Design, Synthesis and Evaluation of Branched RRWQWR-Based Peptides as Antibacterial Agents Against Clinically Relevant Gram-Positive and Gram-Negative Pathogens

Abstract

Multidrug resistance of pathogenic bacteria has become a public health crisis that requires the urgent design of new antibacterial drugs such as antimicrobial peptides (AMPs). Seeking to obtain new, lactoferricin B (LfcinB)-based synthetic peptides as viable early-stage candidates for future development as AMPs against clinically relevant bacteria, we designed, synthesized and screened three new cationic peptides derived from bovine LfcinB. These peptides contain at least one RRWQWR motif and differ by the copy number (monomeric, dimeric or tetrameric) and structure (linear or branched) of this motif. They comprise a linear palindromic peptide (RWQWRWQWR), a dimeric peptide (RRWQWR)2KAhx and a tetrameric peptide (RRWQWR)4K2Ahx2C2. They were screened for antibacterial activity against Enterococcus faecalis (ATCC 29212 and ATCC 51575 strains), Pseudomonas aeruginosa (ATCC 10145 and ATCC 27853 strains) and clinical isolates of two Gram-positive bacteria (Enterococcus faecium and Staphylococcus aureus) and two Gram-negative bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa). All three peptides exhibited greater activity than did the reference peptide, LfcinB (17–31), which contains a single linear RRWQWR motif. Against the ATCC reference strains, the three new peptides exhibited minimum inhibitory concentration (MIC50) values of 3.1–198.0 μM and minimum bactericidal concentration (MBC) values of 25–200 μM, and against the clinical isolates, MIC50 values of 1.6–75.0 μM and MBC values of 12.5–100 μM. However, the tetrameric peptide was also found to be strongly hemolytic (49.1% at 100 μM). Scanning Electron Microscopy (SEM) demonstrated that in the dimeric and tetrameric peptides, the RRWQWR motif is exposed to the pathogen surface. Our results may inform the design of new, RRWQWR-based AMPs.

Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains

“…17,26 The linear, dimeric, and tetrameric peptides were obtained with high chromatographic purity, and the nal products had the expected monoisotopic mass. 17 Previously we reported the antibacterial activity of peptides containing LfcinB (20)(21)(22)(23)(24)(25), LcnB (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), and [Ala 19 ]-LfcinB (17-31) sequences against two E. coli strains (ATCC 25922 and 11 775). 17 It was established that dimeric and tetrameric peptides exhibited higher antibacterial activity than their monomeric analogue peptides.…”

“…Our results conrm that dimeric and tetrameric peptides derived from LfcinB (20-25) exhibited higher antibacterial activity against the evaluated strains than their monomeric counterpart. Furthermore, the tetrameric and dimeric peptides LfcinB (20)(21)(22)(23)(24)(25) 4 and LfcinB (20-25) 2 exhibited high antibacterial activity against all the tested E. coli strains; specically, their MICs ranged from 5 to 22 mM. Additionally, LfcinB (20)(21)(22)(23)(24)(25) 4 exhibited the highest antibacterial activity The results obtained in this investigation, together with our previous reports, indicate that polyvalence enhanced the antibacterial activity of LfcinB-derived sequences.…”

“…[11][12][13][14][15][16] LfcinB-derived short peptides have exhibited higher antibacterial activity than LfcinB and Lactoferrin themselves. 10,[17][18][19][20][21] Previous studies have shown that the inclusion of non-natural amino acids in some LfcinB-derived sequences improved their antibacterial activity 13,[17][18][19][20] Furthermore, dimeric and tetrameric peptides containing the RRWQWR motif exhibited higher antibacterial activity than Lactoferrin or LfcinB. In like manner, tetrameric and dimeric peptides containing 20 RRWQWR 25 or 17 FKARRWQWRMKKLGA 31 sequences exhibited higher antibacterial activity against E. coli strains than their monomeric peptide analogues.…”

“…In like manner, tetrameric and dimeric peptides containing 20 RRWQWR 25 or 17 FKARRWQWRMKKLGA 31 sequences exhibited higher antibacterial activity against E. coli strains than their monomeric peptide analogues. 13,[17][18][19][20] High antibacterial activity was also found when the palindromic sequence RWQWRWQWR was tested. 13,17,19 In the present study, the antibacterial activity of polyvalent peptides derived from LfcinB was evaluated against E. coli ATCC 43827, S. aureus ATCC 25923, E. faecalis ATCC 29212, and P. aeruginosa ATCC 27853.…”

“…The tetrameric peptide containing the RRWQWR motif exhibited a bactericidal effect against the evaluated strains. Additionally, the peptides LfcinB (20)(21)(22)(23)(24)(25) 4 or LfcinB (20-25) 2 combined with vancomycin or ciprooxacin exhibited a signicant synergistic antibacterial effect.…”