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

• 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
• 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

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.

Burkholderia cepacia Complex（BCC）的患病率是一组多药（MDR）病原体 预计肺部疾病患者的预计会显着增加（例如，慢性阻塞性肺 到2024年，疾病（COPD），囊性纤维化（CF）和哮喘。 目前所有推荐的疗法都将出现。不幸的是，针对MDR的新型药物的开发 BCC缺乏我们对这些独特病原体的理解。在回顾性研究中，死亡率为35％ 在获得BCC感染的退伍军人中。另外，退伍军人被证明是 受COPD的影响不成比例，这使他们面临BCC获取感染的风险。 确实，在过去十年中，世界各地的BCC爆发次数增加了一倍。识别小说 在这些高度复杂的生物中克服抗生素耐药性的策略，该生物具有多种潜力 染色体是一个巨大的未满足的医疗需求，也是一个重大的科学挑战。 β-内酰胺是处方最安全，最安全的抗生素之一，通常用于治疗BCC 感染。但是，β-内酰胺酶的产生是最普遍的β-内酰胺耐药机制 BCC的成员，具有两个染色体编码的诱导β-内酰胺酶Blapena和Blaampc。 结果，该应用的主要目的是确定克服β-内酰胺抗性的新方法 BCC。在先前进行的研究的基础上，将使用基于机制的方法进行选择性 抑制BCC中的以下蛋白质：1。Pena，一种多功能碳酸酶酶； 2。AMPC，一种独特的头孢菌素酶； 3。青霉素结合蛋白（PBP），β-内酰胺的生物学靶标，其抑制作用与BLA有关（β- 乳糖酶基因）表达；和4。Penra，BLA基因的转录调节剂。 为了解决这些目标，将使用基于机制的方法来恢复MDR的敏感性 通过单独测试选定的β-内酰胺，并与β-内酰胺酶抑制剂结合进行BCC，进行生化 PENA和AMPC与β-内酰胺和β-内酰胺酶抑制剂的结构分析，分析基因组 MDR BCC，并确定选定组合的体内效率。而且，PBP之间的链接 抑制作用和BLA表达将通过识别哪些β-内酰胺对BLA表达，测量哪些β-内酰胺 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-内酰胺在细胞形态上，构建PBP基因敲除并评估其表型。在 此外，PENRA将通过使用晶体学定义结合来将PENRA作为抑制作用。 Penra效应子结合域（EBD）的口袋，并进行靶向小分子抑制剂库 使用内部高通量荧光测定法进行筛选。 预期的结果包括识别新型组合以抑制高度耐药的BCC 确定哪种化合物靶标PENA，AMPC和/或PBP。而且，对链接有更深入的了解 在PBP抑制和BLA表达之间将获得获得，从而使临床医生可以更好地选择 治疗。 Penra天然配体之间的相互作用以及选定的小分子面板 将确定类似于天然配体的人，从而允许识别“铅”化合物的靶向 PENRA并抑制BLA表达。根据这里进行的研究，退伍军人以及其他人 与当前可用的相比，获得BCC感染将具有替代性治疗选择 使临床医生能够消除生物体并获得临床治疗。

项目成果

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)

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