Phage Display–Mediated Discovery of Novel Tyrosinase-Targeting Tetrapeptide Inhibitors Reveals the Significance of N-Terminal Preference of Cysteine Residues and Their Functional Sulfur Atom

Abstract

Tyrosinase, a key copper-containing enzyme involved in melanin biosynthesis, is closely associated with hyperpigmentation disorders, cancer, and neurodegenerative diseases, and as such, it is an essential target in medicine and cosmetics. Known tyrosinase inhibitors possess adverse side effects, and there are no safety regulations; therefore, it is necessary to develop new inhibitors with fewer side effects and less toxicity. Peptides are exquisitely specific to their in vivo targets, with high potencies and relatively few off-target side effects. Thus, we systematically and comprehensively investigated the tyrosinase-inhibitory abilities of N-and C-terminal cysteine/tyrosine-containing tetrapeptides by constructing a phage-display random tetrapeptide library and conducting computational molecular docking studies on novel tyrosinase tetrapeptide inhibitors. We found that N-terminal cysteine-containing tetrapeptides exhibited the most potent tyrosinase-inhibitory abilities. The positional preference of cysteine residues at the N terminus in the tetrapeptides significantly contributed to their tyrosinase-inhibitory function. The sulfur atom in cysteine moieties of N-and C-terminal cysteine-containing tetrapeptides coordinated with copper ions, which then tightly blocked substrate-binding sites. N-and C-terminal tyrosinecontaining tetrapeptides functioned as competitive inhibitors against mushroom tyrosinase by using the phenol ring of tyrosine to stack with the imidazole ring of His263, thus competing for the substrate-binding site. The N-terminal cysteine-containing tetrapeptide CRVI exhibited the strongest tyrosinase-inhibitory potency (with an IC 50 of 2.7 6 0.5 mM), which was superior to those of the known tyrosinase inhibitors (arbutin and kojic acid) and outperformed kojic acid-tripeptides, mimosine-FFY, and shortsequence oligopeptides at inhibiting mushroom tyrosinase.

Amino acid composition and anti‐polyphenol oxidase of peptide fractions from sericin hydrolysate

“…Lee et al . () demonstrated that the ability of tetra‐peptides to bind tyrosine may be related to the presence of cysteine at the N‐terminal position. Peptides with cysteine located at the N‐terminus showed better ability to inhibit tyrosinase than peptides with cysteine located at the C‐terminus.…”

Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

“…In this study, for the purpose of developing a safe and effective skin-whitening agent, we screened about 400 natural products from 78 different of Chinese herbal plants for compounds with inhibitory activity against mushroom tyrosinase. The natural compound T1 isolated from Gastrodia elata shows outstanding inhibitory potency against tyrosinase (IC 50 = 0.53 μM, K i = 58 ± 6 nM), which is more effective than β-arbutin, kojic acid, short peptides 74 75 and other known natural compounds ( Supplementary Table S1 ). Our docking model demonstrates that the sulfur atom of T1 makes close contacts with the copper ions in the active site of tyrosinase ( Fig.…”

Advancement and applications of peptide phage display technology in biomedical science

“…Combinatorial phage-displayed random peptide libraries are very valuable tools for studying the interaction between peptides and other substances (or materials). In the past, scientists have been focused on identifying B/T cell epitopes [11][12][13][14][15][16][17][18][19], disease-specific antigen mimics [22][23][24][25][26][27][28][29][30][31][32][33][34], receptor agonists/antagonists [35][36][37][38][39][40][41][42][43][44][45], enzyme inhibitors [48][49][50][51][52][53][54] and protein partners [55][56][57][58][59][60][61][62]. In recent years, there is an increasing number of researchers who apply this technique to new areas ...…”

“…TIMP-2 is a broad range inhibitor of matrix metalloproteinases (MMPs) [51]. In addition to protease inhibitors, peptide-based inhibitors against various enzymes, such as human HMG-CoA reductase [52], ubiquitin ligases [53], and tyrosinase [54], have all been identified by phagedisplayed random peptide libraries.…”