第一部分：利福平與Microcin J25共軛物對抑制細菌生長的潛在協同作用 第二部分：利用Phage display技術篩選可結合GFP螢光基團的環狀胜肽

Abstract

第一部分: 利福平與Microcin J25共軛物對抑制細菌生長的潛在協同作用 利福平(rifampicin)為現今廣泛使用的一種半合成廣效型抗生素，其抗菌機制藉由結合RNA聚合酶(RNAP)的β次單元來抑制轉錄。隨著現今抗生素的濫用導致人們面臨多重抗藥性的超級細菌威脅，我們因此急迫需要研發新型抗生素來面對難關。其中抗生素共軛物的研發便是解方之一，透過將抗生素與另一個功能性分子，如胜肽、抗體、不同抗生素等，以化學合成方法連接起來進而得到提升膜破壞力、結合專一性、殺菌廣度等促使抗菌能力顯著增強的協同作用。 此次研究我們選定同樣結合RNA聚合酶的胜肽類抗生素Microcin J25 (MccJ25)當作與利福平共軛的標靶藥物並預期具有協同作用，根據晶體結構兩者僅相距20-35 Å。實驗設計上我們分別對利福平做化學炔基修飾及對MccJ25序列上三個不嚴重影響與RNAP結合的位置做個別定點突變並純化出帶有離胺酸的MccJ25突變物。利用五乙二醇及八乙二醇連接基團兩端的羧基與疊氮化物做醯胺偶連反應及點擊化學以合成出三個利福平-MccJ25共軛物。透過最小抑制濃度測試我們發現三個共軛物均可有效抑制革蘭氏陽性菌的金黃色葡萄球菌(Staphylococcus aureus)及枯草桿菌(Bacillus subtilis)，而其中一個共軛物可以抑制革蘭氏陰性菌的腸道沙門氏菌(Salmonella enterica)。 雖然我們設計的共軛物有顯著的抗菌能力，然而其抑制程度卻均不及相同濃度的利福平與突變MccJ25的混合物。我們認為缺乏協同效應的原因可能源自連接基團長度或抗生素相對位向無法讓兩者同時正確結合在抑制位點，未來我們的優化方向將嘗試選用更多不同長度的連接基團及對不同MccJ25位置做連接，試圖提升兩者同時結合的可能。

第二部分: 利用Phage display技術篩選可結合GFP螢光基團的環狀胜肽 蛋白質螢光標籤技術為研究蛋白表現、功能、相互作用及細胞內外定位與動態變化的重要工具。現行方法多以將螢光蛋白（如GFP）基因融合於目標蛋白之 N 端或 C 端，或利用可與特定螢光分子共價鍵結的蛋白標籤（如Halo Tag、SNAP-tag）進行標記。本研究旨在建立一套新型螢光標籤系統，藉由合成 GFP 螢光基團衍生物，並運用噬菌體展示技術（phage display）篩選可專一性結合此類螢光分子的小分子胜肽，期望未來可將該胜肽之基因序列融合至目標蛋白，並透過添加 GFP 螢光基團衍生物以達到標記之目的。 該研究初步合成了 GFP 螢光核心結構 HBI 及其三個衍生物（DMHBI、DFHBI、DMABI）並與 M13 噬菌體進行混合螢光測試，結果未觀察到螢光訊號確認了此類螢光基團不會與噬菌體產生非特異性結合。接續將 HBI 與 DMHBI 接上生物素標籤，作為與鍊親合素磁珠結合的探針，以利後續噬菌體展示篩選流程之執行。

Part I. Investigation of Potential Antibacterial Synergistic Effects of Rifampicin–Microcin J25 Conjugates in Inhibiting Bacterial Growth Rifampicin is a widely used semi-synthetic broad-spectrum antibiotic that inhibits bacterial transcription by binding to the β-subunit of RNA polymerase (RNAP). However, due to the misuse of antibiotics, the rise of multidrug-resistant "superbugs" presents an urgent need for novel antibiotic strategies. Among these, antibiotic conjugates have emerged as promising candidates. By chemically linking antibiotics to other functional molecules, such as peptides, antibodies, or different antibiotics, conjugates can significantly enhance antibacterial efficacy through synergistic effects, including improved membrane disruption, specificity, and broadened bactericidal spectra. In this study, we covalently linked Microcin J25 (MccJ25), a peptide antibiotic also targeting RNAP, to rifampicin, in hope of creating a conjugate with synergistic effect. The two are in close spatial proximity (~20–35 Å) as revealed by the crystal structure. To facilitate conjugation, rifampicin was chemically modified with an alkyne group, while three specific sites within the MccJ25 sequence, previously determined as non-critical for RNAP binding were individually mutated to lysine-containing variants. Using penta- and octa-ethylene glycol linkers containing carboxylic acid at one end and an azide group at the other, three rifampicin–MccJ25 conjugates were synthesized via amide coupling reactions and click chemistry. Minimum inhibitory concentration (MIC) assays demonstrated that all three conjugates effectively inhibited Gram-positive bacteria, including Staphylococcus aureus and Bacillus subtilis. Notably, one conjugate exhibited activity against the Gram-negative bacteria Salmonella enterica. Although our designed conjugates displayed significant antibacterial activities, their efficacy was inferior to that of mixtures of rifampicin and mutant MccJ25 at equivalent concentrations. We propose that the observed lack of synergistic effects could stem from suboptimal linker length or spatial orientation, preventing simultaneous proper binding of both antibiotics to their respective inhibitory sites. Future optimization will explore different linker lengths and alternative conjugation sites on MccJ25 to enhance simultaneous target binding and overall antibacterial synergy.

Part II. Identifying Cyclic Peptides That Bind the Pro-Fluorescent GFP Chromophore via Phage Display Fluorescent protein labeling is a vital technique for studying protein expression, function, interactions, and spatial–temporal dynamics both inside and outside of cells. Current strategies commonly involve fusing fluorescent proteins (such as GFP) to the N- or C-terminus of the target protein, or using self-labeling protein tags (such as Halo Tag or SNAP-tag) that covalently bind to specific fluorescent ligands. In this study, we aim to develop a novel fluorescent labeling system by synthesizing GFP chromophore derivatives and screening for small peptide ligands with specific binding affinity to these fluorophores using phage display. The selected peptide sequences may subsequently be genetically fused to target proteins, enabling fluorescent labeling through the addition of corresponding GFP chromophore derivatives. To initiate the system, we synthesized the core GFP chromophore HBI and three derivatives (DMHBI, DFHBI, and DMABI), and examined their fluorescence upon mixing with M13 phage. No fluorescence signal was detected, confirming that these fluorophores do not exhibit nonspecific binding to the phage particles. We then conjugated biotin to HBI and DMHBI, generating biotinylated fluorescent probes for immobilization on streptavidin magnetic beads, thereby enabling downstream phage display selection.