探索套索胜肽的潛力：合成 MccJ25 仿生分子及擴展其應用

Abstract

近幾十年來，抗藥性細菌的威脅已成為全球性的醫藥危機。其中包括金黃色葡萄球菌 (Staphylococcus aureus)、鮑氏不動桿菌 (Acinetobacter baumannii) 和大腸桿菌 (Escherichia coli) 等許多種抗藥性病原菌都顯著地提升了相關的死亡率。 Microcin J25 (MccJ25) 是一種針對革蘭氏陰性菌的強效抗生素，屬於核醣體合成及轉錄後修飾胜肽 (RiPPs) 家族。MccJ25 以其透過干擾 RNA 聚合酶(RNAP) 的轉錄功能來抑制細菌生長而備受關注。在結構上，MccJ25 有著特殊的三維套索 (lariat-like) 構型。這種獨特的結構使 MccJ25 對熱及 pH 值的變化以及酵素的降解有著極高的穩定性。然而，也正是因為這種結構的穩定性，對於深入研究及探索 MccJ25 的應用潛力都帶來了挑戰。 在本研究中，我們將透過兩種化學的角度來更進一步探索 MccJ25：(1) 利用固相胜肽合成 (SPPS) 及有機合成的技術來合成 MccJ25 的仿生分子；(2) 結合特定的酵素與化學合成方法來創造 MccJ25 的衍生物。 在第一種策略中，我們將 MccJ25 視為一種「構型受限」的支鏈環狀 (cyclic branched) 胜肽。利用這種概念，我們設計了多種運用共價或非共價作用的方法來模仿 MccJ25 的構型。我所設計的 Y 型仿生分子 (Y mimic) 主要是結合SPPS再利用共價作用的方法來約束從而達到構型受限的目的。運用半胱氨酸 (cysteine) 取代了三個原有序列的胺基酸，使其能夠與1,3,5-三(溴甲基)苯 (TBMB) 進行三次的雙分子親核取代 (SN2) 反應，固定原先可變動的 (flexible) 支鏈環狀胜肽骨架，進而模仿 MccJ25 的結構。在研究成果中，我們所合成的 Y mimics 物能夠有效地抑制 RNAP 的功能，為我們的假設提供了強而有力的驗證。 在第二種方法中，我們希望可以利用 MccJ25 極高的穩定性與其較長的環狀區域 (loop region) 作為一種特殊的骨架載體，結合酵素與化學反應來形成 MccJ25 的衍生物。我們首先利用嗜熱菌蛋白酶 (thermolysin) 將 MccJ25 有選擇性的從[1]輪烷 ([1]rotaxane) 轉變為[2]輪烷。搭配我們進一步的化學合成方法，成功地合成了不同的 MccJ25 衍生物，並證明了 MccJ25 的環狀區域對生物活性方面具有關鍵作用。 我們的下一步是利用 MccJ25 獨特的結構和卓越的穩定性來拓展其骨架的應用。計劃引入治療性配體 (therapeutic ligand)，希望增強其對目標的結合親和力 (binding affinity) 並且提高本身對酵素的穩定性。我們相信這些策略將加深我們對 MccJ25 的理解，並促進更多化學和醫學方面的研究與應用。

Bacterial antimicrobial resistance (AMR) has escalated into a global crisis in recent decades, with several resistant pathogens such as Staphylococcus aureus, Acinetobacter baumannii, and Escherichia coli contributing significantly to mortality rates. Microcin J25 (MccJ25), belongs to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs), is a potent gram-negative bacteria antibiotic. It’s notable for its ability to inhibit bacterial growth by disrupting RNA polymerase (RNAP) function. Structurally, MccJ25 is a lasso peptide characterized with a lariat-like three-dimensional configuration. This unique threaded configuration grants MccJ25 exceptional stability against environmental factors such as heat, pH variations, and enzymatic digestion. However, this same structural stability has posed challenges for detailed characterization and the exploration of MccJ25's potential applications through chemical and biological methods. In this study, we aimed to explore the chemical landscape of MccJ25 through two approaches: (1) synthesize MccJ25 mimics by combining solid-phase peptide synthesis and organic synthesis, and (2) derivatize MccJ25 by combining site specific enzymatic cleavage and chemical synthesis. In the first strategy, we viewed the structure of MccJ25 as a “conformationally-restricted” branched macrocyclic peptide and developed several mimic designs using covalent and noncovalent forces to constrain the backbone conformation. My design, referred to as the Y mimic, was synthesized using SPPS and organic chemistry. This design replaced three residues with cysteine, thereby enabling a three-fold SN2 reaction with 1,3,5-tris(bromomethyl)benzene (TBMB) to anchor the peptide backbone in a manner similar to MccJ25. The resulting Y mimics successfully inhibited RNAP function, providing strong validation for our hypothesis. In the second approach, we exploited MccJ25’s remarkable stability and its long loop regions, hypothesizing that MccJ25 could potentially serve as a robust scaffold to present epitopes by combining enzymatic and chemical reactions. We first demonstrated that thermolysin can cleave MccJ25 site-specifically, converting it from a [1]rotaxane to a [2]rotaxane. We then successfully synthesized derivatives of MccJ25, highlighting the critical role of the loop region in maintaining its bioactivity. Our next step is to further explore the potential of MccJ25 by taking advantage of its unique structure and exceptional stability. Specifically, we plan to incorporate a therapeutic ligand which could potentially enhance its binding affinity and resistance to proteases. We believe these strategies will deepen our understanding of MccJ25 and enable the exploration of many novel chemical and medicinal applications.