Selectivity Modulation of Small Cationic Membrane-Active Cyclic Peptides with Broad-Spectrum Activity against Bacteria and Fungi

To investigate how ring size and backbone flexibility influence antimicrobial potency and cytotoxicity, we synthesized a series of macrocyclic peptides of lead peptide p1 (c[Arg-Arg-arg-arg-dip-Trp-dip]) by incorporating Gly, 2-aminoethoxyacetic acid (EAA), or 2,4-diaminobutyric acid (Dab). Two optimized peptides, 6b and 10b, retained broad-spectrum activity against drug-resistant Gram-positive (MIC, 1.5–6.2 μg/mL) and Gram-negative bacteria (MIC, 4–25 μg/mL), as well as pathogenic fungi, while exhibiting enhanced selectivity for microbial cells. Their therapeutic indices (TI ∼407 and ∼394, respectively) were ∼2-fold higher than p1, indicating improved safety. Both peptides remained effective against Gram-negative pathogens beyond the reach of daptomycin, were rapidly bactericidal, and eradicated bacterial and fungal biofilms. Mechanistic studies (e.g., calcein-leakage and extracellular ATP leakage assays) confirmed a membranolytic mode of action. NMR analysis revealed a distinct “sandwich” conformation in 6b that rationalizes its improved selectivity. Both peptides exhibited high plasma stability (t1/2 ∼ 6–8 h), supporting their therapeutic potential.