Copper Chelating Compounds as Novel Antimicrobial Class

摘要 几个世纪以来，人们就知道铜对细菌是有毒的。在宿主的先天免疫系统中， 被吞噬的细菌暴露在巨噬细胞吞噬酶体内的高水平铜中。作为一名 为了中和这种由先天免疫系统使用的工具，细菌进化出了输出系统 排出进入细菌细胞的铜。研究病原体对这种铜胁迫的反应将会有所帮助 美国设计出新的抗微生物策略。我们的模式生物肺炎链球菌是主要原因。 全世界脑膜炎、中耳炎、肺炎和败血症。对巨噬细胞来源的铜的反应 应激，肺炎链球菌上调COP操纵子基因，该基因用于将铜从细菌中输出 手机。增强铜胁迫超过细菌的输出能力可能是一种新的 抗菌剂。铜的络合化合物将铜引导到巨噬细胞，以便在体内摄取和使用 吞噬酶体会增强铜胁迫。新近开发的抗真菌药物8-羟基氯喹(8-HQ)， 利用铜的螯合和吸收进入巨噬细胞，以提高杀灭效率。在放映了 几种已知的铜络合剂和具有相似结构元素的化合物，我们的实验室已经确定了几种 候选螯合化合物以测试抗菌效果。我推测类似的铜螯合作用 化合物增强杀灭细菌和增强宿主巨噬细胞对吞噬病原体的杀灭作用 提高巨噬细胞内铜浓度。目前我们知识中的差距包括 病原体对这些螯合化合物引起的铜胁迫以及化合物是否起作用做出反应 在体外和体内。为了验证这一假设，我将首先定义络合化合物与铜的亲和力 随后细菌内Cu2+浓度的变化。这一目的的发现评估了螯合作用 化合物可增加细菌中Cu2+的浓度或增加其在细胞内的有效性。另外，我会 确定肺炎链球菌对铜络合物诱导的铜胁迫的反应。发现 从这个目的将表征铜胁迫是如何由这些铜螯合化合物引起的。最后，我会 确定巨噬细胞在我们确认的协同铜络合物抗菌效果中的作用 化合物。这一目标的发现将显示抗菌效果依赖于巨噬细胞或 巨噬细胞非依赖性。虽然大多数商业上可用的抗菌剂都是针对细菌DNA转录的 或mRNA翻译，我们在这项拟议的研究中将使用早已为人所知的铜原理 用于未充分利用的目的的毒性。

Abstract It has been known for centuries that copper is toxic to bacteria. Within the host innate immune system, engulfed bacteria are exposed to high levels of copper within the phagolysosome of macrophages. As a response to counteract this tool used by the innate immune system, bacteria have evolved export systems to pump out copper that enter the bacterial cell. Studying how pathogens respond to this copper stress will help us devise novel antimicrobial strategies. Our model organism, Streptococcus pneumoniae, is a leading cause of meningitis, otitis media, pneumonia, and sepsis worldwide. In response to macrophage-derived copper stress, S. pneumoniae upregulates the cop operon locus, which serves to export copper out from the bacterial cell. Enhancing copper stress above a bacterium’s export capacity can be a mechanism for a novel antimicrobial. Copper chelating compounds that direct copper to macrophages for uptake and use within the phagolysosome will enhance copper stress. A recently developed antifungal, 8-hydroxychloroquine (8-HQ), utilizes copper chelation and uptake into macrophages to enhance killing efficiency. Following screening of several known copper chelators and compounds with similar structural elements, our lab has identified several candidate chelating compounds to test for antimicrobial efficacy. I hypothesize that similar copper chelating compounds enhance kill bacteria and enhance host macrophage killing of engulfed pathogens by increasing the intra-macrophage copper concentration. Current gaps in our knowledge include how pathogens respond to the copper stress induced by these chelating compounds and whether compounds work in vitro and in vivo. To test this hypothesis, I will first define the copper affinity of chelating compounds and the subsequent change in intra-bacterial Cu2+ concentration. Findings from this aim assess whether chelating compounds increase bacterial Cu2+ concentration or increase its intracellular availability. Additionally, I will determine how S. pneumoniae respond to copper stress induced by copper chelating compounds. Findings from this aim will characterize how copper stress is induced by these copper chelating compounds. Lastly, I will determine the role of macrophages in antimicrobial efficacy of our identified synergistic copper chelating compounds. Findings from this aim will show antimicrobial efficacy to be macrophage-dependent or macrophage-independent. While most commercially available antimicrobials target bacterial DNA transcription or mRNA translation, our research in this proposed study will employ the long-known principle of copper toxicity for an under-utilized purpose.