Penicillin-Binding Proteins, Mechanism and Inhibition

青霉素结合蛋白、机制和抑制

• 批准号: 6724937
• 负责人: Shahriar Mobashery
• 金额: $ 22.09万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 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/6724937
• 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：(申请人描述)青霉素结合蛋白(PBPs)是一种 一组参与组装和多项功能的酶 细菌细胞壁的调节。这些酶是β-内酰胺的靶标。 抑制细菌生长的抗生素。多学科方法 已经概述了PBPS的研究，它建立在机理的基础上 该实验室的研究结果是初步结果。倾倒特定的 概述了目标。具体目标1详细说明了克隆、表达和 大规模生产两种多溴联苯，其中一种来自大肠杆菌(革兰氏阴性) 细菌)和金黄色葡萄球菌(革兰氏阳性菌)。 这些蛋白质将被用于生化研究，也将被 提供给康涅狄格大学的朱迪·凯利教授 结晶。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 WALL(多肽聚糖)。这些头孢菌素与一种 被建议作为转肽酶的机械探针。 某些PBP在细胞壁生物合成的最后一步中进行的反应 (细胞壁的交联性)。生化和结构实验是 详细介绍了这些头孢菌素作为多溴联苯作用机制的探针的用途。 转肽酶(A)细胞壁交联度的测定 PBP)在特定目的3中描述。酶反应是生化反应 分成酰化和去酰化两个步骤，每个步骤都有 介绍了一种定量测定方法。这些方法将允许 对这些多氯联苯的机械细节进行调查。此外，一系列 已提出四种肽多糖衍生物来研究 对转肽反应的最小底物的要求 多氯联苯。特定目标4详细说明了寻找新的非f3-内酰胺类抑制剂 多氯联苯。这些分子将被合成，其潜在的PBP抑制作用 并将在体内和体外研究抗菌活性 实验。