Leibniz Institute of Molecular Pharmacology (FMP), Berlin, Germany.
Antimicrob Agents Chemother. 2011 Feb;55(2):788-97. doi: 10.1128/AAC.01098-10. Epub 2010 Nov 22.
The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane of E. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS >> POPC-rough LPS > POPC-lipid A = POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coli activity of the cyclic peptides.
环-RRRWFW（c-WFW）对大肠杆菌的活性已被证明受到芳香基序和细菌外膜中脂多糖（LPS）的调节。为了确定相互作用的位点并阐明 c-WFW 的作用方式，通过用具有改变的疏水性、偶极矩和四极矩、氢键形成能力、两亲性和环大小的类似物替换色氨酸（W）来合成肽。肽对枯草芽孢杆菌和红细胞的活性随着疏水性的增加而增加，而对大肠杆菌的影响则呈现出更为复杂的模式。尽管它们在浓度高于 MIC 时对大肠杆菌内膜没有影响，但根据其抗菌活性模式，肽渗透了外膜，这表明 LPS 在肽穿过细胞壁的运输中起主要作用。对于肽-脂质双层相互作用的等温滴定量热法（ITC）研究，我们使用 POPC（1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱），单独使用或与 1-棕榈酰基-2-油酰基-sn-甘油-3-[磷酸-rac-（1-甘油）]（POPG）混合使用，分别模拟真核和细菌膜的电荷特性，以及与脂质 A、粗糙 LPS 和光滑 LPS 的混合物，作为大肠杆菌外膜的模型。肽积累通过静电和疏水相互作用来确定。脂质系统的敏感性遵循以下顺序：POPC-光滑 LPS >> POPC-粗糙 LPS > POPC-脂质 A = POPC-POPG > POPC。低肽疏水性和增强的灵活性降低了结合能力。其他特性对分配自由能的影响较小，但氢键形成能力和偶极矩的增强导致焓和熵的贡献发生显著变化。在存在粗糙和光滑 LPS 的情况下，这些参数对结合的调节作用降低。对大肠杆菌的高度分化的活性模式在肽与含 LPS 的膜的结合中反映较差。然而，与 POPC-粗糙 LPS 相比，肽在 POPC-光滑 LPS 中的分配更强，这揭示了 O-抗原和外核心寡糖在肽运输和外膜通透性以及环状肽的抗大肠杆菌活性中的重要作用。
