College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
High Throughput Screening Laboratory, University of Kansas, Lawrence, KS, United States.
Front Cell Infect Microbiol. 2018 Dec 18;8:435. doi: 10.3389/fcimb.2018.00435. eCollection 2018.
The enteropathogenic and enterohemorrhagic NleB proteins as well as the SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian -linked -acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed cross-reactivity with the mammalian -linked -acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases.
肠致病性和肠出血性 NleB 蛋白以及 SseK 蛋白是 III 型分泌系统效应物，作为糖基转移酶酶，对精氨酸残基上的宿主底物进行翻译后修饰。这种修饰是不寻常的，因为它发生在胍基的精氨酸上，胍基是较差的亲核试剂，与哺乳动物连接的 -乙酰葡萄糖胺基转移酶的活性不同。我们进行了高通量筛选实验，以鉴定抑制 NleB/SseK 活性的小分子。两种化合物 100066N 和 102644N 均显著抑制了 NleB1、SseK1 和 SseK2 的活性。将这些化合物添加到培养的哺乳动物细胞中足以抑制 NleB1 对肿瘤坏死因子受体 1 相关 DEATH 结构域蛋白的糖基化。这些化合物还能够抑制 株 ATCC 14028 在鼠巨噬样细胞中的复制。这两种抑制剂对哺乳动物细胞均没有明显的毒性，也没有与哺乳动物连接的 -乙酰葡萄糖胺基转移酶发生交叉反应。这些化合物或从药物化学改进中生成的衍生物可能可用作抗生素的潜在替代治疗策略，或用作进一步研究细菌糖基转移酶的试剂。
