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药物产生耐药性，这给治疗带来了巨大的挑战，对人类健康造成了毁灭性的后果。新抗菌药物开发的创新差距进一步加剧了这一问题，部分原因是人们相信新药耐药性是不可避免的。这 日益高涨的抵抗浪潮和缺乏解决该问题的新方法相结合，造成了特别可怕的局面。为了满足这一需求，我们寻求通过针对病原体突变和产生耐药性的途径，从根本上改变对抗耐药性的范式。 铜绿假单胞菌就充分体现了多重耐药病原体的影响，这种生物体在囊性纤维化病程中耐药性不断增强，常常极大地限制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，我们的目标是针对参与这一促诱变途径的关键调节酶和效应酶 LexA 和 DinB。在其基础状态下，调节蛋白 LexA（一种双功能阻遏蛋白蛋白酶）将应激反应维持在“关闭”状态。当感受到压力时，LexA 会自我分裂以“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，促进基因组中引入更高水平的突变。我们的策略是询问并干扰 SOS 响应的两端。我们建议利用化学生物学、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发现可以扰乱这些酶进化的小分子。在建立了 LexA 自动蛋白水解的分子基础后，我们将发现可以防止 SOS 开关“打开”的自裂解抑制剂。与此同时，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。借助现有的化学探针，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加深我们对细菌病原体突变如何产生的理解，这是一个具有基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，我们的研究解决了对抗耐药性祸害的创新替代方法的迫切需求。 公共卫生相关性：鉴于新抗菌药物发现方面的创新差距，耐药病原体日益严重的公共卫生问题变得更具威胁性。我们建议通过针对病原体适应、进化并从而获得耐药性的途径，寻求一种创新方法来解决这个问题。