Structural Studies of AIDS-Responsive Drugs

描述（由申请人提供）：病原体如肺囊虫(P)、刚地弓形虫（Tg）和鸟分枝杆菌（Ma）是免疫功能低下患者（特别是艾滋病患者）机会性感染和死亡的主要原因。肺囊虫是一大类普遍分布的非典型真菌，每一种都有特定哺乳动物宿主的特异性。肺孢子虫（pj）是肺炎肺孢子虫（PcP）的病原体，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联合磺胺甲恶唑和甲氧苄啶，靶向叶酸生物合成。高达50%的艾滋病患者不能长期耐受这种治疗。最近的研究还表明，随着时间的推移，突变在靶酶，二氢叶酸还原酶（DHFR）和二氢叶酸合酶（DHPS）中积累，可能会产生耐药性。这些发现强调了开发更有效治疗方法的关键必要性。该项目的一个主要目标是对pjDHFR及其变体进行结构和生化表征，以便设计有效的抑制剂，作为治疗PcP的潜在药物。我们提出了两个特定的目的来验证这样的假设，即使用抗叶酸盐对抗机会致病菌感染的功效是特定的酶抑制剂与目标DHFR相互作用的结果。具体目的一是研究pjDHFR与特定酶抑制剂复合物的克隆、表达、纯化和结晶。杆状病毒表达系统已经开发出可溶、稳定的酶，初步生化分析显示一种新型抗叶酸盐对pjDHFR具有纳米摩尔抑制作用。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小分子文库的硅筛选，以确定用于合成和测试的新支架。3D QSAR等计算方法将用于预测已知抗叶酸盐与pjDHFR结合的功效。这些数据将用于指导新抑制剂的合成。第二个特定目标的重点是对DHFR进行定点诱变研究，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抑制剂效力和赋予耐药性方面的作用。应用新的蛋白质组学工具和同源建模技术将用于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。将进行诱变研究，以测试这些基于结构的相关性的可能性，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.