合成肽聚醣轉醣酶之Lipid II受質與發展過渡態模擬抑制劑

Abstract

細過去數十年來廣泛的使用抗生素下，許多致病細菌病原體突變出具抗藥性的菌種，引起人與動物嚴重的用藥問題。然而這些抗微生物抗藥性不斷的浮現，促使努力地去獲悉更多有關細菌肽聚醣轉醣酶(PGTs)的結構與機制資訊，以利於開發新型抗微生物劑。肽聚醣是組成細菌細胞壁主要組成，其透過Lipid II受質單體的轉醣化與轉胜肽化來形成似網狀聚合體，以提供胞壁的堅韌與剛性來保持菌體形狀，來抵抗外界滲透壓差。 肽聚醣轉醣酶由於不存在於哺乳類細胞，加上酵素是落於細胞膜的外層，且各菌株轉醣酶胺基酸序列同質性高，對於現今所用的抗生素造成抗性的突變株亦含高保留轉醣酶序列，因而日漸成為抗菌劑研究的關注方向。我們的研究目標針對肽聚醣生合成路徑中的獨特轉醣酶，經由化學合成方法來製備出天然Lipid II受質與其衍生物。以高效能液相層析及螢光檢測轉醣酶活性分析顯示，其受質缺乏四胜肽團基仍是可在轉醣化反應作為受質，但是會造成受質與酵素之間的結合力降低。 另外我們也設計與合成出可能的轉醣酶抑制劑，採用3-羥氮雜戊環作為骨架，並在六號位置修飾多種取代團基；隨後合成出轉醣酶天然抑制劑– moenomycin中的phosphoglycerate衍生物部份，因為這方面的團基似乎在雙磷酸脂質鏈結合區佔據極重要的角色。連結3-羥氮雜戊環與phosphoglycerate兩部份設計出潛在的轉醣酶抑制劑，預期生理條件下質子化的氮雜戊環，與轉醣反應中過渡態的oxocarbenium具有相似的結構與電荷分布。 於高效能液相層析搭配螢光檢測轉醣酶活性，除了化合物JMF2809與JMF2805對轉醣酶呈現微弱的抑制性，其它化合物無任何的抑制效果。依據活性的化合物結構特徵來看，極可能是與雙磷酸脂質結合位置發生了競爭而產生抑制性；未來將這些具活性的結構衍生修飾，是有機會發展出更具轉醣酶抑制效果的抑制劑。

After decades of extensively using antibiotics, many bacterial pathogens have mutated to resist antibiotics, and become an increasingly serious medical problem of human and animal. The rise of antimicrobial resistance problem has promoted the research efforts to understand the detailed structures and mechanistic information on bacterial peptidoglycan glycosyltransferases (PGTs) in order to design new antimicrobial agents. Peptidoglycan, a major component of bacterial cell wall, is formed by transglycosylation and transpeptidation of Lipid II substrate to a mesh-like polymer, which provides strength and rigidity to maintain the shape against variable internal osmotic shock. PGTs are attractive targets because no equivalent exists in the mammalian cell. PGTs are located on the outer surface of membrane with high homology in various strains including those mutated to exhibit resistance against the current antibiotics. Our research is to target the specific enzyme, transglycosylase (TGase) in peptidoglycan biosynthesis pathway, through chemical synthesis to prepare natural Lipid II substrate and derivatives. Our HPLC-based TGase fluorescence assay indicated that the truncated Lipid II lacking tetrapeptide moiety was still a substrate in the tranglycosylation process, nevertheless, with decreased substrate–enzyme binding affinity. We also designed and synthesized some possible TGase inhibitors, using a 3-hydroxyl pyrrolidine as the core structure with various substituents at the 6- position. The derivatives of phosphoglycerate motif in moenomycin, the unique natural inhibitor of TGase, were adopted to mimic the important pyrophosphate binding site of Lipid II. Several 3-hydroxyl pyrrolidine core and phosphoglycerate lipid moieties linked to afford the potential TGase inhibitors. In physiological conditions, the protonated pyrrolidine structure has the geometry and charge distribution similar to an oxocarbenium ion, which is a necessary element of transition-state in the process of transglycosylation. By the HPLC-based TGase fluorescence assay, compounds JMF2809 and JMF2805 presented some TGase inhibition, where most of other compounds were inactive. By comparison of chemical structures, the active compounds likely compete the pyrophosphate lipid portion of Lipid II in the TGase binding site. Modification of these active compounds may eventually lead to efficient inhibitors of TGase.