Segmented In Vitro Translation for Expanding Peptide Library Diversity

mRNA display is a method of rapidly generating large protein-peptide binding datasets and discovering de novo peptide therapeutics. In particular, mRNA display has been the starting point for discovering several macrocyclic peptide drugs addressing a variety of conditions: cholesterol management (MK-0616, Phase III trials), cancer radiotherapy (RYZ101, Phase III), and myasthenia gravis (Zilucoplan, approved). Yet these compounds all took years of additional screening to advance from mRNA display hit to a lead compound. Creating more productive mRNA libraries that yield lead-ready hits would be of great interest for drug discovery practitioners and patients. One key limitation of current mRNA display macrocycle libraries derives from the method of synthesis. The precursor peptides are synthesized with two fixed residues, a chloroacetyl and a thiol amino acid, that spontaneously react to form cycles. However, this results in libraries with zero diversity at two positions, and therefore suboptimal hits that necessarily contain these two residues. Although variants can be manually synthesized and tested post-selection, any synergistic effects with other ring elements cannot be recovered. Onca Bio proposes to diversify these cyclization positions, leading to binding hits that utilize all residues, as well as more complete structure-activity-relationship datasets for use in any optimization work that is needed. The work will leverage a new approach, Segmented In Vitro Translation (SIVT), that allows more flexibility to install non-natural amino acids, including those that drive cyclization. SIVT can reassign mRNA codons to different amino acids multiple times within a single transcript sequence; i.e., the codon table is defined locally by position, rather than globally across the entire sequence. SIVT has been demonstrated in single sequences, but applying it in the context of ultra large libraries will require fundamental technology development in both the methodology of SIVT, the design of the mRNA libraries, and the non-natural substrates used. If successful, the resulting mRNA display libraries will contain diversity at every amino acid position including the cyclization residues, and boost the overall chemical space coverage by an order of magnitude. The ultimate measure of library productivity will be testing the libraries in mRNA display selections against “undruggable” targets and benchmarking against typical mRNA display macrocycle libraries. Successful execution of this SBIR phase one effort will allow Onca to commercialize SIVT-based mRNA display for peptide discovery. Proprietary information – Onca Bio, Inc.