天然产物库。除了针对野生型A进行筛选之外。熏烟，我们将执行一个重新利用 使用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，我们希望能够识别出新型的霉菌活性抗真菌药物，以及可以重新利用的药物， 加速临床应用以治疗真菌感染。

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.