Structural Studies of AIDS-Responsive Drugs

艾滋病反应药物的结构研究

• 批准号: 7888607
• 负责人: Vivian Cody
• 金额: $ 10.44万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-14 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888607
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Affinity; Baculoviruses; Binding; Biochemical; Biological Assay; Cells; Collaborations; Complex; Computer Simulation; Computing Methodologies; Coupled; Crystallization; Data; Development; Dihydrofolate Reductase; Dihydrofolate Reductase Inhibitor; Dihydropteroate Synthase; Drug Design; Drug resistance; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Eukaryota; Folate Biosynthesis Pathway; Folic Acid Antagonists; Goals; Homology Modeling; Human; Immunocompromised Host; Infection; Kinetics; Measures; Methods; Modeling; Molecular; Mutagenesis; Mutation; Mycobacterium avium; Opportunistic Infections; Organism; Patients; Pattern; Pharmaceutical Preparations; Pneumocystis; Pneumocystis carinii; Pneumocystis carinii Pneumonia; Predisposition; Prokaryotic Cells; Proteomics; Quantitative Structure-Activity Relationship; Rattus; Recombinants; Research Personnel; Role; Screening procedure; Site-Directed Mutagenesis; Specificity; Structural Models; Structure; Structure-Activity Relationship; Sulfamethoxazole; System; Techniques; Testing; Therapeutic Agents; Time; Toxoplasma gondii; Trimethoprim; Variant; Work; X ray diffraction analysis; X-Ray Diffraction; base; combat; design; drug candidate; effective therapy; enzyme activity; expression cloning; fungus; inhibitor/antagonist; mortality; novel; pathogen; programs; research study; scaffold; small molecule libraries; tool

Pathogens such as Pneumocystis (P), Toxoplasma gondii (Tg)and Mycobacterium avium (Ma) are major causes of opportunistic infection and mortality in immunocompromised patients, particularly those with AIDS. Pneumocystis organisms represent a large group of species of atypical fungi with universal distribution,each with specificity for a specific mammalian host. Pneumocystis jirovecii (pj) is the causative agent of Pneumocystis pneumonia(PcP), one of the most frequent and severe opportunistic infections in immunocompromised patients. Current treatment for PcP combines sulfamethoxazole with trimethoprim, targeting folate biosynthesis. Up to 50% of AIDS patients do not tolerate this treatment long term. Recent studies also show that mutations accumulate over time in the target enzymes, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), potentially giving rise to drug resistance. These findings underscore the crucial need to develop more effective treatments. A major goal of this project is to structurally and biochemically characterize pjDHFR and its variants in order to design effective inhibitors that have potential as therapeutic agents for the treatment of PcP. Two specific aims are proposed to test the hypothesis that efficacy of antifolate use in combating infections from opportunistic pathogens is the result of specific enzyme-inhibitor interactions with the target DHFR. Specific aim one focuses on cloning, expression, purification and crystallization of pjDHFR in complex with selected enzyme inhibitors. A baculovirusexpression system has been developed to produce soluble, stable enzyme and initial biochemical assays reveal nanomolar inhibitionagainst pjDHFR by a novel antifolate. Structural characterization of this pjDHFR inhibitor complex is underway. Molecular modelingtools will be used for in silico screening of small molecule libraries to define novel scaffolds for synthesis and testing. Computational methods such as 3D QSAR will be used to predict the efficacy of known antifolates for binding to pjDHFR. These data will be used to guide synthesis of novel inhibitors. Th e focus of the second specific aim is to carry out site-directed mutagenesis studies on DHFR to determine the role of specific residues in modulatingpjDHFR inhibitorpotency and in conferring drug-resistance as observed in AIDS patient isolates. Application of novel proteomic tools and homology modeling techniques will be used to determine residues that are critical to enzyme fold and function. These results will help guide the design of species selective inhibitors. Mutagenesis studies will be carried out to test these possibilitiesin the structure-based correlations to help design novel pjDHFRinhibitors.

病原体，例如肺炎（p），弓形虫弓形虫（TG）和鸟分枝杆菌（MA）是主要原因 免疫功能低下的患者，特别是患有艾滋病的患者的机会性感染和死亡率。肺炎藻 有机体代表了一大批具有普遍分布的非典型真菌，每个真菌具有特异性 特定的哺乳动物宿主。肺炎藻（PJ）是肺炎肺炎（PCP）的病因，是之一 免疫功能低下的患者中最常见和严重的机会性感染。当前的PCP治疗 将磺胺甲氧唑与甲氧苄啶相结合，靶向叶酸生物合成。多达50％的艾滋病患者没有 长期容忍这种治疗。最近的研究还表明，突变随着时间的流逝积累在目标酶中， 二氢叶酸还原酶（DHFR）和二氢蛋白酶合酶（DHPS）有可能引起耐药性。这些 发现强调了开发更有效治疗的至关重要的需求。该项目的主要目标是在结构上 并在生化方面表征了PJDHFR及其变体，以设计具有潜力的有效抑制剂 治疗PCP的治疗剂。提出了两个具体目的来检验以下假设 抗叶酸在对抗机会病原体中的感染中使用是特定酶抑制剂的结果 与目标DHFR的相互作用。特定的目标专注于克隆，表达，纯化和结晶 PJDHFR与选定的酶抑制剂相关。已经开发了一种生产的baculovirusexpression系统 可溶性，稳定的酶和初始的生化测定法显示了新型抗叶酸的纳摩尔抑制剂PJDHFR。 该PJDHFR抑制剂复合物的结构表征正在进行中。分子建模工具将用于 对小分子库的硅酸筛选，以定义新的支架进行合成和测试。计算方法 例如3D QSAR将用于预测已知抗叶酸与PJDHFR结合的疗效。这些数据将是 用于指导新型抑制剂的合成。第二个特定目的的重点是进行现场定向 对DHFR的诱变研究，以确定特定残基在调节PJDHFR抑制剂中的作用以及在 在AIDS患者分离株中观察到的抗药性。新颖的蛋白质组学工具和同源性的应用 建模技术将用于确定对酶折叠和功能至关重要的残基。这些结果将会 帮助指导物种选择性抑制剂的设计。将进行诱变研究以测试这些可能性 基于结构的相关性，以帮助设计新型的PJDHFR抑制剂。