Identification of broad-spectrum antifungal efflux pump inhibitors

描述(申请人提供)：真菌感染，例如口腔和侵袭性念珠菌病，在不断增加的人口中导致相当大的发病率和死亡率：免疫功能受损。抗真菌药物耐药性的产生严重阻碍了真菌感染患者的治疗。我们研究的目标是通过发现新的治疗药物来解决这一健康需求，这些药物可以通过抑制真菌细胞膜上的药物外排泵分子来克服临床耐药。先导化合物将通过分子库小分子存储库(MLSMR)的高通量筛选(HTS)进行鉴定，方法是使用新墨西哥大学分子发现中心(UNCMD)的斯克拉尔教授开发的多重分析系统和我们实验室开发的一组模式酵母酿酒酵母菌株。这些菌株表达了一系列单独的真菌外排泵，这些泵将成为拟议的HTS的目标。具体目的是：1.采用多重流式细胞术进行MLSMR的初步筛选：原理证明初步实验表明，该方法将适用于临床相关真菌：白色念珠菌、光滑念珠菌、克鲁斯念珠菌和新生隐球菌外排泵的酵母菌株群。开发的基于酵母细胞的多重方法包括在HTS中使用三个“前哨”菌株，该方法测量荧光泵底物尼罗红的外流，并与其他面板菌株进行后续微量荧光分析，以识别泵抑制剂。2.筛选三个二级屏幕的点击率：一级屏幕的点击率将使用本实验室先前开发的二级屏幕进行确认。二次检测将确认靶标是真菌外排泵，抑制活性的程度，并阐明抑制剂/靶标相互作用的性质。已确定的化合物将指导进一步的研究，以开发克服抗真菌耐药性的药物。 公共卫生相关性：我们的长期目标是通过发现与当前抗真菌药物联合作用的化合物来克服抗真菌耐药性，从而改善机会性真菌感染患者的治疗。

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.