Combating Bacterial Drug Resistance by Targeting the Enzymes of Evolution

通过针对进化酶来对抗细菌耐药性

• 批准号: 8355227
• 负责人: Rahul Manu Kohli
• 金额: $ 222.47万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8355227
• 关键词: Active Sites; Address; Bacterial Drug Resistance; Belief; Biology; Chemicals; Cleaved cell; Cystic Fibrosis; DNA-Directed DNA Polymerase; Development; Disease; Drug resistance; Enzymatic Biochemistry; Enzymes; Equilibrium; Evolution; Genome; Hand; Health; Human; Investigation; Molecular; Multi-Drug Resistance; Mutate; Mutation; Organism; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Polymerase; Proteolysis; Pseudomonas aeruginosa; Public Health; Research; Resistance; Resistance development; SOS Response; Scourge; Shapes; Son of Sevenless Proteins; Stress; Techniques; Therapeutic; Translating; abstracting; antimicrobial; antimicrobial drug; base; biological adaptation to stress; combat; genetic regulatory protein; inhibitor/antagonist; innovation; mortality; novel strategies; nucleotide analog; pathogen; preference; prevent; programs; public health relevance; response; small molecule

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.

描述（由申请人提供） 摘要：病原体能够迅速对我们最好的抗菌剂产生耐药性，这是一项艰巨的治疗挑战，对人类健康造成毁灭性后果。这一问题因新抗菌药物开发方面的创新差距而进一步加剧，部分原因是人们认为对新药的耐药性是不可避免的。的 日益高涨的抵制浪潮加上缺乏解决问题的新办法，造成了一种特别可怕的局面。为了满足这一需求，我们寻求从根本上改变对抗耐药性的模式，瞄准使病原体发生突变和产生耐药性的途径。 多药耐药病原体的这种影响很好地例证了铜绿假单胞菌，铜绿假单胞菌是一种可以在囊性纤维化的病程中变得越来越耐药的生物体，通常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（称为SOS途径）有关。在我们提出的研究计划中，我们的目标是关键的监管和效应酶，莱克萨和DinB，参与这一促突变途径。在其基础状态下，调节蛋白莱克萨，一种双功能阻遏蛋白酶，将应激反应维持在“关闭”状态。当感觉到压力时，莱克萨会分裂自己，“开启”压力反应。作为这种反应的一部分，DinB和其他易错DNA聚合酶被诱导，促进基因组中突变水平的增加。我们的策略是询问和扰乱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干扰这些酶进化的小分子的发现。在建立了莱克萨自身蛋白水解的分子基础之后，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，我们建议定义易错聚合酶DinB的开放活性位点。我们将利用其耐受性来发现可以特异性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如何发生突变的理解，这是一个具有根本科学重要性的问题。通过这种方式，我们的研究将塑造我们如何思考基因组中稳定性和适应性的平衡要求。最重要的是，我们的研究解决了迫切需要创新替代方法来打击耐药性祸害的问题。 公共卫生相关性：鉴于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具威胁性。我们建议寻求一种创新的方法来解决这个问题，通过靶向允许病原体适应，进化并从而获得耐药性的途径。