Combating Bacterial Drug Resistance by Targeting the Enzymes of Evolution

描述(由申请人提供) 摘要：病原体对我们最好的抗菌药迅速产生抗药性的能力构成了一个可怕的治疗挑战，给人类健康带来了毁灭性的后果。新抗菌剂开发方面的创新差距进一步加剧了这个问题，部分原因是人们相信，对新药的耐药性是不可避免的。这个 抵抗浪潮的上升，加上缺乏解决问题的新方法，造成了一种特别可怕的局面。为了回应这一需求，我们寻求通过瞄准允许病原体突变和进化耐药的途径，从根本上改变抗药性的范式。多重耐药病原体的这种影响以铜绿假单胞菌为例，这种微生物在囊性纤维化的疾病过程中会变得越来越耐药，往往极大地限制了治疗选择，并导致死亡。铜绿假单胞菌的进化性和耐药性与控制应激反应的途径有关，称为SOS途径。在我们提出的研究计划中，我们的目标是参与这一促突变途径的关键调节和效应酶LexA和DinB。在它的基础状态下，调节蛋白LexA，一种双功能的抑制-蛋白水解酶，维持压力反应在“关”的状态。当感受到压力时，Lexa会自我分裂，开启压力反应。作为这种反应的一部分，DinB和其他容易出错的DNA聚合酶被诱导，促进了基因组中更高水平的突变的引入。我们的策略是审问和扰乱SOS反应的两端。我们建议使用化学生物学、酶学和生物物理技术来探索这些酶的底物偏好，并将这些发现转化为可以扰乱这些酶进化的小分子的发现。在建立了LexA自我蛋白分解的分子基础后，我们将发现自我切割的抑制剂，它可以防止SOS开关被打开。同时，我们建议定义容易出错的聚合酶DinB的开放活性位点。我们将利用它的耐受性来发现能够特异性地抑制这种进化聚合酶的核苷酸类似物。有了化学探针，我们将直接评估抗进化小分子对铜绿假单胞菌获得耐药性的影响。我们提出的研究将有助于推进我们对细菌病原体中突变是如何产生的理解，这是一个具有基础科学意义的问题。通过这种方式，我们的研究将塑造我们对基因组稳定性和适应性平衡要求的思考方式。最重要的是，我们的研究解决了抗击抗药性祸害的创新替代方法的迫切需要。 公共卫生相关性：考虑到在发现新的抗微生物药物方面的创新差距，耐药病原体日益严重的公共卫生问题更加具有威胁性。我们建议寻求一种创新的方法来解决这个问题，通过瞄准使病原体适应、进化并从而获得耐药性的途径。

DESCRIPTION (Provided by the applicant) Abstract: The ability for pathogens to rapidly develop resistance to our best antimicrobials makes for a formidable treatment challenge, with devastating consequences to human health. The problem has been further exacerbated by the innovation gap in the development of new antimicrobials, partially resulting from the belief that resistance to new drugs is inevitable. The combination of a rising tide of resistance and the lack of novel approaches to the problem makes for a particularly dire situation. In response to this need, we seek to fundamentally alter the paradigm for combating drug resistance by targeting the very pathways that allow pathogens to mutate and evolve drug resistance. This impact of multidrug resistant pathogens is well exemplified by Pseudomonas aeruginosa, an organism that can become increasingly drug resistant through the disease course of cystic fibrosis, often greatly limiting therapeutic options and contributing to mortality. Evolvability and drug resistance in P. aeruginosa are tied to the pathway that governs stress responses, known as the SOS pathway. In our proposed research program, we aim to target key regulatory and effector enzymes, LexA and DinB, involved in this pro-mutagenic pathway. In its basal state, the regulatory protein LexA, a bi-functional repressor-protease, maintains stress response in an ""off"" state. When stress is sensed, LexA cleaves itself to turn ""on"" stress responses. As part of this response, DinB and other error-prone DNA polymerases are induced promoting the introduction of increased levels of mutations in the genome. Our strategy is to interrogate and perturb both ends of the SOS response. We propose to use chemical biology, enzymology and biophysical techniques to explore the substrate preferences for these enzymes and translate these findings into the discovery of small molecules that can perturb these enzymes of evolution. After establishing the molecular basis for LexA auto-proteolysis, we will uncover inhibitors of self-cleavage that can prevent the SOS switch from being flipped ""on"". In parallel, we propose to define the open active site of the error-prone polymerase DinB. We will exploit its tolerance to discover nucleotide analogs which can specifically inhibit this evolutionary polymerase. With chemical probes at hand, we will directly evaluate the impact of anti-evolutionary small molecules on the acquisition of drug resistance in P. aeruginosa. Our proposed studies will help advance our understanding of how mutations arise in bacterial pathogens, a question of fundamental scientific importance. In this manner, our investigations will shape how we think about the balanced requirements for stability and adaptability in the genome. Most importantly, our studies address the exigent need for innovative alternative approaches to combating the scourge of drug resistance. Public Health Relevance: The mounting public health problem of drug-resistant pathogens is yet more menacing given the innovation gap in the discovery of new antimicrobial drugs. We propose to pursue an innovative approach to this problem, by targeting the very pathways that allow a pathogen to adapt, evolve and thereby acquire drug resistance.