Abstract

Background: Multidrug efflux proteins, frequently overexpressed in cancer cells, reduce intracellular drug accumulation and limit chemotherapeutic efficacy. Short drug-binding peptides containing the WXXW motif have been shown to non-covalently bind multidrug resistance (MDR)-associated drugs, potentially masking structural features recognized by efflux transporters. We hypothesized that incorporating this motif into a membrane-active peptide would generate a hybrid analogue capable of reversible drug binding and enhanced cellular uptake. Methods: A melittin-derived peptide (M3) was rationally designed by introducing the WXXW motif into the flexible region adjacent to the conserved proline kink to generate a dual-functional peptide with membrane activity and reversible drug binding. Membrane activity was evaluated using liposome leakage assays, and drug-binding interactions with doxorubicin were characterized using fluorescence quenching and microscale thermophoresis (MST). Molecular dynamics simulations were performed to elucidate binding mechanisms, and functional effects were assessed using calcein AM efflux assays, confocal imaging, and cytotoxicity studies across cancer cell lines. Results: M3 retained membrane activity and exhibited moderate, reversible binding to doxorubicin, with simulations showing binding initiation at the WXXW motif and extension to tryptophan residues W12, W15, and W19, forming a multivalent aromatic interface that suggested shielding of key drug functionalities. Functionally, M3 enhanced intracellular calcein retention and increased doxorubicin accumulation, and combination treatment produced synergistic cytotoxicity in the multidrug-resistant H69AR cell line with reduced toxicity toward normal epithelial cells. Conclusions: M3 acts as a membrane-active, reversible drug-binding peptide that enhances intracellular drug accumulation, supporting its potential as a modular strategy to overcome efflux-mediated MDR.

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