Structural Studies of AIDS-Responsive Drugs

描述(申请人提供)：肺孢子虫(P)、弓形虫(Tg)和禽分枝杆菌(Ma)等病原体是免疫受损患者，特别是艾滋病患者机会性感染和死亡的主要原因。肺孢子虫代表了一大群分布广泛的非典型真菌，每一种都对特定的哺乳动物宿主具有特异性。肺孢子虫肺炎(PCP)是免疫功能低下患者中最常见和最严重的机会性感染之一。目前治疗PCP的方法是将磺胺甲恶唑与甲氧匹林联合使用，靶向是叶酸的生物合成。高达50%的艾滋病患者不能长期忍受这种治疗。最近的研究还表明，随着时间的推移，靶标酶二氢叶酸还原酶(DHFR)和二氢蝶呤酸合酶(DHPS)中的突变会积累，可能会导致耐药性。这些发现强调了开发更有效的治疗方法的迫切需要。该项目的一个主要目标是对pjDHFR及其变异体的结构和生化特征进行表征，以设计具有治疗PCP潜力的有效抑制剂。提出了两个特定的目标来检验这一假设，即抗叶酸在对抗来自机会性病原体的感染方面的有效性是特定的酶抑制剂与靶DHFR相互作用的结果。具体目的一是pjDHFR基因的克隆、表达、纯化及与部分酶抑制剂形成的复合体结晶。一种杆状病毒表达系统已经被开发出来，以产生可溶的、稳定的酶，初步的生化测试表明，一种新型的抗叶酸对pjDHFR具有纳分子抑制作用。该pjDHFR抑制剂复合体的结构表征正在进行中。分子模拟工具将用于小分子文库的电子筛选，以定义用于合成和测试的新型支架。三维定量构效关系等计算方法将被用来预测已知的抗叶酸盐与pjDHFR结合的有效性。这些数据将用于指导新型缓蚀剂的合成。第二个特定目标的重点是对dhfr进行定点突变研究，以确定在艾滋病患者分离株中观察到的特定残基在调节pjdhfr抑制剂效力和增加耐药性方面的作用。应用新的蛋白质组学工具和同源建模技术将被用来确定对酶折叠和功能至关重要的残基。这些结果将有助于指导物种选择性抑制剂的设计。突变研究将在基于结构的相关性中测试这些可能性，以帮助设计新的pjDHFR抑制剂。

DESCRIPTION (provided by applicant): 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 baculovirus expression system has been developed to produce soluble, stable enzyme and initial biochemical assays reveal nanomolar inhibition against pjDHFR by a novel antifolate. Structural characterization of this pjDHFR inhibitor complex is underway. Molecular modeling tools 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. The focus of the second specific aim is to carry out site-directed mutagenesis studies on DHFR to determine the role of specific residues in modulating pjDHFR inhibitor potency 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 possibilities in the structure-based correlations to help design novel pjDHFR inhibitors.