HTS development for targeted anti-fungal small molecules

靶向抗真菌小分子的 HTS 开发

• 批准号: 8134502
• 负责人: PAUL D. KAUFMAN
• 金额: $ 4.11万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8134502
• 关键词: Acetyl Coenzyme A; Acetylation; Acetyltransferase; Acquired Immunodeficiency Syndrome; Active Sites; Affect; Alkaline Phosphatase; Alkylating Agents; Antibodies; Antifungal Agents; Antifungal Therapy; Biological Assay; Candida albicans; Candidiasis; Cells; Complex; DNA; Data; Detection; Development; Dimethyl Sulfoxide; EP300 gene; Enzyme-Linked Immunosorbent Assay; Enzymes; Eukaryotic Cell; Evaluation; Genome Stability; Growth; Histone Acetylation; Histone H3; Hospitals; Human; Hydrogen Peroxide; Immunoassay; Immunocompromised Host; Individual; Infection; Injection of therapeutic agent; Lead; Libraries; Link; Lysine; Mammalian Cell; Massachusetts; Measures; Modeling; Molecular Bank; Molecular Chaperones; Multienzyme Complexes; Mutagens; Pathogenesis; Patients; Pharmaceutical Preparations; Phase; Protocols documentation; Public Health; Reaction; Reactive Oxygen Species; Reagent; Resistance; Screening procedure; Serum; Signal Transduction; Solid; Specificity; Systemic infection; Tail; Temperature; Testing; Therapeutic; Titrations; Toxic effect; United States National Institutes of Health; Universities; Veins; assay development; base; efficacy testing; fungus; high throughput screening; histone acetyltransferase; inhibitor/antagonist; medical schools; mortality; mouse model; mutant; novel; pathogen; protein complex; public health relevance; small molecule

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.

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。 最近发现的一种名为 Rtt109 的真菌组蛋白乙酰转移酶 (HAT) 可以乙酰化组蛋白 H3 赖氨酸 56，对于基因组稳定性和对基因毒性物质的抵抗力非常重要。 Rtt109 与哺乳动物 p300/CBP HAT 酶的关系非常远，但抑制 p300/CBP 的化合物不会抑制 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 在哺乳动物细胞中并不丰富，甚至无法检测到。因此，我们假设可以发现小分子可以抑制 Rtt109，但不会显着影响哺乳动物 HAT 酶的活性，从而对哺乳动物宿主表现出最小的毒性。 我们发现纯合的 rtt109-/- 突变体白色念珠菌缺乏 H3K56 乙酰化，并且对基因毒性剂高度敏感，包括 DNA 烷化剂和活性氧 (ROS) 如过氧化氢。值得注意的是，rtt109-/-突变细胞在尾静脉注射诱导的全身性念珠菌病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 是一个有前途的抗真菌治疗新靶点。 我们特别鼓励开展这些研究，并制定了初步方案，以微量滴定格式测量 Rtt109 的酶活性。目标 1：检测开发。我们将重新优化测定参数（洗涤次数、所用试剂的体积/量、辅助检测试剂）以确定 384 孔板形式的最佳 Z 因子得分。目标 2：HTS 测定的配置。 基于我们优化的检测配置，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、假阳性率以及更大规模筛选工作的最佳检测试剂。 我们计划制定阳性和阴性筛选标准来优先考虑初始候选者，并且我们将为这些标准开发试剂和方案。首先，我们需要抑制 Rtt109-Vps75 蛋白复合物组蛋白乙酰化的化合物在受到 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，以排除广泛抑制乙酰转移酶反应而对 Rtt109 活性位点没有特异性的化合物。 最后，我们将测试已鉴定化合物对白色念珠菌细胞的功效，测量对基因毒性剂的敏感性以及对 H3K56-ac 水平的影响。这将识别最能够渗透细胞的化合物。我们还将鉴定对哺乳动物细胞有毒的 Rtt109 抑制剂，以便优先考虑无毒候选药物。总之，这些研究将为 NIH 分子图书馆更大的图书馆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，对免疫功能低下的个体（包括艾滋病患者）尤其危险。白色念珠菌感染也常见于医院，这使其成为一个重大的公共卫生问题。最近，发现了一种新酶，它对于真菌的正常生长以及白色念珠菌的发病机制很重要。我们正在开发高通量筛选能够抑制这种酶的化合物，因为这些将成为我们寻找新抗真菌药物的候选药物。