Mechanistic studies and inhibition strategies for antibiotic resistance

描述（由申请人提供）：细菌感染对人类健康构成严重威胁。此外，像流感这样的病毒感染经常伴随着细菌感染，这往往是致命的组合。由于耐药性的发展，治疗感染的选择已大大减少。这种耐药性在很大程度上是由于细菌表达的β -内酰胺酶可降解β -内酰胺类抗生素，包括青霉素、头孢菌素和“最后手段”碳青霉烯类抗生素。治疗对内酰胺耐药的革兰氏阴性感染的另一种方法是除青霉素样抗生素外联合使用对内酰胺酶抑制剂。遗憾的是，p-内酰胺酶也进化出了一种抑制剂耐药表型，能够克服这种治疗选择。水解这些抑制剂的对内酰胺酶变体被称为抑制剂耐药[3-内酰胺酶]，水解头孢菌素的被称为扩展谱对内酰胺酶（ESBLs），而水解碳青霉烯类的被称为碳青霉烯酶。本提案的总体目标是了解ESBL-，碳青霉烯酶-和抑制剂耐药p-内酰胺酶表型的结构基础，并开发新的抑制策略。我们的结构-功能研究涉及x射线和拉曼晶体学之间的新型协同作用，这种创新的跨学科方法使我们能够在x射线分析之前识别和跟踪晶体内部的反应中间体，并为实现我们的目标提供了独特的优势。目的1：进一步改进我们新设计的β -内酰胺酶抑制剂SA2-13，通过改变总体电荷和羧基连接来改善吸收和反式烯胺的稳定性。目的2：验证存在于ESBL的SHV-6， -8和-24中的D179或附近的变化已经进化到通过移动omega环来水解头孢他啶，从而延长活性位点以容纳头孢他啶的假设。目的3：验证KPC-2等A类碳青霉烯酶采用较浅的活性位点和柔性的催化870侧链高效水解碳青霉烯类的假设。目的4：验证能够进行双环化的抑制剂（如LN1-255）或fra/is-enamine抑制剂（SA2-13）能够与抑制剂耐药的A类和抑制剂不敏感的D类p-内酰胺酶形成稳定的抑制复合物的假设。β -内酰胺酶介导的抗生素耐药性对人类健康的影响是巨大的，造成数十亿美元的卫生保健费用。需要详细的了解，我们的目标结构知识将为最近在纽约爆发的KPC介导的碳青霉烯耐药肺炎克雷伯菌等病例提供分子见解。这些耐药性的见解将导致新的治疗方法，我们的目标是研究和开发新的广谱β -内酰胺酶抑制剂。

DESCRIPTION (provided by applicant): Bacterial infections pose serious threats to human health. Furthermore, viral infections such as the flu are frequently accompanied by bacterial infections which is often a deadly combination. Due to the development of resistance, the options for treating infections have dwindled substantially. This resistance is in large part due to the bacterial expression of beta-lactamases that degrade beta-lactam antibiotics including penicillins, cephalosporins, and the "last resort" carbapenem antibiotics. An alternative approach for treating p-lactam resistant gram-negative infections is co-administering a p-lactamase inhibitor in addition to penicillin-like antibiotics. Regrettably, p-lactamases have also evolved an inhibitor-resistant phenotype able to overcome this treatment option. The p-lactamase variants that hydrolyze these inhibitors are called inhibitor resistant [3-lactamases, those that hydrolyze cephalosporins are called extended-spectrum p-lactamases (ESBLs), and those that hydrolyze carbapenems are known as carbapenemases. The overarching goals of this proposal are to understand the structural basis of the phenotypes of ESBL-, carbapenemase-, and inhibitor resistant p-lactamases, and to develop novel inhibition strategies. Our structure-function studies involve a novel synergy between X-ray and Raman crystallography and this innovative inter-disciplinary approach allows us to identify and track reaction intermediates inside crystals prior to X-ray analysis and provides a unique advantage to accomplish our Aims. Aim 1: To further improve our novel designed beta-lactamase inhibitor SA2-13 by modifying the overall charge and carboxyl linker to improve uptake and trans-enamine stabilization. Aim 2: To test the hypothesis that the changes in or near D179 which are present in ESBL's SHV-6, -8, and -24 have evolved to hydrolyze ceftazidime by shifting the omega loop thereby extending the active site to accommodate ceftazidime. Aim 3: To test the hypothesis that class A carbapenemases such as KPC-2 have adopted a shallower active site and flexible catalytic 870 side chain to efficiently hydrolyze carbapenems. Aim 4: To test the hypothesis that inhibitors that either can carry out bi-cyclization (such as LN1-255) or fra/is-enamine inhibitors (SA2-13) are capable of forming stable inhibitory complexes with inhibitor-resistant class A and inhibitor-insensitive class D p-lactamases. The impact of beta-lactamase mediated antibiotic resistance on human health is enormous, costing billions of dollars in health care costs. Detailed understanding is needed and our targeted structural knowledge will provide for molecular insights into cases such as the recent outbreaks of KPC mediated carbapenem-resistant K. pneumoniae in New York. These resistance insights will lead to new therapeutic approaches and our goal is to study and develop new broad-spectrum beta-lactamase inhibitors.