Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules

Abstract

Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three-helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid-phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N-terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C-terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface-plasmon-resonance-based (SPR-based) biosensor studies of the protein domains showed that the three-helix structure and high-affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.

Modulation of Coiled-Coil Binding Strength and Fusogenicity through Peptide Stapling

“…Unless otherwise specified, samples were measured at 20 °C in a quartz cuvette with a 2 mm path length. Spectra were recorded from 190 to 260 at 1 nm intervals, with a bandwidth of 1 nm, with the final spectrum consisting of the average of 5 spectra. The mean residue molar ellipticity (θ, deg cm 2 dmol• res −1 ) was calculated according to eq 1:…”

“…Intramolecular cross-linking of peptides, commonly referred to as peptide stapling, is often employed to change or constrain the secondary structure of small peptides and to induce unstructured peptides to mimic complex protein folds and protein–protein interactions (PPIs). − Stapling also contributes to an increased resistance to denaturation and proteolytic degradation, making it a useful technique for the modification of peptide-based therapeutics . Hydrocarbon stapling, a technique which is based on catalyzed olefin metathesis, has seen widespread application with multiple compounds being investigated in academic, preclinical, and clinical studies. − …”

“…1−4 Stapling also contributes to an increased resistance to denaturation and proteolytic degradation, making it a useful technique for the modification of peptide-based therapeutics. 5 Hydrocarbon stapling, a technique which is based on catalyzed olefin metathesis, has seen widespread application with multiple compounds being investigated in academic, preclinical, and clinical studies. 6−10 Peptide stapling techniques can be broadly divided into two categories: single-and two-component strategies.…”

Rational Design of Antigen Incorporation Into Subunit Vaccine Biomaterials Can Enhance Antigen-Specific Immune Responses

“…This suggests that the SLS-N formulation was able to be broken down and processed sufficiently to induce significant levels of DC presentation and maturation. Proteases in endolysosomes and the proteasome in cytosol upon endosomal escape, cleave proteins into minimal epitopes using several mechanisms of amide bond breakage, so it is possible that the amide bonds that form SLS-N PNC are broken down more efficiently by these mechanisms ( 58 , 59 ) SLS-M is stabilized with thioether bonds, which are commonly used to increase metabolic stability ( 60 – 62 ). Although SLS-T PNC are crosslinked with reversible disulfide bonds, these bonds were shown to be difficult to fully reduce ( Supplementary Figure 5 ) and may lead to decreased ability of DCs to break-up SLS-T PNC.…”

Multi-targeted stimuli-sensitive mixed micelles as a strategy for cancer therapy

“…Although some studies report that thioether bonds are sensitive to reductive agents, [125][126] others state that they are irreversible and greatly improved the structural stability of many bioactive peptides. [127][128][129] In fact, the cleavage of miRNA-34a-S-DOPE was not checked again after modification of the synthesis protocol. These observations led to the conclusion that a disulfide bond between lipid and miRNA, as originally proposed in the beginning of this project, might indeed be the best approach to prove GSH-sensitivity in vivo.…”