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

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.

The Cyanopyridine–Aminothiol Click Reaction: Expanding Horizons in Chemical Biology

“…Aminoacyl-tRNA synthetases capable of selectively incorporating Cpa have been independently discovered to facilitate such applications. 17,20 It should be noted that synthetases for two different structural isomers of Cpa have been used, which are referred to as mCpa and pCpa (Figure 2). The incorporation of Cpa into…”

Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display

“…Electrophilic genetically encoded libraries (GELs), such as those in phage and mRNA display, could be significantly useful for covalent inhibitor discovery. GELs can be exceedingly large (>10 13 molecules), are synthesized quickly and accurately by the ribosome, and are increasingly approaching the chemical space of natural products. − Resulting peptide inhibitors have a greater propensity to occupy shallower binding pockets, , disrupt protein–protein interactions, − and have high selectivity for their protein target; − the last characteristic may be particularly desirable in covalent drug discovery, where nonspecific reactivity is a unique concern. Despite these advantages, there are relatively few examples of electrophilic GELs, with a handful of reports in phage display − and only cofactor-targeted examples in mRNA display …”

Synthesis of DNA-Encoded Macrocyclic Peptides via Nitrile-Aminothiol Click Reaction

“…Recently, Iskandar et al published a similar adaption in mRNA display technology further demonstrating the utility of this linkage for the discovery of bioactive molecules. 22 To assess our hypothesis, we synthesized Fmoc protected L-3-(2-cyano-4-pyridyl)-L-alanine (Cpa) according to the published procedures 21 and installed it onto the DNA headpiece (HP) using HATU-mediated amide coupling conditions. Subsequent Fmoc deprotection yielded DNA-conjugated substrate 1 which served as the starting material for peptide elongation (Scheme 2).…”

“…The newly formed aromatic heterocyclic motif provides an SP2-rich moiety that is expected to increase the conformational rigidity of the macrocycles and potentially facilitate protein engagement (Scheme ). Recently, Iskandar et al published a similar adaption in mRNA display technology further demonstrating the utility of this linkage for the discovery of bioactive molecules …”