2. Academic Validation
  3. Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase

  • J Am Chem Soc. 2023 Jan 25;145(3):1512-1517. doi: 10.1021/jacs.2c11294.
Sabrina E Iskandar 1 2,  Jarrett M Pelton 1 2,  Elizaveta T Wick 3 4,  Derek L Bolhuis 4 5,  Albert S Baldwin 4,  Michael J Emanuele 3 4,  Nicholas G Brown 3 4,  Albert A Bowers 1 2 6
Affiliations

Affiliations

  • 1 Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
  • 2 Center for Integrative Chemical Biology and Drug Discovery, Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
  • 3 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
  • 4 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
  • 5 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
  • 6 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
PMID: 36630539 DOI: 10.1021/jacs.2c11294
Abstract

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide Binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical Amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the Deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over Other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

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