Recent advances in the development of therapeutic peptides

Abstract

Peptides have unique characteristics that make them highly desirable as therapeutic agents. The physicochemical and proteolytic stability profiles determine the therapeutic potential of peptides. Multiple strategies to enhance the therapeutic profile of peptides have emerged. They include chemical modifications, such as cyclization, substitution with d-amino acids, peptoid formation, N-methylation, and side-chain halogenation, and incorporation in delivery systems. There have been recent advances in approaches to discover peptides having these modifications to attain desirable therapeutic properties. We critically review these recent advancements in therapeutic peptide development.

Keywords: N-methylation; cyclization; d-amino acid; peptides; peptoid; stability.

Figure 1:. Approaches for discovering and modifying therapeutic peptides.

Therapeutic peptides can be identified through screening phage display libraries, mRNA display libraries, and peptide libraries. Peptides can also be discovered from natural sources or through computer-aided design (utilizing artificial intelligence and machine learning). The bioactivity and stability of the lead peptides discovered can be further improved by implementing various chemical modifications, including cyclization, conversion to D-amino acids, peptoid formation, and N-alkyl peptide formation.

Figure 2.. Schematic diagram of the biopanning procedure using a peptide phage display library.

First, the target protein is immobilized and then incubated with a phage library. The unbound phages are removed by washing, leaving only the phages that have bound to the target. The bound phages are eluted and amplified using host bacteria. The amplified phages are then used for subsequent rounds of biopanning, which is repeated 3-4 times. Finally, DNA sequencing is performed to identify the sequences of the peptides that bind to the target.

Figure 3:. Major cyclization formats of therapeutic peptides.

When creating a cyclic peptide, several important parameters, such as length, stability, and linkage rigidity must be considered. There are various methods for cyclizing peptides. (A) end-to-end cyclic peptide. (B) end-to-side chain cyclic peptide. (C) stapled peptide. (D) bicyclic peptide. (E) side chain-to-side chain cyclic peptide. (F) Macrocyclization scanning. The two terminal amino acids in a linear peptide are replaced with cysteine. Then, the position of one or both cysteine residues is sequentially altered, followed by the formation of a disulfide bond between the two cysteine residues, leading to the formation of a macrocyclic peptide.

Figure I.. Alanine scanning and truncation to study the structure-activity relationship of the H3K4me3 peptide.

(A) Alanine scanning of the wild type H3K4me3 10-mer peptide. The amino acid residues within the peptide sequence are systematically replaced with alanine. It helps to identify which amino acids play a crucial role in the structure, interaction, stability, and function of the peptide. (B) The flanking amino acids of the peptide sequence are removed from the C-terminus. Truncation helps to identify the shortest sequence that maintains its functional activity. (C) The inhibition constant (K_i) of the peptides derived from alanine scanning or truncation. Reproduced from the reference [75] with permission from the Royal Society of Chemistry.

Figure II.. Cellular uptake mechanisms of bioactive peptides.

(A) Peptides binds to the extracellular domain of receptors. After binding to the receptor, the downstream function is mediated depending on the nature of the peptide drug. Peptides with blocking effect mediate inhibitory signal to inhibit normal physiological functions. (B) Peptides with agonistic effect enhance downstream functions. (C) Direct translocation. Cell-penetrating peptides cross the cell barrier via direct translocation. (D) Receptor-mediated endocytosis. Binding of peptides to the surface receptor forms a receptor-peptide complex, which is then internalized into the cell. (E) Clathrin-mediated endocytosis. Peptides enter the cells through clathrin-mediated endocytosis.