Development of AK-based assays for antimicrobial screening

描述(由申请人提供)：从历史上看，传统的全细胞、基于生长的分析一直是识别抑制细菌增殖的抗感染药物的唯一最有效的方法。然而，基于生长的分析方法有许多重要的局限性，包括相对分子分析相对较低的灵敏度/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，如生物膜，表现不佳。为了解决这些限制，一个 已经开发了一些改进的基于细胞的筛查方法，每一种方法都有其自身的成功，但每一种方法也都有一套固有的局限性。例如，测量有机体呼吸活动的阿拉马蓝染料可以说是全细胞抗菌筛选的黄金标准技术。但是，这种染料不能用于有效地筛选呼吸缺陷细菌种群，例如小菌落变种。染料也不能区分抑菌剂和杀菌剂。我们开发了一种新的抗菌活性测定方法，通过测量TH细胞内酶腺苷酸激酶(AK)作为细胞死亡报告进入培养基中的释放，直接检测细菌的杀灭。我们最近的出版物验证了该分析在抗菌筛选中的使用，并确定了它有三个关键特征。首先，该检测方法适用于几乎所有细菌的筛选。 人口。其次，该检测方法具有极高的灵敏度。第三，该方法丰富了杀菌剂的鉴定。因此，我们认为AK试验为抗菌药物的发现提供了一个非常强大的新的筛查选择，特别是对病原体或细菌生长状态，这些病原体或细菌生长状态是巨大的医疗保健问题，不能使用传统的基于生长的方法进行筛选，并且需要杀菌抗生素；三个例子包括非复制(和生长缓慢的)结核分枝杆菌、已建立的生物膜内的细菌和细菌的小菌落变体。因此，本申请是根据PA-10-213“开发用于探针和治疗前发现的高通量筛查分析”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检测的高通量筛查策略，这些策略使用基于生长的检测是不可能的，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：1)非复制(和缓慢生长)的结核分枝杆菌；2)已建立的生物膜内的细菌；以及3)细菌的小菌落变体。

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.