C‐Terminal Modification and Multimerization Increase the Efficacy of a Proline‐Rich Antimicrobial Peptide

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Two series of branched tetramers of the proline‐rich antimicrobial peptide (PrAMP), Chex1‐Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram‐negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C‐terminal‐cysteine bearing dimers that also incorporated C‐terminal peptide chemical modification. DTM‐linked tetrameric peptides containing a C‐terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49–2.33 μm. A second series of tetrameric analogues with C‐terminal hydrazide modification was prepared by using alternative conjugation linkers including trans‐1,4‐dibromo‐2‐butene, α,α’‐dibromo‐p‐xylene, or 6‐bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram‐negative nosocomial pathogens, particularly the butene‐linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL−1) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C‐terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.