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的人类真菌病原体，在全身感染期间会导致高死亡率（念珠菌病）。由于真菌是真核细胞，因此对人类无毒的抗真菌治疗化合物的发展是具有挑战性的。 最近发现的一种真菌组蛋白乙酰转移酶（HAT）酶，称为RTT109，乙酰酸盐组组蛋白H3赖氨酸56，对基因组稳定性和对遗传毒性剂的抗性很重要。 RTT109与哺乳动物P300/CBP HAT酶密切相关，但是抑制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H3-K56AC在哺乳动物细胞中不丰富，甚至不可检测。因此，我们假设可以发现抑制RTT109的小分子，但不会显着影响哺乳动物帽子酶的活性，从而表现出对哺乳动物宿主的最小毒性。 我们已经发现纯合RTT109 - / - 突变体白色念珠菌缺乏H3K56乙酰化，并且对包括DNA烷基化剂和反应性氧（ROS）（如氢氧化氢）对遗传毒性剂高度敏感。值得注意的是，在尾静脉注射引起的全身性念珠菌模型中，RTT109 - / - 突变细胞的致病性要小得多。总之，这些数据支持我们的假设，即RTT109是抗真菌治疗的新颖靶标。 特别鼓励我们进行这些研究，该研究生成了初步方案，以微量尺形式测量RTT109的酶活性。目标1：测定开发。我们将重新优化测定参数（洗涤次数，所使用的试剂的体积/量，次级检测试剂）以确定384孔板格式的最佳z因子得分。目标2：HTS测定法的配置。 根据我们优化的测定配置，我们将在马萨诸塞大学医学院的小分子筛查设施上进行30,000种化合物的试验屏幕。该试验屏幕的结果将确定命中率，误报率以及大规模筛选工作的最佳检测试剂。 我们计划有积极的和负面筛查标准来优先考虑初始候选人，我们将为这些候选人开发试剂和协议。首先，我们将要求RTT109-VPS75蛋白复合物抑制组蛋白乙酰化的化合物在被ASF1而不是VPS75刺激时，RTT109也会抑制RTT109的乙酰化。作为阴性选择，我们将测试初步RTT109抑制剂对无关的PICNUA4 HAT酶复合物的影响，以排除化合物，这些化合物可广泛抑制乙酰基转移酶反应，而对于RTT109活性位点而没有特异性。 最后，我们将测试鉴定化合物对白色念珠菌细胞的疗效，测量对遗传毒性剂的敏感性以及对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，以便将无毒的候选者优先考虑。总之，这些研究将提供阳性对照RTT109抑制剂，以进一步筛选NIH分子文库的较大文库。 公共卫生相关性：白色念珠菌是一种致病的真菌，对包括艾滋病患者在内的免疫功能低下的个体特别危险。白色念珠菌感染也通常在医院中获得，使其成为主要的公共卫生问题。最近，发现一种新酶对真菌的正常生长以及白色念珠菌的发病机理很重要。我们正在为可以抑制这种酶的化合物开发高通量筛选，因为这些将是我们寻找新的抗真菌药物的候选者。