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

Abstract

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 ) 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.

Advantage of a Narrow Spectrum Host Defense (Antimicrobial) Peptide Over a Broad Spectrum Analog in Preclinical Drug Development

“…While the reported activity of the hydrazide analog to A. baumannii is considered very weak (MIC = 130 mg/L) (Li et al, 2015 , 2017a ), even at this low activity level in the current study we could not repeat the superiority of the hydrazide over the amide (64 mg/L and >512 mg/L in 25% and full-strength MHB for both). The reasons for the lack of activity differences might be either alterations in the assay protocol (Lambert and Pearson vs. broth microdilution) or the identity of the actual A. baumannii strain.…”

“…In previous studies, the Chex1-Arg20 amide (ARV-1502) was shown to inhibit the growth of E. coli and K. pneumoniae strains with ~2-fold reduction in MIC values (6.3 vs. 10 mg/L and 2 vs. 4.2 mg/L respectively) compared to the hydrazide analog (Li et al, 2015 , 2017a ). At the same time, the hydrazide was twofold more active than the amide against A. baumannii and P. aeruginosa (130 vs. >250 mg/L and 72 vs. 140 mg/L, respectively).…”

IUAPC 2017 Distinguished Women in Chemistry or Chemical Engineering

“…Separovic and her research group are interested in structural biology and biophysical chemistry, with a focus on biological solid‐state NMR spectroscopy and membrane‐active peptides, including antimicrobial and amyloid peptides and toxins. She has reported in Chemistry—A European Journal on a proline‐rich antimicrobial peptide …”

Covalent conjugation of cationic antimicrobial peptides with a β‐lactam antibiotic core

“…We have also previously shown the synergistic combination of proline‐rich AMP (PrAMP) to recover the activity of conventional antibiotics . Our laboratories have also recently further developed this PrAMP and found a class of multimerized PrAMP with C ‐terminal functionalization that showed a broader spectrum or activity and increased potency as potential antibiotic alternatives …”