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

洋葱伯克霍尔德氏菌复合体(BCC)是一组多药耐药(MDR)病原体，其流行率为 预计肺部疾病患者(例如慢性阻塞性肺疾病)的发病率显著增加 疾病(COPD)、囊性纤维化(CF)和哮喘)。此外，耐药的BCC分离株对 目前推荐的所有疗法都在涌现。不幸的是，抗多药耐药新药的开发 BCC缺乏，我们对这些独特的病原体的了解也是如此。在一项回顾研究中，35%的死亡率 在获得基底细胞癌感染的退伍军人中观察到。此外，退伍军人被证明是 不成比例地受到慢性阻塞性肺病的影响，这使他们感染BCC的风险增加。 事实上，在过去十年中，世界各地的BCC疫情数量翻了一番。辨别小说 在这些高度复杂的生物中克服抗生素耐药性的策略 染色体是一个重大的未得到满足的医学需求，也是一个重大的科学挑战。 β-Lactam是处方最多、最安全的抗生素之一，经常用于治疗基底细胞癌 感染。然而，β-内酰胺酶的产生是β-内酰胺类药物最普遍的耐药机制 BCC的成员，拥有两种染色体编码的诱导型β-内酰胺酶，blapenA和blaampC。 因此，这项应用的主要目的是寻找克服β-内酰胺类药物耐药性的新方法。 密件抄送。在以前进行的研究的基础上，将有选择地使用基于机制的方法 抑制BCC中的下列蛋白：1.多功能碳青霉烯酶Pena，2.独特的头孢菌素酶AmpC； 3.青霉素结合蛋白(PBPS)，是β-内酰胺类药物的生物靶点，其抑制作用与BLA(β- 内酰胺酶基因)的表达；4.BLA基因的转录调控因子PENRA。 为实现这些目标，将使用一种基于机制的办法来恢复对多药耐药性的敏感性 通过单独检测选定的β-内酰胺类药物并与β-内酰胺酶抑制剂联合检测，进行生化 用β-内酰胺类和β-内酰胺酶抑制剂对Pena和AmpC进行结构分析，对基因组进行分析 MDR BCC，并测定所选组合的体内疗效。此外，PBP之间的联系 抑制和bla表达将通过识别哪些β-内酰胺类药物影响bla表达，测量 β-内酰胺类药物与PBPS的结合，通过显微镜观察暴露于β-内酰胺类药物的细胞 β-内酰胺类药物对细胞形态的影响，构建pBP基因敲除系统并对其表型进行鉴定。在……里面 此外，通过使用结晶学来定义结合，PENRA将成为多食性芽孢杆菌的抑制靶点。 PENRA效应结合域(EBD)的口袋并进行靶向小分子抑制物库 使用内部高通量荧光分析进行筛查。 预期的结果包括确定新的组合，通过以下方式抑制高度耐药的基底细胞癌 确定哪些化合物针对Pena、AMPC和/或PBPs。此外，对这种联系的更好的理解 将在PBP抑制和BLA表达之间获得，从而允许临床医生对 心理治疗。PENRA天然配体与一组选定的小分子之间的相互作用 将确定与天然配基相似的化合物，从而能够识别目标为“铅”的化合物 PENRA并抑制bla的表达。根据这里进行的研究，退伍军人以及其他个人 与目前可用的治疗方法相比，感染基底细胞癌的患者将有其他治疗选择， 使临床医生能够根除这种微生物，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.