Cooperative Oligomeric Peptide Combinations Enhance the Predicted Therapeutic Profile of SET-M33

Background/Objectives: Antimicrobial peptides (AMPs) are promising candidates against multidrug-resistant bacteria, although their clinical translation is frequently limited by cytotoxicity. In this study, we investigated whether combinations of structurally related oligomeric analogs could cooperatively enhance bacterial membrane targeting while redistributing the associated cytotoxic burden. Methods: Monomeric, dimeric, and tetrameric AMPs were evaluated through antimicrobial susceptibility testing, checkerboard interaction assays, RAW 264.7 macrophage cytotoxicity assays, and all-atom molecular dynamics simulations, including biased membrane insertion and umbrella sampling analyses. In addition, we introduced the Combinatorial Therapeutic Index (CTI) as an exploratory metric to estimate the predicted reduction in cytotoxic burden associated with peptide combinations. Results: Cytotoxicity varied substantially among oligomeric forms, with larger and more hydrophobic peptides, particularly tetramers, exhibiting the highest cytotoxicity. Additive effects were observed in checkerboard assays involving linear, dimeric, and tetrameric forms, supporting the redistribution of the toxic burden and enabling the beneficial membrane-interaction properties of hydrophobic linear peptides to be leveraged at lower cytotoxic cost. Predicted therapeutic improvement ranged from approximately twofold for the SET-M33:L33 combination to nearly ninefold for the SET-M33:DIM-33:L8 triple combination. Molecular dynamics simulations revealed non-redundant membrane interaction behaviors, with smaller peptides exhibiting deeper membrane insertion and the dimeric form favoring interfacial membrane engagement. Conclusions: These findings support a cooperative formulation strategy in which structurally related SET-M33 oligomers contribute complementary antibacterial functions while reducing the predicted cytotoxic burden. Further experimental validation using direct cytotoxicity assays of complete peptide mixtures will be necessary to confirm the therapeutic potential of these formulations.