Determining the mechanism of inhibition of metallo-b-lactamase inhibitors

项目摘要/摘要 金属-b-内酰胺酶(MBLS)是使病原菌对最大类耐药的细菌酶。 抗生素。尽管已经发表了许多MBL抑制剂，但明确的结构-活性关系 非典型肺炎(SARS)往往难以辨别，这些抑制剂都没有进入临床使用。我们建议 抑制剂的发现和临床有用化合物的开发之间的脱节是由于， 部分原因是缺乏对这些化合物起作用的机制的了解，以及由于 金属剥离机制本身就是非专属性的，并有相关的毒性问题。直接 为了解决这些问题，我们建议辨别几种定义不足的MBL抑制剂的抑制机制。 具体目标#1将描述几类MBL抑制剂的抑制机制。许多人报告了MBL 缓蚀剂含有可能结合金属离子的官能团，但其作用机制尚不清楚。 在这里，我们使用多学科方法来确定特定类别的MBL抑制剂是否通过金属起作用- 通过形成三元络合物(缓蚀剂：金属离子：蛋白质)或通过其他机制进行剥离。此信息 将使研究更有效，并能更好地选择更适合临床的候选人 申请。 具体目标#2将开发两种新的技术来探测结合到MBL的抑制剂。一种技术是EPR- ，并被预测为产生关于驻留在活动的MBL上的不变b发夹循环的信息。 网站。第二种技术是基于本地MS的，预计将产生关于该机制的直接信息 使用较低浓度的酶/抑制剂进行抑制。 这项研究计划的长期目标是确定最有希望的MBL抑制剂支架， 可与现有的含有抗生素的β-内酰胺类药物联合使用，以治疗耐药细菌。 感染。通过明确有前景的MBL抑制剂支架的作用机理，我们将 为这些抑制剂进入临床试验提供了更清晰的途径。

PROJECT SUMMARY/ABSTRACT Metallo-b-lactamases (MBLs) are bacterial enzymes that render pathogenic bacteria resistant to the largest class of antibiotics. Although a number of MBL inhibitors have been published, clear structure-activity relationships (SARs) are often difficult to discern, and none of these inhibitors has advanced to clinical use. We propose that the disconnect between the discovery of inhibitors and the development of clinically-useful compounds is due, in part, to a lack of understanding the mechanisms whereby these compounds work, and by the predominance of metal-stripping mechanisms, which are inherently non-specific and have associated toxicity issues. To directly address these issues, we propose to discern the mechanism of inhibition of several under-defined MBL inhibitors. Specific aim #1 will characterize inhibition mechanisms of several MBL inhibitor classes. Many reported MBL inhibitors contain functional groups with potential to bind metal ions, but their mechanisms remain undefined. Here, we use a multidisciplinary approach to determine if selected classes of MBL inhibitors work by metal- stripping, by ternary complex formation (inhibitor:metal ion:protein) or by other mechanisms. This information will enable more effective SAR studies and allow better selection of candidates more suitable for clinical applications. Specific aim #2 will develop two novel techniques to probe inhibitor binding to MBLs. One technique is EPR- based and is predicted to yield information about an invariant b-hairpin loop that resides over the MBL active sites. The second technique is native MS-based and is predicted to yield direct information about the mechanism of inhibition using lower concentrations of enzyme/inhibitor. The long-term goal of this research program is to identify the most promising scaffolds for MBL inhibitors, which can be given in combination with existing b-lactam containing antibiotics to treat antibiotic resistant bacterial infections. Through clearly determining the mechanism of action of promising MBL inhibitor scaffolds, we will provide a clearer pathway for these inhibitors to proceed to clinical trials.