RaPID development of macrocyclic peptides for challenging targets Catherine Hurd 1,2 , Jacob Bush 1 , Andrew Powell 1 , Louise Walport 2 1 Crick-GSK Biomedical LinkLabs, UK, 2 Protein-Protein Interaction Laboratory, UK mRNA display is a powerful tool to screen libraries of >10 12 peptides against a protein of interest. It has been used successfully to generate high affinity peptide ligands for a wide range of protein targets. Despite this wide scope, the methodologies have previously been limited to targets that can be expressed and purified recombinantly. Consequently, work is often carried out on isolated protein domains and difficult-to-purify targets such as full- length membrane receptors cannot be used. This work aims to apply an adaptation of mRNA display, known as RaPID, to identify peptide tools for challenging targets that cannot be purified recombinantly. RaPID display uses genetic code reprogramming to incorporate unnatural amino acids into peptide libraries, enabling the screening of covalently cyclized peptides. 1 A proof- of-concept selection has been performed against LgBiT protein, in which the protein was overexpressed in mammalian cells and immunoprecipitated via a tag, bypassing the requirement for target purification prior to selection. This cell lysate-based approach was successful in identifying high affinity peptide ligands for LgBiT ( K i < 1 nM). We have since used this method to perform selections against large proteins (>100 kDa) containing intrinsically disordered regions, that cannot be purified in their full-length forms. We are also aiming to apply this technology to targets expressed at the cell surface using photoaffinity crosslinking peptide libraries, as was recently reported by the Walport group. 2 In summary, this approach will enable the screening of challenging protein targets in a cellular context, expanding the possibilities for novel tool discovery. References 1. Huang, Y., Wiedmann, M. M. & Suga, H. RNA Display Methods for the Discovery of Bioactive Macrocycles. Chem. Rev. 119 , 10360–10391 (2019). 2. Wu, Y. et al. Identification of photocrosslinking peptide ligands by mRNA display. ChemRxiv 1–4 (2021).
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