Koehbach Johannes, Gani Jurnorain, Hilpert Kai, Craik David J
Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
Institute for Infection and Immunity, St. George's University, London SW17 0RE, UK.
Microorganisms. 2021 Jun 8;9(6):1249. doi: 10.3390/microorganisms9061249.
According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.
根据世界卫生组织（WHO）的数据，微生物对抗生素产生耐药性是最紧迫的健康问题之一。由于目前只有少数制药公司在开发新型抗菌化合物，这种情况将加剧。迫切需要发现和开发具有新作用模式的新型抗菌化合物。抗菌肽（AMPs）已知能够杀死多重耐药细菌，因此有兴趣被开发成抗菌药物。这些肽的蛋白水解稳定性和毒性是需要克服的挑战，而经常用于解决稳定性问题的一种策略是环化。在这里，我们引入了一个二硫键来环化一种强效且无毒的9聚体肽，此外，作为概念验证研究，将该肽嫁接到环肽MCoTI-II的环6中。这是首次将抗菌肽成功嫁接到环肽支架上。二硫键环化和嫁接的环肽在肉汤中显示出中等活性，在1/5肉汤中对临床相关耐药病原体显示出强活性。线性肽在两种条件下均显示出优异的活性。测定了线性肽在100%人血清中的半衰期为13分钟，简单二硫键环化肽为9分钟，嫁接环肽为7小时15分钟。添加10%人血清会导致不同生物体的抗菌活性丧失，环肽的丧失倍数为1至>8倍。对于二硫键环化版本和线性版本，活性也分别下降到不同程度，分别为2至18倍和1至30倍。尽管稳定性存在巨大差异，但线性肽仍显示出优异的抗菌活性。环肽和二硫键环化版本的杀菌效果比线性版本慢。所有三种肽在高pH和低pH下均稳定，并且具有非常低的溶血和细胞毒性活性。
