Enzyme Targets-Sterol Synthesis-Opportunistic Pathogens

描述（由申请人提供）：有一个长期的兴趣， 表征介导生产和加工的分子事件 麦角甾醇生物合成作为选择性化学靶向的策略 用于治疗学发现和开发的抗真菌剂。的重点 建议将在麦角甾醇合成和家庭的甾醇甲基 转移酶（SMT），来自白色念珠菌，新型隐球菌， 荚膜组织胞浆菌和卡氏肺孢子虫。概述了四个项目： 1)在试验真菌中建立麦角甾醇的从头途径， 13 C标记的中间体。 2)评价供试真菌的生长、甾醇组成和SMT活性 用我们实验室合成的一系列药物治疗， 单作用（抑制SMT活性）或双作用（抑制SMT和 14 α-脱甲基酶活性）麦角甾醇合成的抑制剂。的动力学 将探索响应于底物类似物的SMT活性作为一种手段， 新型抑制剂。 3)卡氏毕赤酵母的甾醇组成与植物相似，具有24-甲基和 24-乙基甾醇和这些“植物甾醇”被认为是 诊断目的。我们最近通过基于PCR的方法克隆了卡氏肺孢子虫SMT， 由合作者提供的EST的同源性策略。我们将决定 卡氏毕赤酵母SMT的性质与C. 白色念珠菌C.白色念珠菌和卡氏疟原虫SMT将 将相关cDNA亚克隆到大肠杆菌表达中后实现 系统化学亲和和光亲和标记将用于鉴定 甾醇和二甲双胍结合亚结构域，分别在活性中心。 将采用定点突变，然后进行活性测定来探测 参与催化作用的选择残基的功能重要性。 4)SMT的三维结构将借助于 合作者。 这些研究的结果将有助于设计治疗方案， 策略旨在提供物种特异性的抑制歧视， 麦角固醇生物合成途径。

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.