Enzyme Targets-Sterol Synthesis-Opportunistic Pathogens

酶靶点-甾醇合成-机会性病原体

• 批准号: 6636683
• 负责人: William David Nes
• 金额: $ 18.18万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636683
• 关键词: AIDS; Candida albicans; Cryptococcus neoformans; Escherichia coli; Pneumocystis carinii; active sites; affinity labeling; carbon; complementary DNA; enzyme activity; enzyme inhibitors; enzyme structure; ergosterol; lanosterol; methyltransferase; mycosis; nuclear magnetic resonance spectroscopy; opportunistic infections; polymerase chain reaction; protein purification; site directed mutagenesis; sitosterols; stable isotope; steroid biosynthesis

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.

描述（由申请人提供）：长期存在的兴趣 表征介导的分子事件，介导的生产和加工 麦角固醇生物合成作为选择性化学靶向的策略 治疗剂发现和发育的抗真菌群体。重点 建议将是关于麦角固醇的合成和固醇甲基的家族 来自白色念珠菌的转移酶（SMT）酶，新生虫的加密赛车， 组织囊肿囊膜和肺炎藻菌。概述了四个项目： 1）使用使用 13C标记的中间体。 2）评估测试真菌的生长，固醇组成和SMT活性 用在我们的实验室中合成的一组药物治疗 单个动作（抑制SMT活性）或双动作（抑制SMT和 14a-二甲基酶活性）麦角固醇合成的抑制剂。动力学 响应底物类似物的SMT活动将被探讨 时尚小说抑制剂。 3）P。carinii的固醇成分在具有24-甲基和 24-乙基固醇和这些“植物固醇”被视为签名脂质 诊断目的。我们最近通过基于PCR的P. carinii SMT克隆 合作者提供的EST的同源策略。我们将确定是否 P. carinfi SMT的性质与C的性质相似。 白色唱片SMT。白色念珠菌和Carinii smt的纯化将是 将相关cDNA取代到大胆管表达中后实现 系统。化学亲和力和光性标签将用于识别 活性中心的固醇和ADOMET结合子域分别。 定点诱变，然后进行活动测定法进行探测 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A定义 合作者。 这些研究的结果将有助于治疗的设计 旨在提供抑制物种特定于物种的策略 麦角固醇生物合成途径。