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

摘要（项目1） 球孢子菌病是一种由球孢子菌和球孢子菌引起的真菌感染。是 据估计，美国每年有15万新感染病例。这种感染的发生率 在流行地区继续上升。迫切需要开发更好的治疗药物 对抗球孢子菌病目前，球孢子菌病的治疗包括抗真菌药物， 如阿替霉素B、氟康唑和伊曲康唑。然而，它们的毒性和副作用，以及对 唑类耐药临床分离株的出现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，我们建议筛选再利用库，以寻找具有新活性的药物 对寄生的C. posadasii，并应用我们新开发的真菌细胞学分析（FCP） 鉴定潜在抗CM候选药物的方法。球孢子菌属有一个独特 二型生活史的特征是节孢子转变成小球进入寄生阶段。 与腐殖孢子/菌丝相比，使用小球筛选药物库更具医学相关性。 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、多基因表达） 核形成）与球孢子虫寄生虫周期发展相关，可以通过FCP评估初始 药物库筛选以及随后的药物细胞作用发现。药物库中的先导化合物 筛选将在体外表征其对相关临床球孢子菌分离株的抗真菌活性 两个C。posadasii和C.包括唑类耐药菌株。随后的体内抗真菌功效将 使用我们新开发的Galleria mellonella球孢子菌病模型进行，该模型提供了 在测试之前，我们提供了一个强大且具有成本效益的预筛选系统， 昂贵的啮齿动物模型将进一步表征主要化合物，以深入了解其 分子作用机制。全球转录组学分析将确定推定的药物靶向 基因/途径，这将通过基因特异性缺失和互补进一步评估。这 分子评估将通过真菌表型分析包括显微镜（共聚焦， TEM）和FCP，以进一步了解每种候选药物的作用方式。目标是确定在 在本项目完成时，至少有4种领先的可重新定位化合物用于临床前研究 开发抗球孢子菌病的化学疗法。

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.