Design and mechanistic studies of mimics of antimicrobial peptides

说明(申请人提供)：抗菌肽(AMP)是天然免疫系统的重要组成部分，提供保护免受细菌感染的作用。然而，AMPS作为静脉注射的发展。抗生素由于体内毒性和疗效有限而遇到了困难。因此，在之前的资助期间，我们设计了具有强大抗金黄色葡萄球菌活性的AMP的小分子模拟物。其中一种化合物(PMX30063)现已在人体第二阶段试验中被证明是安全有效的(由Polymedix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。从细菌对亚致死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结论。 这些药剂的浓度。经过数百万年与宿主的共同进化，细菌已经对AMP产生了精心的反应。其中许多反应依赖于细菌组氨酸激酶(HKS)，这是一种跨膜蛋白激酶。这些蛋白质与它们的伙伴反应调节器一起组成双组分系统(TCS)，介导抗菌耐药性、群体感应、营养利用和毒力。我们将描述金黄色葡萄球菌和大肠杆菌对AMPS、环脂肽抗生素和AMP模拟物的转录和翻译反应。这些研究不仅将提供更好的抗菌机制的了解，而且还将定义新的靶点，以提高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：1)AMPs和模拟物在大肠杆菌中引起与膜应激和周质蛋白错误折叠应激相关的基因表达的巨大变化。我们将扩展这一分析，并比较金黄色葡萄球菌的相应反应。在体外，将通过确定过度表达或敲除已识别基因的菌株的细菌生长和生存能力，来评估每个基因对AMP和AMP模拟物的保护程度或敏感性。目的2)我们将在小鼠模型中检验在目标1中检测到的已鉴定基因产物对毒力、定植和抗菌药敏感性的影响程度。目的3)我们将在AMP传感HK，PhoQ中测试抗菌素识别和信号转导的机制。这一机制将通过定量的二硫化物测绘、氢-重离子交换和X射线结晶学进行测试。目的4)我们将发现并鉴定HKS的多肽和小分子调节剂。PhoQ与小的内源调节蛋白相互作用，这种调节的机制将被阐明。我们还将发现该HKS和其他HKS的小分子抑制剂，并利用它们来探索HKS在动物模型中的定植和毒力作用。

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.