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。最近发现的一种真菌组蛋白乙酰转移酶（HAT）酶，称为Rtt109，可使组蛋白H3赖氨酸56乙酰化，对基因组稳定性和对基因毒性药物的抗性至关重要。Rtt109是哺乳动物p300/CBP HAT酶的远亲，但抑制p300/CBP的化合物不抑制Rtt109。在真菌中，H3-K56乙酰化发生在所有新合成的分子上，但在哺乳动物细胞中，H3-K56ac要么不丰富，要么甚至无法检测到。因此，我们假设可以找到抑制Rtt109但不实质性影响哺乳动物HAT酶活性的小分子，从而对哺乳动物宿主显示最小的毒性。我们发现纯合子rtt109-/-突变体白色念珠菌缺乏H3K56乙酰化，对DNA烷基化剂和活性氧（ROS）如过氧化氢等遗传毒性物质高度敏感。值得注意的是，rtt109-/-突变细胞在尾静脉注射诱导的全身念珠菌感染小鼠模型中的致病性要低得多。总之，这些数据支持我们的假设，即Rtt109是抗真菌治疗的一个有希望的新靶点。我们特别受鼓舞地继续进行这些研究，并制定了以微滴度形式测量Rtt109酶活性的初步方案。目标1：试验开发。我们将重新优化检测参数（洗涤次数，所用试剂的体积/数量，二次检测试剂），以确定384孔板格式的最佳z因子评分。目标2:HTS检测方法的配置。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30,000种化合物进行试点筛选。该试验筛选的结果将确定命中率、假阳性率和最佳检测试剂，以进行更大规模的筛选工作。我们已经制定了阳性和阴性筛选标准，计划优先考虑初始候选物，我们将为此开发试剂和方案。首先，我们将要求抑制Rtt109-Vps75蛋白复合物的组蛋白乙酰化的化合物在Rtt109受到Asf1而不是Vps75刺激时也能抑制Rtt109的乙酰化。作为阴性选择，我们将测试初步的Rtt109抑制剂对不相关的picNuA4 HAT酶复合物的影响，以排除对Rtt109活性位点没有特异性的广泛抑制乙酰转移酶反应的化合物。最后，我们将测试鉴定的化合物对白色念珠菌细胞的功效，测量对基因毒性药物的敏感性和对H3K56-ac水平的影响。这将确定最能渗透细胞的化合物。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。

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.