MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles

Abstract

Macrocyclic peptides represent attractive scaffolds for targeting protein−protein interactions, making methods for the diversification and functional selection of these molecules highly valuable for molecular discovery purposes. Here, we report the development of a novel strategy for the generation and high-throughput screening of combinatorial libraries of macrocyclic peptides constrained by a nonreducible thioether bridge. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive noncanonical amino acid was integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. This platform, named MOrPH-PhD, was successfully applied to produce and screen 10 5 -to 10 8 -member libraries of peptide macrocycles against three different protein targets, resulting in the discovery of a high-affinity binder for streptavidin (K D : 20 nM) and potent inhibitors of the therapeutically relevant proteins Kelchlike ECH-associated protein 1 (K D : 40 nM) and Sonic Hedgehog (K D : 550 nM). This work introduces and validates an efficient and general platform for the discovery and evolution of functional, conformationally constrained macrocyclic peptides useful for targeting proteins and protein-mediated interactions.

Expanded toolbox for directing the biosynthesis of macrocyclic peptides in bacterial cells

“…To investigate this aspect, we selected as testbed two bioactive cyclopeptides we have recently isolated from the screening of phage displayed libraries of O2beY-based macrocycles. 45 These correspond to Strep-m3 , a ( i / i – 8)-linked macrocycle with nanomolar affinity for streptavidin ( K D : 20 nM), and KDD-m1 , a ( i / i + 7)-linked macrocyclic peptide capable of targeting Kelch-like ECH-associated protein 1 (Keap1) ( K D : 107 nM), which is implicated in regulating cytoprotective responses to oxidative stress in human cells. 64 To examine the impact of the thioether linkage on the function of these peptides, a series of macrocyclic peptides were generated by introducing each of the new eUAAs in place of O2beY within these peptide sequences ( Table 1 , entries 14 and 15).…”

“…Furthermore, our results show how swapping of the eUAA cyclization module in two bioactive cyclopeptides had a profound and scaffold-dependent effect on the target binding properties, highlighting the value of this strategy toward modulating the protein recognition properties of a functional macrocyclic peptide. Overall, the peptide cyclization strategies reported here are expected to expand opportunities for the creation of structurally and functionally diverse libraries of peptide macrocycles which can be produced in bacterial cells and can be explored through functional assays 42 , 44 or in combination with high-throughput display platforms, 45 toward the discovery of chemical agents for targeting and modulating biomolecular interactions.…”

“…macrocyclic organo-peptide hybrids or MOrPHs) 39 in living cells via the spontaneous, post-translational cyclization of recombinant polypeptides by means of genetically encoded non-canonical amino acids (ncAA). 40 – 43 These methodologies have enabled the creation and screening of macrocyclic peptide libraries which can be produced in cellulo 42 , 44 or displayed on phage particles 45 for the discovery of chemical agents capable of disrupting protein–protein interactions. So far, however, only a few ncAAs 40 , 42 were made available for these applications, thus limiting opportunities for structural diversification of the resulting macrocyclic peptides through variation of the ncAA module.…”

Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

“…The thioester transient bond at the connection with the intein makes possible the cyclization of the recombinant peptide to organopeptide macrocycle through a hydrazide bond. Another very recent example describes the generation of 10 5 - to 10 8 -member libraries constrained through non-reducible thioether bridge [ 54 ]. Further screening against different protein–protein interaction, led to the identification of potent peptide macrocycles to disrupt the Keap1/Nrf2 (best component K D : 40 nM), a PPI associated to the upregulation of cytoprotective enzymes of interest for inflammatory processes, and the interaction between Hedgehog interacting protein (HHIP) and Patched-1 (PTCH1) transmembrane receptor (K D : 550 nM), of interest for treating various human malignancies [ 54 ].…”

Strategies to expand peptide functionality through hybridisation with a small molecule component

“…This cyclisation strategy has recently been integrated with phage display with the displayed macrocyclic libraries successfully applied to identify a high-affinity binder for streptavidin (K D = 20 nM), and potent inhibitors of Keap 1 (K D = 40 nM) and Sonic Hedgehog (K D = 550 nM). 68 Of the large variety of macrocyclisation chemistries currently available, the majority focus only on cross-links that constrain the peptide fragment of the molecule. [17][18][19]69 For example, constraining the peptide through stapling can promote secondary structure formation in the form of alpha helices.…”