Degtyareva Natalya N, Gong Changjun, Story Sandra, Levinson Nathanael S, Oyelere Adegboyega K, Green Keith D, Garneau-Tsodikova Sylvie, Arya Dev P
NUBAD, LLC , Greenville, South Carolina 29605, United States.
Department of Chemistry, Clemson University , Clemson, South Carolina 29634, United States.
ACS Infect Dis. 2017 Mar 10;3(3):206-215. doi: 10.1021/acsinfecdis.6b00176. Epub 2017 Feb 17.
The antibacterial effects of aminoglycosides are based on their association with the A-site of bacterial rRNA and interference with the translational process in the bacterial cell, causing cell death. The clinical use of aminoglycosides is complicated by resistance and side effects, some of which arise from their interactions with the human mitochondrial 12S rRNA and its deafness-associated mutations, C1494U and A1555G. We report a rapid assay that allows screening of aminoglycoside compounds to these classes of rRNAs. These screening tools are important to find antibiotics that selectively bind to the bacterial A-site rather than human, mitochondrial A-sites and its mutant homologues. Herein, we report our preliminary work on the optimization of this screen using 12 anthraquinone-neomycin (AMA-NEO) conjugates against molecular constructs representing five A-site homologues, Escherichia coli, human cytosolic, mitochondrial, C1494U, and A1555G, using a fluorescent displacement screening assay. These conjugates were also tested for inhibition of protein synthesis, antibacterial activity against 14 clinically relevant bacterial strains, and the effect on enzymes that inactivate aminoglycosides. The AMA-NEO conjugates demonstrated significantly improved resistance against aminoglycoside-modifying enzymes (AMEs), as compared with NEO. Several compounds exhibited significantly greater inhibition of prokaryotic protein synthesis as compared to NEO and were extremely poor inhibitors of eukaryotic translation. There was significant variation in antibacterial activity and MIC of selected compounds between bacterial strains, with Escherichia coli, Enteroccocus faecalis, Citrobacter freundii, Shigella flexneri, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermidis, and Listeria monocytogenes exhibiting moderate to high sensitivity (50-100% growth inhibition) whereas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellla pneumoniae, and MRSA strains expressed low sensitivity, as compared to the parent aminoglycoside NEO.
氨基糖苷类抗生素的抗菌作用基于它们与细菌核糖体RNA（rRNA）的A位点结合，并干扰细菌细胞内的翻译过程，从而导致细胞死亡。氨基糖苷类抗生素的临床应用因耐药性和副作用而变得复杂，其中一些副作用源于它们与人类线粒体12S rRNA及其与耳聋相关的突变（C1494U和A1555G）的相互作用。我们报告了一种快速检测方法，可用于筛选氨基糖苷类化合物与这些类型rRNA的结合情况。这些筛选工具对于找到选择性结合细菌A位点而非人类线粒体A位点及其突变同源物的抗生素非常重要。在此，我们报告了我们的初步工作，即使用12种蒽醌 - 新霉素（AMA-NEO）偶联物，通过荧光置换筛选试验，针对代表五种A位点同源物（大肠杆菌、人类胞质、线粒体、C1494U和A1555G）的分子构建体优化此筛选方法。还测试了这些偶联物对蛋白质合成的抑制作用、对14种临床相关细菌菌株的抗菌活性以及对使氨基糖苷类抗生素失活的酶的影响。与新霉素（NEO）相比，AMA-NEO偶联物对氨基糖苷类修饰酶（AMEs）的耐药性显著提高。与NEO相比，几种化合物对原核生物蛋白质合成的抑制作用显著更强，并且对真核生物翻译的抑制作用极差。所选化合物在不同细菌菌株之间的抗菌活性和最低抑菌浓度（MIC）存在显著差异，与亲本氨基糖苷类抗生素NEO相比，大肠杆菌、粪肠球菌、弗氏柠檬酸杆菌、福氏志贺菌、粘质沙雷氏菌、奇异变形杆菌、阴沟肠杆菌、表皮葡萄球菌和单核细胞增生李斯特菌表现出中度至高敏感性（生长抑制率为50 - 100%），而鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌和耐甲氧西林金黄色葡萄球菌（MRSA）菌株敏感性较低。
