Assays for Non-selective Cation Channel Inhibitors

非选择性阳离子通道抑制剂的测定

基本信息

批准号：
8102469
负责人：
KYLE W CUNNINGHAM
金额：
$ 15.81万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8102469
关键词：
Adverse effects Animals Antibiotics Antifungal Agents Antifungal Antibiotics Antifungal Therapy Autoimmune Diseases Azole resistance Azoles Biological Assay Ca(2+)-Calmodulin Dependent Protein Kinase Calcineurin Calcium Channel Calcium Signaling Calcium/calmodulin-dependent protein kinase Candida albicans Cations Cell Death Cell surface Cells Cessation of life Culture Media Cyclosporine Data Defense Mechanisms Development Effectiveness Enzymes Exposure to FK506 Flow Cytometry Fungal Components Genetic Genome Goals Graft Rejection Growth Homologous Gene Human Hypersensitivity Immunosuppression Immunosuppressive Agents In Vitro Industrial fungicide Lead Life Measures Methods Modeling Mycoses Necrosis Organ Transplantation Organism Pathway interactions Pharmaceutical Preparations Phosphoric Monoester Hydrolases Population Proteins Research Saccharomyces cerevisiae Screening procedure Sodium Chloride Specificity Staining method Stains Testing Yeast Model System Yeasts base cell growth combat fungus genetic regulatory protein high throughput screening improved in vivo inhibitor/antagonist killings mutant novel overexpression pathogen prevent research study resistant strain response small molecule libraries sodium ion voltage

项目摘要

DESCRIPTION (provided by applicant): Summary Azole-class compounds are widely employed as antifungals in the treatment of nosicomial and life-threatening fungal infections. The effectiveness of azole-class antibiotics is limited by their fungistatic rather than fungicidal mechanism of action resulting in the emergence of azole-resistant strains of many pathogenic species of fungi. Both of these problems can be averted by disabling the calcium signaling network of fungal cells, which becomes activated in response to antibiotics and acts as a defense mechanism that promotes survival of fungal cells during long-term exposure to the antibiotics. Remarkably, inhibitors of the calcium signaling network convert azole-class antibiotics from fungistats to fungicides. Two natural compounds that inhibit the fungal calcium signaling network (Cyclosporine and FK506) are known to be highly effective fungicidal co-drugs but they cannot be used to combat fungal infections because of their potent immunosuppressive effects in humans. Compounds that inhibit other fungi-specific components of the calcium signaling network would be ideal for improvement of antifungal therapies. We have recently identified a non-selective cation channel (NSCC) that functions at the apex of the calcium signaling network and we demonstrated that NSCCs are essential in yeasts for survival when exposed to azoles and other antibiotics. Being located at the cell surface and expressed only in fungi, the NSCC represents an ideal target for development of novel fungicidal co-drugs. Here we will develop cell-based assays that can be used for high-throughput screening of chemical libraries for compounds that specifically target fungal NSCCs. Specifically; we will express Nsc1 proteins from various species of pathogenic fungi in Nsc1-deficient mutants of Saccharomyces cerevisiae non-pathogenic model yeast) and determine the optimal environmental conditions in which the NSCCs inhibit yeast cell growth. Preliminary data show that NSCCs confer sensitivity to high sodium ions in the medium. Thus, NSCC inhibitors should restore yeast growth in simple culture media containing high salt. We will also develop optimal secondary assays using fluorescent stains and flow cytometry that directly measure cell death caused by NSCC-deficiency. The combination of cell-based growth and death assays will provide a powerful platform for the discovery of NSCC inhibitors. PUBLIC HEALTH RELEVANCE: A commonly prescribed class of antibiotics used to combat fungal infections can slow the growth of fungal pathogens but cannot kill them. By disabling fungal defenses with a secondary drug, these same antibiotics become potent fungicides. The research proposed here will develop primary and secondary assay methods that can be used for the identification of novel compounds that disable fungal defenses to common antibiotics. The assays focus on a highly drugable target enzyme - the NSCC - which occurs only in fungi. Therefore, the compounds identified by such an approach would have few side-effects in humans and massively increase the effectiveness of current antifungal antibiotics.

描述（由申请人提供）：概述唑类化合物广泛用作抗真菌剂，用于治疗医院感染和危及生命的真菌感染。唑类抗生素的有效性受到其抑制真菌而非杀真菌作用机制的限制，导致许多致病真菌物种出现唑类抗性菌株。这两个问题都可以通过禁用真菌细胞的钙信号网络来避免，该网络会响应抗生素而被激活，并作为一种防御机制，在长期暴露于抗生素的过程中促进真菌细胞的存活。值得注意的是，钙信号网络的抑制剂将唑类抗生素从抑真菌剂转变为杀菌剂。已知两种抑制真菌钙信号网络的天然化合物（环孢素和 FK506）是高效的杀菌联合药物，但由于它们对人类具有强大的免疫抑制作用，因此不能用于对抗真菌感染。抑制钙信号网络中其他真菌特异性成分的化合物将是改进抗真菌疗法的理想选择。我们最近发现了一种非选择性阳离子通道（NSCC），它在钙信号网络的顶端发挥作用，并且我们证明，当酵母接触唑类和其他抗生素时，NSCC对于酵母的生存至关重要。 NSCC 位于细胞表面并仅在真菌中表达，是开发新型杀菌联合药物的理想靶标。在这里，我们将开发基于细胞的检测方法，可用于高通量筛选化学库中专门针对真菌 NSCC 的化合物。具体来说;我们将在酿酒酵母非致病模型酵母的 Nsc1 缺陷突变体中表达来自各种致病真菌的 Nsc1 蛋白，并确定 NSCC 抑制酵母细胞生长的最佳环境条件。初步数据表明，NSCC 对培养基中的高钠离子具有敏感性。因此，NSCC 抑制剂应该可以恢复酵母在含有高盐的简单培养基中的生长。我们还将使用荧光染色和流式细胞术开发最佳的二次测定，直接测量由 NSCC 缺陷引起的细胞死亡。基于细胞的生长和死亡检测的结合将为 NSCC 抑制剂的发现提供强大的平台。公共卫生相关性：用于对抗真菌感染的常用抗生素类别可以减缓真菌病原体的生长，但不能杀死它们。通过使用辅助药物禁用真菌防御，这些相同的抗生素成为有效的杀菌剂。这里提出的研究将开发初级和次级测定方法，可用于鉴定使真菌对常见抗生素失去防御能力的新型化合物。这些检测的重点是一种高度可药物化的目标酶——NSCC——它只存在于真菌中。因此，通过这种方法鉴定的化合物对人类几乎没有副作用，并且大大提高了当前抗真菌抗生素的有效性。