Interruption of Signaling-Mediated Bacterial Persistent Infections

描述（由申请人提供）：由于非抗生素耐药突变体细胞亚群的抗生素耐受性，持续和慢性感染通常对抗生素难治性，而是“休眠”细胞在抗生素杀死后存活。我们的研究结果表明，铜绿假单胞菌和伯克霍尔德菌会分泌一种小分子，作为一种持久性“信息化学物质”，表明这些耐抗生素持久性（AT/P）细胞的积累和变化对病原体适应和慢性感染至关重要。我们的目标是通过引入一种使用我们已经确定的化合物破坏诱导AT/P细胞形成的细菌信号的治疗方法，实现持续感染干预的范式转变；为了实现这一目标，我们将在体内使用适应的小鼠感染模型来改进和验证先导化合物。我们的方法与传统的抗微生物疗法有着根本的不同，因为它专门针对在抗生素治疗（和宿主防御杀伤机制）中存活的AT/P细胞亚群，这些细胞最终导致持续和复发性感染。我们建议通过使用铜绿假单胞菌（P. aeruginosa）的实验来发展这种方法，这是一种顽固性革兰氏阴性细菌，可以抵抗抗生素的根除，形成生物膜，并举例说明当前临床有问题的病原体。在R21期Aim 1研究中，我们将使用构效关系（SAR）数据来改进我们已经确定的特别有前途的第一代化合物的化学成分。这种方法的可行性已经通过我们上一代的一系列结构相关的药物来建立，这些药物可以阻断pr-AT/P信号分子的合成并降低体内毒力。在目标2中，我们将对第二代化合物进行一系列微生物学、细胞学和生化评估，以评估它们与不同类别的抗生素联合使用或单独使用时的相对IC50值及其对几种临床分离株（包括泛耐药和多重耐药分离株）的功效。以及它们破坏信号分子合成的能力，并防止由此导致的DNA拓扑失衡和翻译效应，我们已经证明，这些失衡发生在已经过渡到AT/P状态的细胞中。R33阶段（目标3和4）将在我们明确定义的里程碑实现后进行。在Aim 3中，我们将评估在R21期鉴定的最有希望的第二代化合物的药理学效率特性。在Aim 4中，将在单微生物和多微生物浮游生物和生物膜环境中测试化合物对耐药、耐药病原体的功效，这些病原体与人类感染中的铜绿假单胞菌共存，并形成类似的AT/P细胞（即鲍曼不动杆菌、肺炎克雷伯菌和伯克霍尔德菌）。联合药物试验将进行，以确定我们的铅分子是否提高抗生素对生物膜的清除。高度优先的高级线索将在我们开发的已建立的小鼠模型中进行验证。这些研究的总体目标是仔细评估靶向AT/P细胞的铅小分子作为干预迄今无法治疗的慢性和持续性感染的新方法的潜在效用。这些抗at /P分子可与传统抗生素疗法联合使用以获得最佳效果。

DESCRIPTION (provided by applicant): Persistent and chronic infections are often refractory to antibiotics due to antibiotic tolerance of a subpopulation of cells that are not antibiotic resistant mutants, but rather are "dormant" cells that survive antibiotic killing. Our findings show that Pseudomonas aeruginosa and Burkholderia species excrete a small molecule that serves as a persistence "infochemical" that signals the accumulation of these antibiotic tolerant persister (AT/P) cells and changes that are critical for pathogen adaptation and important for chronic infection. Our aim is to achieve a paradigm shift in persistent infection interventions by introducing a treatment that disrupts the bacterial signaling that induces AT/P cell formation using compounds we have identified; to achieve this aim we will refine and validate lead compounds in vivo, using adapted mouse models of infection. Our approach is fundamentally different from traditional antimicrobial therapies as it specifically targets the AT/P subpopulation of cells that survive antibiotic treatment (and host defense killing mechanisms), and that are ultimately responsible for persistent and relapsing infections. We propose to develop this approach through experiments employing P. aeruginosa, a recalcitrant Gram-negative bacterium that defies eradication by antibiotics, forms biofilms, and exemplifies current clinical problematic pathogens. In the R21 phase Aim 1 studies, we will use structure-activity relationship (SAR) data to refine the chemical compositions of the particularly promising 1st generation compounds we have identified. The feasibility of this approach has been established by our prior generation of a series of structurally related agents that block the synthesis of a pr-AT/P signaling molecule and reduce virulence in vivo. In Aim 2, we will perform a series of microbiological, cellular, and biochemical evaluations of the 2nd generation compounds to assess their relative IC50 values and their efficacy against several clinical isolates (including pan-resistant and multi-drug resistant isolates) when used in combination with different classes of antibiotics or alone, as well as their ability to disrupt the synthesis of the signaling molecule and prevent the resultant imbalance in DNA topology and translational effects that we have demonstrated to occur in cells that have transitioned to the AT/P state. The R33 phase (Aims 3 and 4) will be undertaken if our well-defined milestones are achieved. In Aim 3, we will assess the pharmacological efficiency properties of the most promising 2nd generation compounds identified in the R21 phase. In Aim 4, the compounds' efficacies against drug resistant, tolerant pathogens that co-exist with P. aeruginosa in human infections and similarly form AT/P cells (i.e. Acinetobacter baumannii, Klebsiella pneumoniae, and Burkholderia species) will be tested in mono- and polymicrobial planktonic and biofilm settings. Combination drug assays will be performed to determine whether our lead molecules improve antibiotic clearance of biofilms. Highly prioritized advanced leads will then be validated in established mouse models that we have developed. The overall goal of these studies is to carefully assess the potential utility of lead small molecules that target AT/P cells as a new way to intervene against chronic and persistent infections that have thus far been untreatable. These anti-AT/P molecules may be combined with traditional antibiotic therapies for optimal effectiveness.