Targeting Burkholderial β-lactamases: Structure, function, and regulation

洋葱伯克霍尔德菌复合体（Bcc）是一组多药耐药（MDR）病原体， 预测在患有肺部疾病的患者中显著增加（例如，慢性阻塞性肺 疾病（COPD），囊性纤维化（CF）和哮喘）。此外，耐药的MDR Bcc分离株 所有目前推荐的疗法都在出现。不幸的是，开发新的抗MDR药物， 我们缺乏对这些独特病原体的了解。在一项回顾性研究中， 在退伍军人中观察到获得BCC感染。此外，退伍军人被证明是 不成比例地受到COPD的影响，这使他们获得Bcc感染的风险增加。 事实上，在过去十年中，世界各地爆发的BCC数量翻了一番。鉴定新 在这些高度复杂的生物体中克服抗生素耐药性的策略， 染色体是一个重大的未满足的医疗需求和重大的科学挑战。 β-内酰胺类抗生素是处方最多、最安全的一类抗生素，通常用于治疗基底细胞癌 感染.然而，β-内酰胺酶的产生是细菌中最普遍的β-内酰胺耐药机制。 Bcc的成员，其具有两种染色体编码的诱导型β-内酰胺酶blapenA和blaampC。 因此，本申请的主要目的是鉴定克服大肠杆菌中的β-内酰胺抗性的新方法。 密送在以前进行的研究的基础上，基于机制的方法将被用来选择性地 抑制Bcc中的以下蛋白：1.佩纳，一种多功能碳青霉烯酶; 2. AmpC酶是一种独特的头孢菌素酶; 3.青霉素结合蛋白（PBP）是β-内酰胺类药物的生物学靶点，其抑制作用与bla（β-内酰胺酶）有关。 内酰胺酶基因）表达;和4. PenRA，bla基因的转录调节因子。 为了实现这些目标，将使用基于机制的方法来恢复对MDR的易感性 通过检测选定的β-内酰胺类药物单独使用以及与β-内酰胺酶抑制剂联合使用，进行生化 用β-内酰胺类和β-内酰胺酶抑制剂对佩纳和AmpC进行结构分析， 的MDR Bcc，并确定所选组合的体内功效。此外，PBP之间的联系 通过鉴定哪种β-内酰胺影响bla表达，测量 β-内酰胺与PBPs的结合，通过显微镜观察暴露于β-内酰胺的细胞，以揭示β-内酰胺对PBPs的影响。 β-内酰胺类抗生素对细胞形态的影响，构建pbp基因敲除体并进行表型分析。在 此外，PenRA将在B中靶向抑制。通过使用晶体学来定义结合 PenRA效应子结合结构域（EBD）的口袋，并进行靶向小分子抑制剂文库 使用内部高通量荧光测定进行筛选。 预期的结果包括鉴定新的组合，以抑制高度耐药的BCC， 确定哪种化合物靶向佩纳、AmpC和/或PBP。此外，更好地了解 PBP抑制和bla表达之间的关系，从而使临床医生能够更好地选择 疗法PenRA的天然配体以及一组选定的小分子之间的相互作用， 类似于天然配体的化合物将被确定，从而允许鉴定靶向的“先导”化合物。 PenRA和抑制bla表达。根据本文进行的研究，退伍军人以及其他个人 与目前可用治疗相比，获得BCC感染将具有替代治疗选择， 使临床医生能够根除该微生物并获得临床治愈。

The prevalence of the Burkholderia cepacia complex (Bcc), a group of multidrug-resistant (MDR) pathogens, is predicted to significantly increase in patients with pulmonary disorders (e.g., chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and asthma) by 2024. Moreover, MDR Bcc isolates that are resistant to all currently recommended therapies are emerging. Unfortunately, the development of novel drugs against MDR Bcc is lacking as is our understanding of these unique pathogens. In a retrospective study, a 35% mortality rate in Veterans that acquired a Bcc infection was observed. Additionally, Veterans are shown to be disproportionately affected by COPD, which puts them at an increased risk of acquiring infections by Bcc. Indeed, the number of Bcc outbreaks around the world has doubled over the last decade. Identifying novel strategies to overcome antibiotic resistance in these highly complex organisms that possess multiple chromosomes is a significant unmet medical need and a substantial scientific challenge. β-Lactams are one of the most prescribed and safest class of antibiotics and are often used to treat Bcc infections. However, the production of β-lactamases is the most prevalent β-lactam-resistance mechanism in members of the Bcc, which possess two chromosomally-encoded inducible β-lactamases, blapenA and blaampC. As a result, the main objective of this application is to identify novel ways of overcoming β-lactam resistance in Bcc. Building upon studies performed previously, mechanism-based approaches will be used to selectively inhibit the following proteins in Bcc: 1. PenA, a versatile carbapenemase; 2. AmpC, a unique cephalosporinase; 3. Penicillin binding proteins (PBPs), the biological target of β-lactams and whose inhibition is linked to bla (β- lactamase gene) expression; and 4. PenRA, the transcription regulator of bla genes. To address these objectives, a mechanism-based approach will be used to restore susceptibility to MDR Bcc by testing selected β-lactams alone and in combination with β-lactamase inhibitors, performing biochemical and structural analysis of PenA and AmpC with the β-lactams and β-lactamase inhibitors, analyzing the genomes of MDR Bcc, and determining the in vivo efficacy of selected combinations. Moreover, the link between PBP inhibition and bla expression will be deciphered by identifying which β-lactams effect bla expression, measuring the binding of β-lactams to PBPs, visualizing cells exposed to β-lactams via microscopy to reveal the impact of β-lactams on cell morphology, and constructing pbp gene knockouts and assessing their phenotypes. In addition, PenRA will be targeted for inhibition in B. multivorans by using crystallography to define the binding pocket of the PenRA effector binding domain (EBD) and conducting a targeted small molecule inhibitor library screen using an in-house high-throughput fluorescence assay. The anticipated outcomes include identifying novel combinations to inhibit highly drug resistant Bcc by determining which compounds target PenA, AmpC, and/or PBPs. Moreover, a greater understanding of the link between PBP inhibition and bla expression will be gained, thus allowing clinicians to make better choices for therapy. The interactions between native ligand of PenRA as well as a selected panel of small molecules which resemble the native ligand will be determined, thus allowing for the identification of “lead” compounds to target PenRA and inhibit bla expression. Based on the studies conducted herein, Veterans as well as other individuals that acquire a Bcc infection will have alternative therapeutic options compared to what is currently available, enabling clinicians to eradicate the organism and obtain clinical cure.