Identifying novel small molecules for improved antifungal drug treatment

描述(由申请人提供)：侵袭性真菌感染(IFI)与高发病率和死亡率有关，并对严重免疫功能低下的患者构成严重的健康问题。由致病性念珠菌引起的感染尤其普遍，影响到近50名重症监护病房患者，并导致北美所有ICU获得性血液感染的近10%。防止这些致命感染受到这些致病物种中持续发展的抗真菌药物耐药性的阻碍。多药耐药主要是通过膜外排泵的转录上调和麦角固醇生物合成途径中药物靶基因的过度表达而获得的。真菌特异性锌簇转录因子家族的成员主要负责上调这些外排泵和麦角甾醇生物合成酶，从而介导酵母的多效性耐药(PDR)。识别以这些转录因子为靶点的小分子药物，并潜在地抑制或改变它们的DNA结合活性，从而降低它们上调参与PDR的靶基因的能力，可能会显著改善目前的抗真菌药物治疗。本项目的主要目标是确定参与酿酒酵母抗真菌耐药性的PDR转录因子的小分子抑制剂以及致病菌种白色念珠菌和光滑念珠菌。各种化合物的文库将通过小分子微阵列(SMM)进行筛选，以识别那些能够与这些TF直接相互作用的化合物。蛋白质结合微阵列(PBM)将被用来表征这些化合物改变或抑制锌簇TFDNA结合活性的能力。同时，还将进行体内药物敏感性测试，以检测SMM筛选中的小分子“HITS”对酿酒酵母、白色念珠菌和光滑葡萄球菌的存活和对这些化合物的敏感性的体内影响；化合物将在体内单独和相互结合以及与现有的抗真菌药物一起进行测试。这项研究的成功完成将有助于开发一种新的方法来鉴定能够防止序列特异性DNA结合的转铁蛋白抑制剂，并将扩大转铁蛋白作为潜在药物靶点的作用。最终，该项目中确定的小分子可能会作为先导化合物，用于开发治疗侵袭性真菌感染的改进的抗真菌药物。 公共卫生相关性：改进的抗真菌药物的开发以及新药物靶点的扩展和发现是未来侵袭性真菌感染治疗的最重要需求之一。该项目致力于寻找新的小分子，这些小分子靶向于几个关键的序列特异性转录因子，可能通过抑制或改变它们的DNA结合活性来控制多效性耐药，从而降低它们上调酿酒酵母、白色念珠菌和光滑念珠菌中多药外排泵和麦角甾醇生物合成基因的能力。这项研究的成功完成将：(A)开发一种新的方法来确定转录因子与序列特异性DNA结合的抑制剂；(B)扩大转录因子作为潜在药物靶标的一般作用；以及(C)确定潜在的先导化合物，用于开发治疗侵袭性真菌感染的新型抗真菌药物。

DESCRIPTION (provided by applicant): Invasive fungal infections (IFIs) are associated with high rates of morbidity and mortality and pose a serious health concern for severely immunocompromised patients. Infections caused by pathogenic Candida species are especially prevalent, affecting nearly one in fifty intensive care unit patients and causing nearly 10% of all ICU-acquired bloodstream infections in North America. Preventing these deadly infections is hindered by the continued development of antifungal drug resistance in these pathogenic species. Multidrug resistance is primarily acquired by transcriptional upregulation of membrane efflux pumps, and by overexpression of drug target genes within the ergosterol biosynthesis pathway. Members of the fungal-specific zinc cluster family of transcription factors (TFs) are primarily responsible for the upregulation of these efflux pumps and ergosterol biosynthesis enzymes and thereby mediating pleiotropic drug resistance (PDR) in yeast. The identification of small molecule drugs that target these TFs, and potentially inhibit or alter their DNA-binding activity, and thus reduce their ability to upregulate target genes involved in PDR could result in significant improvements to current antifungal drug therapies. The overarching goal of this project is to identify small molecule inhibitors of PDR TFs involved in antifungal drug resistance in Saccharomyces cerevisiae as well as the pathogenic species Candida albicans and Candida glabrata. Libraries of diverse compounds will be screened by small molecule microarrays (SMMs) to identify those compounds capable of direct interaction with these TFs. Protein binding microarrays (PBMs) will be used to characterize these compounds' ability to alter or inhibit zinc cluster TF DNA binding activity. In parallel, in vivo drug susceptibility tests will be performed to examine the in vivo effects of the small molecule 'hits' from the SMM screen on S. cerevisiae, C. albicans and C. glabrata survival and susceptibility to these compounds; compounds will be tested in vivo both individually and in combination with each other as well as with existing antifungal drugs. Successful completion of this study will allow for the development of a new methodology for identifying TF inhibitors capable of preventing sequence-specific DNA binding, and will expand the role of TFs as potential drug targets. Ultimately, small molecules identified in this project may serve as lead compounds for development of improved antifungal drugs in the treatment of invasive fungal infections. PUBLIC HEALTH RELEVANCE: The development of improved antifungal drugs and the expansion and discovery of novel drug targets is one of the most important needs for the future treatment of invasive fungal infections. This project is focused on identifying novel small molecules that target several key sequence-specific transcription factors controlling pleiotropic drug resistance, potentially by inhibiting or altering their DNA-binding activity, and thus reduce their ability to upregulate multidrug efflux pumps and ergosterol biosynthesis genes in the species Saccharomyces cerevisiae, Candida albicans and Candida glabrata. Successful completion of this research will: (a) develop a new methodology for identifying inhibitors of sequence-specific DNA binding by transcription factors; (b) expand the general role of transcription factors as potential drug targets; and (c) identify potential lead compounds for development of novel antifungal drugs in the treatment of invasive fungal infections.