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，病毒感染，如流感，经常伴随着细菌感染，这往往是一个致命的组合。由于耐药性的发展，治疗感染的选择大大减少。这种耐药性在很大程度上是由于细菌表达降解β-内酰胺抗生素的β-内酰胺酶，所述β-内酰胺抗生素包括青霉素、头孢菌素和“最后手段”碳青霉烯类抗生素。治疗β-内酰胺耐药革兰氏阴性菌感染的另一种方法是除青霉素样抗生素外，还联合使用β-内酰胺酶抑制剂。令人遗憾的是，β-内酰胺酶也进化出了能够克服这种治疗选择的耐药表型。水解这些抑制剂的β-内酰胺酶变体称为抑制剂耐药β-内酰胺酶，水解头孢菌素的β-内酰胺酶称为超广谱β-内酰胺酶（ESBL），水解碳青霉烯类的β-内酰胺酶称为碳青霉烯酶。本提案的总体目标是了解ESBL、碳青霉烯酶和抑制剂耐药β-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。目标1：为了进一步改进我们新设计的β-内酰胺酶抑制剂SA 2 -13，通过修饰总电荷和羧基接头来改善摄取和反式烯胺稳定性。目标二：为了验证这样的假设：ESBL的SHV-6、-8和-24中存在的D179或其附近的变化已演变为通过移动欧米茄环来水解头孢他啶，从而扩展活性位点以容纳头孢他啶。目标3：为了验证A类碳青霉烯酶（如KPC-2）采用较浅的活性位点和灵活的催化870侧链来有效水解碳青霉烯类的假设。目标4：为了检验以下假设，即可以进行双环化的抑制剂（如LN 1 -255）或反式-异烯胺抑制剂（SA 2 -13）能够与耐药的A类和不敏感的D类β-内酰胺酶形成稳定的抑制复合物。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，花费了数十亿美元的医疗保健费用。我们需要详细的了解，我们有针对性的结构知识将提供分子见解的情况下，如最近爆发的KPC介导的碳青霉烯耐药K。纽约的肺炎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.