Identification of Novel Anti- non-small cell lung cancer (NSCLC) Peptide in toad Skin（Chan Pi） Extract through Integrated Peptidomics and in silico Screening.

Toad skin (Bufo bufo gargarizans Cantor), known as Chan Pi in traditional Chinese medicine, has long been recognized for its therapeutic potential, particularly in cancer treatment. While the pharmacological activities of small molecules in toad skin such as bufadienolides have been extensively studied, peptides-another class of bioactive molecules-remain largely unexplored. In this study, we employed a peptidomics-based approach combined with in silico screening and experimental validation to identify novel bioactive peptides with potential anticancer properties from toad skin extracts. Approximately 2500 distinct peptides were identified using both database searching and de novo sequencing. Computational screening with two predictive models identified 26 candidate peptides with bioactivity scores exceeding 0.75. Among them, three peptides with potential anti-lung cancer activity were synthesized for in vitro evaluation against NSCLC cell lines, A549 and H1975. Peptide STPECLLGMWK exerts selective anti-proliferative effects on TKI resistant H1975 lung cancer cells in a dose-dependent manner. These findings provide new insights into the peptide composition of toad skin extracts and highlight their potential as a source of novel anticancer agents, laying the groundwork for future cancer therapeutics development.