Institute for Infection and Immunity, St. George's University of London, London, UK.
TiKa Diagnostics Ltd, London, UK.
Biochim Biophys Acta Biomembr. 2020 Aug 1;1862(8):183195. doi: 10.1016/j.bbamem.2020.183195. Epub 2020 Mar 1.
The global health threat surrounding bacterial resistance has resulted in antibiotic researchers shifting their focus away from 'traditional' antibiotics and concentrating on other antimicrobial agents, including antimicrobial peptides. These low molecular weight "mini-proteins" exhibit broad-spectrum activity against bacteria, including multi-drug resistant strains, viruses, fungi and protozoa and constitute a major element of the innate-immune system of many multicellular organisms. Some naturally occurring antimicrobial peptides are lipidated and/or glycosylated and almost all antimicrobial peptides in clinical use are either lipopeptides (Daptomycin and Polymyxin E and B) or glycopeptides (Vancomycin). Lipidation, glycosylation and PEGylation are an option for improving stability and activity in serum and for reducing the rapid clearing via the kidneys and liver. Two broad-spectrum antimicrobial peptides NH-RIRIRWIIR-CONH (A1) and NH-KRRVRWIIW-CONH (B1) were conjugated via a linker, producing A2 and B2, to individual fatty acids of C, C, C and C and in addition, A2 was conjugated to either glucose, N-acetyl glucosamine, galactose, mannose, lactose or polyethylene glycol (PEG). Antimicrobial activity against two Gram-positive strains (methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE)) and three Gram-negative strains (Salmonella typhimurium, E. coli and Pseudomonas aeruginosa) were determined. Activity patterns for the lipidated versions are very complex, dependent on sequence, bacteria and fatty acid. Two reciprocal effects were measured; compared to the parental peptides, some combinations led to a 16-fold improvement whereas other combinations let to a 32-fold reduction in antimicrobial activity. Glycosylation decreased antimicrobial activity by 2 to 16-fold in comparison to A1, respectively on the sugar-peptide combination. PEGylation rendered the peptide inactive. Antimicrobial activity in the presence of 25% human serum of A1 and B1 was reduced 32-fold and 8-fold, respectively. The longer chain fatty acids almost completely restored this activity; however, these fatty acids increased hemolytic activity. B1 modified with C8 increased the therapeutic index by 2-fold for four bacterial strains. Our results suggest that finding the right lipid-peptide combination can lead to improved activity in the presence of serum and potentially more effective drug candidates for animal studies. Glycosylation with the optimal sugar and numbers of sugars at the right peptide position could be an alternative route or could be used in addition to lipidation to counteract solubility and toxicity issues.
全球范围内的细菌耐药性威胁导致抗生素研究人员将研究重点从“传统”抗生素转移到其他抗菌剂上，包括抗菌肽。这些低分子量的“迷你蛋白”对细菌具有广谱活性，包括多药耐药菌株、病毒、真菌和原生动物，是许多多细胞生物固有免疫系统的重要组成部分。一些天然存在的抗菌肽是脂化和/或糖基化的，几乎所有在临床上使用的抗菌肽都是脂肽（达托霉素和多粘菌素 E 和 B）或糖肽（万古霉素）。脂质化、糖基化和聚乙二醇化是提高稳定性和活性、减少肾脏和肝脏快速清除的一种选择。两种广谱抗菌肽 NH-RIRIRWIIR-CONH（A1）和 NH-KRRVRWIIW-CONH（B1）通过连接子连接，产生 A2 和 B2，分别与 C、C、C 和 C 的单个脂肪酸连接，此外，A2 与葡萄糖、N-乙酰葡萄糖胺、半乳糖、甘露糖、乳糖或聚乙二醇（PEG）连接。测定了两种革兰氏阳性菌株（耐甲氧西林金黄色葡萄球菌（MRSA）和万古霉素耐药肠球菌（VRE））和三种革兰氏阴性菌株（鼠伤寒沙门氏菌、大肠杆菌和铜绿假单胞菌）的脂化版本的抗菌活性。脂化版本的活性模式非常复杂，取决于序列、细菌和脂肪酸。测量了两个相互影响的效果；与亲本肽相比，一些组合导致抗菌活性提高了 16 倍，而其他组合则导致抗菌活性降低了 32 倍。与 A1 相比，糖肽组合使糖苷化降低了 2 到 16 倍的抗菌活性。聚乙二醇化使肽失去活性。A1 和 B1 在 25%人血清中的抗菌活性分别降低了 32 倍和 8 倍。较长链脂肪酸几乎完全恢复了这种活性；然而，这些脂肪酸增加了溶血活性。用 C8 修饰的 B1 使四种细菌的治疗指数提高了 2 倍。我们的结果表明，找到合适的脂质-肽组合可以提高在血清存在下的活性，并为动物研究提供更有效的候选药物。与最佳糖基化和正确肽位置的糖基化可以是替代途径，或者可以与脂质化结合使用，以对抗溶解度和毒性问题。
