Kondejewski L H, Farmer S W, Wishart D S, Kay C M, Hancock R E, Hodges R S
Department of Biochemistry and the Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta, T6G 2S2.
J Biol Chem. 1996 Oct 11;271(41):25261-8. doi: 10.1074/jbc.271.41.25261.
We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in beta-sheet content, with rings containing 6, 10, and 14 residues exhibiting beta-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Gram-positive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gram-negative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.
我们评估了短杆菌肽S类似物的环大小对二级结构、脂质结合、脂质破坏、抗菌和溶血活性的影响。设计了环大小从4到14个残基的环状类似物，以保持短杆菌肽S中发现的两亲性特征，并通过固相肽合成法进行合成。这些肽的二级结构在β-折叠含量上呈现出一定的周期性，含有6、10和14个残基的环呈现出β-折叠结构，而含有8或12个残基的环大多是无序的。含有4或6个残基的肽不结合脂多糖，而较长的肽显示出随着长度增加对脂多糖结合亲和力增加的趋势。仅在含有10个或更多残基的肽中观察到大肠杆菌外膜的不稳定。含有少于10个残基的肽完全无活性且无溶血活性。10个残基的肽显示出与短杆菌肽S本身相似的活性谱，对革兰氏阳性和革兰氏阴性微生物以及酵母有活性，但也显示出高溶血活性。通过将环的大小增加到12或14个残基获得了不同的活性。14个残基的肽没有抗生素活性，但溶血活性增加。12个残基的肽失去了对革兰氏阳性细菌的活性，保留了对革兰氏阴性微生物和酵母的活性，但溶血活性降低。通过对包含疏水和碱性位点的残基进行一系列取代，优化了12个残基肽的生物学活性，得到了一种肽，其对革兰氏阴性微生物和酵母的活性与短杆菌肽S相当，但溶血活性大大降低。与短杆菌肽S相比，通过治疗指数测定，最佳类似物对革兰氏阴性微生物的抗生素特异性提高了10倍，对酵母相对于人红细胞的特异性提高了7倍。这些结果表明，通过改变环大小可以调节环状短杆菌肽S类似物的结构和活性，并进一步显示了基于短杆菌肽S开发临床有用抗生素的潜力。
