Mono‐Substituted Hydrocarbon Diastereomer Combinations Reveal Stapled Peptides with High Structural Fidelity

Abstract

Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide.

Effects of chiral center on an all‐hydrocarbon tethered peptide

“…As shown in Figure C, peptide 1R with longer retention time on HPLC showed more higher helical contents than peptide 1S , which is in agreement with our previous work, demonstrating that the CIH concept could be translated into an all‐hydrocarbon tethered peptide. Notably, the α‐methyl group in the olefinic amino acid is not necessary for constructing CIH helical peptides . Furthermore, comparing with the thioether tethered CIH peptide previously reported by our group, the all hydrocarbon CIH peptide 1R showed higher helical content than its thiolether tethered counterpart 2R , shown in Figure 3C …”

Stapled Helical Peptides Bearing Different Anchoring Residues

“…Among all obtained stapled peptides, mS5-mS5 mediated Cks1 and CK1α peptides and mS5-mR5 αSyn showed the highest helicity based on CD analysis, indicating that the helixstabilizing effect in this mS5/mR5 stapling was sequencedependent. 202 This result was subtly different from the application of regular α,α-disubstituted amino acids, in which S5-S5 paired anchoring residues were optimal in most cases.…”

“…The enantiomer of the mS5 nonnatural amino acid, termed mR5, was next prepared by Hulme and co-workers. 202 All possible diastereomers of paired anchoring residues mS5-mS5, mR5-mR5, mR5-mS5, and mS5-mR5 were incorporated at the appropriate i, i+4 positions of three peptide candidates, including a 14-mer from the protein alpha-synuclein (αSyn), a 12-mer from the accessory protein cyclin-dependent kinase regulatory subunit 1 (Cks1), and a 10-mer from casein kinase 1 alpha (CK1α), to yield the desired stapled peptides. 202 The on-resin RCM reaction proceeded efficiently under the more helix-promoting solvent TFE in DCM but not in the commonly used DCE solvent.…”

Designing stapled peptides to inhibit protein‐protein interactions: An analysis of successes in a rapidly changing field

“…Indeed, McWhinnie et al have reported three hydrocarbon-stapled peptide series which reproduce the secondary structure of the native protein they were extracted from (αSyn, CKS1 and CK1α) with high fidelity, whereas the unstapled natural sequences displayed almost no secondary structure content ( Figure 3A). [64] Similarly, Wang et al have isolated a minimum helical motif from the coiled-coil region identified in the crystal structure of the respiratory syncytial virus fusion (RSV-F) protein, [65] and then enhanced the helicity of this initial peptide sequence using computational mutagenesis and all-hydrocarbon peptide stapling, achieving a corresponding 2-fold improvement in binding affinity.…”