Identification of CDP-DAG and serine binding sites in Candida albicans phosphatidylserine synthase, an antifungal drug target

抗真菌药物靶标白色念珠菌磷脂酰丝氨酸合酶中 CDP-DAG 和丝氨酸结合位点的鉴定

• 批准号: 9300114
• 负责人: Todd B Reynolds
• 金额: $ 18.59万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-16 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9300114
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Active Sites; Adopted; Adrenal Cortex Hormones; Alanine; Alcohols; Amino Acid Sequence; Amino Acids; Antifungal Agents; Arginine; Binding; Binding Sites; Biochemical; Biological Assay; Candida; Candida albicans; Catalysis; Chemicals; Chemotherapy-Oncologic Procedure; Consensus; Conserved Sequence; Dentures; Development; Diglycerides; Drug Combinations; Drug Design; Drug Targeting; Drug resistance; Drug toxicity; Effectiveness; Enzyme Inhibitor Drugs; Enzymes; Future; Goals; Head; Homology Modeling; Human; Immunocompromised Host; In Vitro; Infection; Inositol; Lead; Lecithin; Lipids; Manuscripts; Mass Spectrum Analysis; Mutagenesis; Mycoses; Organ Transplantation; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phosphatidylethanolamine; Phosphatidylinositols; Phosphatidylserine Synthase; Phosphatidylserines; Phospholipids; Phosphotransferases; Preparation; Production; Proteins; Role; Saccharomyces cerevisiae; Scanning; Sepsis; Sequence Alignment; Sequence Homology; Serine; Structure; Systemic infection; Testing; Toxic effect; Virulence; Water; Work; analog; antineoplastic antibiotics; base; computerized tools; crosslink; design; drug development; enzyme substrate; fungus; genetic analysis; in vivo; inhibitor/antagonist; mortality; mouse model; mutant; new therapeutic target; novel; oral infection; pathogen; predictive modeling

Candida albicans is among the most common causes of fungal infections in humans. Candida causes oral infections in immunocompromised people such as those with AIDS, and it can cause even more serious invasive bloodstream infections in neutropenic patients such as those undergoing cancer chemotherapy and organ transplants. Although there are three classes of antifungals to treat invasive fungal infections, a combination of drug resistance and toxicity limit their effectiveness. Thus, there is a need for new classes of antifungals. The Cho1 phosphatidylserine (PS) synthase represents a potential new drug target for three reasons: 1) Loss of PS synthase blocks virulence, and causes clearance of the fungus in mouse models of systemic or oral infection. 2) PS synthase is not homologous to mammalian (i.e. human) PS synthases, which use a different mechanism to synthesize PS. Mammalian PS synthases exchange head groups from phosphatidylethanolamine (PE) or phosphatidylcholine (PC) for serine to make PS. Fungal PS synthase condenses cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine to make PS. 3) Fungal PS synthases are conserved in all fungal pathogens, so PS synthase inhibitors could be broad spectrum. One approach to developing enzyme inhibitors is to look for competitive inhibitors of the enzyme's natural substrates. This approach would be enhanced by a more comprehensive understanding of Cho1's interactions with its substrates: CDP-DAG and serine. Elucidation of the binding motifs for both substrates would allow us to better design potential competitive inhibitors, such as serine analogs, to act as lead compounds for antifungal development. A common CDP-binding consensus motif that is present in CDP-alcohol phosphotransferases is [D-(X)2-D-G-(X)2-A-R-(X)8-G-(X)3-D-(X)3-D], and this is found in Cho1. However, the serine-binding motif for Cho1 is unknown. Using homology sequence alignments and computational tools that superimpose Cho1 on the solved structure of a prokaryotic CDP-alcohol phosphotransferase, a putative serine binding motif was identified. Two aims will be pursued to confirm the CDP-DAG binding motif and discover the serine-binding motif. Aim 1. Genetic analysis will be used to identify residues important for substrate binding in Cho1. Based on the known CDP binding motif, alanine-scanning mutagenesis will be used to test the importance of this motif in PS synthesis. Alanine-scanning mutagenesis will also be used to test the function of the putative serine binding motif. Aim 2: Chemical cross-linking with serine analogs will be used to identify the serine binding domain. In a less-biased biochemical approach, serine analogs have been synthesized that compete with native L-serine for PS synthesis. This set of analogs will be further developed and used to cross-link the substrates to the PS synthase, and follow this with mass spectrometry to identify amino acids with which they interact. Interacting amino acids will then be genetically analyzed using alanine-scanning mutagenesis.

白色念珠菌是人类真菌感染的最常见原因之一。念珠菌引起口腔 免疫功能低下的人（例如艾滋病患者）感染，可能会导致更严重的情况 中性粒细胞减少症患者（例如接受癌症化疗的患者）的侵袭性血流感染 器官移植。尽管有三类抗真菌药物可治疗侵袭性真菌感染，但 耐药性和毒性的结合限制了它们的有效性。因此，需要新的类别 抗真菌剂。 Cho1 磷脂酰丝氨酸 (PS) 合酶代表了三种药物的潜在新靶标 原因： 1) PS合酶的损失会阻断毒力，并导致小鼠模型中的真菌被清除 全身或口腔感染。 2) PS合酶与哺乳动物（即人类）PS合酶不同源， 使用不同的机制来合成PS。哺乳动物 PS 合酶交换头基 磷脂酰乙醇胺 (PE) 或磷脂酰胆碱 (PC) 生成丝氨酸以制造 PS。真菌PS合酶 缩合胞苷二磷酸二酰基甘油 (CDP-DAG) 和丝氨酸生成 PS。 3) 真菌PS合酶是 在所有真菌病原体中都是保守的，因此 PS 合酶抑制剂可能是广谱的。一种方法 开发酶抑制剂就是寻找酶天然底物的竞争性抑制剂。这 通过更全面地了解 Cho1 与其相互作用，可以增强该方法 底物：CDP-DAG 和丝氨酸。阐明两种底物的结合基序将使我们能够更好地 设计潜在的竞争性抑制剂，例如丝氨酸类似物，作为抗真菌药物的先导化合物 发展。 CDP-醇磷酸转移酶中存在的常见 CDP 结合共有基序是 [D-(X)2-D-G-(X)2-A-R-(X)8-G-(X)3-D-(X)3-D]，这是在 Cho1 中发现的。然而，丝氨酸结合基序 Cho1 未知。使用同源序列比对和计算工具将 Cho1 叠加在 原核CDP-醇磷酸转移酶的已解析结构，推定的丝氨酸结合基序是 确定。我们将追求两个目标：确认 CDP-DAG 结合基序并发现丝氨酸结合 主题。目标 1. 将使用遗传分析来鉴定对 Cho1 中的底物结合重要的残基。基于 在已知的CDP结合基序上，将使用丙氨酸扫描诱变来测试该基序的重要性 在PS合成中。丙氨酸扫描诱变也将用于测试假定的丝氨酸的功能 结合基序。目标 2：与丝氨酸类似物的化学交联将用于鉴定丝氨酸结合 领域。在一种偏差较小的生化方法中，已经合成了丝氨酸类似物，可以与 用于 PS 合成的天然 L-丝氨酸。这组类似物将被进一步开发并用于交联 PS 合酶的底物，然后通过质谱分析来鉴定它们所用的氨基酸 相互影响。然后将使用丙氨酸扫描诱变对相互作用的氨基酸进行遗传分析。