Strömstedt Adam A, Pasupuleti Mukesh, Schmidtchen Artur, Malmsten Martin
Department of Pharmacy, Uppsala University, Sweden.
Antimicrob Agents Chemother. 2009 Feb;53(2):593-602. doi: 10.1128/AAC.00477-08. Epub 2008 Nov 24.
Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.
对提高EFK17（EFKRIVQRIKDFLRNLV）蛋白水解稳定性的方法进行了评估，EFK17是一种源自LL-37的具有抗菌特性的新肽序列。EFK17在已知蛋白酶切割位点通过四个d-对映体或色氨酸（W）取代进行修饰，同时进行末端酰胺化和乙酰化。对肽变体进行了蛋白水解抗性、抗菌效力和细胞毒性方面的研究，还研究了它们在模型脂质膜上的吸附、脂质体泄漏产生以及二级结构行为。W取代导致人中性粒细胞弹性蛋白酶、金黄色葡萄球菌溶血素和V8蛋白酶引起的蛋白水解降解显著降低，但对铜绿假单胞菌弹性蛋白酶引起的降解没有影响。对于前两种内切蛋白酶，末端的酰胺化和乙酰化也降低了蛋白水解降解，但仅在与W取代结合使用时才有效。d-对映体取代使肽对所有四种蛋白酶都难以消化；然而，这些肽几乎没有抗菌效力。另一方面，与天然肽序列相比，W修饰和末端修饰的肽显示出更高的杀菌效力，同时具有适度的细胞毒性，且在血清中基本不存在。杀菌、细胞毒性和脂质体裂解特性相互关联，也与肽在脂质膜上的吸附量以及与吸附相关的螺旋形成程度相关。W取代肽的裂解特性受离子强度增加的影响较小，可能是由于W介导的主要两亲性螺旋构象的稳定作用以及W对双层界面的非静电亲和力的综合作用。总体而言，W取代是一种有趣的方法，可降低EFK17的蛋白水解敏感性，同时提高抗菌性能。
