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

靶向伯克霍尔德β-内酰胺酶：结构、功能和调节

基本信息

批准号：
10515668
负责人：
Curtis Donskey
金额：
--
依托单位：
LOUIS STOKES CLEVELAND VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-10-01 至 2023-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10515668
关键词：
Address Affect Affinity Antibiotic Resistance Antibiotic Therapy Antibiotics Asthma Award Bacteremia Binding Biochemical Biological Biological Assay Burkholderia Infections Burkholderia cepacia complex Ceftazidime Cells Cellular Morphology Cephalosporinase Cessation of life Chromosomes Chronic Chronic Obstructive Pulmonary Disease Clinical Complex Crystallization Crystallography Cystic Fibrosis Development Disease Outbreaks Drug resistance Electrophoretic Mobility Shift Assay Exposure to Fluorescence Gene Proteins General Population Genes Genetic Transcription Genome Goals Healthcare Systems Imipenem Individual Infection Isoelectric Focusing Kinetics Knock-out Lead Libraries Ligands Link Lung diseases Measures Mediating Medical Membrane Microscopy Modeling Multi-Drug Resistance Mutation New Agents Organism Outcome PPBP gene Patients Penicillin-Binding Proteins Persons Phase-Contrast Microscopy Phenotype Piperacillin Pneumonia Predisposition Preparation Prevalence Production Proteins Recommendation Regulation Repression Resistance Retrospective Studies Risk Spectrometry, Mass, Electrospray Ionization Structure Testing Therapeutic VDAC1 gene Veterans Visualization Western Blotting beta-Lactam Resistance beta-Lactamase beta-Lactams carbapenemase drug resistant pathogen evaporation in vivo in vivo evaluation inhibitor knockout gene member molecular mass molecular modeling mortality multi-drug resistant pathogen novel novel strategies novel therapeutics pathogen resistance mechanism small molecule small molecule inhibitor whole genome

项目摘要

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.

洋葱伯克霍尔德菌复合体 (Bcc) 是一组多重耐药 (MDR) 病原体，其流行率是预计患有肺部疾病（例如慢性阻塞性肺疾病）的患者数量将显着增加到 2024 年，疾病（COPD）、囊性纤维化（CF）和哮喘）。此外，耐多药 Bcc 分离株对不幸的是，目前所有推荐的治疗方法都在不断涌现，针对MDR的新药正在开发中。我们缺乏对 BCC 的了解，在一项回顾性研究中，死亡率为 35%。在退伍军人中观察到了 Bcc 感染。受慢性阻塞性肺病 (COPD) 的影响尤为严重，这使得他们感染密件抄送 (Bcc) 的风险增加。事实上，在过去十年中，全球密件抄送事件的爆发数量增加了一倍。克服这些高度复杂的生物体中的抗生素耐药性的策略，这些生物体具有多种染色体是一项重大的未满足的医疗需求，也是一项重大的科学挑战。 β-内酰胺是最常使用且最安全的一类抗生素，通常用于治疗基底细胞癌然而，β-内酰胺酶的产生是最普遍的β-内酰胺抗性机制。 Bcc 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，本申请的主要目的是确定克服β-内酰胺耐药性的新方法密件抄送根据之前进行的研究，将有选择地使用基于机制的方法抑制 Bcc 中的以下蛋白质： 1. PenA，一种多功能碳青霉烯酶； 2. AmpC，一种独特的头孢菌素酶； 3. 青霉素结合蛋白 (PBP)，β-内酰胺的生物靶标，其抑制作用与 bla (β- 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 的敏感性通过单独测试选定的 β-内酰胺以及与 β-内酰胺酶抑制剂组合进行 BCC，进行生化检查使用 β-内酰胺和 β-内酰胺酶抑制剂对 PenA 和 AmpC 进行结构分析，分析基因组 MDR Bcc，并确定所选组合的体内功效此外，PBP 之间的联系。通过鉴定哪些 β-内酰胺影响 bla 表达、测量 β-内酰胺与 PBP 的结合，通过显微镜观察暴露于 β-内酰胺的细胞，以揭示 β-内酰胺对细胞形态的影响，构建 pbp 基因敲除并评估其表型。此外，PenRA 将通过使用晶体学来定义结合来抑制 B. multivorans PenRA 效应器结合域 (EBD) 的口袋并构建靶向小分子抑制剂库使用内部高通量荧光测定进行筛选。预期结果包括确定新的组合，通过以下方式抑制高度耐药的 Bcc：确定哪些化合物以 PenA、AmpC 和/或 PBP 为目标此外，更好地了解其中的联系。将获得 PBP 抑制和 bla 表达之间的差异，从而可以做出更好的选择 PenRA 的天然配体与一组选定的小分子之间的相互作用。类似于天然配体将被确定，从而允许识别“先导”化合物来靶向 PenRA 并抑制 bla 表达，基于本文进行的研究，退伍军人以及其他个体。与目前可用的治疗方案相比，获得 BCC 感染的患者将有其他治疗选择，使暴徒能够根除该生物体并获得临床治愈。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

类鼻疽伯克霍尔德氏菌的膜结合 PenA β-内酰胺酶。

DOI：
10.1128/aac.02444-15
发表时间：
2015
期刊：
Antimicrobial agents and chemotherapy
影响因子：
4.9
作者：
Randall,LinnellB;Dobos,Karen;Papp-Wallace,KrisztinaM;Bonomo,RobertA;Schweizer,HerbertP
通讯作者：
Schweizer,HerbertP

The Class A β-Lactamase Produced by Burkholderia Species Compromises the Potency of Tebipenem against a Panel of Isolates from the United States.

DOI：
10.3390/antibiotics11050674
发表时间：
2022-05-17
期刊：
Antibiotics (Basel, Switzerland)
影响因子：
0
作者：
通讯作者：

Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans.

DOI：
10.1016/j.diagmicrobio.2018.06.005
发表时间：
2018-11
期刊：
Diagnostic microbiology and infectious disease
影响因子：
2.9
作者：
Becka SA;Zeiser ET;Marshall SH;Gatta JA;Nguyen K;Singh I;Greco C;Sutton GG;Fouts DE;LiPuma JJ;Papp-Wallace KM
通讯作者：
Papp-Wallace KM

New β-Lactamase Inhibitors in the Clinic.

DOI：
10.1016/j.idc.2016.02.007
发表时间：
2016-06
期刊：
Infectious disease clinics of North America
影响因子：
4.4
作者：
Papp-Wallace KM;Bonomo RA
通讯作者：
Bonomo RA

Assessing the Potency of β-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.

DOI：
10.1021/acsinfecdis.0c00682
发表时间：
2021-04-09
期刊：
ACS infectious diseases
影响因子：
5.3
作者：
Nukaga M;Yoon MJ;Taracilia MA;Hoshino T;Becka SA;Zeiser ET;Johnson JR;Papp-Wallace KM
通讯作者：
Papp-Wallace KM