1 Department of Structural Biology, Jacobs School of Medicine & Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA.
2 The Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA.
SLAS Discov. 2018 Dec;23(10):1070-1082. doi: 10.1177/2472555218787140. Epub 2018 Jul 10.
Acquiring sufficient quantities of iron to support survival is often a critical limitation for pathogenic bacteria. To meet this demand, bacteria have evolved unique strategies to scavenge iron and circumvent the nutritional immunity exerted by their hosts. One common strategy, which is often a key virulence factor for bacterial pathogens, involves the synthesis, secretion, and reuptake of iron chelators known as siderophores. In vitro and in vivo studies have demonstrated that the siderophore aerobactin is critical for virulence in the hypervirulent pathotype of Klebsiella pneumoniae (hvKP). Given the high rate of multidrug resistance in K. pneumoniae, and in light of the ever-increasing demand for novel Gram-negative therapeutic targets, we identified aerobactin production as a promising antivirulence target in hvKP. Herein, we describe the development of a high-throughput biochemical assay for identifying inhibitors of the aerobactin synthetase IucA. The assay was employed to screen ~110,000 compounds across several commercially available small-molecule libraries. IucA inhibitors with activity at micromolar concentrations were identified in our screening campaigns and confirmed using secondary orthogonal assays. However, the most potent compounds also exhibited some properties commonly observed with promiscuous/nonspecific inhibitors, including incubation time and target enzyme concentration dependence, as well as the potential to antagonize unrelated enzymes.
获取足够数量的铁以支持生存通常是致病细菌的一个关键限制因素。为了满足这一需求，细菌已经进化出独特的策略来掠夺铁并规避其宿主施加的营养免疫。一种常见的策略，通常是细菌病原体的关键毒力因素，涉及铁螯合剂（称为 siderophores）的合成、分泌和再摄取。体外和体内研究表明，铁载体 aerobactin 对于高毒力肺炎克雷伯菌（hvKP）的毒力至关重要。鉴于肺炎克雷伯菌的多药耐药率很高，并且鉴于对新型革兰氏阴性治疗靶点的需求不断增加，我们将 aerobactin 产生鉴定为 hvKP 中的一种有前途的抗毒力靶标。在此，我们描述了一种用于鉴定 aerobactin 合成酶 IucA 抑制剂的高通量生化测定法的开发。该测定法用于筛选来自几个商业上可获得的小分子文库中的约 110,000 种化合物。在我们的筛选活动中发现了在微摩尔浓度下具有活性的 IucA 抑制剂，并使用二级正交测定法进行了确认。然而，最有效的化合物也表现出与混杂/非特异性抑制剂常见的一些特性，包括孵育时间和靶酶浓度依赖性，以及有潜力拮抗不相关的酶。
