Screening Repurposing Libraries for the Identification of Drugs with Novel anti-Coccidioidal Activity

摘要(项目1) 球孢子菌病是由球孢子虫和球孢子虫感染引起的一种真菌感染。它是 据估计，美国每年新增感染病例15万人。这种感染的发病率 在流行地区继续上升。迫切需要开发更好的治疗药物。 治疗球孢子菌病。目前，球孢子菌病的治疗包括抗真菌药物，如 如两性霉素B、氟康唑和伊曲康唑。然而，它们的毒性和副作用，以及对 对唑类耐药的临床分离株的增加表明迫切需要开发新的药物来对抗这种情况 疾病。在这个项目中，我们建议筛选可重新利用的文库，以寻找具有新活性的药物 应用我们最新研制的真菌细胞学图谱(FCP) 确定潜在的抗CM药物候选药物的方法学。球藻(Coccidioidesspp.)拥有独一无二的 二态生活史，特征是节分生孢子转变为球状孢子进入寄生阶段。 与腐生孢子/菌丝相比，使用球体筛选药物文库具有更高的医学相关性。 关键因子和表型的改变(例如，各向同性生长，细胞壁完整性，细胞壁重塑，多细胞 与球孢子虫寄生循环发育相关的核形成)可以通过FCP进行初步评估 药物文库的筛选和后来对药物细胞作用的发现。药品库中的先导化合物 将在体外筛选其对相关临床球虫分离株的抗真菌活性 包括对唑类耐药菌株。随后的体内抗真菌疗效将 使用我们新开发的球孢子菌病的梅隆格列氏菌模型进行研究，该模型提供了 强大且经济高效的预筛选系统在测试之前更加费力、耗时和 昂贵的啮齿动物模型。将对主要化合物进行进一步的表征，以获得对其 分子作用机制。全球转录分析将确定推定的药物靶向 基因/途径，将通过特定基因的缺失和互补进一步评估。这 分子评估将得到真菌表型分析的补充，包括显微镜(共聚焦， TEM)和FCP，以进一步了解每个候选药物的作用模式。我们的目标是在 本项目完成时，至少有4种领先的可重定位化合物进入临床前阶段 球孢子菌病的化疗进展。

Summary (Project 1) Coccidioidomycosis is a fungal infection caused by Coccidioides posadasii and Coccidioides immitis. It is estimated that 150,000 new infections occur in the United States each year. The incidence of this infection continues to rise in endemic regions. There is an urgent need for the development of better therapeutic drugs against coccidioidomycosis. Currently, the management of coccidioidomycosis includes antifungal agents such as amphotericin B, fluconazole and itraconazole. However, their toxicity and side effects, and concerns over the rise of azole-resistant clinical isolates point to an urgent need to develop new agents to combat this disease. In this project, we propose to screen repurposing libraries in search for drugs with novel activity against parasitic spherules of C. posadasii, and to apply our newly developed fungal cytological profiling (FCP) methodology for the identification of potential anti-CM drug candidates. Coccidioides spp. have a unique dimorphic life cycle characterized by the conversion of arthroconidia to spherules to enter parasitic phase. Using spherules to screen drug library is more medically relevant compare to saprobic spores/hyphae. Alteration of critical factor and phenotype (e.g. isotropic growth, cell wall integrity, cell wall remodeling, multi- nuclear formation) associated with coccidioidal parasitic cycle development can be assessed by FCP for initial drug library screening and later for drug cellular action discovery. Lead compounds from the drug library screening will be characterized in vitro for their antifungal activity against relevant clinical Coccidioides isolates of both C. posadasii and C. immitis, including azole-resistant strains. Subsequent in vivo antifungal efficacy will be conducted using our newly developed Galleria mellonella model of coccidioidomycosis, which provides a robust and cost-effective pre-screening system before testing in a more laborious, time-consuming and expensive rodent models. The leading compounds will be further characterized to gain insights into their molecular mechanisms of action. Global transcriptomic analyses will identify putative drug-targeted genes/pathways, which will be further assessed by gene-specific deletion and complementation. This molecular assessment will be complemented by fungal phenotypic analyses including microscopy (confocal, TEM) and FCP to gain further insights into the mode of action of each drug candidate. The goal is to identify at least 4 leading repositionable compounds at the completion of this project for advancing to preclinical development for chemotherapy against coccidioidomycosis.