Enhancement of Anti-Melanogenic Activity of Pinctada martensii Nacre-Derived Peptide Through C-Terminal Amidation: Mechanism Investigation in B16F10 Cells.

Excessive melanin accumulation induced by UV exposure and hormonal changes poses significant challenges to skin health, necessitating safe and effective tyrosinase inhibitors. Pearl and nacre powder have long been utilized in traditional Chinese medicine for skin whitening. Our previous study identified a tyrosinase inhibitory peptide (Ala-His-Tyr-Tyr-Asp) from Pinctada martensii nacre in silico; however, chemical modification strategies for enhancing bioactive peptide efficacy remain underexplored. The present study investigated the structure-activity relationship of NP-TIP-1 (Tyrosinase Inhibitory Peptide of Nacre Powder-1) through three chemical modifications (C-terminal amidation, N-terminal acetylation, and N-terminal palmitoylation). C-terminal amidation significantly improved tyrosinase inhibitory potency, reducing the IC50 value from 2.012 ± 0.088 mM to 0.979 ± 0.028 mM, while retaining thermal stability and copper ion chelating capacity comparable to the native peptide. In B16F10 melanoma cells, NP-TIP-1-NH2 (C-terminally amidated NP-TIP-1) exhibited no cytotoxicity at concentrations up to 600 μM and concentration-dependently suppressed intracellular tyrosinase activity and melanin content. Mechanistic investigation revealed that NP-TIP-1-NH2 treatment downregulates the mRNA and protein expression of TYR and TRP-1 without affecting MITF (Microphthalmia-associated Transcription Factor) or TRP-2, suggesting a potential regulatory mechanism independent of MITF-mediated transcriptional control. Non-targeted metabolomics further identified nucleotide metabolism, purine metabolism, and glycine/serine/threonine metabolism as the most significantly perturbed pathways, with cytosine and GMP downregulated while guanosine and guanine were upregulated. This study establishes NP-TIP-1-NH2 as a promising candidate for multifunctional food preservatives and nutraceuticals, providing a strategic framework for C-terminal amidation as a structure-activity optimization approach for marine-derived anti-melanogenic peptides.