Sanchez Peggy A, Toney Jeffrey H, Thomas Janice D, Berger John M
Department of Chemistry and Biochemistry, Montclair State University, Montclair, New Jersey, USA.
Assay Drug Dev Technol. 2009 Apr;7(2):170-9. doi: 10.1089/adt.2008.167.
Antibiotic-resistant bacteria continue to threaten human health through multiple mechanisms, including hydrolytic inactivation of beta-lactam antibiotics by metallo-beta-lactamases (MBLs). The SPM-1 enzyme, originally identified from a Pseudomonas aeruginosa clinical isolate, is a Class B beta-lactamase responsible for resistance in bacteria against antibiotics such as penicillins, cephalosporins, and carbapenems. Unlike Class A, C, and D beta-lactamases, which employ a serine residue in their active site, Class B enzymes possess one or two Zn atoms in the active site that play both a structural and catalytic role. A beta-lactamase inhibitor with co-administration of a beta-lactam antibiotic has proven to be an effective treatment against antibiotic-resistant bacteria whose resistance is due to serine-based beta-lactamases (e.g., amoxicillin/clavulanic acid). A similar clinical approach has not yet been developed for resistant bacteria possessing MBLs. The identification and development of specific and effective MBL inhibitors to combat this resistance could extend the utility of currently prescribed antibiotics such as cephalosporins and carbapenems. To discover MBL inhibitors, compound libraries are screened typically by enzymatic hydrolysis of a chromogenic substrate such as nitrocefin monitored by absorbance. Spectrophotometric assays, while valuable, lack the sensitivity and selectivity to screen natural product extract libraries because of the strongly absorbing nature of some extracts and the dilute concentrations of active components. An assay is described herein that monitors the SPM-1-catalyzed hydrolysis of penicillin G by high-performance (high-pressure) liquid chromatography-electrospray mass spectroscopy, which permits investigations with greater sensitivity and selectivity allowing the screening of natural product extracts for inhibitors of MBLs.
抗生素耐药细菌通过多种机制继续威胁人类健康，包括金属β-内酰胺酶（MBLs）对β-内酰胺类抗生素的水解失活。SPM-1酶最初是从一株铜绿假单胞菌临床分离株中鉴定出来的，它是一种B类β-内酰胺酶，负责细菌对青霉素、头孢菌素和碳青霉烯类等抗生素的耐药性。与在活性位点使用丝氨酸残基的A类、C类和D类β-内酰胺酶不同，B类酶在活性位点含有一个或两个锌原子，这些锌原子兼具结构和催化作用。已证明，β-内酰胺酶抑制剂与β-内酰胺类抗生素联合使用是治疗由丝氨酸β-内酰胺酶引起耐药性的抗生素耐药细菌的有效方法（例如阿莫西林/克拉维酸）。对于具有MBLs的耐药细菌，尚未开发出类似的临床方法。鉴定和开发特异性有效的MBL抑制剂以对抗这种耐药性，可能会扩大目前处方使用的抗生素如头孢菌素和碳青霉烯类的效用。为了发现MBL抑制剂，通常通过监测吸光度的显色底物（如硝基头孢菌素）的酶促水解来筛选化合物库。分光光度法虽然有价值，但由于一些提取物具有强吸收性且活性成分浓度较低，缺乏筛选天然产物提取物库的灵敏度和选择性。本文描述了一种通过高效（高压）液相色谱 - 电喷雾质谱监测SPM-1催化青霉素G水解的方法，该方法允许以更高的灵敏度和选择性进行研究，从而能够筛选天然产物提取物中的MBL抑制剂。
