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首页> 美国卫生研究院文献>Antimicrobial Agents and Chemotherapy >Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus
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Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus

机译：基于微量滴定板的抗甲氧西林金黄色葡萄球菌青霉素结合蛋白2a抑制剂的测定

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摘要

Penicillin-binding protein 2a (PBP2a), the molecular determinant for high-level β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), is intrinsically resistant to most β-lactam antibiotics. The development and characterization of new inhibitors targeting PBP2a would benefit from an effective and convenient assay for inhibitor binding. This study was directed toward the development of a fluorescently detected β-lactam binding assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were tested as tagging reagents for fluorescence detection by using a streptavidin-horseradish peroxidase conjugate. Both bound surprisingly well to PBP2a, with binding constants of 1.6 ± 0.4 μM and 13.6 ± 0.8 μM, respectively. Two forms of the assay were developed, a one-step direct competition form of the assay and a two-step indirect competition form of the assay, and both forms of the assay gave comparable results. This assay was then used to characterize PBP2a binding to ceftobiprole, which gave results consistent with previous studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening for PBP2a inhibitors by screening a set of 13 randomly selected β-lactams for PBP2a inhibition at 750 μM. Meropenem was observed to give substantial inhibition in this screen, and a follow-up titration experiment determined its apparent Ki to be 480 ± 70 μM. The availability of convenient and sensitive microtiter-plate based assays for the screening and characterization of PBP2a inhibitors is expected to facilitate the discovery and development of new PBP2a inhibitors for use in combating the serious public health problem posed by MRSA.
机译：青霉素结合蛋白2a（PBP2a）是耐甲氧西林的金黄色葡萄球菌（MRSA）中高水平β-内酰胺耐药性的分子决定因素，对大多数β-内酰胺抗生素具有内在耐药性。靶向PBP2a的新型抑制剂的开发和表征将受益于抑制剂结合的有效便捷分析。这项研究的目的是开发荧光检测的MRSA中PBP2a的β-内酰胺结合测定法。通过使用链霉亲和素-辣根过氧化物酶偶联物，对生物素化的氨苄青霉素和生物素化的头孢氨苄作为荧光检测的标记试剂进行了测试。两者均与PBP2a结合得非常好，结合常数分别为1.6±0.4μM和13.6±0.8μM。开发了两种形式的测定，一种是测定的一步直接竞争形式，另一种是测定的两步间接竞争形式，两种形式的测定都给出了可比的结果。然后，该测定法用于表征PBP2a与头孢比普利的结合，其结果与头孢比普利-PBP2a结合的先前研究一致。还通过筛选一组13种随机选择的β-内酰胺以抑制750μM的PBP2a，证明了该分析方法可用于筛选PBP2a抑制剂。在该筛选中观察到美罗培南具有明显的抑制作用，随后的滴定实验确定其表观Ki为480±70μM。用于筛选和表征PBP2a抑制剂的方便，灵敏的基于微量滴定板的检测方法的实用性有望促进新的PBP2a抑制剂的发现和开发，以解决由MRSA引起的严重公共卫生问题。

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