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.

病原体如肺孢子虫(P)、弓形虫(Tg)和禽分枝杆菌(Ma)是引起 免疫受损患者，特别是艾滋病患者的机会性感染和死亡率。肺孢子虫病 生物体代表了一大群分布广泛的非典型真菌，每一种都有其特有的 特定的哺乳动物宿主。吉罗夫氏肺孢子虫(PJ)是引起肺孢子虫肺炎(PCP)的病原体。 免疫受损患者中最常见和最严重的机会性感染。PCP的当前治疗方法 将磺胺甲恶唑与甲氧苄啶结合，靶向叶酸的生物合成。高达50%的艾滋病患者没有 长期耐受这种治疗。最近的研究还表明，随着时间的推移，靶标酶中的突变会累积， 二氢叶酸还原酶(DHFR)和二氢翼酸合成酶(DHPS)，可能会导致耐药性。这些 这些发现强调了开发更有效的治疗方法的迫切需要。这个项目的一个主要目标是在结构上 并对pjDHFR及其变异体进行生化表征，以设计具有潜在的 用于治疗PCP的治疗药物。提出了两个具体的目标来检验这一假设 抗叶酸用于对抗机会性病原体的感染是特定酶抑制剂的结果 与目标DHFR的交互。具体目的一集中在克隆、表达、纯化和结晶方面 PjDHFR与选定的酶抑制剂形成复合体。一种杆状病毒表达系统已经被开发出来，以生产 可溶性的、稳定的酶和初步的生化分析表明，一种新型的抗叶酸对pjDHFR具有纳米分子抑制作用。 该pjDHFR抑制剂复合体的结构表征正在进行中。分子建模工具将用于 电子筛选小分子文库以确定用于合成和测试的新型支架。计算方法 例如3D QSAR将被用来预测已知的抗叶酸盐与pjDHFR结合的有效性。这些数据将是 用于指导新型缓蚀剂的合成。第二个具体目标的重点是开展现场定向 DHFR的突变研究以确定特定残基在调节pjDHFR抑制剂效力和在 在艾滋病患者分离株中观察到的耐药性。蛋白质组学新工具及其同源性的应用 建模技术将被用来确定对酶折叠和功能至关重要的残基。这些结果将 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 基于结构的相关性有助于设计新型的pjDHFR抑制剂。