Î²-Peptoid Hybrids with Potent Anti-inflammatory

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Lipidated #-Peptide/#-Peptoid Hybrids with Potent Anti-inflammatory Activity Sarah Line Skovbakke, Camilla Josephine Larsen, Peter Mikael Helweg Heegaard, Lise Moesby, and Henrik Franzyk J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Dec 2014 Downloaded from http://pubs.acs.org on December 10, 2014

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Journal of Medicinal Chemistry

Lipidated α-Peptide/β-Peptoid Hybrids with

Potent Anti-inflammatory Activity Sarah L. Skovbakke†, Camilla J. Larsen†, Peter M. H. Heegaard‡, Lise Moesby†§, Henrik Franzyk†* † Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark ‡ Innate Immunology Group, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark, Bülowsvej 27, DK-1870, Frederiksberg C, Denmark *Correspondence: [email protected], phone: +45 3533 6255.

ACS Paragon Plus Environment


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ABSTRACT

In this study we investigated, optimized, and characterized a novel subclass of host defense peptide (HDP) mimics based on α-peptide/β-peptoid hybrid oligomers with an alternating cationic/hydrophobic design with respect to their ability to modulate the pro-inflammatory response by human primary leukocytes upon exposure to bacterial components. Structureactivity studies revealed that certain lipidated α-peptide/β-peptoid hybrid oligomers possess antiinflammatory activities in the submicromolar range with low cytotoxicity, and that the antiinflammatory activity of the HDP mimics is dependent on the length and position of the lipid element(s). The resulting lead compound, Pam-(Lys-βNSpe)6-NH2, blocks LPS-induced cytokine secretion with a potency comparable to that of polymyxin B. The mode of action of this HDP mimic does not involve direct LPS interaction since it, in contrast to polymyxin B, displayed only minor activity in the Limulus amebocyte lysate assay. Flow cytometry data showed specific interaction of a fluorophore-labeled lipidated α-peptide/β-peptoid hybrid with monocytes and granulocytes indicating a cellular target expressed by these leukocyte subsets.


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INTRODUCTION The emergence of multidrug-resistant and even pan-resistant bacteria constitutes an increasing risk for patients to contract virtually incurable infections, and we may be on the verge of entering the “post-antibiotic era” in which infections again become a major health issue.1-3 Even with the currently available optimal treatments, sepsis remains one of the most common life-threatening complications for patients admitted to intensive care units with mortality rates as high as 2080%, for severe sepsis and septic shock.4,5 Moreover, the number of deaths caused by septic shock is increasing due to a continuing rise in the incidence of sepsis.6,7 Thus, development of novel treatment strategies against sepsis is urgently needed. Sepsis is defined as infection combined with a systemic inflammatory response syndrome involving an exacerbated host response. The pathogenesis involves elevated concentrations of a range of pro-inflammatory host factors in the circulatory system, as well as an ensuing imbalance between pro- and antiinflammatory responses giving rise to cellular dysfunction, dysregulated coagulation, and finally multiple-organ failure.6, 8, 9 Bacterial membrane components are recognized by pattern-recognition receptors (PRRs) on host cells, thereby activating the innate immune system by inducing synthesis and release of a wide range of host response factors, including pro-inflammatory cytokines and chemokines such as tumor necrosis factor-α (TNF-α) and interleukins 1β, 6, and 8 (IL-1β, IL-6, and IL-8).10-12 Lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria is a very potent inducer of inflammation, and it is believed to be of paramount importance for development of sepsis.13,14 In recent years it has become clear that Gram-positive bacteria are equally common inducers of sepsis4,5,7 as their thick cell walls are rich sources of immunostimulatory components such as lipoteichoic acid (LTA)15,16 as well as peptidoglycan (PGN) and lipoproteins.17, 18



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Blocking of the pro-inflammatory effects of LPS and other bacterial constituents has been proposed as a novel approach for treating sepsis.19,20 Especially, naturally occurring host defense peptides (HDPs) and their synthetic mimetics have been suggested as potential drug leads due to their dual mode of action involving both immunomodulatory and bactericidal effects.19-23 However, the therapeutic value of HDPs has so far been limited, typically because of problems with toxicity and limited bioavailability due to in vivo proteolytic degradation.23 We have previously shown that oligomers of alternating cationic α-amino acids and lipophilic β-peptoid residues mimic naturally occurring HDPs with respect to antimicrobial activity, while showing enhanced stability towards proteolysis.24,25 Recently, we reported on HDM-4, possessing an α-peptide/α-peptoid backbone, with a capability to block LPS-induced proinflammatory

host

responses,

showing

that

such

peptidomimetics

may

display

immunomodulatory activities.26 Here, we describe the discovery of a novel subclass of proteolytically stable peptidomimetics comprising lipidated α-peptide/β-peptoid hybrid oligomers that block the pro-inflammatory response induced by LPS or LTA. Via investigation of structure-activity relationships (compounds 1-16, Figure 1) the ratio between anti-inflammatory activity and cytotoxicity was optimized. Acylation of the N-terminus with fatty acid moieties was found to be essential for a potent anti-inflammatory activity, and a clear correlation between fatty acid chain length and activity was found. On the other hand, the anti-inflammatory activity proved to be less dependent on the structural features of the peptidomimetic such as chirality and type of cationic residues. The resulting lead compound (4, Pam-(Lys-βNSpe)6-NH2) was found to block LPS-induced IL-6 secretion with a potency comparable to that of polymyxin B (PmB), but via a different mechanism, since compound 4, in contrast to PmB, exhibited a poor LPS-neutralizing effect in


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the Limulus amebocyte lysate (LAL) assay. Compound 4 also inhibited IL-6, TNF-α, and IL-8 secretion induced by UV-inactivated Gram-positive and -negative bacteria. Flow cytometry data using a fluorescently labeled analog indicate that cellular target(s) may be present on monocytes and granulocytes.

Figure 1. Chemical structures of investigated α-peptide/β-peptoid hybrids and generic structures of α-amino acids, α-peptoid, and β-peptoid residues.



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RESULTS α-Peptide/β-peptoid hybrid oligomers block the pro-inflammatory effect of LPS We hypothesized that peptidomimetics with an α-peptide/β-peptoid hybrid design might possess anti-inflammatory properties characteristic of certain HDPs. Indeed, we found that compound 1, a 12-mer consisting of alternating lysine (Lys) and βNphe (Figure 1), blocks LPSinduced but not LTA-induced IL-6 secretion from human primary leukocytes without affecting cell viability (Figure 2). However, the potency of compound 1 was low compared to that of the human

cathelicidin-derived

peptide

LL-37P

(IGKEFKRIVQRIKDFLRNLVPRTE-NH2)

previously reported as a potent LPS- and LTA-neutralizing peptide.27 Surprisingly, in our hands, LL-37P had only minor effects on the LTA-induced release of IL-6 in the concentrations used. Moreover LL-37P was found to be mildly toxic at a concentration of 10 µM (Figure 2).


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Figure 2. α-Peptide/β-peptoid oligomer 1 blocks the pro-inflammatory effect of LPS. Freshly prepared human leukocytes were stimulated with compound 1 (1 µM) or the truncated human cathelicidin peptide LL37P (1 µM) alone or in combination with LPS (1 EU/mL) or LTA (1×106 pg/mL). The bar graph in the upper panel shows the mean + 95% CI of IL-6 concentrations determined in six independent experiments. The bar graph in the lower panel shows mean + 95% CI of the percentage of viable cells, using untreated leukocytes as control for 100% viable cells in six independent experiments. The dotted line indicates 100%, and asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. Lipidation improves the anti-inflammatory activity of α-peptide/β-peptoid hybrids Inspired by a recent report on immunomodulatory properties of the ultrashort cationic lipopeptide and lipopeptoid, Pam-Lys3 and Pam-NLys3,28 respectively, the effect of N-terminal lipidation of compound 1 on the anti-inflammatory properties was examined. No effect of the palmitoylated compound 3 on the basal release of IL-6 was observed (Supporting information, page S11). However, as shown in Figure 3, N-terminal palmitoylation resulted in a significantly increased potency in blocking LPS-induced IL-6 secretion. In contrast to the non-lipidated compound 1, the palmitoylated analog 3 also inhibited IL-6 secretion induced by LTA although to a lower degree than seen for stimulation with LPS. The viability of leukocytes was not affected upon exposure to compound 3 at 1 µM, though cytotoxicity was observed at 10 µM, which was in contrast to the non-lipidated peptidomimetic. To test whether the antiinflammatory effects were specific to the subclass of lipidated peptidomimetics with an alternating cationic/hydrophobic design, previously reported immunomodulatory ultrashort cationic lipopeptides and lipopeptoids (i.e Pam-Lys3 and Pam-NLys3)28 were tested for their



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effect on LPS- and LTA-induced IL-6 secretion, however, no such effects were observed for these compounds (Supporting information, page S12).

Figure 3. N-terminal palmitoylation of α-peptide/β-peptoid peptidomimetics increases blocking of LPS- and LTA-induced responses. Freshly prepared human leukocytes were treated with compound 1 or 3 (both at 1 µM) alone or in combination with LPS or LTA. The bar graphs show the mean and 95% CI of IL-6 secretion and viability from six independent experiments. The dotted line indicates 100%, and asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001.


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Anti-inflammatory activity of lipidated α-peptide/β-peptoid hybrids depends on chirality, type of cationic units, and oligomer length Next, more detailed structure-activity relationships for these lipidated anti-inflammatory αpeptide/β-peptoids were investigated. For this purpose an array of peptidomimetics, with variations in structural features of the parent compound 3, was designed and synthesized. The structures of the investigated hybrids are shown in Figure 1. The peptidomimetics are all composed of cationic α-amino acid residues [Lys or guanidinylated Lys = homoarginine (hArg)] and hydrophobic β-peptoid aromatic residues. Chiral (βNSpe) or achiral (βNphe) β-peptoid residues were incorporated (to give 3 or 4), and constructs with a different number of repeats (i.e. 9-12) were included. Finally, lipid length and position were varied (i.e. compounds 5-8, 13 and 14). Each compound was tested independently for its effect on LPS- and LTA-induced IL-6 secretion, assessed at two different concentrations (0.1 µM and 1 µM) in experiments with human primary leukocytes from six different donors. Cytotoxicity of the compounds was tested on the same batches of leukocytes at concentrations 10-fold higher than those used for cytokine studies (i.e. 1 µM and 10 µM). The main results of this structure-function study are presented in Figure 4 and Figure 5. The significance of chirality for anti-inflammatory and cytotoxic properties of lipidated αpeptide/β-peptoid hybrids was investigated by comparing compounds 3 and 4, of which the latter display α-chiral hydrophobic β-peptoid residues. The β-peptoid residue (βNSpe) incorporated into peptidomimetic 4 has a methylated side chain, and thus contains both an additional chirality center and a methyl group as compared to the slightly less hydrophobic achiral βNphe present in compound 3. As shown in Figure 4A, analog 4 exhibited more potent LPS neutralization than



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compound 3. In contrast, the introduction of chirality and slightly enhanced hydrophobicity affected neither LTA neutralization nor cytotoxicity at the concentrations tested. Guanidinium functionalities (i.e. arginine-like side chains) have previously been shown to confer improved antibacterial properties to this class of peptidomimetics, but concomitantly augmented cytotoxicity towards mammalian cell lines.24 Thus, we hypothesized that guanidinylation might also affect the anti-inflammatory effects of lipidated α-peptide/β-peptoid hybrids. Hence, keeping the charges constant three specific ratios of Lys versus hArg were investigated: Lys only (i.e. 4), Lys-hArg 1:1 (i.e. 11), and hArg only (i.e. 5). As seen in Figure 4B a high content of Lys residues was most favorable as it resulted in slightly improved LPS and LTA neutralization, but somewhat unexpectedly without a significant effect on cytotoxicity. The correlation between anti-inflammatory potency and length of the α-peptide/β-peptoid oligomers was investigated by comparison of the effects found for the series 9-12 (Figure 4C). The results show unambiguously that the LPS- and LTA-neutralizing capacity increases with oligomer length. On the other hand, increasing oligomer length was associated with increased cytotoxicity, but the correlation was less pronounced when the oligomer length exceeded 12 residues. Thus, the optimal oligomer length was estimated to be between 12 and 16 residues, but for ease of synthesis and purification an oligomer length of 12 residues was selected for further investigations on structural variants.


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Figure 4. The anti-inflammatory activity of the peptidomimetics is dependent on chirality, degree of guanidinylation, and oligomer length. Freshly prepared human leukocytes were treated with peptidomimetics, alone or in combination with LPS or LTA. The bar graphs show the mean and 95% CI of IL-6 secretion and viability from six independent experiments. The dotted lines indicate 100% on the y-axis, and asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. A: Chirality of the β-peptoid residues increases the LPS-neutralizing activity. The bar graphs display the LPS- and LTA- neutralizing activity (left and middle panel, respectively) and cytotoxicity (right panel) of compounds 3 and 4



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that differ only in the nature of the β-peptoid residues. B: Guanidinylation of the α-amino acids decreases LPS-neutralizing activity. The bar graphs depict the LPS- and LTA-neutralizing activity (left and middle panel, respectively) and cytotoxicity (right panel) of compounds 4, 5, and 11 that differ only in the nature of the cationic α-amino acid. C: increasing oligomer length increases the LPS- and LTA-neutralizing activity. The bar graphs depict the LPS- and LTAneutralizing activity (left and middle panel, respectively) and cytotoxicity (right panel) of compounds 9-12 that differ only in length. Lipid size and positioning determine the anti-inflammatory properties of HDP mimics Since palmitoylation was found to significantly enhance the anti-inflammatory activity (Figure 3) we decided to study the influence of both length and positioning of the lipid moiety on the anti-inflammatory activity of the peptidomimetics. This involved comparison of compounds 4 and 6-8. As seen in Figure 5A, reducing the length of the fatty acid from C16 (i.e. 4) to C8 (i.e. 7) significantly diminished both the LPS- and LTA-neutralizing activity. Notably, addition of two shorter fatty acids via an N-terminal lysine residue (to give 8) was less efficient than attachment of a single longer fatty acid as displayed by compound 4. The cytotoxicity of the compounds was likewise demonstrated to depend on the length of the lipid moiety (Figure 5A) with longer fatty acids associated with increased cytotoxicity. Interestingly, compound 8 carrying two shorter fatty acids (Oct and Lau) exhibited similar cytotoxicity as the compounds with a single longer fatty acid (Lau or Pam), indicating distinct structure-activity relationships for cytotoxicity and antiinflammatory potency. Alternatively, a lipid moiety may be introduced via attachment of an unnatural α-amino acid displaying an aliphatic C6 side chain (i.e. 2-aminooctanoic acid: 2-Aoc) as the N-terminal residue (i.e. 13 and 14). In comparison to the non-lipidated compound 2 incorporation of 2-Aoc (to give 13) resulted only in a slightly enhanced LPS-neutralizing activity


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(Figure 5B). Compared to compound 7, displaying an N-terminal fatty acid of comparable length to 2-Aoc, the LPS-neutralizing activity of compound 13 was significantly lower, again implying that both positioning and length of the longest lipid moiety are determinants for the antiinflammatory activities of lipidated peptidomimetics. Furthermore, when 2-Aoc was combined with N-terminal conjugation to lauric acid (i.e. 14), no additional positive contribution to the activity was found (Supporting information, page S13). Thus, simple N-terminal lipidation seems to be the most favorable approach to increase the blocking of LPS- and LTA-induced proinflammatory effects. However, lipidation concomitantly confers increased cytotoxicity to these peptidomimetics albeit with a less pronounced correlation to the positioning of lipid moieties.



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Figure 5. The anti-inflammatory properties of the peptidomimetics are dependent on lipid length and positioning. Freshly prepared human leukocytes were treated with peptidomimetics, alone or in combination with LPS or LTA. The bar graphs show the mean and 95% CI of IL-6 secretion and viability from six independent experiments. The dotted lines indicate 100% on the y-axis, and asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. A: The bar graphs depict the LPS- (left panel) and LTA- (middle panel) neutralizing activity, and toxicity (right panel) of compounds 2, 4 and 6-8 that differ only by the nature of the fatty acid conjugation. B: the bar graphs depict the LPS- and LTA-neutralizing activity (left and middle panel, respectively), and cytotoxicity (right panel) of compounds 2, 7 and 13, of which the last two display different modes of lipidation. Immunomodulatory activity of the most promising peptidomimetic To supplement the above described structure–function relationships, IC50 values for LPS and LTA neutralization, as well as the 50% lethal concentrations (LC50) for primary leukocytes and for the hepatocellular carcinoma cell line HepG2 were determined for selected compounds and compared to those of LL-37P and PmB (Table 1). Overall, these data support the structure– function study, and the general trend is that leukocytes and HepG2 cells are equally sensitive to treatment with these peptidomimetics. Although chirality in the β-peptoid residues appeared to have a minimal effect on the LTA-neutralizing activity of the lipidated compounds in the initial screening assay, a lower IC50 value for LTA blocking was found for compound 4 as compared to compound 3. Altogether, compound 4 was found to display superior ratios between LC50 and IC50 for inhibition of IL-6 secretion induced by LPS and LTA. The dose-response curves of compound 4 for blocking of LPS- and LTA-induced responses as well as for cytotoxicity against leukocytes are shown in Figure 6A. Notably, its potency in LPS neutralization is comparable to


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that of PmB and LL-37P which are the golden standards of LPS-sequestering peptides. The therapeutic index (TI) of compound 4 for LPS neutralization is however inferior to that of PmB due a higher toxicity. However, since none of the two standard LPS-sequestering peptides have any effect on the LTA-induced cytokine secretion, compound 4 exerts a broader antiinflammatory activity. Based on the above encouraging preliminary results the anti-inflammatory properties of compound 4 were investigated in a setup in which human primary leukocytes were exposed to intact UV-irradiated bacteria. As seen in Figure 6B, compound 4 inhibited secretion of the pro-inflammatory cytokines IL-6 and TNF-α as well as of the pro-inflammatory chemokine IL-8 in a dose-dependent manner, with potency in the submicromolar to micromolar range for E. coli and S. aureus, respectively.

Figure 6. Peptidomimetic 4 exhibits broad anti-inflammatory activity. A: Dose response curves for freshly prepared human leukocytes treated with compound 4 in concentrations ranging from 0.01 µM to 100 µM alone or in combination with LPS or LTA. The graph shows % of induced



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IL-6 (left y-axis) or % viable cells (right y-axis) as a function of the concentration of compound 4. B: Compound 4 inhibits cytokine secretion induced by E. coli and S. aureus. Freshly prepared human leukocytes were treated with compound 4 in concentrations ranging from 0.01 µM to 10 µM alone or in combination with UV-inactivated E. coli or S. aureus. The graphs show mean ± SD of the % of induced IL-6 (left panel), TNF-α (middle panel), and IL-8 (right panel) as a function of the compound concentration from three independent experiments. Studies on the selectivity and mode of action Since some HDPs and their mimics in addition to immunomodulatory activity also have direct bactericidal activity, we investigated the antimicrobial activity of the lead compound 4 and a few selected analogs (Table 2). The MIC values of these lipidated peptidomimetics were in the range of 8-16 µM against E. coli and S. aureus, which are orders of magnitude higher than the concentration range in which they exert their anti-inflammatory activity. To gain insight into the mode of action for its anti-inflammatory activity, the effect of compound 4 on the leukocyte responses to a range of activating ligands with selective binding affinity for a number of known PRRs was investigated. As shown in Figure 7A, compound 4 blocked the pro-inflammatory response to LPS with the highest potency, however, it was also capable of blocking IL-6 secretion induced by LTA or the synthetic TLR2/1 agonist Pam3CSK4. Interestingly, inhibition of the pro-inflammatory response induced by the TLR2/6 agonist Pam2CSK4, the NOD2 ligand PGN, or the TLR5 ligand flagellin was not observed at concentrations below 5 µM. The high potency of peptidomimetic 4 in blocking LPS-induced cytokine secretion indicated that its mode of action might involve direct binding to LPS. This was analyzed in a Limulus amebocyte lysate (LAL) assay. Here, the standard LPS-neutralizing peptide polymyxin B and compound 4 were compared directly. The LAL assay is based solely


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on reactions of soluble enzymes, and thus no immune cell interactions are involved. In this assay, polymyxin B completely blocked LPS activity at a concentration of 0.1 µM, whereas compound 4 exhibited no LPS-neutralizing activity (Figure 7B: left panel). By contrast, when the LPSneutralizing properties of polymyxin B and compound 4 were determined in the cellular IL-6 secretion assay they demonstrated similar antagonistic activity (Figure 7B: right panel). This clearly shows that different mechanisms are involved in the LPS-neutralizing activity of Polymyxin B and compound 4. At higher molar ratios of compound 4 to LPS, moderate LPSneutralizing activities were found in the LAL-assay (Supporting information, page S16). To investigate whether a cellular target is involved in the anti-inflammatory action of the lipidated peptidomimetics, the interaction of fluorescently labeled derivatives of compound 4 (i.e. compound 15) and of the corresponding non-lipidated analog 2 (i.e. compound 15) with human primary leukocytes was explored by flow cytometry. Importantly, introduction of the CF label did not abolish the anti-inflammatory activity (i.e. compound 15 versus compound 2) although the potency for LTA neutralization was lowered as a result of the labelling. In agreement with the structure-activity relationships for the non-labeled peptidomimetics, the CFlabelled non-lipidated compound 16 showed only very low LPS-neutralizing activity. By gating specific leukocyte subsets based on forward-side scattering properties, it was found that compound 15 displayed selective affinity for monocytes and granulocytes at concentrations as low as 0.01 µM (Figure 7C). A significantly higher binding to monocytes than to granulocytes was observed, indicating differential expression of the target(s) in these cell populations. Furthermore, the CF-labeled non-lipidated compound 16 showed significantly less binding to monocytes and granulocytes, in agreement with its expected lower anti-inflammatory activity.



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Figure 7. Cell specificity and mechanism of action for peptidomimetic 4. A: Freshly prepared human leukocytes were treated with compound 4 in concentrations ranging from 0.01 µM to 10


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µM alone or in combination with LPS (1 EU/mL), LTA (1×106 pg/mL), peptidoglycan (200 ng/mL), Pam2CSK4 (5 ng/mL), Pam3CSK4 (10 ng/mL), or flagellin (200 ng/mL). The graphs show the mean percentage of induced IL-6 concentration as a function of the compound concentration from three independent experiments. B: LPS-binding and -neutralizing activities of polymyxin B and compound 4. The bar graphs depicts the LPS-neutralizing activity of polymyxin B and compound 4 at a concentration of 0.1 µM in the Limulus amebocyte lysate (LAL) assay and in the cellular cytokine induction assay. LAL results are shown as mean ± SD of the % recovery of LPS from two independent experiments, whereas the cell-based neutralization results are shown as mean + 95% CI of six independent experiments. C: Evaluation of the effect of fluorescent labeling on the biological activities of peptidomimetics. Freshly prepared human leukocytes were treated with non-labeled peptidomimetics (i.e. the acetylated 2 or the palmitolylated 4) or the corresponding CF-labelled derivatives (i.e. 16 and 15, respectively), alone or in combination with LPS or LTA. The bar graphs show the mean and 95% CI of IL-6 secretion and viability from six independent experiments. The dotted lines indicate 100% on the y-axis, and asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001. D: Specific binding of the fluorescently labeled Npalmitoylated derivative of compound 4 (i.e. 15) and of N-acetylated analog 2 (i.e. 16) to human leukocytes. Representative dot plots show the binding of 0.01 µM CF-labelled peptidomimetic 15 to leukocyte subsets as determined by forward-side scatter. The bar graphs summarize the results from three independent experiments, with 95% CI intervals indicated. Asterisks indicate statistically significant differences. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001.



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DISCUSSION AND CONCLUSIONS In this study we investigated, optimized, and characterized a novel class of HDP mimics, based on lipidated α-peptide/β-peptoid oligomers with an alternating cationic/hydrophobic design, with respect to their ability to modulate the pro-inflammatory response by human primary leukocytes upon exposure to bacterial membrane components. We found that the immunomodulatory capacity of these peptidomimetics is critically dependent on lipidation. The non-lipidated α-peptide/β-peptoid oligomers generally exhibited only low to moderate effect on cytokine secretion induced by LPS, while no significant blocking effect towards LTA-induced responses was seen at concentrations below 10 µM. However, Nterminal conjugation to fatty acids resulted in enhanced LPS blocking and broadened the antiinflammatory effect to include LTA-induced responses. Previously, lipidation has been shown to improve the anti-inflammatory properties of antimicrobial peptides, such as the neutrophilderived lactoferricin and the synthetic diastereomeric Lysine-Leucine (K,L) peptides,29,30 showing its importance as a structural feature in optimizing immunomodulatory activities of both HDPs and their mimics. High net positive charge is a characteristic of HDPs that contributes to their amphiphatic nature and is generally accepted to be of major importance for their antimicrobial as well as immunomodulatory activity.27, 31 Also, the nature of the cationic residues has previously been found to have a major impact on their activity profile as guanidinium functionalities (e.g. in arginines) confer increased antibacterial properties to peptidomimetics, but at the same time augment cytotoxicity towards mammalian cell lines.24 Surprisingly, in the present study, the degree of guanidinylation had only a minor effect on the LPS/LTAneutralizing potential of the peptidomimetics. On the other hand, we found that oligomers containing chiral β-peptoid residues (i.e. βNSpe; Figure 1) were significantly more potent


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blockers of LPS-induced responses than oligomers based on the achiral βNphe. Oligomers displaying βNSpe residues were earlier shown by CD spectroscopy to have a higher degree of secondary structure than the corresponding βNphe-containing compounds,32 suggesting that secondary structure might be important for the anti-inflammatory activity. However, another effect of incorporating several βNSpe residues rather than βNphe residues is a slightly increased overall hydrophobicity due to the accumulated contributions from the additional side-chain methyl groups. In fact this might well account for the increased activity since it has been shown that even small increments in hydrophobicity may optimize the LPS-neutralizing activity of the human cathelicidin LL-37.33 Although the immunomodulatory activity of the lipidated αpeptide/β-peptoid oligomers correlated positively with fatty acid length this appeared not to be a direct consequence of increased hydrophobicity, as the position of the lipid chain(s) was more important than the overall hydrophobicity in determining activity. This is in line with a previous report on synthetic lipidated diastereomeric Lysine-Leucine (K, L) peptides.29 Thus, Rosenfeld and coworkers found a direct correlation between hydrophobicity and antimicrobial activities of K, L-peptides, but no clear correlation of hydrophobicity to LPS neutralization. In addition to conferring significantly improved anti-inflammatory activity lipidation also gave rise to increased cytotoxicity of the peptidomimetics. However, the ratio between LC50 and IC50 values could be raised 3-fold by introducing the chiral and more hydrophobic β-peptoid residue βNSpe instead of βNphe (i.e. 3 → 4). This highlights the importance of assessing not only the primary pharmacological effect (LPS/LTA neutralization in the current context), but also the potential toxic effect in structural optimization of HDP mimics.34 Investigation of structure-activity relationships identified Pam-(Lys-βNSpe)6-NH2 (4) as having the most favorable pharmacological profile as estimated by the ratio between LC50 and



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IC50 values. The LPS-neutralizing activity (IC50 = 0.06 µM) of hybrid 4 was comparable to that of both polymyxin B (IC50 = 0.04 µM) and LL-37P (IC50 = 0.09 µM). As opposed to polymyxin B and LL-37P, compound 4 also blocked LTA-induced cytokine secretion (IC50 = 0.85 µM) within the tested concentration range. The lack of LTA-neutralizing activity of LL-37P is in contrast to the original report in which an IC50 value in the range of 0.5 µM was found.27 However, the extraction method, purity, and concentration of the LTA used differ between the two studies. The extraction method and purity of LTA has been shown to be of paramount importance for its ability to stimulate leukocytes via TLR2.35 Specifically, Morath et al. found that some commercial LTA preparations extracted by using phenol, including the one used by Nell et al.,27 are partly degraded and contaminated with endotoxin as well as by other nonendotoxin immunostimulatory entities.36 The LTA used in this study, on the other hand, was purified by butanol extraction, which has been shown to be the most effective method in avoiding degradation,35 and it is certified as Endofit™ corresponding to an endotoxin level below 0.001 EU/µg. Thus, the use of different LTA preparations might explain the diverging results regarding the LTA-neutralizing potential of LL37P. Compound 4 was further characterized for its ability to block cytokine secretion induced by whole UV-inactivated Gram-positive and Gram-negative bacteria, and it was found that the active concentration differed almost 100-fold in the neutralization of IL-6 secretion induced by E. coli as compared to that elicited by S. aureus. These findings are consistent with the higher potency of compound 4 for inhibition of LPS-induced cytokine secretion as compared to its inhibitory effect on the pro-inflammatory response to LTA. Besides suppressing the IL-6 induction, compound 4 inhibited release of the pro-inflammatory cytokine TNF-α as well as of the neutrophil chemoattractant IL-8 that has been associated with development of sepsis.11 TNF-


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α is a potent anaphylatoxin that upon intravenous injection in experimental animals causes widespread inflammatory alterations as well as tissue damage similar to the pathogenesis of sepsis.37,38 Furthermore, an increased plasma concentration of TNF-α is associated with higher mortality in septic patients.39 Elevation of the plasma IL-6 concentration has been firmly established as a clinical biomarker for sepsis, and it is often used for stratifying patients according to severity.40-42 The capability of the examined lipidated α-peptide/β-peptoid oligomers to inhibit release of several inflammatory mediators suggests that they may be potential leads for development of novel therapeutic agents to target sepsis provided their effect and low toxicity can be verified in vivo. However, proof of the beneficial effect of limiting release of IL-8, IL-6, and/or TNF-α in septic patients remains to be established. Also, other inflammatory mediators are obviously of importance for the development of sepsis. The question of how lipidated α-peptide/β-peptoid oligomers exert their anti-inflammatory effect was addressed by investigating whether a direct interaction with LPS in the LAL assay takes place as well as by applying flow cytometry to examine the interaction of human leukocytes with fluorescently labelled peptidomimetics. Compound 4, in contrast to polymyxin B, displayed only low activity in the LAL assay, although the two compounds exhibited similar ability to inhibit cytokine release in a cellular assay. This suggests that the anti-inflammatory activity of compound 4 is not mediated by a direct interaction with LPS. At higher molar ratios of compound 4 to LPS, moderate LPS-neutralizing activity was found in the LAL assay, indicating that direct LPS binding most likely occurs only at substantially higher concentrations of compound 4, and thus is secondary to its primary effect observed at low concentrations. The flow cytometry data showed selective interaction of a carboxyfluorescein (CF)-labelled lipidated α-peptide/β-peptoid oligomer with monocytes and granulocytes indicating the presence of



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cellular targets on these leukocyte subsets. However, the specific cellular target(s) was not identified in the present study, and given the N-terminal lipidation as well as the amphiphatic nature of the compounds they may also interact directly with lipid membranes. However, several studies have shown specific interactions of amphiphatic HDPs with receptors highly expressed in monocytes and granulocytes. Examples include the formyl-peptide receptors (FPRs) which have been found to interact with HDPs of the innate defense regulator (IDR) family, and with the human cathelicidin LL-37.43-46 Furthermore, LL-37 has been proposed to interact with CD14,47 CXCR2,48 P2X7,46 and the intracellular protein GAPDH.49 These and other receptors (e.g. TLRs) are candidates for being specific receptors for peptidomimetic 4 and related oligomers. In conclusion, we have identified and characterized a novel class of host defense peptide mimics with potent anti-inflammatory activity. We suggest that the mechanism of action may be dual involving direct interaction with leukocytes belonging to the monocytic and granulocytic subsets at low concentrations, while direct LPS binding appear to occur at higher concentrations. Due to their anti-inflammatory activity and low cytotoxicity, lead compounds belonging to this subclass of peptidomimetics may have potential for development of drugs for treatment of infection-induced inflammation as well as for management of sepsis.


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EXPERIMENTAL SECTION Starting materials and solvents were purchased from commercial suppliers (Iris Biotech, Sigma-Aldrich, Bachem, VWR, AppliChem, LabScan) and used without further purification. Water used during analytical and preparative HPLC was filtered through a 0.22 µm membrane filter. Analytical HPLC (Schimadzu system) was used to determine purity and was performed on a Phenomenex Luna C18(2) (3 µm) column (150 × 4.6 mm) using binary mixtures of eluent A (water–MeCN–TFA 95:5:0.1) and eluent B (water–MeCN–TFA 5:95:0.1) for elution with a flow rate of 0.8 mL/min. For peptidomimetics linear gradients of 10-60% B during 30 min, 20-80% B during 30 min, 30-80% B during 30 min or 20-100% B during 30 min were used; UV detection at λ = 220 nm. All compounds had a purity of at least 95% as determined by analytical HPLC. Elution gradients and retention times (tR) are given for each compound. Preparative HPLC was carried out on a Phenomenex Luna C18 (2) (5 µm) column (250 mm × 21.2 mm) using an Agilent 1100 LC system with a multiple-wavelength UV detector. Elution was performed with linear gradients of 10-40% B during 20 min (non-lipidated analogs) or 20-80% B during 20 min (lipidated analogs) at a flow rate of 20 mL/min. LC-HRMS was performed with a Phenomenex Luna C18(2) (3 µm) column (150 mm × 4.6 mm) using binary mixtures of eluent C (water– MeCN–formic acid 95:5:0.1) and eluent D (water–MeCN–formic acid 5:95:0.1); elution was performed with a linear gradient of 10-60% D or 20-100% D during 3 min with a flow rate of 0.5 mL/min. HRMS spectra were obtained by using a Bruker MicroTOF-Q II MS detector. The analyses were performed as ESI-MS (m/z): [M + nH]n+ for all peptidomimetics. General

Procedure

for

Synthesis

of

Peptidomimetics.

The

α-peptide/β-peptoid

peptidomimetics 1-16 were synthesized on a Rink amide resin (loading, 0.5-0.7 mmol/g; 0.050.1 mmol scale) in Teflon reactors (10 mL) by standard Fmoc solid-phase synthesis using the



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appropriate dimeric building blocks.50 Fmoc deprotection was performed with 20% piperidine in DMF (2 × 10 min, each time with 5 mL; shaking at room temperature). After Fmoc deprotection and coupling the resin was washed with DMF, MeOH, and DCM (each 3 × 3 min with 5 mL). Coupling of building blocks was performed with PyBOP:DIPEA 1:2 in DMF (2-3 mL DMF; PyBOP: 2.0 equiv for loading, 2.5 equiv for the first two elongations, and 3.0 equiv for subsequent elongations; >2 h under shaking at room temperature). Capping was applied after coupling no. 4 with Ac2O–DIPEA–NMP 1:2:3 (5 mL, 10 min at room temperature).50 After the final Fmoc deprotection the N-terminus was acetylated (conditions as for capping) or acylated with fatty acids (or 2-Aoc) via coupling of the corresponding acid (5 equiv, 16 h; octanoic acid or commercial Fmoc-Lys(Pam)-OH) using PyBOP (5 equiv) as coupling reagent, or via coupling with the N-hydroxysuccinimidyl esters (5 equiv, 16 h; Lau-OSu and Pam-OSu; 5 equiv DIPEA). The fluorophore in compounds 15 and 16 was introduced as previously described.51 Following cleavage from the resin with TFA–water 95:5 (5 mL, 1 h at room temperature) all peptidomimetics were purified to homogeneity by preparative HPLC. The identity of the compounds was verified by HRMS (∆M < 10 ppm), and the purity was determined by analytical HPLC (> 95% at 220 nm). After lyophilization target compounds were stored at −20 °C until use. Peptidomimetic 1. Analytical HPLC (10% → 60% B during 30 min): tR = min. HRMS: calcd for [M + 4H]4+ 449.7865, found 449.7886 ; ∆M = 6.1 ppm. Peptidomimetic 2.24 Analytical HPLC (10% → 60% B during 30 min): tR = 19.35 min. Peptidomimetic 3. Analytical HPLC (20% → 80% B during 30): tR = 22.79 min. HRMS: calcd for [M + 4H]4+ 498.8413, found 498.8446; ∆M = 6.6 ppm.


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Peptidomimetic 4. Analytical HPLC (20% → 80% B during 30 min): tR = 24.05 min. HRMS: calcd for [M + 4H]4+ 519.8647, found 519.8681; ∆M = 6.5 ppm. Peptidomimetic 5. Analytical HPLC (10% → 60% B during 30 min then rising to 100% B during 10 min): tR = 32.06 min. HRMS: calcd for [M + 5H]5+ 466.5195, found 466.5197 ; ∆M = 0.4 ppm. Peptidomimetic 6. Analytical HPLC (20% → 80% B during 30 min): tR = 20.34 min. HRMS: calcd for [M + 4H]4+ 505.8491, found 505.8520; ∆M = 5.7 ppm. Peptidomimetic 7. Analytical HPLC (20% → 80% B during 30 min): tR = 16.98 min. HRMS: calcd for [M + 4H]4+ 491.8334, found 491.8367; ∆M = 6.7 ppm. Peptidomimetic 8. Analytical HPLC (20% → 80% B during 30 min): tR = 23.55 min. HRMS: calcd for [M + 4H]4+ 569.3990, found 569.3982 ; ∆M = 1.4 ppm. Peptidomimetic 9. Analytical HPLC (20% → 100% B during 30 min): tR = 24.38 min. HRMS: calcd for [M + 2H]2+ 452.8415, found 452.8443; ∆M = 6.1 ppm. Peptidomimetic 10. Analytical HPLC (10% → 60% B during 30 min then rising to 100% B during 10 min): tR = 33.43 min. HRMS: calcd for [M + 4H]4+ 389.0275, found 389.0293; ∆M = 4.6 ppm. Peptidomimetic 11. Analytical HPLC (20% → 100% B during 30 min): tR = 20.18 min. HRMS: calcd for [M + 5H]5+ 441.3064, found 441.3053; ∆M = 2.4 ppm. Peptidomimetic 12. Analytical HPLC (20% → 100% B during 30 min): tR = 19.96 min. HRMS: calcd for [M + 6H]6+ 475.9919, found 475.9901; ∆M = 3.7 ppm. Peptidomimetic 13. Analytical HPLC (10% → 60% B during 30 min): tR = 23.20 min. HRMS: calcd for [M + 5H]5+ 405.0726, found 405.0721; ∆M = 1.2 ppm.



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Peptidomimetic 14. Analytical HPLC (10% → 60% B during 30 min then rising to 100% B during 10 min): tR = 31.63 min. HRMS: calcd for [M + 5H]5+ 433.1039, found 433.1030; ∆M = 2.0 ppm. Peptidomimetic 15. Analytical HPLC (10% → 60% B during 30 min): tR = 21.98 min. HRMS: calcd for [M + 5H]5+ 513.3219, found 513.3253; ∆M = 6.6 ppm. Peptidomimetic 16.52 Analytical HPLC (20% → 80% B during 30 min): tR = 24.27/24.79 min (separation into 5- and 6-isomers of CF). Peptide LL-37P. HRMS: calcd for [M + 5H]5+ 591.9576, found 591.9565; ∆M = 1.8 ppm. Cells and bacteria. To remove endotoxin, all solutions and media used for cell culturing were passed through a 20 kDa cut-off filter (Ultrasart D 20, Sartorius, Germany) immediately after preparation. HepG2 cells were cultured in Eagle’s minimal essential medium (EMEM) supplemented with 10% low endotoxin heat-inactivated fetal bovine serum (FBS) (Biological industries, Israel), 1% glutamax (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA), nonessential amino acids (Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (SigmaAldrich, USA). Anonymized buffy coats from healthy blood donors were obtained from the blood bank at Copenhagen University Hospital (Rigshospitalet), in agreement with Danish legislation. Human primary leukocytes were isolated from buffy coats by lysis of erythrocytes. Briefly, buffy coat was mixed with four times the volume of red blood cell lysis buffer (NH4Cl 8.26 mg/mL, KHCO3 1 mg/mL, EDTA 37 µg/mL), gently rotated for 5 min or until lysis was visible, and gently centrifuged (100×g for 7 min). The lysis procedure was repeated, and the leukocytes were washed once in endotoxin-free Hanks’ balanced salt solution (HBSS) (Gibco, USA), before suspension in complete culture medium i.e. low endotoxin RPMI-1640 (Biological Industries, Israel) supplemented with 5% heat-inactivated FBS, 1% glutamax, 100 U/mL


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penicillin, and 100 µg/mL streptomycin. The leukocytes were maintained in complete culture medium in a humidified atmosphere (5% CO2, 95% air) at 37 °C. Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 29213) were obtained from the American Type Culture Collection (ATCC). To standardize suspensions of bacteria for inactivation a single colony from a fresh overnight TGY (0.5% trypton, 0.3% yeast extract, 0.1% glucose, 0.5% NaCl) agar plate was suspended in 10 mL endotoxin-free RPMI, and incubated overnight at 37 oC. The cells were centrifuged (1000×g for 5 min), washed, and resuspended to an optical density (OD) of 1 at 450 nm in endotoxin-free RPMI. The cell density in the suspensions was determined by total vital counts on TGY-plates. Inactivation was performed by exposure of bacterial suspensions to UV irradiation for 5 min. The inactivation was confirmed after overnight incubation on TGY-plates at 37 oC. To avoid clusters of bacteria in the suspension the UV-inactivated S. aureus was subjected to cycles of 15 sec sonication followed by 15 sec pause of treatment over a period of 5 min. An ultrasonicator with a microtip (Wibra Cell 400 W, Sonics & Materials Inc., CT, USA) with an effect of 130 W was used. Stimulation of Leukocytes with Toll-like Receptor Ligands and UV-Irradiated Bacteria. E. coli O55:B5 LPS was purchased from Lonza whereas purified S. aureus LTA, recombinant flagellin from Salmonella typhimurium, PGN from Bacillus subtilis, Pam2CSK4, and Pam3CSK4 were all purchased from Invivogen, USA. Polymyxin B sulfate was obtained from SigmaAldrich. Freshly isolated leukocytes were seeded in 24-well culture plates to a cell density of 1×106/mL in complete endotoxin-free culture medium. Immediately after seeding, the cells were exposed to Toll-like receptor (TLR) agonists in concentrations inducing significant, but submaximal, IL-6 responses: E. coli LPS (1 EU/mL), S. aureus LTA (1×106 pg/mL), Bacillus subtilis PGN (1×108 pg/mL), flagellin from Salmonella typhimurium (200 ng/mL), Pam2CSK4 (5



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ng/mL), Pam3CSK4 (10 ng/mL), or with UV-irradiated bacteria E. coli (100 CFU/mL), or S. aureus (1×104 CFU/mL) followed by addition of the peptidomimetics to be tested. After 10 h (TNF-α measurement) or 20-24 h (IL-6, and IL-8 measurement) cell-free supernatants were harvested by centrifugation (2000×g for 3 min). Cytokine Measurement. IL-6 and IL-8 in cell-free supernatants, obtained from human primary leukocytes after 20-24 h culture in the presence of agonist/peptidomimetics, were measured by time-resolved dissociation-enhanced lanthanide fluoro-immunoassay (DELFIA; a non-competitive sandwich immunoassay), as previously described.53 Antibodies and standards: anti-human IL-6 capture monoclonal antibody (mAb) (MAB206), biotinylated polyclonal antihuman IL-6 detection antibody (BAF206), recombinant human IL-6 standard (206-IL-050), antihuman IL-8 capture mAb (MAB208), biotinylated polyclonal anti-human IL-8 detection antibody (BAF208) and recombinant human IL-8 standard (208-IL-050) were all obtained from RnD systems. To avoid interference of fluorescent peptidomimetics in the EN detection of cytokines, 3,3',5,5'-tetramethylbenzidine (TMB) Plus (obtained from Kem-En-Tec) was used as substrate for the horseradish peroxidase-conjugated streptavidin (RnD Systems) for detection in experiments in which fluorescently labelled peptidomimetics was used. The reaction was stopped by addition of sulfuric acid, and then the response was detected at 450 nm. All other reagents and settings were identical to the DELFIA procedure. TNF-α in supernatants obtained after 10 h leukocyte culture, was measured by DIAplex multiplex for flow cytometry (Diaclone) according to the manufacturer´s instructions. The bead recording was performed on a Beckman Coulter Gallios flow cytometer. All samples were analyzed in triplicate. Viability Assays. For assessment of cell viability the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used as a measure of mitochondrial activity. In this


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assay the mitochondrial activity is determined as the capacity to convert MTT into formazan crystals which can be solubilized for homogeneous OD measurement at 570 nm. Freshly prepared human leukocytes, suspended in complete culture medium, were seeded in a flatbottomed 96-well plate (1×105 cells/well) before addition of α-peptide/β-peptoid hybrid suspended in complete culture medium and a final concentration of 0.1 µg/mL MTT (SigmaAldrich) in a final volume of 200 µL. The cells were incubated for 24 h in a humidified atmosphere (5% CO2, 95% air) at 37 °C before lysis to release formazan crystals by addition of lysis buffer (100 µL; 10% sodium dodecyl sulfate, 50% N,N-dimethyl formamide adjusted to pH 4.5). Samples were incubated for 2 h at 37 °C before measurement of OD570. Adherent layers of HepG2 cells at approximately 50% confluence were treated with peptidomimetics in fresh culture medium for 20 h before addition of a final concentration of 0.1 µg/mL MTT in a final volume of 200 µL. The cells were incubated for 4 h in a humidified atmosphere (5% CO2, 95% air) at 37 °C before lysis to release formazan crystals by addition of lysis buffer. All samples were analyzed in triplicate. MIC determination. MIC was determined by using the microdilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI). The lipidated peptidomimetics were diluted in 1:2 serial dilutions in sterile water from a 256 µM stock solution to give a final range of 0.5–128 µM in the wells. A Polymyxin B 2-fold dilution series was prepared from a 1024 µM stock solution in sterile water to give a final range of 0.0625-256 µM in the wells. Ciprofloxacin 1:2 serial dilutions in sterile water were prepared from a 2048 µM stock solution to give a final range of 0.0625-256 µM in the wells. Bacteria from frozen stocks were suspended in cation-adjusted Mueller Hinton II broth (MHB) to give a turbidity of 0.13 at OD546 (approx. 1 × 108 CFU/ml), and then diluted in MHB pH 7.4 to a final concentration of 5 ×



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105 CFU/ml in each well. Polypropylene plates (Nunc 442587) were used to minimize peptide binding, and the incubation time was 20-24 h at 37 °C. All samples were analyzed in triplicate, and MIC was determined in two biological replicates as the lowest concentration of the peptide analog at which no visible growth was observed. Limulus Amebocyte Lysate Assay. For the Limulus amebocyte lysate (LAL) assay the Pyrotell® T kit (Associates of Cape Codd, inc., USA) was used, and the procedure was performed according to the manufacturer´s instructions. Briefly, endotoxin standards and samples of polymyxin B and of compound 4 were prepared in endotoxin-free water and dispensed (total volume of 100 µL) into a sterile microtiter plate (Nunc, Roskilde, Denmark) before preheating for 10 min at 37 °C. Then LAL reagent (100 µL) was added, and the kinetic (1 measurement/min for 1 h) turbidimetric (340 nm) measurement was started immediately by using a VersaMax spectrophotometer. An endotoxin standard curve ranging from 0.05 EU/mL to 5 EU/mL was used to evaluate the samples. Flow cytometry. Freshly prepared human leukocytes were suspended in PBS supplemented with 1% human serum isolated from buffy coats by centrifugation (3000×g for10 min), and then incubated in the dark with fluorescently labelled α-peptide/β-peptoid hybrids, FITC-labeled mouse anti-human CD14 (clone TÜK4, Miltenyi Biotech), or an isotype control (FITC labeled mouse IgG2a, clone S43.10, Miltenyi Biotech) for 30 min on ice. After the incubation, cells were washed twice with PBS containing 1% human serum, before analysis by flow cytometry using a Beckman coulter Gallios flow cytometer. Gating of leukocyte subsets was performed in a forward-side scatter plot, and confirmed by staining of separate cell samples with anti-CD14. Granulocytes were CD14low, monocytes CD14high, and lymphocytes CD14-.


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Statistical Analysis. All statistical analyses were performed with GraphPad Prism 6. Data are represented as mean + 95% confidence intervals (CI) unless stated otherwise. Statistical comparison of treatments was performed by one- or two-way analysis of variance (ANOVA) followed by the recommended adjustment for multiple comparisons; p< 0.05 was considered statistically significant. The ANOVA test assumes normal distribution of dependent variables within groups and that the variance in the different groups is identical.



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TABLES Table 1: Dose-response data (in µM) and therapeutic indices (TIs) for selected compounds. Cmp.a

IC50 b

IC50 b

LC50 b

LPS

LTA

Leukocytes

2

0.43 (0.28-0.66)

-

3

0.17 (0.11-0.26)

4

LC50 b

TI (LPS)c

TI (LTA)d

> 100

>230

-

> 100

1.28 (0.76-2.17)

23 (16-32)

135

18

25 (10-60)

0.06 (0.04-0.08)

0.85 (0.50-1.43)

24 (19-30)

400

28

28 (23-37)

5

0.06 (0.04-0.08)

1.88 (1.08-3.31)

21 (16-29)

350

11

22 (16-29)

6

0.13 (0.08-0.21)

1.84 (1.20-2.82)

27 (18-40)

207

15

24 (14-42)

7

0.30 (0.22-0.42)

5.23 (2.95-9.29)

65 (42-100)

216

12

21 (16-28)

9

0.14 (0.07-0.28)

-

48 (38-61)

343

-

61 (47-80)

11

0.07 (0.05-0.10)

1.35 (0.68-2.66)

18 (16-21)

257

13

24 (19-30)

LL37P

0.09 (0.05-0.16)

-

65 (51-84)

722

-

70 (61-80)

PmB

0.04 (0.03-0.06)

-

> 100

>2500

-

> 100

HepG2

a

Raw data dose-response curves are found in Supporting information ( pages S14-15)

b

Brackets indicate the 95% confidence interval

c

Based on LPS-neutralizing activity and cytotoxicity to human leukocytes

d

Based on LTA-neutralizing activity and cytotoxicity to human leukocytes


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Table 2: Antimicrobial activity (MIC in µM) for selected compounds. Compound

E. coli

S. aureus

4

8

8

5

16

16

11

8

8

Polymyxin B

0.5

20

Ciprofloxacin a

0.125

0.125

a

Results are given in µg/mL.



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ASSOCIATED CONTENT Supporting Information. Analytical HPLC chromatograms of target compounds 1-16, as well as other supporting data regarding the biological effects of peptidomimetics. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +45 3533 6255. Present Addresses §

Lise Moesby, Quality control manager for Biomaterials, Ferrosan medical devices A/S,

Sydmarken 5, 2860 Søborg, Denmark. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. H.F., P.M.H.H., and S.L.S. designed and wrote the manuscript together. H.F., L.M., and S.L.S. designed the experiments. S.L.S and C.J.L. performed the experiments, and analyzed the data together with H.F. and L.M. Funding Sources Sarah Line Skovbakke is supported by generous research grants from Department of Drug Design and Pharmacology (Faculty of Health and Medical Sciences, University of Copenhagen), Aase and Ejnar Danielsens foundation, and King Christian 10th foundation. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT We would like to thank Janne Møgelhold Colding, Betina Vidtfeld Schøler, Katrine Juhl Krydsfeldt, and Birgitte Simonsen for their outstanding technical assistance, Kenneth T. Kongstad and Nils Nyberg for recording the HRMS spectra, and Hanne Mørck Nielsen for kindly providing the HepG2 cell line.

ABBREVIATIONS ATCC, American Type Culture Collection; 2-Aoc, 2-aminooctanoic acid; βNphe, N-phenyl-βalanine; βNSpe, N-(S)-1-phenylethyl-β-alanine; CF, 5(6)-carboxyfluorescein; CFU; colonyforming units; CI, confidence interval; CLSI, Clinical and Laboratory Standards Institute; Cmp., compound;

DELFIA,

dissociation-enhanced

lanthanide

fluoro-immunoassay;

DIPEA,

diisopropylethylamine; EMEM, Eagle’s minimal essential medium; EU, endotoxin units; FBS, fetal bovine serum; HDP, host defense peptide; hArg, homoarginine; IL, interleukin; kDa, kilo Dalton; LAL, Limulus amebocyte lysate; Lau, lauryl; LC50, the concentration at which 50% of the cells die; LL37-P, LL37-peptide; LTA, lipoteichoic acid; Lys(Pam); N-ε-palmitoyl-lysine; mAb, monoclonal antibody; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; NLys, α-peptoid lysine; Oct, octanoyl; OSu, N-hydroxysuccinimidyl esters; Pam, palmitoyl; PGN, peptidoglycan; PmB, Polymyxin B; PRR, pattern recognition receptor; PyBOP, (benzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate TGY, trypton, yeast, glucose medium; TI, therapeutic index; TMB, 3,3′,5,5′-tetramethylbenzidine; tR, retention time in analytical HPLC; U, units.



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TABLE OF CONTENT GRAPHICS



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Page 47Journal of 53 of Medicinal Chemistry

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115x55mm (300 x 300 DPI)


Î²-Peptoid Hybrids with Potent Anti-inflammatory

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