Integrated isothermal shift assay and multi-omics identify melittin as a novel EGFR-targeting peptide to suppress NSCLC

BACKGROUND: Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, highlighting the urgent need for novel therapeutic agents. Melittin (MEL), a bioactive peptide, exhibits potent antitumor activity; however, its clinical translation is limited by an incomplete understanding of its molecular mechanism. Furthermore, the development of peptide-based therapeutics is often constrained by the lack of systematic approaches to identify their specific cellular targets. METHODS: To address these challenges, we established a multi-dimensional framework integrating biophysical validation with omics analysis. We evaluated the specific anti-NSCLC efficacy of MEL in vitro and in a PC9 xenograft mouse model. The direct molecular target of MEL was deconvoluted using a combination of isothermal shift assay (iTSA), cellular thermal shift assay (CETSA), and bio-layer interferometry (BLI), supported by AlphaFold3-based structural modeling. Mechanistic insights were obtained through quantitative proteomic and phosphoproteomic profiling of tumor tissues, followed by validation using shRNA-mediated gene knockdown. RESULTS: MEL demonstrated selective cytotoxicity against NSCLC cells and suppressed tumor growth in vivo, with minimal toxicity toward normal human bronchial epithelial cells. Isothermal shift assay and CETSA revealed that MEL treatment significantly enhanced the thermal stability of epidermal growth factor receptor (EGFR), indicating direct target engagement. This interaction was further confirmed by BLI, which showed high binding affinity (KD = 18.6 nM), and structural modeling predicted a specific interaction interface. Proteomic and phosphoproteomic analyses indicated that MEL binding inhibited EGFR phosphorylation, thereby attenuating the downstream ERK and STAT3 signaling cascades and inducing autophagy-dependent apoptosis. Importantly, the antitumor effects of MEL were significantly attenuated in EGFR-knockdown cells, confirming its dependency on this target. CONCLUSIONS: This study identifies EGFR as a direct molecular target of MEL in NSCLC, supporting the potential of MEL as a novel peptide-based EGFR inhibitor. Furthermore, our integrated biophysical and omics strategy provides a valuable methodological reference for systematic peptide target identification, thereby facilitating clinical translation.