Penicillin-Binding Proteins, Mechanism and Inhibition

青霉素结合蛋白、机制和抑制

• 批准号: 6324997
• 负责人: Shahriar Mobashery
• 金额: $ 24.27万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6324997
• 关键词: Escherichia coli; Staphylococcus aureus; antibiotics; bacterial proteins; beta lactam antibiotic; binding proteins; cell wall; cephalosporins; crystallization; drug design /synthesis /production; drug discovery /isolation; microorganism metabolism; molecular assembly /self assembly; molecular cloning; pharmacokinetics; transpeptidation

DESCRIPTION: (Applicant's Description) Penicillin-binding proteins (PBPs) are a group of enzymes involved in a number of functions in the assembly and regulation of bacterial cell wall. These enzymes are the targets of beta-lactam antibiotics for inhibition of bacterial growth. A multidisciplinary approach has been outlined for the study of PBPs, which builds on the mechanistic findings from this laboratory presented as Preliminary Results. Pour Specific Aims are outlined. Specific Aim 1 details the plans for cloning, expression and large-scale production of two PBPs, one from Escherichia coli (a Gram-negative bacterium) and another from Staphylococcus aureus (a Gram-positive bacterium). These proteins will be used in the biochemical studies and also will be provided to Professor Judy Kelly of the University of Connecticut for crystallization. Specific Aim 2 describes our design and proposed syntheses for two cephalosporins that are incorporated with structural components of the cell wall (peptidogylcan). These cephalosporins, in conjunction with one that is already synthesized, are proposed as mechanistic probes for the transpeptidase reaction carried out by certain PBP in the last step of cell wall biosynthesis (cross-linking of cell wall). Biochemical and structural experiments are detailed for the use of these cephalosporins as probes of mechanisms for PBPs. An assay for the cell wall cross-linking reaction of the transpeptidases (a PBP) is described in Specific Aim 3. The enzymic reaction is biochemically dissected into the acylation and deacylation steps, for each of which a quantitative assay method is described. These methodologies will allow investigations of the mechanistic details of these PBPs. Furthermore, a series of four peptidoglycan derivatives have been proposed to investigate the requirements for a minimal substrate for the transpeptidation reaction of the PBPs. Specific Aim 4 details the search for novel non-f3-lactam inhibitors for PBPs. These molecules will be synthesized and their potential PBP inhibitory and antibacterial activities will be investigated in both in vivo and in vitro experiments.

描述：（申请人的描述）青霉素结合蛋白（PBP）是一种 一组酶，参与组装中的许多功能， 调节细菌细胞壁。这些酶是β-内酰胺的目标 抑制细菌生长的抗生素。多学科方法 已经概述了PBPs的研究，它建立在机械的基础上， 该实验室的研究结果作为初步结果呈现。具体倾注量 目标已概述。具体目标1详细说明了克隆、表达和 两种PBP的大规模生产，一种来自大肠杆菌（革兰氏阴性菌）， 细菌）和另一种来自金黄色葡萄球菌（革兰氏阳性细菌）。 这些蛋白质将用于生物化学研究，也将用于 提供给康涅狄格大学的朱迪·凯利教授 晶化具体目标2描述了我们的设计和建议的合成， 与细胞结构成分结合的两种头孢菌素 壁（肽聚糖）。这些头孢菌素，与一种 已经合成，提出作为转肽酶的机制探针 在细胞壁生物合成的最后一步由某些PBP进行的反应 （细胞壁的交联）。生化和结构实验是 详细介绍了这些头孢菌素作为PBPs机制探针的用途。 转肽酶（a）的细胞壁交联反应的测定 具体目标3中描述了PBP）。酶反应是生物化学反应 分解为酰化和脱酰化步骤，对于每个步骤， 描述了定量测定方法。这些方法将允许 这些PBP的机制细节的调查。此外，A系列 四种肽聚糖衍生物已被提出来研究 用于酶的转肽反应的最小底物的要求 PBPs。具体目标4详细介绍了寻找新型非f3-内酰胺抑制剂， PBPs。这些分子将被合成并具有潜在的PBP抑制作用 并在体内和体外研究其抗菌活性 实验