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是抗生素难治性的，这是由于细胞亚群的抗生素耐受性，这些细胞不是抗生素耐药突变体，而是在抗生素杀死后存活的“休眠”细胞。我们的研究结果表明，铜绿假单胞菌和伯克霍尔德氏菌分泌一种小分子，作为持久性“信息化学品”，信号这些抗生素耐受持留（AT/P）细胞的积累和变化，是病原体适应的关键和重要的慢性感染。我们的目标是通过引入一种治疗方法来实现持续性感染干预的范式转变，该治疗方法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传导;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完善和验证先导化合物。我们的方法从根本上不同于传统的抗微生物疗法，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，并且最终导致持续性和复发性感染。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耐药性革兰氏阴性细菌，无法通过抗生素根除，形成生物膜，并消除当前临床上有问题的病原体。在R21阶段目标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组成。这种方法的可行性已经通过我们的前代一系列结构相关的药物建立，这些药物阻断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、细胞和生化评价，以评估其相对IC 50值及其对几种临床分离株的疗效（包括泛耐药和多重耐药分离株）与不同类别的抗生素联合使用或单独使用时，以及它们破坏信号分子的合成并防止DNA拓扑结构和翻译效应中产生的不平衡的能力，我们已经证明这种不平衡发生在已经转变为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。在目标3中，我们将评估在R21阶段确定的最有前途的第二代化合物的药理学有效性。在目的4中，将在单微生物和多微生物共生和生物膜环境中测试化合物对与铜绿假单胞菌共存于人类感染中并类似地形成AT/P细胞的耐药、耐受病原体（即鲍曼不动杆菌、肺炎克雷伯氏菌和伯克霍尔德氏菌属）的功效。将进行组合药物测定以确定我们的先导分子是否改善生物膜的抗生素清除。高度优先的先进的线索，然后将在建立的小鼠模型，我们已经开发的验证。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分子可以与传统的抗生素疗法组合以获得最佳效果。

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.