Macrocyclic peptides containing non-canonical amino acids have huge potential as therapeutics including targeting oncogenic proteins. However, the development of peptide like drugs with non-canonical amino acids has been hindered by the difficulty of synthesising and testing large libraries. The flexible in vitro translation (FIT) system utilizes flexible tRNA-acylation ribozymes known as flexizymes, to readily facilitate genetic code reprogramming. Substituting Methionine from the translation reaction to vacate the initiation AUG codon for either a non-standard N-chloroacetyl-L-Tyrosine or N-chloroacetyl- D-Tyrosine facilitates the selection of two separate libraries of macrocyclic peptides. The FIT system in combination with mRNA display, termed RaPID (Random nonstandard Peptides Integrated Discovery) is a powerful drug discovery platform that can elucidate macrocyclic peptide inhibitors for difficult to target proteins and protein-protein interactions from libraries of 1012-1013 sequences.
The receptor tyrosine kinase-like orphan receptor 1 ROR1 facilitates the interactions of cavin-1 and caveolin-1 (CAV1), which are two essential structural components of the lipid rafts caveolae on the plasma membrane. ROR1 knockdown has been shown to act as an ‘Achilles heel’ inhibiting lung adenocarcinoma cell lines, even those with resistance to gefitinib, an EGFR tyrosine kinase inhibitor, (Yamaguchi et al. 2015). Utilizing the RaPID system we isolated peptides with high affinity for the oncoprotein ROR1 from two libraries consisting of either L or D conformation. MiSeq deep sequencing elucidated the peptide sequences, and their low nanomolar binding affinity for ROR1 was validated.