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

项目总结/摘要 金属-β-内酰胺酶（MBL）是使病原菌对最大类抗生素耐药的细菌酶。 抗生素。虽然已经发表了许多MBL抑制剂，但明确的结构-活性关系 （SAR）通常难以辨别，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上有用的化合物的开发之间的脱节是由于， 部分原因是对这些化合物的作用机制缺乏了解， 的金属剥离机制，这是固有的非特异性，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将表征几种MBL抑制剂类别的抑制机制。许多人报告MBL 抑制剂含有具有结合金属离子潜力的官能团，但它们的机理仍不清楚。 在这里，我们使用多学科的方法来确定是否选择类MBL抑制剂的工作，通过金属- 通过三元复合物形成（抑制剂：金属离子：蛋白质）或通过其它机制进行剥离。这些信息 将实现更有效的SAR研究，并允许更好地选择更适合临床的候选人 应用. 具体目标#2将开发两种新技术来探测抑制剂与MBL的结合。一种技术是EPR- 基于并预测产生关于位于MBL活性区上的不变b-发夹环的信息。 网站.第二种技术是基于原生MS的，预计将产生有关机制的直接信息 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前途的MBL抑制剂支架， 可以与现有的含有β-内酰胺的抗生素联合给药， 感染.通过明确有前途的MBL抑制剂支架的作用机制，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.