Enzyme Targets-Sterol Synthesis-Opportunistic Pathogens

描述(由申请人提供)：有长期的兴趣 描述调节生产和加工的分子事件的特征 麦角甾醇生物合成作为选择性化学靶向的策略 用于治疗药物发现和开发的抗真菌药物。这件事的重点是 提案将涉及麦角甾醇的合成和甲基甾醇家族 白色念珠菌、新生隐球菌、 组织胞浆菌和卡氏肺孢子虫。概述了四个项目： 1)在试验真菌中建立了麦角甾醇的从头合成途径 13C标记的中间体。 2)对供试真菌的生长、甾醇组成和SMT活性进行评价 用我们实验室合成的一套药物治疗，作为 单作用(抑制SMT活性)或双作用(抑制SMT和 14A-去甲基酶活性)麦角甾醇合成的抑制剂。反应的动力学 SMT活性对底物类似物的响应将作为一种手段来探索 时尚新奇的抑制剂。 3)卡氏拟青霉的甾醇成分与植物相似，含有24-甲基和 24-乙基类固醇和这些“植物类固醇”被认为是 诊断目的。我们最近通过基于聚合酶链式反应的方法克隆了卡氏肺孢子虫SMT。 来自合作者提供的EST的同源策略。我们将确定是否 卡氏假单胞菌SMT的特性与C. 白念珠菌SMT。白念珠菌和卡氏肺假单胞菌SMT的纯化将是 亚克隆相关基因后实现了原核表达 系统。将使用化学亲和标记和光亲和标记来识别 分别位于活性中心的甾醇和ADOMet结合亚域。 将采用定点突变和活性分析相结合的方法进行探测 与催化有关的精选残基的功能重要性。 4)SMT的三维结构将借助 合作者。 这些研究的结果将有助于设计治疗性的 旨在提供针对特定物种的抑制歧视的战略 麦角固醇的生物合成途径。

DESCRIPTION (provided by applicant): There is a long-standing interest to characterize the molecular events that mediate the production and processing of ergosterol biosynthesis as a strategy for selective chemical targeting of antifungals for therapeutics discovery and development. The focus of this proposal will be on ergosterol synthesis and the family of sterol methyl transferase (SMT) enzymes from Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Pneumocystis carinii. Four projects are outlined: 1) Establish the de novo pathway to ergosterol in the test fungi using 13C-labeled intermediates. 2) Evaluate the growth, sterol composition and SMT activity of the test fungi treated with a set of drugs synthesized in our laboratory to serve as single-action (inhibit SMT activity) or dual-action (inhibit SMT and 14a-demethylase activities) inhibitors of ergosterol synthesis. The kinetics of SMT activity in response to substrate analogs will be explored as a means to fashion novel inhibitors. 3) The sterol composition of P. carinii is plant-like in having 24-methyl and 24-ethyl sterols and these "phytosterols" are regarded as signature lipids for diagnostic purposes. We recently cloned the P. carinii SMT by a PCR-based homology strategy from ESTs provided by a cooperator. We will determine whether the properties of the P. carinfi SMT are similar to the properties of the C. albicans SMT. Purification of the C. albicans and P. carinii SMTs will be achieved after subcloning the relevant cDNA into a Escherichia coil expression system. Chemical affinity and photoaffinity labeling will be used to identify the sterol and AdoMet-binding subdomains, respectively, in the active center. Site-directed mutagenesis followed by activity assay will be employed to probe the functional importance of select residues involved with catalysis. 4) The three-dimensional structure of a SMT will be defined with the aid of a cooperator. The results from these studies will facilitate the design of therapeutic strategies aimed to provide species-specific discrimination of inhibition in the ergosterol biosynthetic pathway.