Intramolecular Thioether Crosslinking of Therapeutic Proteins to Increase Proteolytic Stability

Abstract

Protein-based pharmaceuticals typically display high selectivity and low toxicity, but are also characterized by low oral availability, mainly because of enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal wall. One way to increase the proteolytic stability of peptides and proteins is by intramolecular crosslinking, such as the introduction of disulfide bridges. However, disulfide bridges are at risk of thiol-disulfide exchange or reduction during production, purification, and/or therapeutic use, whereas thioether bridges are expected to be stable under the same conditions. In this study, thioether crosslinking was investigated for a 46 aa albumin-binding domain (ABD) derived from streptococcal protein G. ABD binds with high affinity to human serum albumin (HSA) and has been proposed as a fusion partner to increase the in vivo half-lives of therapeutic proteins. In the study, five ABD variants with single or double intramolecular thioether bridges were designed and synthesized. The binding affinity, secondary structure, and thermal stability of each protein was investigated by SPR-based biosensor analysis and CD spectroscopy. The proteolytic stability in the presence of the major intestinal proteases pepsin (found in the stomach) and trypsin in combination with chymotrypsin (found in pancreatin secreted to the duodenum by the pancreas) was also investigated. The most promising crosslinked variant, ABD_CL1, showed high thermal stability, retained high affinity in binding to HSA, and showed dramatically increased stability in the presence of pepsin and trypsin/chymotrypsin, compared to the ABD reference protein. This suggests that the intramolecular thioether crosslinking strategy can be used to increase the stability towards gastrointestinal enzymes.

Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules

“…Through the use of the chemical crosslinking strategy presented here the substitution of the amino acid residues that potentially are part of the binding site is avoided, and, as shown by the CD and SPR experiments, the intramolecular crosslinks do not affect the conformation of the protein domain or interfere with the binding. Together with our earlier results on stabilization of an albumin‐binding domain, this study shows that intramolecular chemical crosslinking can be used to increase the stability of small, helical proteins towards gastrointestinal proteases without negatively affecting their function. Even an oral bioavailability as low as 1 % has been shown to result in oral activity for biologically active peptides, and so we envision that the presented strategy could be a step towards orally available protein drugs.…”

“…However, the discrepant shape of the melting curves suggests that the unfolding of the K58 and K4K58 variants is a gradual transition rather than a cooperative two‐state transition. This result was not unexpected, because we had previously observed that intramolecular crosslinking of a different three‐helix protein resulted in variants that showed non‐cooperative unfolding in variable‐temperature measurements, making it impossible to determine a melting temperature reliably …”

“…Along these lines we have previously applied a thioether‐based intramolecular crosslinking strategy to a small (46 aa) albumin‐binding domain . It was shown that the introduction of a single intramolecular thioether bridge in this small protein domain dramatically increased its resistance to degradation by the major digestive enzymes of the gastrointestinal tract—that is, pepsin, trypsin, and chymotrypsin—thus suggesting improved potential for oral uptake.…”

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity

“…However, the similar result for the C91S mutant excludes this possibility since an internal disulfide could not occur with the mutant protein. An alternative possibility to account for the observed mass of Prdx6 is the loss of 2 H + resulting in the formation of a thioether, but that appears to be relatively rare in non-engineered proteins and a mechanism for its possible formation is not clear (30). The most likely possibility for the observed molecular mass of non-reduced Prdx6 is the auto-oxidation of Prdx6 to the sulfenic acid (-Cys S-OH) and its subsequent dehydration with formation of a sulfenylamide bond between the sulfur atom of C47 and the nitrogen of an adjacent amino acid.…”

Peroxiredoxin 6 in the repair of peroxidized cell membranes and cell signaling

“…Our mass spectroscopic evaluation of the recombinant protein following storage (that presumably was accompanied by auto-oxidation of the protein) indicated a molecular mass that was 2 Da less than the predicted mass of the reduced protein [35]. Based on this finding, we have proposed that a sulfenylamide forms through dehydration of oxidized Prdx6 (reduced protein +16 for “O” −18 for H 2 O =−2) [35]; an alternative possibility for the measured mass is a thioether, but that is extremely rare in nature [67]. As noted in the review by Forman in this volume, sulfenylamide formation of a protein in vivo requires the reactivity of its sulfhydryl group toward peroxides that is in the range demonstrated by Prdx6.…”