HTS development for targeted anti-fungal small molecules

描述(由申请人提供)：白色念珠菌是一种广泛存在的人类真菌病原体，在全身感染(念珠菌病)期间会导致高死亡率。由于真菌是真核细胞，开发对人类无毒的抗真菌治疗化合物具有挑战性。新近发现的一种真菌组蛋白乙酰转移酶(HAT)，命名为Rtt109，乙酰化组蛋白H3赖氨酸56，对基因组稳定性和对遗传毒物的抗性具有重要作用。Rtt109与哺乳动物的p300/CBP HAT酶有很远的亲缘关系，但抑制p300/CBP的化合物并不抑制Rtt109。在真菌中，H3-K56乙酰化发生在所有新合成的分子上，但H3-K56ac在哺乳动物细胞中不丰富，甚至检测不到。因此，我们假设可以找到抑制Rtt109的小分子，但不会实质上影响哺乳动物HAT酶的活性，从而对哺乳动物宿主显示出最小的毒性。我们发现纯合子rtt109-/-突变的白色念珠菌缺乏H3K56乙酰化，并且对包括DNA烷化剂和过氧化氢等活性氧的遗传毒性物质高度敏感。值得注意的是，在尾静脉注射诱导的系统性念珠菌病小鼠模型中，rtt109-/-突变细胞的致病性要低得多。总之，这些数据支持我们的假设，即Rtt109是一个有希望的抗真菌治疗的新靶点。我们尤其被鼓励继续进行这些研究，已经产生了一个初步的方案，以微滴度的形式测量Rtt109的酶活性。目的1：检测方法的发展。我们将重新优化检测参数(洗涤次数、试剂用量/用量、二次检测试剂)，以确定384孔板格式的最佳Z因子分数。目标2：HTS检测系统的配置。基于我们优化的分析配置，我们将在马萨诸塞大学医学院的小分子筛选设施中对目前存在的30,000种化合物进行中试筛选。试验筛查的结果将确定更大规模筛查工作的命中率、假阳性率和最佳检测试剂。我们有计划的阳性和阴性筛选标准，以确定初步候选人的优先顺序，我们将为这些标准开发试剂和方案。首先，我们将要求抑制Rtt109-Vps75蛋白复合体的组蛋白乙酰化的化合物也将在ASF1而不是Vps75的刺激下抑制Rtt109的乙酰化。作为否定选择，我们将测试初步的Rtt109抑制剂对无关的picNuA4 HAT酶复合体的影响，以排除那些没有Rtt109活性部位特异性的化合物，这些化合物广泛地抑制乙酰基转移酶反应。最后，我们将测试已鉴定的化合物对白色念珠菌细胞的有效性，测量对遗传毒性药物的敏感性，以及对H3K56-Ac水平的影响。这将确定最能渗透到细胞中的化合物。我们还将确定对哺乳动物细胞有毒的Rtt109抑制剂，以便优先考虑无毒的候选药物。总之，这些研究将为NIH分子文库中更大文库的进一步筛选提供阳性对照Rtt109抑制剂。 公共卫生相关性：白色念珠菌是一种致病真菌，对包括艾滋病患者在内的免疫功能受损的人特别危险。白念珠菌感染通常也是在医院感染的，这使它们成为一个主要的公共卫生问题。最近，人们发现了一种新的酶，它对真菌的正常生长和白色念珠菌的致病起着重要的作用。我们正在开发能够抑制这种酶的化合物的高通量筛选，因为这些化合物将成为我们寻找新的抗真菌药物的候选药物。

DESCRIPTION (provided by applicant): Candida albicans is a widespread human fungal pathogen that causes high rates of mortality during systemic infections (candidiasis). Because fungi are eukaryotic cells, development of antifungal therapeutic compounds that are non-toxic to humans is challenging. A recently discovered fungal histone acetyltransferase (HAT) enzyme, termed Rtt109, acetylates histone H3 lysine 56, and is important for genome stability and resistance to genotoxic agents. Rtt109 is very distantly related the mammalian p300/CBP HAT enzyme, but compounds that inhibit p300/CBP do not inhibit Rtt109. In fungi, H3-K56 acetylation occurs on all newly synthesized molecules, but H3-K56ac is either not abundant or even detectable in mammalian cells. We therefore hypothesized that small molecules can be found that inhibit Rtt109 but do not substantially affect the activity of mammalian HAT enzymes, and thereby display minimal toxicity to mammalian hosts. We have discovered that homozygous rtt109-/- mutant C. albicans lack H3K56 acetylation, and are highly sensitive to genotoxic agents including DNA alkylating agents and reactive oxygen species (ROS) such as hydrogen peroxide. Notably, rtt109-/- mutant cells are much less pathogenic in a mouse model of systemic candidaisis induced by tail vein injection. Together, these data support our hypothesis that Rtt109 is a promising novel target for antifungal therapy. We are particularly encouraged to pursue these studies having generated a preliminary protocol to measure the enzymatic activity of Rtt109 in a microtiter format. Aim 1: Assay Development. We will re-optimize the assay parameters (number of washes, volumes/amounts of reagents used, secondary detection reagent) to determine the optimal Z-factor score in a 384-well plate format. Aim 2: Configuration of Assays for HTS. Based on our optimized assay configuration, we will perform a pilot screen of 30,000 compounds present here at the University of Massachusetts Medical School's Small Molecule Screening Facility. The results of this trial screen will establish the hit rate, the rate of false positives, and the best detection reagent for larger scale screening efforts. We have positive and negative screening criteria planned to prioritize initial candidates, and we will develop the reagents and protocols for these. First, we will require that compounds that inhibit histone acetylation by the Rtt109-Vps75 protein complex will also inhibit acetylation by Rtt109 when it is stimulated by Asf1 rather than Vps75. As a negative selection, we will test preliminary Rtt109 inhibitors for effects on the unrelated picNuA4 HAT enzyme complex, to rule out compounds that broadly inhibit acetyltransferase reactions without specificity for the Rtt109 active site. Finally, we will test the efficacy of identified compounds on C. albicans cells, measuring sensitivity to genotoxic agents and effects on H3K56-ac levels. This will identify compounds best able to permeate cells. We will also identify Rtt109 inhibitors that are toxic to mammalian cells, so that non-toxic candidates can be prioritized. Together, these studies will provide positive control Rtt109 inhibitors for further screening of the larger libraries at the NIH Molecular Libraries. PUBLIC HEALTH RELEVANCE: Candida albicans is a pathogenic fungus that is particularly dangerous to immunocompromised individuals, including AIDS patients. C. albicans infections are also commonly acquired in hospitals, making them a major public health problem. Recently, a new enzyme was discovered that is important for normal growth of fungi, and for pathogenesis by C. albicans. We are developing high-throughput screens for compounds that can inhibit this enzyme, because these will be candidates in our search for new anti-fungal drugs.