Phage Selection of Chemically Stabilized α-Helical Peptide Ligands

Abstract

Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding β-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

Tuning the Binding Affinity and Selectivity of Perfluoroaryl‐Stapled Peptides by Cysteine‐Editing

“…This topic has been recently reviewed by Fairlie . Examples of thiol cross‐linking include the use of dibromomaleimide, dichloroacetone, 1‐,4‐dichlorotetrazine, 1,2,4,5‐tetrabromodurene, α,α′‐dibromo‐ m ‐xylene, trans ‐1,4‐dibromo‐2‐butene and cis ‐1,4‐dichloro‐2‐butene and perfluoroaryl reagents . Although significant attention has focussed on the nature of the cross‐linking electrophile, comparatively little, if any, attention has focussed on the cysteine residues, with the single exception of introducing homocysteine .…”

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of CaV Function

“…59,60 This cysteine alkylation strategy has the advantage of not requiring unnatural amino acids. To date, all strategies for stapled peptide synthesis have focused on introduction of linkers along one α-helix face, an approach that can buttress the structure but that does not restrain the α-helix polar ends.…”

“…Incorporation of an m -xylyl staple, a strategy used previously to stabilize the protease inhibitor calpastatin 59 and β-catenin, 60 enhanced AID helix formation when placed at either N-terminal (AID-CAP) or central (AID-CEN) positions (Figure 1C). The AID-CAP configuration proved superior for inducing helical content.…”

“…Previously, we and others demonstrated that chemical cross-linking of i and i + 4 cysteines could be useful for α-helical peptide stabilization. 59,60 Here, we expand this cysteine cross-linking strategy to constrain an N-terminal capping motif 61,62 appended to the AID. Our studies demonstrate that stapling AID peptides with a meta -xylyl bridge 59,63 between two engineered cysteines creates AID peptides having enhanced helical content that bind Ca V β in a native-like manner.…”

Targeting intracellular protein–protein interactions with cell-permeable cyclic peptides

“…D-amino acids) can be readily incorporated into chemically synthesized libraries to generate more structurally diverse cyclic peptides. The rational design and combinatorial library approached have also been combined to generate stapled peptide inhibitors of exceptionally high potencies [32,33]. …”