| 摘要: | Indolicidin (IL)是一條有廣效抗生活性的鹼性抗生胜肽，包含對抗許多的病原體，可惜的是其細胞毒性大大的限制了其在臨床之使用。我們曾以置換胺基酸的方式來設計出九條IL的類似胜肽，以期具有低細胞毒性且高抗生活性。本研究探討IL及其類似胜肽之結構與生物活性間的關係，並進一步地提出設計抗生胜肽方面的準則，我們將研究帶正電性、親疏水性、疏水矩與抗菌、抗腫瘤、溶血、正常細胞毒性間的關聯。此外也將胜肽進行螢光標定並以共軛聚焦顯微鏡與螢光定量的方式探討其穿膜特性。結果顯示IL具有抗菌及抗腫瘤活性但卻有高溶血性，而IL類似物(IL-K7F89及IL-RF89)則是除了具有高抗菌及抗腫瘤活性，且同時展現低細胞毒性與溶血性，因此這兩條抗生胜肽在胜肽藥物臨床測試與新藥開發方面具有相當的潛力。另一方面，由結果可推測疏水性(Interface scale)與溶血活性有正相關性，但卻與疏水性(Water-octanol scale)和疏水矩並非直接相關，這可能是由於IL及其類似物與生物活性間的關連十分複雜，因此抗生胜肽之設計原則與胜肽之帶正電性、親疏水性與疏水矩，還需要更進一步的分析與探究才能歸納出彼此間的關聯性。在穿膜特性方面，由實驗結果顯示IL能協助FITC進入細胞內而IL-R57F89則不會，推測兩者的穿膜特性可能有些不同，IL可能擾動膜的程度比IL-R57F89還大，也可能是IL能在膜上形成孔洞或造成膜部分溶解，故能協助小分子物質FITC進入細胞中。另一方面，我們將螢光物質FITC以共價鍵結接枝在IL和具有臨床發展潛力的抗菌胜肽IL-R57F89上，結果顯示IL與IL-R57F89會經由胞吞和直接穿膜之形式進入纖維母細胞內。本研究證實IL-R57F89具有雙功能生物特性:一是具有低細胞毒性的鹼性抗生胜肽，二是可做為讓IL-R57F89攜帶小分子物質進入細胞當中的細胞穿膜胜肽。所以IL-R57F89不僅能作為胜肽抗生藥物，也能當載體運送分子進入細胞內，這在藥物的開發方面也開啟一個新的里程。Indolicidin (IL) is a cationic antimicrobial peptide with broad-spectrum against many kinds of pathogens; however, the cytotoxicity of IL limits its clinical application. Nine IL analogues with lower hemolysis or cytotoxicity have been designed by engineering the critical amino acids for IL biological action. In this study, the structure-function relationships of IL and its analogues were discussed to provide important implications on antimicrobial IL analogues design. The effects of hydrophobicity, electropositivity, hydrophobic moment of peptides on their biological activities, including antibacterial activity, normal cell toxicity, hemolytic activity, and antitumor cell activity, were examined. Furthermore, transmembranes of peptides were also monitored by the confocal microscopy analysis via image the localization of FITC-conjugated peptides, and quantified through the FITC fluorescence. IL has effective antibacterial and antitumor activities, but it is harmful to human erythrocytes. In conji.;jltrast, IL-K7F89 and IL-R57F89 peptides exhibit not only high antibacterial and antitumor activities but also lower cytotoxicity and hemolytic activity. These two peptides are potential antimicrobial peptides for clinical use. Furthermore, we observed that the hydrophobicity based on interface scale is well correlated to the hemolytic activity rather than water-octanol scale and hydrophobic moment. Apparently, the biological action of IL and its analogues are trickier. Moreover, we found free IL peptide can facilitate FITC into NIH-3T3 cells but IL-R57F89 doesn’t, indicating that IL peptide could make the membrane perturbation, pore forming or membrane local disruption, to transport the FITC into cell. On the other hand, we conjugated the FITC to IL peptide and potential antimicrobial peptide candidate, IL-R57F89 peptide, through the thiourethane linking. We observed that IL and IL-R57F89 peptides could enter the cell via direct transmembrane and endocytosis. Consequently, we suggested that IL-R57F89 peptide has the dual-functional biological actions: one is a lower cytotoxicity cationic antimicrobial peptide; another is a cell penetrating peptide for transporting the small molecule into cell. |
