Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes

[Image: see text] Chemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications. Chemical protein synthesis allows a wide range of changes to the protein’s chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, d-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic d-peptide binders. Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates. Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48- and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field.