Generation of New-to-Nature Lasso Peptides using Chemical and Synthetic Biology

Lasso肽是一种结构独特的天然产物。N-末端大环内酰胺环通过线性C-末端尾部穿线，并且该穿线折叠仅通过环与尾部中具有庞大侧链的氨基酸之间的空间相互作用来维持。Lasso肽通常表现出有趣的生物学特性，特别是抗微生物活性。然而，这些天然存在的肽[1]轮烷不能通过化学合成获得，使得细菌中的（异源）生产目前是获得这些分子的唯一途径。虽然编码套索肽前体的基因的突变使得能够产生新的变体，只要生物合成酶耐受引入的变化，但是前体的核糖体合成限制了可以掺入的氨基酸。在这里，建议利用这样的事实，即套索肽微菌素J25（MccJ 25）的环的部分可以通过蛋白水解去除，而环和尾之间的空间相互作用使这些现在非共价连接的分子仍然彼此缔合。蛋白水解后，得到的[2]轮烷具有新的N-和C-末端，可用于指导位点特异性化学偶联反应。因此，可以用任何其他肽序列以及非肽分子替换环的去除部分。这种方法是完全新颖的，并且将允许产生用于各种应用的新天然套索肽：1）抗微生物套索肽MccJ 25的蛋白水解稳定性可以通过掺入非蛋白质氨基酸和引入更稳定的N-甲基化肽键来改善。因此，MccJ 25用于药物开发的效用将大大增加。2)通过掺入RGD序列，MccJ 25先前被转变为α v β 3整联蛋白受体的nM-亲和性拮抗剂，这是某些肿瘤中令人感兴趣的靶点。通过采用所提出的半合成策略，可以极大地扩展携带RGD的MccJ 25变体的结构-活性-关系研究，并且还包括非蛋白原性和非肽残基。通过在结合基序附近引入偶氮苯部分，将试图获得可以以光诱导方式在高亲和力构象和低亲和力构象之间切换的受体结合剂。3)MccJ 25与利福平的偶联将通过不同的互补机制将联合收割机两种抑制细菌RNA聚合酶（RNAP）的分子结合。通过筛选具有不同缀合位点和接头基团的多种缀合物，将尝试产生双功能的协同RNAP抑制剂。两者合计，提出了一个及时的项目结合基础和应用驱动的研究目标。德国和台湾的两个小组之间的密切合作，以其独特的专门知识相互补充，为计划的工作方案奠定了基础。

Lasso peptides are natural products with a remarkable structure. An N-terminal macrolactam ring is threaded by a linear C-terminal tail and this threaded fold is maintained merely by steric interactions between the ring and amino acids with bulky side chains in the tail. Lasso peptides often exhibit interesting biological properties, especially antimicrobial activities. These naturally occurring, peptidic [1]rotaxanes can however not be accessed by chemical synthesis, making the (heterologous) production in bacteria currently the only route to obtain these molecules. While mutation of the genes encoding the lasso peptide precursors enables the generation of new variants as long as the biosynthetic enzymes tolerate the changes introduced, the ribosomal synthesis of the precursors limits what amino acids can be incorporated. Here, it is proposed to leverage the fact that parts of the loop of the lasso peptide microcin J25 (MccJ25) can be removed proteolytically, while the steric interactions between ring and tail keep these now non-covalently connected molecules still associated with each other. After proteolysis, the resulting [2]rotaxane has a new N- and C-terminus that can be used to guide site specific chemical coupling reactions. Thereby, it becomes possible to replace the removed portion of the loop with any other peptide sequence but also with non-peptidic molecules. This approach is completely novel and will allow the generation of new-to-nature lasso peptides for various applications: 1) Proteolytic stability of the antimicrobial lasso peptide MccJ25 can be improved by incorporation of non-proteinogenic amino acids and introduction of more stable, N-methylated peptide bonds. Thus, the utility of MccJ25 for drug development would be vastly increased. 2) By incorporation of the RGD sequence, MccJ25 was previously turned into a nM-affinity antagonist of the v3 integrin receptor, which is an interesting target in certain tumors. By employing the proposed semi-synthetic strategy, structure-activity-relationship studies of RGD-carrying MccJ25 variants can be vastly extended and also include non-proteinogenic and non-peptidic residues. By introducing an azobenzene moiety in proximity to the binding motif, it will be attempted to obtain receptor binders that can be toggled between high- and low-affinity conformations in a light-inducible manner. 3) Conjugation of MccJ25 with rifampicin would combine two molecules inhibiting the bacterial RNA polymerase (RNAP) by distinct, complementary mechanisms. By screening a variety of conjugates with different conjugation sites and linker groups, it will be attempted to generate bifunctional, synergistic RNAP inhibitors. Taken together, a timely project is proposed combining basic and application-driven research goals. The close collaboration between the two groups in Germany and Taiwan, who strongly complement each other with their unique expertise, lays the foundation for the planned work programme.