Discovery of Hsp100-selective inhibitors for targeting multiple microbial pathogens

项目摘要/摘要 病原微生物是世界各地各种感染和疾病的原因。新技术的出现 耐药菌株增加了感染率和死亡率，需要新的方法和努力 抗菌药物的发现。我们研究的长期目标是了解分子的作用 伴侣在病原菌生长、存活和毒力中的作用，并将这些知识应用于开发分子 防治传染病的方法。这项应用的目的是发现小分子 利用高通量文库筛选Hsp100分子伴侣的抑制物。Hsp100分子伴侣重新激活 聚集的细胞蛋白。Hsp100的丧失对许多细菌的传染性和生存都是有害的 和原生动物病原体。重要的是，在后生动物蛋白质组中没有发现明显的Hsp100同源基因。AS 抑制剂开发和测试的主要目标，我们将使用Hsp100伴侣ClpB，来自 志贺氏菌/大肠杆菌。实现以下具体目标：1.分两步走 小分子文库的筛选，它将询问ClpB的两个正交功能：依赖于ATP 底物结合和底物诱导激活ATPase活性。2.我们将验证并确定优先顺序 使用药物化学方法抑制ClpB的化合物Hit化学类型。3.我们将验证 选择的HIT化合物的抑制效力和测试其对ClpB的体外选择性 正交生化分析的次数。4.我们将对其抗菌活性进行初步测试 革兰氏阴性菌大肠杆菌、福氏志贺氏菌和宋内志贺菌中的主要化合物，以及 革兰氏阳性金黄色葡萄球菌。拟议研究的预期结果将是发现 经过验证的小分子Hsp100抑制剂候选药物，用于未来的药物化学优化和生物 测试。这种方法是创新的，因为还没有实施以伴侣为基础的抗菌剂。

PROJECT SUMMARY/ABSTRACT Pathogenic microorganisms are the cause of diverse infections and diseases worldwide. The emergence of drug resistant strains increases the infection and mortality rates and demands new approaches and efforts in the antimicrobial drug discovery. The long-term goal of our research is to understand the role of molecular chaperones in pathogen growth, survival, and virulence and to apply that knowledge in developing molecular approaches to combat infectious diseases. The objective of this application is to discover small-molecule inhibitors of Hsp100 chaperones using high-throughput library screening. The Hsp100 chaperones reactivate aggregated cellular proteins. A loss of Hsp100 is detrimental for infectivity and survival of a number of bacterial and protozoan pathogens. Importantly, no apparent Hsp100 orthologs are found in metazoan proteomes. As the primary target for inhibitor development and testing, we will use the Hsp100 chaperone, ClpB, from Shigella/Escherichia coli. The following Specific Aims will be pursued: 1. We will perform a sequence of two screens of a small-molecule library that will interrogate two orthogonal functionalities of ClpB: ATP-dependent substrate binding and substrate-induced activation of the ATPase activity. 2. We will validate and prioritize compound hit chemotypes that inhibit ClpB using medicinal chemistry approaches. 3. We will validate the inhibitory potency of selected hit compounds and test their selectivity towards ClpB in vitro by performing a number of orthogonal biochemical assays. 4. We will perform preliminary testing of the antimicrobial activity of the hit compounds in Gram-negative bacteria Escherichia coli, Shigella flexneri, and Shigella sonnei, and Gram-positive Staphylococcus aureus. The expected outcome of the proposed studies will be the discovery of validated small-molecule Hsp100-inhibitor candidates for future medicinal chemistry optimization and biological testing. This approach is innovative because no chaperone-based antimicrobials have been implemented yet.