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

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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.