Development of AK-based assays for antimicrobial screening

开发基于 AK 的抗菌药物筛查检测方法

基本信息

批准号：
8631305
负责人：
Paul Dunman
金额：
$ 37.6万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-20 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8631305
关键词：
Acinetobacter baumannii Address AlamarBlue Animal Model Anti-Bacterial Agents Anti-Infective Agents Antibiotic Resistance Antibiotic Therapy Antibiotics Attenuated Bacteria Bacterial Infections Biological Assay Biomass Cell Death Cells Cessation of life Culture Media Data Detection Development Devices Disease Dyes Enterobacter Enterococcus faecium Enzymes Genus Mycobacterium Goals Gold Gram-Negative Bacteria Growth Healthcare Implant Infection Klebsiella pneumonia bacterium Laboratories Legal patent Libraries Life Measurable Measures Medical Medical Device Medicine Membrane Microbial Biofilms Modern Medicine Molecular Mycobacterium smegmatis Mycobacterium tuberculosis NIH Program Announcements Operative Surgical Procedures Organism Performance Pharmaceutical Preparations Phase Population Process Pseudomonas aeruginosa Publications Pulmonary Tuberculosis Reading Recurrence Relative (related person)Reporter Reporting Staphylococcus aureus Techniques Technology Testing Therapeutic Tuberculosis Variant adenylate kinase antimicrobial antimicrobial drug assay development bactericide base cell growth drug discovery global health high throughput screening improved interest killings meetings novel pathogen public health relevance research study respiratory response scaffold screening small molecule tuberculosis drugs

项目摘要

DESCRIPTION (provided by applicant): Historically, traditional whole cell, growth-based assays have been the single most productive approach to the identification of anti-infectives that inhibit bacterial proliferation. None the less, growth-based assays have a number of important limitations including relatively low sensitivity/dynamic range relative to molecular assays, inability to distinguish bacteriostatic from bactericidal molecules, and poor performance in clinically important phases of bacterial disease, such as biofilms. To address these limitations, a number of improved cell-based screening approaches have been developed, each successful in its own right, but each also with an inherent set of limitations. For instance, Alamar blue dye, which measures the respiratory activity of organisms, is arguably the gold-standard whole cell antimicrobial screening technique. But, the dye cannot be used to effectively screen respiratory defective bacterial populations, such as small colony variants. Nor does the dye allow for distinction between bacteriostatic and bactericidal agents. We have developed a novel assay of antibacterial activity that directly detects the killing of bacteria by measuring the release of th intracellular enzyme adenylate kinase (AK) into the culture medium as a reporter of cell death. Our recent publication validated the use of the assay in antimicrobial screening and established that it has three key features. First, the assay is applicable to screening virtually any bacterial population. Second, the assay performs with exquisite sensitivity. Third, the assay enriches for the identification of bactericidal agents. As such, we believe the AK assay provides a very powerful new screening option for antimicrobial drug discovery, particularly for pathogens or bacterial growth states that are of immense healthcare concern that cannot be readily screened using conventional growth- based approaches and for which bactericidal antibiotics are desired; three examples include non-replicating (and slow growing) Mycobacterium tuberculosis, bacteria within established biofilms, and bacterial small colony variants. Accordingly, this application is being submitted in response to PA-10-213, "Development of Assays for High Throughput Screening for use in Probe and Pre-therapeutic Discovery". Consistent with the goals of this program announcement, we propose to further develop and validate three AK-assay based high throughput screening strategies that would not be possible using growth based assays and for which bactericidal agents are needed. We will develop AK-based high throughput screens for agents that kill: 1) non-replicating (and slow growing) M. tuberculosis; 2) bacteria within established biofilms; and 3) bacterial small colony variants.

描述（由申请人提供）：从历史上看，传统的全细胞、基于生长的测定一直是鉴定抑制细菌增殖的抗感染剂的最有效的方法。尽管如此，基于生长的测定具有许多重要的局限性，包括相对于分子测定而言相对较低的灵敏度/动态范围、无法区分抑菌分子和杀菌分子，以及在细菌疾病的临床重要阶段（例如生物膜）中表现不佳。为了解决这些限制，已经开发了许多改进的基于细胞的筛选方法，每种方法本身都取得了成功，但每种方法也都有一系列固有的局限性。例如，用于测量生物体呼吸活动的阿拉玛蓝染料可以说是金标准的全细胞抗菌筛选技术。但是，该染料不能用于有效筛查呼吸道缺陷细菌群体，例如小菌落变异体。该染料也无法区分抑菌剂和杀菌剂。我们开发了一种新颖的抗菌活性测定方法，通过测量细胞内酶腺苷酸激酶 (AK) 释放到培养基中作为细胞死亡报告者来直接检测细菌的杀灭情况。我们最近的出版物验证了该测定在抗菌药物筛选中的使用，并确定它具有三个关键特征。首先，该测定法适用于筛选几乎任何细菌人口。其次，该检测具有极高的灵敏度。第三，该测定丰富了杀菌剂的鉴定。因此，我们相信 AK 测定为抗菌药物的发现提供了一种非常强大的新筛选选择，特别是对于那些引起巨大医疗保健关注的病原体或细菌生长状态，这些病原体或细菌生长状态无法使用传统的基于生长的方法轻松筛选，并且杀菌抗生素是针对这些病原体或细菌生长状态的。想要的；三个例子包括非复制（且生长缓慢）的结核分枝杆菌、已建立的生物膜内的细菌以及细菌小菌落变体。因此，本申请是为了响应 PA-10-213“用于探针和治疗前发现的高通量筛选测定的开发”而提交的。与本计划公告的目标一致，我们建议进一步开发和验证三种基于 AK 测定的高通量筛选策略，而使用基于生长的测定无法实现这些策略，并且需要杀菌剂。我们将开发基于 AK 的高通量筛选，用于杀死以下物质： 1) 非复制（且生长缓慢）结核分枝杆菌； 2) 已建立的生物膜内的细菌； 3) 细菌小菌落变体。