Design and development of anticancer peptide-conjugated daunorubicin: Smart targeting systems for multidrug...

Anticancer peptides represent a promising alternative to conventional chemotherapy by selective targeting cancer cell membranes, thereby enhancing specificity and reducing off-target effects. Here, computational modeling and experimental validation were integrated to design novel anticancer peptides and assess their efficacy as daunorubicin conjugations for overcoming multidrug resistance. From an initial library of 5400 anticancer peptides, 20 candidates were selected based on optimized physicochemical and structural properties. Molecular dynamics simulations, including steered molecular dynamics and umbrella sampling, were employed to analyze peptide-membrane interactions using realistic cancer and normal membrane models. Two top-performing peptides were synthesized, conjugated to daunorubicin via a disulfide linker, and evaluated for cytotoxicity, drug accumulation and efflux, and apoptosis induction in drug-sensitive EPG85.257 and MDR malignant EPG85.257RDB lines. Computational analysis revealed that the lead peptides preferentially penetrated cancer cell membranes, exhibiting lower free energy barriers compared to normal membranes. Conjugation significantly enhanced cytotoxicity, increasing potency by 79 % (peptide-05 conjugate) and 44 % (peptide-20 conjugate) in drug-sensitive cells and by over 35 % in MDR cells. Flow cytometry confirmed near-complete daunorubicin retention in MDR cells with minimal necrosis. Furthermore, conjugated peptide-05 and peptide-20 induced apoptosis up to 70 % in EPG85.257 cells and 67.54 % and 39.06 % in EPG85.257RDB cells, respectively, suggesting receptor-independent mechanisms and effective MDR evasion. These findings highlight the potential of anticancer peptide-drug conjugates to circumvent MDR, with peptide-05 emerging as a particularly promising candidate for targeted chemotherapy.