Structural Studies of AIDS-Responsive Drugs

病原体如肺孢子虫（P）、刚地弓形虫（Tg）和鸟分枝杆菌（Ma）是引起 机会性感染和免疫功能低下的患者，特别是艾滋病患者的死亡率。肺孢子 生物体代表了一大群普遍分布的非典型真菌，每种真菌对一种 特定哺乳动物宿主。耶氏肺孢子虫（Pneumocystis jirovecii，pj）是肺孢子虫肺炎（Pneumocystis pneumonia，PcP）的病原体， 免疫功能低下患者中最常见和最严重的机会性感染。目前对PcP的治疗 结合磺胺甲恶唑和甲氧苄啶，靶向叶酸生物合成。高达50%的艾滋病患者没有 长期忍受这种治疗。最近的研究还表明，随着时间的推移，突变在靶酶中积累， 二氢叶酸还原酶（DHFR）和二氢蝶酸合酶（DHPS），潜在地引起耐药性。这些 研究结果强调了开发更有效的治疗方法的迫切需要。该项目的主要目标是从结构上 并对pjDHFR及其变体进行生物化学表征，以设计有效的抑制剂， 用于治疗PcP的治疗剂。提出了两个具体的目标来检验这一假设， 抗叶酸剂用于对抗机会性病原体感染是特异性酶抑制剂的结果， 与目标DHFR的相互作用。具体目标一是对重组蛋白进行克隆、表达、纯化和结晶， pjDHFR与选定的酶抑制剂复合。杆状病毒表达系统已被开发用于生产 可溶性、稳定的酶和初始生化测定揭示了新型抗叶酸剂对pjDHFR的纳摩尔抑制。 这种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 小分子文库的计算机筛选以定义用于合成和测试的新支架。计算方法 例如3DQSAR将用于预测已知抗叶酸剂结合pjDHFR的功效。这些数据将 用于指导新型抑制剂的合成。第二个具体的重点目标是开展现场指导 对DHFR的诱变研究，以确定特定残基在调节pJDHFR的生物学效价和 如在AIDS患者分离物中观察到的那样赋予耐药性。新蛋白质组学工具的应用与同源性 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 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，以帮助设计新的pjDHF抑制剂。

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.