Interruption of Signaling-Mediated Bacterial Persistent Infections

描述(由申请人提供)：持续性和慢性感染通常对抗生素耐药，这是由于一群细胞对抗生素的耐受性，这些细胞不是抗生素耐药突变株，而是在抗生素杀灭中幸存下来的“休眠”细胞。我们的发现表明，铜绿假单胞菌和伯克霍尔德氏菌分泌一种小分子，作为一种持久性的信息化学物质，传递这些耐药持久(AT/P)细胞的积累和变化，这些变化对病原体适应至关重要，对慢性感染很重要。我们的目标是通过引入一种治疗方法来实现持续感染干预措施的范式转变，这种治疗方法使用我们已经确定的化合物来扰乱诱导AT/P细胞形成的细菌信号；为了实现这一目标，我们将使用适应的小鼠感染模型在体内提炼和验证先导化合物。我们的方法与传统的抗菌疗法有根本的不同，因为它专门针对在抗生素治疗中幸存下来的AT/P亚群细胞(和宿主防御杀伤机制)，这些细胞最终要对持续和复发的感染负责。我们建议通过使用铜绿假单胞菌的实验来开发这种方法，铜绿假单胞菌是一种顽固的革兰氏阴性细菌，无法被抗生素根除，形成生物膜，并例证目前临床上存在问题的病原体。在R21阶段的目标1研究中，我们将使用结构-活性关系(SAR)数据来精炼我们已经确定的特别有希望的第一代化合物的化学成分。我们上一代的一系列结构相关的药物已经证实了这种方法的可行性，这些药物可以阻断Pr-AT/P信号分子的合成，并降低体内的毒力。在目标2中，我们将对第二代化合物进行一系列微生物、细胞和生化评估，以评估它们的相对IC50值，以及它们与不同类别的抗生素联合使用或单独使用时对几种临床菌株(包括泛耐药和多药耐药菌株)的有效性，以及它们扰乱信号分子合成和防止由此导致的DNA拓扑失衡和翻译效应的能力，我们已经证明这些效应发生在转换到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的情况下进行。在目标3中，我们将评估在R21阶段确定的最有希望的第二代化合物的药理效率特性。在目标4中，将在单一和多菌浮游和生物膜环境中测试化合物对与铜绿假单胞菌共存并类似形成AT/P细胞(即鲍曼不动杆菌、肺炎克雷伯氏菌和伯克霍尔德氏菌)的抗药性和耐受性病原体的有效性。将进行联合药物分析，以确定我们的先导分子是否提高了生物膜对抗生素的清除能力。然后，我们将在我们开发的已建立的鼠标模型中验证高度优先的高级线索。这些研究的总体目标是仔细评估以AT/P细胞为靶标的铅小分子作为一种新的方法来干预到目前为止无法治疗的慢性和持续性感染的潜在效用。这些抗AT/P分子可以与传统的抗生素治疗相结合，以达到最佳效果。

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.