細菌肽聚醣衍生物之合成與功能性探討

Abstract

有別於直接作用於細菌的抗生素，細菌細胞壁之降解分子片段被證實可以誘發宿主免疫機制並清除細菌，具有抵禦病原體感染、甚至是具抗藥性的細菌的潛力，是一個亟需發展的領域。細菌細胞壁組成多元且特性複雜，而其共通主要成分為肽聚醣。為了研究這些與肽聚醣相關的議題，在本文中，透過多步驟化學合成的設計，製備了系統性結構改質之肽聚醣分子群及其前驅物。 本論文研究分為兩個部分： 第一部分、針對胞壁肽系統性化學結構改質之自噬活化劑篩選以應用於殺菌能力之探討 為了對抗細菌感染，透過小分子引發宿主先天性免疫機制作為新的治療策略開始受到關注。透過天然肽聚醣特定片段──胞壁二肽(MDP)引發自噬進而清除細菌的研究，證明了分子誘導之細胞自噬現象與殺菌能力有高度相關。為了解具有殺菌能力之分子結構，這裡我們延續實驗室所開發的化學合成及天然物純化策略，由細菌純化出天然成分如肽聚醣及磷壁酸，以及由(i)單醣中間體、(ii)正交保護雙醣中間體，(iii)二胺基庚二酸(meso-DAP)改質骨架之三肽作衍生化的化學方法，系統性製備一系列胞壁肽衍生物之分子庫。透過細胞實驗建立自噬表現分析之細胞平台，以檢測細胞壁相關衍生物分子庫潛在的自噬活化劑。接著進一步經由沙門氏桿菌清除實驗篩選出相較天然物MDP具有更佳殺菌能力之化合物6、 9及 15，並以微量熱泳動技術探討配體-受器作用力鑑定其模式識別受體及機制，我們初步選擇以與肽聚醣相關性高的人體核苷酸結合寡聚化區域受器(NOD-like receptors)進行實驗，發現化合物15是經由NOD1引發自噬及細菌清除，而化合物6及9則透過其他機制達到細菌清除的效果，透過NOD1及NOD2專一性NF-κB活性測試驗證，提出分子結構、NF-κB及自噬三者之可能關聯性。 第二部分、發展以胞壁肽為基礎之二聚體衍生物應用於人類受體NOD2活性探討 MDP及其衍生物經研究證實具備生物活性，並應用於對抗細菌感染或腫瘤。受到人類受體NOD2引發寡聚化機制的模型啟發，我們以胞壁肽為骨架設計了一系列具有不同位向和長度的新型同源二聚體MDP複合物，並評估其對NOD2活性的影響。經由改善現有的合成方法在MDP醣六號位及胜肽引進胺基，並進行不同長度衍生化得到同源二聚MDP複合物，同時透過細胞實驗分析其長度對NOD2的活性影響，發現分子量1000的間隔為最佳長度，並在後續以微量熱泳動進行配體－受體作用力測試，我們觀察到二聚體複合物36c (1000)與NOD2的解離常數約在12 μM，略優於MDP。此外，我們初步示範將此分子設計方法應用於嫁接複合物及螢光基團並證實其對NOD2的作用力及功能，以期未來應用於其他藥物的嫁接。

Unlike antibiotics that directly act on bacteria, the bio-degraded fragments of bacterial cell wall induce host immune responses to generate pathogen clearance. This is another emerging field for developing a new treatment against bacterial infection even including drug-resistant bacteria. The bacterial cell wall has diverse composition and complex characteristics, of which primary common component is peptidoglycan (PGN). To further study these PGN-related issues, in this thesis, the key lies in the design and synthesis of PGN-associated molecules and their precursors through multi-step synthesis to get systematically modified muropeptides with well-defined structures to investigate the structural requirements for their bioactivity. This research is divided into two parts: Part I. Discovery of muropeptide-derived molecules as autophagy inducers by cellular phenotypic approach to study bacterial clearance (Chapter 2) To develop new therapeutic strategies against bacterial infection, the small molecule-based immunobiotics that can induce host immune responses have gained attention. Through the mechanism studies of muramyl dipeptide (MDP), the strong correlation between autophagy and antibacterial ability was demonstrated in autophagy-mediated bacterial clearance. To elucidate the structural requirements for antibacterial ability, a series of bacterial cell wall-related and muropeptide-derived molecules was prepared by isolation methods or chemical approaches. These PGN derivatives were evaluated toward autophagic responses via a cellular phenotypic approach, and 7 potential autophagy inducers were found through derived from the modification of 3 primary hits, including 2 muropeptides and 1 tripeptide-based molecule. Further in vitro studies demonstrated that compounds 6, 9, and 15, showed a more efficient clearance of intracellular S. typhimurium than the current inducer MDP. In mechanism studies by microscale thermophoresis and receptor-specific NF-κB assay, compound 15 induced autophagy and subsequent bacterial clearance via NOD1 signaling pathway. However, compounds 6 and 9 might activate additional pathways leading to subsequent autophagy-mediated bacterial clearance. These results provide initial insights into the interplay among molecular structures, NF-κB, and autophagy.

Part II. Preparation of dimeric MDP analogs with a variant spacer and their functional study toward NOD2 (Chapter 3) MDP and its analogs has received particular attention due to their antibacterial or anti-tumor activities. Inspired by NOD2-mediated oligomerization, we designed novel homo-dimeric MDP conjugates with different orientations and spacer lengths using a reverse genetic approach. It is revealed that 36c with proper orientation and optimized spacer of 1000 Da in a molecular weight exhibited the best activity toward NOD2-specific NF-κB activity. In the subsequent experiments, we conducted ligand-receptor interaction studies using microscale thermophoresis (MST). Through this technique, we observed that the binding affinity between dimeric 36c and NOD2 is approximately 12 μM, slightly better than that of MDP. This molecular design approach shows potential for future applications in conjugation of other drugs, such as antibody drug conjugates.