Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents
Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents
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DOI：
10.1128/aac.01640-12
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发表时间：
2013-01-01
影响因子：
4.9
通讯作者：
Dunman, Paul M.
中科院分区：
文献类型：
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作者：
Jacobs, Anna C.;DiDone, Louis;Dunman, Paul M.
腺苷酸激酶（AK）是一种普遍存在的细胞内酶，在细胞裂解时释放到细胞外空间。我们已经表明，AK释放作为一个有用的报告杀菌剂活性，并可用于抗菌筛选的目的。AK检测试剂盒的灵敏度优于基于生长的检测试剂盒，并且可以检测出对临床相关生长状态下的细菌具有活性的试剂，这些细菌难以使用常规方法进行筛选，例如小菌落变体（SCV）和已建立的生物膜内的细菌。AK检测试剂盒的有效性通过筛选非专利药物库中对多种细菌物种（包括大肠埃希菌和“ESKAPE”病原体的所有成员（屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌属））具有抗菌特性的药物进行验证。该测定在库中检测到了预计对所筛选的生物体有活性的抗生素。此外，38种以前没有报道过抗菌活性的药物引起AK释放。获得了其中4个，并且通过标准敏感性测试验证了所有这些都表现出抗微生物活性。这些分子中的两个被进一步表征。抗组胺药特非那定对S.金黄色葡萄球菌增殖、SCV群体和生物膜相关细胞。雌激素受体拮抗剂三苯氧胺对E. faecium的体外培养，并降低E.大蜡螟感染模型中的屎肠致病性。我们的数据表明，AK试验提供了一个有吸引力的筛选方法，以确定新的抗菌药物。此外，特非那定和他莫昔芬可能代表新的抗微生物药物开发支架。
Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.