Improved Antibacterial Activity of the Marine Peptide N6 against Intracellular Salmonella Typhimurium by Conjugating with the Cell-Penetrating Peptide Tat11 via a Cleavable Linker

Article abstract of DOI:10.1021/acs.jmedchem.8b01079

The poor penetration ability of antimicrobial agents limits their use in the treatment of intracellular bacteria. In this study, the conjugate CNC (6) was generated by connecting the cell-penetrating peptide Tat₁₁ (1) and marine peptide N6 (2) via a cathepsin-cleavable linker, and the C-terminal aminated N6 (7) and CNC (8) were first designed and synthesized to eliminate intracellular Salmonellae Typhimurium. The cellular uptake of 6 and stability of 7 were higher than those of 2, and conjugates 6, 8, and 7 had almost no hemolysis and cytotoxicity. The antibacterial activities of 6, 8, and 7 against S. Typhimurium in RAW264.7 cells were increased by 67.2-76.2%, 98.6-98.9%, and 96.3-97.6%, respectively. After treatment with 1-2 μmol/kg of 6, 8, or 7, the survival of the S. Typhimurium-infected mice was 66.7-100%, higher than that of 2 (33.4-66.7%). This result suggested that 6, 8, and 7 may be excellent candidates for novel antimicrobial agents to treat intracellular pathogens.

Full text of DOI:10.1021/acs.jmedchem.8b01079

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Article
Improved Antibacterial Activity of the Marine Peptide N6
against Intracellular Salmonella Typhimurium by Conjugating
with the Cell-penetrating Peptide Tat11 via a Cleavable Linker
Zhanzhan Li, Da Teng, Ruoyu Mao, Xiao Wang, Ya Hao, Xiumin Wang, and Jianhua Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01079 • Publication Date (Web): 10 Aug 2018
Downloaded from http://pubs.acs.org on August 11, 2018
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Journal of Medicinal Chemistry
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Improved Antibacterial Activity of the Marine Peptide N6 against
Intracellular Salmonella Typhimurium by Conjugating with the Cell-penetrating Peptide
Tat11 via a Cleavable Linker
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Zhanzhan Li,†, ‡, § Da Teng,†, ‡, § Ruoyu Mao,†, ‡ Xiao Wang,†, ‡, § Ya Hao,†, ‡ Xiumin Wang,†, ‡* and
Jianhua Wang,†, ‡
*
†Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, People’s Republic of
China
‡Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences,
Beijing 100081, People’s Republic of China
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Journal of Medicinal Chemistry
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ABSTRACT: The poor penetration ability of antimicrobial agents limits their use in the treatment of
intracellular bacteria. In this study, the conjugate–CNC (6) was generated by connecting the
cellꢀpenetrating peptide–Tat11 (1) and marine peptide–N6 (2) via a cathepsinꢀcleavable linker, and the
Cꢀterminal aminated N6 (7) and CNC (8) were first designed and synthesized to eliminate intracellular
Salmonellae Typhimurium. The cellular uptake of 6 and stability of 7 were higher than those of 2, and
conjugates–6/8/7 had almost no hemolysis and cytotoxicity. The antibacterial activities of 6/8/7 against
S. Typhimurium in RAW264.7 cells were increased by 67.2–76.2%, 98.6–98.9% and 96.3–97.6%,
respectively. After treatment with 1–2 ꢁmol/kg of 6/8/7, the survival of the S. Typhimurium–infected
mice was 66.7–100%, higher than that of 2 (33.4–66.7%). This result suggested that 6/8/7 may be
excellent candidates for novel antimicrobial agents to treat intracellular pathogens.
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Keywords: Cellꢀpenetrating peptides; Tat11; antimicrobial peptide; N6; amination; intracellular activity
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Journal of Medicinal Chemistry
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INTRODUCTION
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The high worldwide incidence of Salmonella infection and the rapid emergence of antibioticꢀresistant
strains highlight the urgent need for new approaches to therapeutic intervention.1 Another challenge is
that Salmonellae can invade and survive in a specialized membraneꢀbound compartment named the
Salmonellaꢀcontaining vacuole (SCV) in phagocytic cells, wherein pathogens can adapt to intracellular
acidic conditions to evade host immune responses, and commonly used drugs such as aminoglycosides
limit access to intracellular targets due to their poor cellular uptake.2 Phagocytic cells become a
reservoir for recurrence and reinfections of S. Typhimurium, resulting in a wide range of diseases in
humans and animals, including gastroenteritis, bacteremia, enteric fever and focal infections.3 Until
now, there has been no effective way to eradicate intracellular Salmonellae.
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Due to the advantages of the broadꢀspectrum activity, rapidꢀkilling effect and low resistance potential,
antimicrobial peptides (AMPs) may open a new avenue for the development of antibacterial agents.4
Our preliminary study has demonstrated that N6 (2) exhibited potent antimicrobial activity against
Gramꢀnegative bacteria, especially Salmonellae and Escherichia coli.5 However, 2 displayed poor
ability to traverse eukaryotic cell membranes and kill intracellular S. Typhimurium, and it is very
susceptible to acidic pH values between pH 2.0 and pH 6.0.5 Recently, cellꢀpenetrating peptides (CPPs)
have been widely used as delivery vectors to deliver traditional antibiotics and drugs into cells to target
intracellular pathogens.6, 7 However, most of the current delivery vectorsꢀCPPs cannot effectively
release “cargoes” or lack a target, leading to a partial loss of efficacy. The human immunodeficiency
virus type 1 (HIVꢀ1) transꢀactivator of transcription Tat11 (1) peptide (residues 47–57), one of the most
commonly used CPPs, can deliver various drug molecules into eukaryotic cells, including antibodies,
oligonucleotides and peptides;8, 9 thus, 1 is a promising tool to improve the nonꢀinvasive cellular
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