Knappe Daniel, Ruden Serge, Langanke Stefanie, Tikkoo Tarun, Ritzer Jennifer, Mikut Ralf, Martin Lisandra L, Hoffmann Ralf, Hilpert Kai
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
Amino Acids. 2016 Jan;48(1):269-80. doi: 10.1007/s00726-015-2082-2. Epub 2015 Sep 3.
The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4-8 and 0.5 µg/mL, respectively.
开发新型抗菌药物以鉴定针对多重耐药菌的先导分子是一项紧迫任务。富含脯氨酸的抗菌肽在体外和体内均具有高活性，但仅针对少数革兰氏阴性人类病原体，对铜绿假单胞菌和金黄色葡萄球菌的活性较弱。这种疗效降低可能与摄取机制不足或细胞内靶蛋白（即70S核糖体或伴侣蛋白DnaK）的结构差异有关。在这里，我们使用可裂解连接子在纤维素膜上合成肽阵列，以释放游离的单个肽用于进一步的抗菌测试。因此，通过用所有其他19种标准氨基酸替换每个残基，产生了一个单取代癌蛋白类似物文库，得到一组361个单独的肽，用于针对发光铜绿假单胞菌菌株进行评估。13种取代显示出前景，在大规模合成这些类似物后，它们对不同细菌的抗菌活性得到了证实。通过将两种有利的取代组合到一个肽中，我们最终获得了一种癌蛋白类似物，其对铜绿假单胞菌的活性比原始癌蛋白高10倍，对金黄色葡萄球菌的活性甚至高100倍，最小抑菌浓度分别为4-8和0.5μg/mL。
