Design and mechanistic studies of mimics of antimicrobial peptides

抗菌肽模拟物的设计和机理研究

• 批准号: 8706638
• 负责人: WILLIAM DEGRADO
• 金额: $ 57.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8706638
• 关键词: Affect; Animal Model; Antibiotic Resistance; Antibiotics; Antimicrobial susceptibility; Bacteria; Bacterial Drug Resistance; Bacterial Infections; Binding; Cytoplasmic Tail; Deuterium; Development; Disulfides; Escherichia coli; Evolution; Funding; Gene Expression; Genes; Growth; Heat shock proteins; Human; Hydrogen; Immune system; In Vitro; Infection; Intestines; Knock-out; Maps; Mediating; Membrane; Morphology; Motion; Mus; Nutrient; Peptides; Periplasmic Proteins; Permeability; Pharmaceutical Preparations; Phase; Phase II Clinical Trials; Predisposition; Protein Kinase; Proteins; RNA; Regulation; Role; Signal Transduction; Staphylococcus aureus; Stress; System; Testing; Toxic effect; Transmembrane Domain; Virulence; X-Ray Crystallography; antimicrobial; antimicrobial peptide; crosslink; design; genetic regulatory protein; in vivo; inhibitor/antagonist; interest; killings; mimetics; mouse model; mutant; novel; overexpression; protein misfolding; protein-histidine kinase; quorum sensing; resistant strain; response; small molecule; stress protein; synthetic drug

DESCRIPTION (provided by applicant): Antimicrobial peptides (AMPs) represent an essential component of the innate immune system that provides protection from bacterial infections. However, the development of AMPs as i.v. antibiotics has encountered difficulties from in vivo toxicity and limited efficacy. Therefore, in the previous funding periods we designed small molecule mimics of AMPs with robust activity against Staphyococcal aureus. One of these compounds (PMX30063) has now proven safe and efficacious in phase II trials in humans (conducted by PolyMedix). Membrane-binding appears to be an essential part of their antimicrobial mechanism. However, the precise mechanism by which these compounds and many AMPs kill bacteria has not been determined. Much can be inferred concerning the modes of action from studies of the transcriptional and translational response of bacteria to sub-killing concentrations of these agents. Bacteria have developed an elaborate response to AMPs over millions of years of co-evolution with their hosts. Many of these responses rely on bacterial histidine kinases (HKs), which are membrane-spanning protein kinases. These proteins, together with their partner response regulators, make up two-component systems (TCSs) that mediate antibacterial resistance, quorum sensing, nutrient utilization, and virulence. We will profile the transcriptional and translational responses of S. aureus and E. coli to AMPs, cyclic lipopeptide antibiotics, and AMP mimetics. These studies will not only provide a better understanding of the antimicrobial mechanisms, but will also define new targets to enhance the susceptibility of bacteria to synthetic drugs as well as our own endogenous AMPs. Our aims are: Aim 1) AMPs and mimetics cause large changes in expression of genes associated with membrane stress and periplasmic protein misfolding stress in E. coli. We will extend this analysis and compare the corresponding response of S. aureus. The degree of protection or sensitivity to AMPs and AMP mimetics imparted by each gene will be evaluated in vitro by determining bacterial growth and viability of strains that over-express or have the identified genes knocked out. Aim 2) We will examine the degree to which the identified gene products examined in Aim 1 affect virulence, colonization, and antimicrobial-susceptibility in mouse models. Aim 3) We will test a mechanism of antimicrobial recognition and signal transduction in the AMP sensing HK, PhoQ. The mechanism will be tested using quantitative disulfide mapping, hydrogen-deuterium exchange, and X-ray crystallography. Aim 4) We will discover and characterize peptide and small molecule modulators of HKs. PhoQ interacts with small endogenous regulatory proteins, and the mechanism of this regulation will be elucidated. We will also discover small molecule inhibitors of this and other HKs, and use them to probe the roles of HKs in colonization and virulence in animal models.

描述（由申请人提供）：抗菌肽（AMP）是先天免疫系统的重要组成部分，可提供保护，免受细菌感染。然而，AMP作为静脉内抗生素的开发遇到了来自体内毒性和有限功效的困难。因此，在之前的资助期间，我们设计了对金黄色葡萄球菌具有稳健活性的AMP的小分子模拟物。其中一种化合物（PMX 30063）现已在人体II期试验中证明安全有效（由PolyMeidine进行）。膜结合似乎是其抗菌机制的重要组成部分。然而，这些化合物和许多AMP杀死细菌的确切机制尚未确定。从细菌对亚杀伤的转录和翻译反应的研究中可以推断出许多关于作用模式的信息 这些药剂的浓度。在与宿主的数百万年的共同进化中，细菌已经对AMP产生了复杂的反应。许多这些反应依赖于细菌组氨酸激酶（HK），这是跨膜蛋白激酶。这些蛋白质与它们的伴侣反应调节剂一起构成介导抗菌剂抗性、群体感应、营养利用和毒力的双组分系统（TCS）。我们将分析S. aureus和E.大肠杆菌对AMP、环脂肽抗生素和AMP模拟物。这些研究不仅可以更好地了解抗菌机制，还可以确定新的靶点，以增强细菌对合成药物以及我们自己的内源性AMP的敏感性。我们的目标是：目的1）AMP及其模拟物引起大肠杆菌膜应激和周质蛋白错误折叠应激相关基因的表达变化。杆菌我们将扩展这一分析，并比较S的相应响应。金黄色。通过测定过表达或敲除鉴定基因的菌株的细菌生长和活力，在体外评价由每种基因赋予的对AMP和AMP模拟物的保护或敏感性程度。目的2）我们将研究目的1中鉴定的基因产物在多大程度上影响小鼠模型中的毒力、定殖和抗菌药物敏感性。目的3）探讨AMP敏感HK、PhoQ的抗菌识别和信号转导机制。将使用定量二硫键图谱、氢-氘交换和X射线晶体学测试该机制。目的4）发现和鉴定HK的多肽和小分子调节剂。PhoQ与小的内源性调节蛋白相互作用，这种调节的机制将被阐明。我们还将发现这种和其他HK的小分子抑制剂，并使用它们来探测HK在动物模型中的定殖和毒力中的作用。