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 <b>p1</b> (c[Arg-Arg-arg-arg-dip-Trp-dip]) by incorporating Gly, 2-aminoethoxyacetic acid (EAA), or 2,4-diaminobutyric acid (Dab). Two optimized peptides, <b>6b</b> and <b>10b,</b> 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 <b>p1</b>, 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 <b>6b</b> that rationalizes its improved selectivity. Both peptides exhibited high plasma stability (<i>t</i>₁/₂ ∼ 6-8 h), supporting their therapeutic potential.