Development of AK-based assays for antimicrobial screening

描述（由申请人提供）：历史上，传统的全细胞、基于生长的测定法一直是鉴定抑制细菌增殖的抗感染药物的最有效方法。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的相对低的灵敏度/动态范围，不能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）中的差的性能。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，每种方法本身都是成功的，但每种方法也具有固有的一组限制。例如，测量生物体呼吸活性的Alcohol蓝染料可以说是金标准的全细胞抗菌筛选技术。但是，染料不能用于有效地筛选呼吸缺陷细菌群体，例如小菌落变体。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的报告分子，直接检测细菌的杀伤。我们最近的出版物验证了该测定法在抗菌剂筛选中的应用，并确定其具有三个关键特征。首先，该测定适用于筛选几乎任何细菌 人口第二，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测定为抗微生物药物发现提供了非常强大的新筛选选项，特别是对于具有巨大医疗保健关注的病原体或细菌生长状态，其不能使用常规的基于生长的方法容易地筛选并且需要杀菌抗生素;三个例子包括非复制型（和缓慢生长的）结核分枝杆菌、已建立的生物膜内的细菌和细菌小菌落变体。因此，本申请是根据PA-10-213“用于探针和治疗前发现的高通量筛选试验的开发”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，这些策略不可能使用基于生长的测定，并且需要杀菌剂。我们将开发基于AK的高通量筛选剂，杀死：1）非复制（和缓慢生长）M。结核病; 2）已建立的生物膜内的细菌;和3）细菌小菌落变体。

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.