College of Medicine, Department of Pharmacology and Therapeutics, Center for Inflammation Science and Systems Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, University of Florida, 130 Scripps Way, Jupiter, Florida 33458, United States.
College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, 1703 E. Mabel Street, P.O. Box 210207, Tucson, Arizona 85721, United States.
J Am Chem Soc. 2024 Jul 31;146(30):20845-20856. doi: 10.1021/jacs.4c05057. Epub 2024 Jul 23.
We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent -sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, . A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.
我们最近报道了 GroES/GroEL 伴侣系统的小分子抑制剂作为潜在的抗生素对抗 和 ESKAPE 病原体，但未能确定 GroES/GroEL 是导致细胞死亡的细胞靶标。在这项研究中，我们使用两种最有效的 -磺酰胺基-2-苯基苯并恶唑（PBZ），通过冷冻电镜确定了 PBZ 分子的结合位点，并发现 GroEL 是负责作用模式的细胞靶标。冷冻电镜显示 PBZ1587 结合在 GroEL 环环界面（RRI）上。细胞报告基因检测证实 PBZ1587 在细胞中与 GroEL 结合，但细胞拯救实验显示出潜在的脱靶效应。这促使我们探索一种密切相关的类似物 PBZ1038，它也结合在 RRI 上。生化特性研究表明，它对革兰氏阴性伴侣蛋白具有很强的抑制作用，但对革兰氏阳性生物的伴侣蛋白的抑制作用要低得多。细胞报告基因检测显示 PBZ1038 也能在细胞中与 GroEL 结合，并且细胞毒性表型可以通过染色体拷贝的 GroEL/GroES 或表达抗性 RRI 突变体来挽救。这些数据表明，PBZ1038 的抗菌作用是通过抑制 GroES/GroEL 发挥的，验证了该伴侣系统作为抗生素靶标。
