Yang Lihua, Zhai Hang, Tian Tingting, Liu Botong, Ni Xianpu, Xia Huanzhang
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China.
Front Pharmacol. 2025 Apr 24;16:1575840. doi: 10.3389/fphar.2025.1575840. eCollection 2025.
Aminoglycosides (AGs) are one of the initial classes of antibiotics that have been used clinically and possess broad spectrum of activity. Nevertheless, their clinical utilization is restricted by safety issues associated with nephrotoxicity and ototoxicity.
Glycosyltransferase (GT) KanM2 was introduced into to produce the gentamicin derivatives, in which a kanosamine sugar ring was introduced to replace the garosamine. The premature termination codon (PTC) readthrough activity of genkamicins (GKs) was compared using dual luciferase reporter assay. The toxicity of GK was assessed in HEK-293 and NCI-H1299 cells and determined based on cell viability calculated after 48 h of treatment with different concentrations of the compounds. The NCI-H1299 cells harbouring the R213X nonsense mutation were treated with different concentrations of the derivatives to compare their expression of p53 proteins. The expression of p53 and its downstream targets p21 and BAX was detected using Western blotting and qRT-PCR in NCI-H1299 cells containing the R213X nonsense mutation treated with different concentrations of GK-Ae and G418. Finally, immunofluorescence and flow cytometry were used to determine the subcellular localization of full-length p53 protein induced by GK-Ae treatment and its effect on apoptosis in cancer cells.
Eight gentamicin derivatives were obtained in this study. GK-Ae displayed similar PTC readthrough activity and reduced toxicity compared to natural aminoglycoside G418. Moreover, GK-Ae increased the levels of both p53 and its downstream targets p21 and BAX, and promoted apoptosis of cancer cells.
These results demonstrate the potential of combinatorial biosynthesis to increase the diversity of structures of AGs and provide directions for the development of new AGs with low toxicity and high PTC readthrough activity.
氨基糖苷类抗生素（AGs）是最早用于临床的一类抗生素，具有广谱抗菌活性。然而，其临床应用受到肾毒性和耳毒性等安全性问题的限制。
将糖基转移酶（GT）KanM2导入以生产庆大霉素衍生物，其中引入肌醇胺糖环以取代加洛糖胺。使用双荧光素酶报告基因检测法比较庆大霉素（GK）的提前终止密码子（PTC）通读活性。在HEK-293和NCI-H1299细胞中评估GK的毒性，并根据用不同浓度化合物处理48小时后计算的细胞活力来确定。用不同浓度的衍生物处理携带R213X无义突变的NCI-H1299细胞，以比较其p53蛋白的表达。在含有R213X无义突变的NCI-H1299细胞中，用不同浓度的GK-Ae和G418处理后，使用蛋白质免疫印迹法和qRT-PCR检测p53及其下游靶点p21和BAX的表达。最后，使用免疫荧光和流式细胞术确定GK-Ae处理诱导的全长p53蛋白的亚细胞定位及其对癌细胞凋亡的影响。
本研究获得了8种庆大霉素衍生物。与天然氨基糖苷类抗生素G418相比，GK-Ae表现出相似的PTC通读活性且毒性降低。此外，GK-Ae增加了p53及其下游靶点p21和BAX的水平，并促进了癌细胞的凋亡。
这些结果证明了组合生物合成在增加AGs结构多样性方面的潜力，并为开发低毒性和高PTC通读活性的新型AGs提供了方向。
