Optimization Strategies for the Stability of Peptides In Vivo
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Peptides are a class of molecules that play an important role in living organisms, and are widely used in drug delivery and disease treatment due to their low toxicity, low immunogenicity, high tissue permeability, and easy synthetic modification. However, peptides are easily decomposed by a variety of proteases and have a high clearance rate, which leads to their fast metabolism, poor stability and short half-life in vivo, which brings challenges to the development and application of peptide-related drugs. The purpose of this article is to briefly summarize the strategies to improve the stability of peptides in vivo, in order to provide a reference for the application of peptides.
The reason why peptides have poor stability and short half-life in vivo is closely related to their special molecular structure. The amide groups located on the molecular surface of the peptide backbone are prone to protease-catalyzed and non-enzyme-catalyzed deamidation reactions, and the amino acid residues on the chain are also prone to conformational changes, such as the dissociation of α-helix and the cleavage of salt bridges. On the other hand, peptides have a relatively small molecular size and are therefore easily filtered out of the bloodstream by the kidneys. In order to improve the stability of the peptide in vivo, it is possible to enhance its stability by decreasing the activity of the peptide structure involved in the degradation reaction, or to increase the molecular weight of the peptide to avoid being removed. Specific optimization strategies can be carried out from the following aspects:
The structural modification of the N-terminus and C-terminus of peptides can evade the recognition of aminopeptidase and carboxypeptidase, which is an effective strategy to protect peptides from protease degradation.
A certain degree of lengthening of the peptide chain from the N-terminus or C-terminus can enhance the electrostatic effect, thereby improving the stability of the peptide. Moreover, if the amino acid sequence that is thermally stable or insensitive to enzymes is extended along the N-terminus or C-terminus along the peptide chain backbone for a certain length, the degradation of aminopeptidase and carboxypeptidase from the N-terminus or C-terminus can be inhibited to a certain extent. In addition, modifications such as methylation, N-terminal acetylation, and C-terminal amidation can also prevent enzymatic hydrolysis of exopeptidase.
Fig.1 Neurotensin terminal modification.
Table 1 Peptide modification services at Creative Peptides.
Amino acid replacement: Reduced affinity with proteases
D-amino acids
The hydrolysis reaction of proteases is generally stereospecific. The introduction of D-amino acids will change the configuration of the peptide, which in turn makes the modified peptide less susceptible to hydrolysis by proteolytic enzymes. Available methods are to replace the C- and N-terminus amino acids of the peptide chain with D-amino acids, or to replace amino acid residues with poor peptide bond stability, or to replace all amino acids. When the peptide is a special sequence that cannot be replaced, it can be linked to a D-peptide to improve the protease stability of the peptide. (D Amino Acid Peptide Synthesis Service at Creative Peptides)
Fig.2 Lutein hormone releasing hormone (LHRH) introduces D-amino acids.
Artificial amino acids
Substitution of β- and γ- amino acids and other unnatural amino acids. Taking β-amino acids as an example, it can change the overall conformation of the peptide, and the water molecules in the original protease cleavage center cannot form hydrogen bonds with the amide bonds, which is not conducive to the cleavage of amide bonds by proteases, so it has stronger hydrolysis resistance.
Fig.3 β-amino acids resist hydrolysis.
Change single or multiple amino acids
The structure of the peptide chain is mainly maintained by electrostatic action between the main chain and the main chain, and between the side chains. The electrostatic interaction between the peptide chains can be enhanced by replacing several of these amino acids. In addition, selective fluorination of peptides improves chemical and thermal stability while enhancing hydrophobicity. Peptide glycosylation is also an ideal way to stabilize peptides.
Alter peptide bonds: Avoid protease recognition and hydrolysis
Pseudopeptidization
Peptide bonds (-CONH-) are characteristic of peptide molecules, and peptide bonds are easily recognized and degraded by proteases in vivo. Pseudopeptides replace one or more of the two atoms in the peptide bond with other atoms, which essentially changes the chemical structure of the amide bond, which is different from the homologous structure of proteins or peptides, so it can avoid the recognition and hydrolysis of proteases in vivo, thereby improving the stability and activity of peptide molecules.
Fig.4 Examples of pseudo-peptide bonds.
Reverse peptidization
The change of peptide bond direction can also change the recognition of substrate by protease, so as to achieve the effect of anti-degradation. Such structural modification strategies of peptides that change the direction of peptide bonds are called reverse peptidization modification, and related peptides are called reverse peptides or inverse peptides.
Cyclization or bicyclization: Increases compound rigidity
Peptide cyclization
Compared with straight-chain peptides, peptide cyclization reduces the flexibility of the peptide chain and protects it from the influence of peptide chain terminal lyases, so the cyclic peptide has better structural stability. Cyclization can use amide bonds, disulfide bonds, lactone bonds, ether bonds, thioether bonds, olefins, carbamate bonds and other ways to connect peptides end to tail, head to side chains, tail to side chains and side chains to connect between side chains, etc., among which amide bonds and disulfide bonds are commonly used.
Fig.5 An example of cyclic peptide was formed.
Table 2 Cyclic peptide services at Creative Peptides.
Stapled peptides
Most of the peptide fragments will not be able to stably form the secondary structure required for binding after leaving the overall structure of the protein, and are prone to form irregular coiled conformations, resulting in decreased binding activity, and are more susceptible to degradation by peptidases, and cannot be directly druggable. The active conformation of the α-helix peptide was stabilized by applying a full carbon backbone or other linkers to form a side-chain cyclic structure, i.e., the stapled peptide. At present, ruthenium-catalyzed metathesis of olefins is a relatively mature method for the formation of staple peptides. Staple peptides are a good choice to improve stability and stabilize the helical structure, but their toxicity cannot be ignored according to the literature. (Stapled Peptide Synthesis at Creative Peptides)
Fig.6 An example of a stapled peptide.
Dimer construction
The formation of α-helical dimers by intramolecular or intermolecular disulfide bonds, α-helix, and hydrophobic association of peptides can not only maintain the peptide in the α-helix conformation, but also regulate hydrophobic interactions, and place sites that are easily recognized by proteases on the inner side.
Fig.7 The peptide molecule forms a dimer.
Increase molecular weight: Avoid rapid metabolism
Advanced fatty acid modification
Specific sites of peptides are chemically introduced into higher fatty acid chains in the form of covalent bonds. In addition, the introduction of aliphatic chains can enhance the structural stability of peptides by increasing hydrophobicity. Studies have shown that the aliphatic chain can promote the non-covalent binding of peptide derivatives to serum albumin, and the bound complex is not easy to transport due to the large molecule, which can prolong the circulation time of peptides in vivo.
Fig.8 Advanced fatty acid modification peptide.
PEG is highly water-soluble, low toxic, non-immunogenic, and easily cleared from the body. The terminal modification with PEG can protect amino acid residues, significantly increase molecular weight and steric hindrance, so it can improve the stability of peptide molecules, enhance solubility, reduce the degradation of proteases, and is not easy to be filtered by glomeruli, thereby prolonging the half-life of peptides.
Fig.9 PEG modified Interferon α.
In addition to PEG, there are polymers composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA). Ngambenjawong used the polymer to prolong the stability of macrophage-targeting peptide (M2pep).
Protein fusion: Extended half-life
Albumin (Alb) and immunoglobulin G (IgG) in plasma have a relatively long half-life compared to other proteins. Using genetic engineering technology, the peptide molecule is fused with an immunoglobulin Fc fragment or serum albumin HSA. The size of the peptide molecule after fusing Fc or HSA fragment is significantly increased, which reduces the renal clearance rate of the peptide, thereby prolonging the half-life of the peptide.
Physical embedding: Creates protective barrier
In addition to modifying the peptide itself, a polymer substance can be used to encapsulate the peptide to form a barrier to protect the peptide from protease cleavage. Commonly used embedding methods include nanoparticles, liposomes, microspheres, microemulsions, etc. In addition, some researchers use self-assembling active hydrophobic proteins to encapsulate peptides in the cavity, thereby preventing them from being degraded by proteases.
Fig.10 Hydrophobic proteins envelop GLP-1.
Multiple methods used in combination
The combination of multiple methods to modify peptides can not only enhance the stability of peptides, but also enhance the ability of cell binding, anti-tumor ability and ability to overcome the blood-brain barrier.
Peptides as therapeutic molecules and drug/gene carriers are limited in many applications in multiple fields due to their instability. Although some progress has been made in the structural modification of peptides to improve stability, they still face challenges such as difficult synthesis and possible impact on biological activity. Future studies need to improve stability while taking into account the physiological activity of peptides. Therefore, according to the characteristics of each peptide, choosing the appropriate modification or modification method may be a more reliable and ideal strategy at present.
