Rational design of an N-terminal cysteine-containing tetrapeptide that inhibits tyrosinase and evaluation of its mechanism of action

Abstract

There has been a resurgence of interest in bioactive peptides as therapeutic agents. This is particularly interesting for tyrosinase, which can be inhibited by thiol-containing peptides. This work demonstrates that an N-terminal cysteine-containing tetrapeptide can be rationally designed to inhibit tyrosinase activity in vitro and in cells. The tetrapeptide cysteine (C), arginine (R), asparagine (N) and leucine (L) or CRNL is a potent inhibitor of tyrosinase activity with an IC50 value of 39.62 ± 6.21 μM, which is comparable to currently used tyrosinase inhibitors. Through structure-activity studies and computational modeling, we demonstrate the peptide interacts with the enzyme via electrostatic (R with E322), hydrogen bonding (N with N260) and hydrophobic (L with V248) intermolecular interactions and that a combination of these is required for potent activity. Moreover, copper chelating activity might be one of the mechanisms of tyrosinase inhibition by CRNL. Kinetic studies show that tetrapeptide is a competitive inhibitor with two-step irreversible inhibition. In addition, CRNL had no toxicity and could reduce melanin levels in the murine melanoma cell line (B16F1). Overall, CRNL is a very promising candidate for hyperpigmentation treatment.

Keywords

• Depigmentation
• Enzyme kinetics
• Modified peptide
• Molecular docking
• Tyrosinase inhibitor
• Tetrapeptide