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.