Assays for Non-selective Cation Channel Inhibitors

描述（由申请人提供）：概述唑类化合物被广泛用作抗真菌药物，用于治疗危害生命的真菌感染。唑类抗生素的有效性受到其抑菌而非杀真菌作用机制的限制，导致许多致病性真菌物种出现耐唑菌株。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，钙信号网络在对抗生素的反应中被激活，并作为一种防御机制，在长期暴露于抗生素时促进真菌细胞的存活。值得注意的是，钙信号网络抑制剂将唑类抗生素从抑菌剂转化为杀菌剂。两种抑制真菌钙信号网络的天然化合物（环孢素和FK506）被认为是非常有效的杀真菌联合药物，但它们不能用于对抗真菌感染，因为它们在人体中具有强大的免疫抑制作用。抑制钙信号网络中其他真菌特异性成分的化合物将是改善抗真菌治疗的理想选择。我们最近发现了一个非选择性阳离子通道（NSCC），它在钙信号网络的顶端起作用，我们证明了NSCC在暴露于唑类和其他抗生素时对酵母的生存至关重要。NSCC位于细胞表面，仅在真菌中表达，是开发新型杀真菌联合药物的理想靶点。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文库，以寻找特异性靶向真菌NSCCs的化合物。明确;我们将从多种致病真菌中提取Nsc1蛋白，在酿酒酵母（非致病模型酵母）的Nsc1缺失突变体中表达，并确定Nsc1抑制酵母细胞生长的最佳环境条件。初步数据表明，nscc对介质中的高钠离子具有敏感性。因此，NSCC抑制剂应该能恢复酵母在含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分析，直接测量由nscc缺乏症引起的细胞死亡。基于细胞的生长和死亡试验的结合将为发现NSCC抑制剂提供一个强大的平台。

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.