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

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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.