Zhu Wan Long, Hahm Kyung-Soo, Shin Song Yub
Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Korea.
J Pept Sci. 2007 Aug;13(8):529-35. doi: 10.1002/psc.882.
Recently, we designed a novel cell-selective antimicrobial peptide (TPk) with intracellular mode of action from Pro --> Nlys (Lys peptoid residue) substitution in a noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptide, tritrpticin-amide (TP; VRRFPWWWPFLRR-NH(2)) (Biochemistry 2006; 45: 13007-13017). In this study, to elucidate the effect of Pro --> Nlys substitution on therapeutic index and mode of action of other noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptides and develop novel short antimicrobial peptides with high cell selectivity/therapeutic index, we synthesized Nlys-substituted antimicrobial peptides, TPk, STPk and INk, in which all proline residues of TP, symmetric TP-analogue (STP; KKFPWWWPFKK-NH(2)) and indolicidin (IN; ILPWKWPWWPWRR-NH(2)) were replaced by Nlys, respectively. Compared to parent Pro-containing peptides (TP, STP and IN), Nlys substituted peptides (TPk, STPk and Ink) had 4- to 26-fold higher cell selectivity/therapeutic index. Parent Pro-containing peptides induced a significant depolarization of the cytoplasmic membrane of intact Staphylococcus aureus at their MIC, whereas Nlys-substituted antimicrobial peptides did not cause visible membrane depolarization at their MIC. These results suggest that the antibacterial action of Nlys-substituted peptides is probably not due to the disruption of bacterial cytoplasmic membranes but the inhibition of intracellular components. Taken together, our results showed that Pro --> Nlys substitution in other noncell-selective Trp/Pro-rich antimicrobial peptides such as STP and IN as well as TP can improve the cell selectivity/therapeutic index and change the mode of antibacterial action from membrane-disrupting to intracellular targeting. In conclusion, our findings suggested that Pro --> Nlys substitution in noncell-selective Trp/Pro-rich antimicrobial peptides is a promising method to develop cell-selective antimicrobial peptides with intracellular target mechanism.
最近，我们在一种非细胞选择性的、源自cathelicidin的富含色氨酸/脯氨酸的抗菌肽tritrpticin-酰胺（TP；VRRFPWWWPFLRR-NH₂）中，通过将脯氨酸（Pro）替换为N-赖氨酸（Lys类肽残基），设计了一种具有细胞内作用模式的新型细胞选择性抗菌肽（TPk）（《生物化学》，2006年；45: 13007 - 13017）。在本研究中，为了阐明Pro替换为N-赖氨酸对其他非细胞选择性的、源自cathelicidin的富含色氨酸/脯氨酸的抗菌肽的治疗指数和作用模式的影响，并开发具有高细胞选择性/治疗指数的新型短抗菌肽，我们合成了N-赖氨酸取代的抗菌肽TPk、STPk和INk，其中TP、对称TP类似物（STP；KKFPWWWPFKK-NH₂）和吲哚杀菌素（IN；ILPWKWPWWPWRR-NH₂）的所有脯氨酸残基分别被N-赖氨酸取代。与含脯氨酸的亲本肽（TP、STP和IN）相比，N-赖氨酸取代的肽（TPk、STPk和Ink）的细胞选择性/治疗指数高4至26倍。含脯氨酸的亲本肽在其最低抑菌浓度（MIC）时可诱导完整金黄色葡萄球菌的细胞质膜发生显著去极化，而N-赖氨酸取代的抗菌肽在其MIC时未引起可见的膜去极化。这些结果表明，N-赖氨酸取代肽的抗菌作用可能不是由于破坏细菌细胞质膜，而是抑制细胞内成分。综上所述，我们的结果表明，在其他非细胞选择性的富含色氨酸/脯氨酸的抗菌肽（如STP和IN以及TP）中进行Pro替换为N-赖氨酸，可以提高细胞选择性/治疗指数，并将抗菌作用模式从破坏膜转变为靶向细胞内。总之，我们的研究结果表明，在非细胞选择性的富含色氨酸/脯氨酸抗菌肽中进行Pro替换为N-赖氨酸是开发具有细胞内靶向机制的细胞选择性抗菌肽的一种有前景方法。
