Ultrashort Cationic Lipopeptides and Lipopeptoids Selectively Induce Cytokine Production in Macrophages

<div><p>A series of ultrashort lipopeptides and lipopeptoids were tested for their ability to induce cytokine production in macrophages. Fourteen compounds were found to strongly induce production of chemokines Groα and IL-8, with a structural bias that was absent from previous antibacterial activity investigations. Compounds based on LysGlyLys and <em>N</em>LysGly<em>N</em>Lys sequences did not induce cytokine production, whereas those based on LysLysLys and <em>N</em>Lys<em>N</em>Lys<em>N</em>Lys were active only when linked to a lipid tail at least sixteen carbons long. Three lipopeptides induced high levels of IL-8 production, above that of equivalent concentrations of cathelicidin LL-37, while no compound induced production of the pro-inflammatory cytokine TNF-α at or below 100 µM. Two compounds, peptoids C16OH-<em>N</em>Lys<em>N</em>Lys<em>N</em>Lys and C16OH-<em>N</em>Har<em>N</em>Har<em>N</em>Har, were selective for IL-8 production and did not induce TNF-α or IL-1β. These compounds may prove beneficial for in vivo treatment of infectious disease, with improved bioavailability over LL-37 due to their protease-resistant scaffold.</p> </div

Structures for the cationic amphiphiles used in this study.

<p>Har = homoarginine; <i>N</i>Lys = lysine peptoid; <i>N</i>Har = homoarginine peptoid.</p

Cytokine production and LDH release by human macrophage-like THP-1 cells following incubation with amphiphiles 1–21.

<p>A) IL-8 production. TC supernatants were monitored for IL-8 production by ELISA, and results were recorded in pg/mL. B) Groα production, in pg/mL. Inset: Expanded values for amphiphiles 5–7 and 21 at 5 µM and 10 µM. C) Cytotoxicity following incubation with amphiphiles 1–21. TC supernatants were monitored for LDH release as a measure of cellular toxicity, and results shown represent percent cytotoxicity over un-stimulated cells. All studies were performed in two independent biological replicates with two technical replicate each, with the data here presented as the mean plus standard error of the mean (sem) and with LL-37 data included as a positive control.</p

IL-1β production.

<p>Human macrophage-like THP-1 cells were exposed to amphiphiles <b>4–11</b> and <b>14–21</b>, for twenty-four hours. TC supernatants were monitored for IL-1β by ELISA, with results shown in pg/mL. Studies were performed in two independent biological replicates with two technical replicate each, with the data here presented as the mean plus sem. Inset: Expanded results for the negative control and amphiphiles <b>4–7</b> at 5 µM and 10 µM.</p

Guanidylation and Tail Effects in Cationic Antimicrobial Lipopeptoids

<div><h3>Background</h3><p>Cationic antimicrobial peptides (CAMPs) are attractive scaffolds for the next generation of antimicrobial compounds, due to their broad spectrum of activity against multi-drug resistant bacteria and the reduced fitness of CAMP-insensitive mutants. Unfortunately, they are limited by poor <em>in vivo</em> performance, including ready cleavage by endogenous serum proteases.</p> <h3>Methodology/Principal Findings</h3><p>To explore the potential for peptoid residues to replace well studied CAMP scaffolds we have produced a series of antimicrobial lipopeptoids, with sequences similar to previously reported lipopeptides. The activity of the peptoids was assessed against a panel of clinically relevant and laboratory reference bacteria, and the potential for non-specific binding was determined through hemolytic testing and repeating the antimicrobial testing in the presence of added bovine serum albumin (BSA). The most active peptoids displayed good to moderate activity against most of the Gram positive strains tested and moderate to limited activity against the Gram negatives. Antimicrobial activity was positively correlated with toxicity towards eukaryotic cells, but was almost completely eliminated by adding BSA.</p> <h3>Conclusion/Significance</h3><p>The lipopeptoids had similar activities to the previously reported lipopeptides, confirming their potential to act as replacement, proteolytically stable scaffolds for CAMPs.</p> </div

Antimicrobial testing of N_lysN_lysN_lys based lipopeptoids.

a<p>MIC, reported in µg/mL.</p>b<p>ATCC 29213.</p>c<p>ATCC 33592.</p>d<p>81388 CANWARD 2008.</p>e<p>CAN-ICU 61589.</p>f<p>ATCC 29212.</p>g<p>ATCC 27270.</p>h<p>ATCC 49619.</p>i<p>ATCC 25922.</p>j<p>CAN-ICU 61714.</p>k<p>CAN-ICU 63074.</p>l<p>ATCC 27853.</p>m<p>CAN-ICU 62308.</p>n<p>CAN-ICU 62584.</p>o<p>CAN-ICU 63169.</p>p<p>ATCC 13883.</p>q<p>Percent haemolysis at 100µg/mL of compound.</p

Peptoid residues with comparison amino acids.

<p>Lipid tails were attached at the N-terminus while all peptoids were amidated at their C-terminus.</p

Lipopeptoids under consideration.

<p>Lipopeptoids under consideration.</p

Antimicrobial testing of N_lysN_lysN_lys based lipopeptoids in the presence of 4% bovine serum albumin.

a<p>MIC, reported in µg/mL.</p>b<p>ATCC 29213.</p>c<p>ATCC 33592.</p>d<p>81388 CANWARD 2008.</p>e<p>CAN-ICU 61589.</p>f<p>ATCC 29212.</p>g<p>ATCC 27270.</p>h<p>ATCC 49619.</p>i<p>ATCC 25922.</p>j<p>CAN-ICU 61714.</p>k<p>CAN-ICU 63074.</p>l<p>ATCC 27853.</p>m<p>CAN-ICU 62308.</p>n<p>CAN-ICU 62584.</p>o<p>CAN-ICU 63169.</p>p<p>ATCC 13883.</p

Antimicrobial testing of N_lysGN_lys based lipopeptoids.

a<p>MIC, reported in µg/mL.</p>b<p>ATCC 29213.</p>c<p>ATCC 33592.</p>d<p>81388 CANWARD 2008.</p>e<p>CAN-ICU 61589.</p>f<p>ATCC 29212.</p>g<p>ATCC 27270.</p>h<p>ATCC 49619.</p>i<p>ATCC 25922.</p>j<p>CAN-ICU 61714.</p>k<p>CAN-ICU 63074.</p>l<p>ATCC 27853.</p>m<p>CAN-ICU 62308.</p>n<p>CAN-ICU 62584.</p>o<p>CAN-ICU 63169.</p>p<p>ATCC 13883.</p>q<p>Percent haemolysis at 100µg/mL of compound.</p