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

利用化学和合成生物学生成新的天然套索肽

• 批准号: 528244377
• 负责人: Dr. Julian Hegemann
• 金额: --
• 依托单位: National Science and Technology Council (NSTC)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/528244377?language=en
• 关键词: Generation; New; Nature; Lasso; Peptides

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.

套索肽是一种结构独特的天然产物。N-末端的大内酰胺环由线性的C-末端尾部缠绕，这种螺纹状折叠仅通过环与尾部带有巨大侧链的氨基酸之间的空间相互作用来维持。套索多肽经常表现出有趣的生物学特性，特别是抗菌活性。然而，这些自然产生的多肽轮烷不能通过化学合成获得，这使得在细菌中(异源)生产目前是获得这些分子的唯一途径。虽然编码套索多肽前体的基因的突变能够产生新的变体，只要生物合成酶容忍引入的变化，但前体的核糖体合成限制了可以结合的氨基酸。在这里，它被提议利用这样一个事实，即套索多肽microcin J25(MccJ25)的环的部分可以被蛋白质水解性移除，而环和尾部之间的立体相互作用使这些现在非共价连接的分子仍然相互关联。蛋白分解后，生成的[2]轮烷有一个新的N-端和C-端，可用于指导特定部位的化学偶联反应。因此，可以用任何其他肽序列替换环的去除部分，也可以用非肽分子替换。这种方法是完全新颖的，将允许产生新的自然套索多肽用于各种应用：1)抗菌套索多肽MccJ25的蛋白分解稳定性可以通过加入非蛋白生成氨基酸和引入更稳定的N-甲基化多肽键来改善。因此，MccJ25在药物开发中的效用将大大增加。2)通过引入RGD序列，MccJ25先前被转变为v3整合素受体的NM亲和力拮抗剂，而整合素受体是某些肿瘤的有趣靶点。通过采用所提出的半合成策略，携带RGD的MccJ25变异体的结构-活性-关系研究可以得到极大的扩展，还可以包括非蛋白来源和非肽残基。通过在结合基序附近引入偶氮苯部分，将尝试获得能够以光诱导的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。3)MccJ25与利福平的结合将通过不同的、互补的机制将抑制细菌RNA聚合酶(RNAP)的两个分子结合在一起。通过筛选具有不同连接位点和连接基团的各种结合物，将试图产生双功能、协同的RNAP抑制剂。综上所述，提出了一个结合基础研究目标和应用驱动研究目标的适时项目。德国和台湾的两个小组之间的密切合作为计划中的工作方案奠定了基础，这两个小组以其独特的专长有力地相互补充。