Identification of broad-spectrum antifungal efflux pump inhibitors

描述（由申请人提供）：真菌感染，以口腔和侵袭性念珠菌病为例，在不断增加的人群中引起相当大的发病率和死亡率：免疫功能低下。真菌感染患者的治疗受到抗真菌药物耐药性发展的严重阻碍。我们的研究目标是通过发现新的治疗药物，通过抑制抗真菌耐药性的一个主要原因，即真菌细胞膜中的药物外排泵分子，来克服临床耐药性，从而解决这一健康需求。先导化合物将通过分子库小分子库（MLSMR）的高通量筛选（HTS）进行鉴定，使用由新墨西哥大学分子发现中心（UNMCMD）的Sklar教授开发的多重检测系统和我们实验室开发的一组酿酒酵母模型菌株。菌株表达了一系列单独的真菌外排泵，这将是所提出的HTS的目标。具体目标是：1。使用多重流式细胞术试验对MLSMR进行初步筛选：原理证明初步实验表明，该试验将适用于表达以下临床相关真菌的外排泵的酵母菌菌株组：白色念珠菌、光面念珠菌、克鲁念珠菌和新型隐球菌。开发的基于酵母细胞的多重方法涉及在HTS中使用三种“前哨”菌株，测量荧光泵底物尼罗红的外排，并与其他面板菌株进行后续微滴荧光测定，以确定泵抑制剂。2. 用三个辅助筛选面板筛选获得的命中值：在主筛选中获得的命中值将使用我们实验室先前开发的辅助筛选进行确认。二级分析将确认目标是真菌外排泵，抑制活性的程度，并阐明抑制剂/目标相互作用的性质。确定的化合物将指导进一步研究开发克服抗真菌耐药性的药物。

DESCRIPTION (provided by applicant): Fungal infections, exemplified by oral and invasive candidiasis, cause considerable morbidity and mortality in an ever-increasing human population: the immunocompromised. Treatment of patients with fungal infections is severely hampered by the development of antifungal drug resistance. The goal of our research is to address this health need by discovering novel therapeutic agents that overcome clinical drug resistance by inhibiting a major cause of antifungal resistance, the drug efflux pump molecules in the fungal cell membrane. Lead compounds will be identified by high throughput screening (HTS) of the Molecular Libraries Small Molecule Repository (MLSMR) using a multiplex assay system developed by Professor Sklar at the University of New Mexico Center for Molecular Discovery (UNMCMD) and a panel of strains of the model yeast Saccharomyces cerevisiae, developed in our laboratory. The strains express a range of individual fungal efflux pumps that will be the targets for the proposed HTS. The specific aims are to: 1. Undertake a primary screen of the MLSMR using a multiplex flow cytometry assay: Proof of principle preliminary experiments has indicated that the assay will be applicable to the panel of yeast strains expressing efflux pumps from the following clinically relevant fungi: Candida albicans, Candida glabrata, Candida krusei and Cryptococcus neoformans. The yeast cell-based multiplex approach developed involves the use of three 'sentinel' strains in an HTS which measures efflux of the fluorescent pump substrate Nile Red and follow-up microtitre fluorescent assays with the other panel strains to identify pump inhibitors. 2. Screen the hits obtained with a panel of three secondary screens: The hits obtained in the primary screen will confirmed using secondary screens previously developed in our laboratory. The secondary assays will confirm that the targets are fungal efflux pumps, the extent of inhibitory activity, and elucidate the nature of the inhibitor/target interaction. The compounds identified will guide further research to develop drugs that overcome antifungal resistance. PUBLIC HEALTH RELEVANCE: Our long-term goal is to improve the treatment of patients with opportunistic fungal infections by discovering compounds that will act in combination with current antifungal drugs to overcome antifungal drug resistance.