Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library

Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of “natural product-like” macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.