Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with

Article pubs.acs.org/jmc

Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection Emilie Teyssières,† Jean-Philippe Corre,† Stephanie Antunes,‡,§ Catherine Rougeot,∥ Christophe Dugave,⊥ Grégory Jouvion,#,∇ Paul Claudon,‡,§ Guillain Mikaty,† Céline Douat,‡,§ Pierre L. Goossens,*,†,# and Gilles Guichard*,‡,§ †

Pathogénie des Toxi-Infections Bactériennes, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France Univ. Bordeaux, CBMN, UMR 5248, Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac, France § CNRS, CBMN, UMR 5248, F-33600 Pessac, France ∥ Laboratoire de Pharmacologie de la Douleur, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France ⊥ Institut de Biologie et de Technologie de Saclay (iBiTec-S), Service de Chimie Bio-organique et de Marquage, CEA-Saclay, bâtiment 547, 91191 Gif-sur-Yvette, France # Institut Pasteur, Histopathologie Humaine et Modèles Animaux, 75724 Paris, France ∇ Paris Descartes Université, PRES Sorbonne-Paris-Cité, 75006 Paris, France ‡

S Supporting Information *

ABSTRACT: The synthesis of bioinspired unnatural backbones leading to foldamers can provide effective peptide mimics with improved properties in a physiological environment. This approach has been applied to the design of structural mimics of membrane active antimicrobial peptides (AMPs) for which activities in vitro have been reported. Yet activities and pharmacokinetic properties in vivo in animal models have remained largely unexplored. Here, we report helical oligourea AMP mimics that are active in vitro against bacterial forms of Bacillus anthracis encountered in vivo, as well as in vivo in inhalational and cutaneous mouse models of B. anthracis infection. The pharmacokinetic profile and the tissue distribution were investigated by βradio imager whole-body mapping in mice. Low excretion and recovery of the native oligourea in the kidney following intravenous injection is consistent with high stability in vivo. Overall these results provide useful information that support future biomedical development of urea-based foldamer peptide mimics.

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INTRODUCTION

defensins or cathelicidins provides protection in murine models of infection.6,7 The protective effects were also mediated through modulation of the innate immune response.7−9 There is a growing interest in the development of cationic membrane-active AMPs as alternative to conventional antibiotics for the treatment of bacterial infections, and several cationic AMPs are currently in clinical trials.10 However, a number of issues such as susceptibility to proteolytic degradation and toxicity following systemic application need to be addressed for AMPs to meet their full potential in the clinic. Today, there is also increasing evidence that pathogenic bacteria can develop resistance to endogenous AMPs through a variety of mechanisms often involving biophysical and

Endogenous AMPs are essential components of the innate immune system of eukaryotic organisms, from plants to mammals, serving as a first line of defense against a broad range of micro-organisms, including Gram-negative and Grampositive bacteria.1−3 AMPs form a large and highly diverse family of molecules ranging in size from 15 to >50 amino acid residues; they do share common physicochemical features though. AMPs are largely cationic and display an amphiphilic character, thus allowing them to interact preferentially with negatively charged bacterial membranes. Although a large fraction of AMPs have been described to exert their bactericidal effect by direct perturbation of the bacterial membrane (permeabilization and lysis), some have been shown to enter the bacteria and to specifically block intracellular processes (e.g., certain proline-rich AMPs4,5). In vivo administration of © 2016 American Chemical Society

Received: February 1, 2016 Published: August 16, 2016 8221

DOI: 10.1021/acs.jmedchem.6b00144 J. Med. Chem. 2016, 59, 8221−8232

Journal of Medicinal Chemistry

Article

Figure 1. Design of amphiphilic cationic oligoureas assuming idealized helical structures and formulas of oligomers 1−7 investigated in this work.

biochemical changes to the bacterial cell wall.11,12 Considerable efforts have been made to circumvent some of these drawbacks by generating and optimizing synthetic analogues of AMPs. 13−19 Among these, membrane active synthetic oligomers made of artificial monomeric units but recapitulating essential features of amphiphilic AMPs are of increasing importance. The emergence of multiple families of AMP mimics with artificial backbones coincides with recent advances in foldamer chemistry and the ability to design synthetic oligomers with well-defined and predictable folded structures.20−22 Linking structure with function in these systems is greatly facilitated by the exquisite control over the monomer sequence and secondary structure as well as by the diversity of the building block repertoire. The improved resistance to proteolysis demonstrated for several foldamer backbones is another significant advantage.23−26 Foldamer AMP mimics with various degrees of similarity to natural peptides (e.g., helical βpeptides,27−30 peptoids,31−33 and related α-peptide hybrids,24,34,35 aliphatic oligoureas36,37 and sulfono-γ-AApeptides38) and even dramatically different structures (e.g., arylamide foldamers39−42) have been described. They usually demonstrate broad-spectrum activity against bacteria in vitro including clinically relevant strains, yet few reports have described activities in animal models, such examples being restricted to arylamide foldamers active in a Staphylococcus aureus thigh infection model.40 Animal models of bacterial infection are important to assay the potential of AMP mimics under physiological conditions, with the mouse widely used as the model of choice.43 However,

many infections cannot be easily reproduced in this animal model due to species restrictions for various key steps of the infectious process. To overcome this limitation, unnatural routes of infection, alterations of the immunological status, or genetically modified mice have been used,44−46 but the relevance of these models to infection is questionable. An infection model using Bacillus anthracis is highly informative to explore the crosstalk between host innate defenses, subversion mechanisms, and potential therapeutics. This Gram-positive sporulating bacillus is at the origin of recurrent outbreaks of anthrax worldwide with severe economic and health consequences. It is also a potential bioterrorism threat, classified as a chemical, biological, radiological, and nuclear (CBRN) threat.47−49 B. anthracis exists under two differentiation forms, the spore (the dormant infecting form surviving for extended periods outside its host) and the encapsulated bacillus (that emerges from the spore during germination after entry into its host).47,49 It provokes a toxiinfection, combining an infection (essentially due the presence of the capsule) and a toxemia (due to the production of two toxins); both virulence factors are induced in vivo.49 B. anthracis is able to naturally infect immuno-competent animals (mouse, rat, guinea pig, rabbit, non-human primates) by natural routes (cutaneous, inhalational, and digestive) without any immunological manipulations.47 It is therefore possible to explore cutaneous and inhalational infections, two main types of infection processes that need to be addressed in human therapeutics. 8222



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Scheme 1. Dimerization of 5 into 6 or 7

Figure 2. Bactericidal efficiency of oligourea foldamers against the various differentiation forms of B. anthracis. Increasing concentrations of 1 (A, left), 2 (B, left), 6 (C), and γ-peptide 3 (D) were incubated for 30 min with germinated spores, encapsulated bacilli of the 9602P strain, or nonencapsulated bacilli of the capsule-deficient 9602PR strain. Bactericidal activity for increasing incubation times with a saturating concentration of 1 (10 μg/mL, A, right) or 2 (20 μg/mL, B, right). Dotted line represents 50% bacterial killing. Data are expressed as mean ± SEM, n ≥ 9.

S. aureus and methicillin-resistant S. aureus (MRSA)) with selectivity for prokaryotic versus mammalian red blood cell membranes.36,37 The sequence of 1 was designed assuming idealized helical structures, by sequestration of cationic residues on one face of the helix (Figure 1).36 Previous findings suggest that oligoureas are more resistant to protease degradation than

Here, this animal model of B. anthracis bacterial infection was applied to evaluate the activity of cationic amphipathic N,N′linked oligoureas, a class of nonpeptide helical foldamers50 mimicking AMPs. Oligourea 8-mer 1 with protein-like side chains was previously found to display in vitro broad antibacterial activity (Escherichia coli, Pseudomonas aeruginosa, 8223



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cognate α-peptides, which bodes well for in vivo applications. To note, available data on the in vivo biodistribution properties of synthetic foldamers remain extremely limited thus far51,52 and documenting their pharmacokinetic (PK) properties is certainly of importance for possible future development of these new classes of medium size molecules. In this study, the bactericidal efficiency of 1 on the various differentiation forms of B. anthracis was evaluated in vitro, and further tested for protective activity in vivo in cutaneous and inhalational models of infection. Compound 1 was compared with various analogues containing isosteric backbone mutations (2 and 3) and 6 and 7, two covalent dimers designed to amplify bactericidal effects. To gain additional insight into the in vivo behavior of cationic oligoureas, which may differ from that of natural AMPs, we have characterized the PK properties of 3Hradiolabeled 1 and its tissue distribution in our animal model.

poly(γ-D-glutamic acid) (PDGA) capsule surrounding B. anthracis does not hinder the bactericidal activity of 1. Nonencapsulated bacilli were also susceptible to 1, albeit with a slight reduction in efficiency (EC50 at 6 μg/mL). Anthracidal activity was extremely rapid, as the incubation time needed to reach 50% killing (t1/2) was estimated at 60 s for germinating spores and 30−35 s for bacilli (Figure 2A, right; Table 1). The encapsulated form of B. anthracis was thus highly susceptible to 1, as more than 95% of the bacterial population was killed in less than 2 min (Figure 2A, right). As an initial measure of selectivity for bacteria over mammalian cells, we previously determined the lytic activity of 1 in an erythrocyte lysis assay and found a selectivity ratio of 8.0 for the tested bacteria (S. aureus ATCC 25923, E. coli ATCC 25922).37 Here, we confirm this selectivity on nucleated cells, that is, the macrophage RAW 264.7 cell line. The toxic dose inducing 50% cell lethality was 31.1 ± 0.5 μg/mL (mean ± SEM, n = 3), similar to defensins,56 which gives a selectivity ratio of 9.5 for B. anthracis germinated spores. Changes in the Helical Scaffold Alters Bactericidal Activity. To evaluate the importance of the oligourea helical backbone on the anthracidal activity of 1, we investigated the activities of related hybrid oligomer 2 and cognate γ-peptide 3 (Figure 1). Hybrid 2 contains a single isosteric γ-amino-acid substitution in the middle of its sequence (Valu4 → γ-Val4), a substitution that was shown previously to be well tolerated in terms of both secondary structure and antibacterial activity (comparable MBC against S. aureus).37 Here, we show that this single modification slightly altered the bactericidal activity (Figure 2B). Though the three bacterial B. anthracis differentiation forms were efficiently killed by 2, the EC50 (7−10 μg/ mL, Figure 2B) and the t1/2 (1−2 min, Figure 2B, right) were higher for germinating spores and encapsulated bacilli. Interestingly, 3, the isosteric γ-peptide analogue of 1 bearing the same side chain distribution but an oligoamide backbone, did not exhibit efficient bactericidal activity (Figure 2D). The two backbones are largely isostructural, yet this result is consistent with previous observations showing weaker membrane-disruption properties of 3 compared with 1.37 Resistance of Oligourea 1 to Enzymatic Degradation in Vitro. Prior to in vivo studies, we have evaluated the resistance to proteolysis of urea oligomer 1 using different enzymes, namely, trypsin, chymotrypsin, and pronase E.25 Trypsin and chymotrypsin cleave the peptide bonds adjacent to positively charged (Lys or Arg) and hydrophobic (Phe, Tyr, or Trp) residues, respectively, whereas pronase E is a mixture of enzymes from Streptomyces griseus that generally leads to extensive protein degradation. An α-peptide containing Lys, Trp, and Val residues arranged in the same order as in 1 (AcVal-Lys-Trp-Val-Lys-Trp-Lys-Val-NH2, 8) was used for direct comparison and to set up the assay. Figure 3 shows that peptide 8 was completely cleaved by all three enzymes in less than 3 min. Under the same conditions, oligourea 1 was found to be fully resistant to all three enzymes with no detectable degradation after 24 h. These experiments demonstrate the very high stability of the oligourea backbone to enzyme digestion. Time Course of Radiolabeled 1 Blood Distribution in the Mouse. To date N,N′-linked aliphatic oligoureas have not been characterized for their pharmacodynamic and tissular distribution properties. Although oligoureas adopt a folded helical structure that has overall similarity with the α-helix, the peptide and oligourea backbones physicochemically differ to a

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RESULTS Oligourea Synthesis. Oligoureas 1, 2, 4, and 5 (Figure 1) were assembled stepwise by microwave assisted solid-phase synthesis on a 4-methylbenzhydrylamine resin using Osuccinimidyl-(tert-butyloxycarbonylamino)-ethyl carbamate derivatives as building blocks.37,53,54 The cognate γ-peptide 3 was prepared as previously described using standard Fmoc chemistry.37 Covalent dimers 6 and 7 were prepared as shown in Scheme 1 by reaction of 5, an analogue of 1 bearing a terminal cysteamine moiety with N,N′-(ethane-1,2-diyl)bis(2bromoacetamide)55 or by dithiol condensation of 5, respectively. All oligomers were purified by reversed-phase HPLC (C18) to a final purity of >95% and lyophilized. Efficient Bactericidal Activity of 1 on B. anthracis. The bactericidal activity of 1 was assayed on the different bacterial forms of B. anthracis that exist in vivo, that is, the spore, the germinating spore, and the encapsulated bacillus (9602P strain).47,48 The nonencapsulated form of B. anthracis (9602PR strain) was also tested as a surrogate for unencapsulated Gram-positive bacteria. No activity was detected on the nongerminated spore (5.26 ± 0.04 colony forming units (CFU) versus 5.28 ± 0.02 CFU for the 1-treated and control, respectively, mean CFU ± standard error of the mean (SEM), n = 12). Efficient bactericidal activity was observed on spores as soon as germination was triggered and on encapsulated bacilli (Figure 2A). The estimated EC50 was similar for both bacterial forms, at 3.3 μg/mL (Table 1). This shows that 1 was able to gain access to its bacterial targets in the germinating spore, as soon as the external protective structures (exosporium, coat) become permeable, and that the Table 1. Effector Concentrations (EC50) and Incubation Times (t1/2) Giving 50% Bacterial Killing for 1, 2 and 6 on the Different Bacterial Forms of B. anthracis 1 bacterial form (strain) germinated spores (9602P) encapsulated bacilli (9602P) nonencapsulated bacilli (9602PR) a

2

3

6

EC50 (μg/ mL)

t1/2 (s)

EC50 (μg/ mL)

t1/2 (s)

EC50 (μg/ mL)

EC50 (μg/ mL)

3.3

60

10

120

>20

0.2

3.25

30

7

75

a

0.5

6

35

8.5

45

a

a

Not determined. 8224



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Figure 3. Resistance to proteolysis of oligourea 1 (●) and control peptide 8 (◇) upon treatment with trypsin (A), α-chymotrypsin (B), and Pronase E (C).

Figure 4. Time course of [3H]1 blood concentrations after in vivo injection. After intravenous bolus administration of 5 μCi/100 μg 1 in conscious mouse, the total [3H]1 molecular populations circulating in the bloodstream of adult mice was quantified over a 240 min time period by direct measure of blood radioactivity. Results are expressed as mean ± SD of cpm/mL blood for 2 mice and are represented in log scale.

significant extent, thus making extrapolation of the in vivo behavior of cationic antimicrobial oligoureas difficult. To address this issue, a radioactive 1 derivative was prepared by tritiation on the Trpu residues using catalytic isotopic exchange in TFA. The distribution of [3H]1 into the blood was measured over time after a single intravenous (iv) injection (0.5 × 106 cpm/(100 μg/mouse)) into conscious mice. PK parameters of 1 were established from the total [3H]1 molecular populations (native and potential plasma-induced hydrolysis or chemical changes) circulating into the bloodstream of mouse over the time course of the 240 min postinjection period. The plasma concentration of 1 was found to follow a biphasic pattern (Figure 4): (i) after reaching a maximal level within 2 min, an α-initial phase from 2 to 20 min postinjection of rapid decrease, primarily attributed to compound distribution from the circulation into the peripheral body tissues and (ii) a βphase of gradual decrease, primarily attributed to drug metabolism and excretion. PK analyses were then performed by two-compartmental model with iv bolus administration using Kinetica software (ThermoFisher). Such analysis helps predict, from the concentration−time profile, the t1/2-life α-distribution and t1/2-life β-elimination of 1 into the bloodstream. Globally, 1related molecular populations circulating in the bloodstream compartment were rapidly distributed into the body tissue

compartments with a half-time α-distribution evaluated at 3−5 min for n = 2 mice (R2 = 0.86 and 0.83, respectively). Then, all 1-related populations circulating in the bloodstream were eliminated from this compartment with a half-time βelimination evaluated at 99−103 min, n = 2 mice (R2 = 0.98 and 0.97, respectively). The radioactivity excreted over the time course of the experiment was estimated at about 2−4% eliminated over a 240 min period. The extent to which cationic oligourea 1 is actually binding to plasma proteins is an important parameter when assessing its PK profile as it can influence its half-life in the body. For example, binding of a peptide or a drug to plasma protein may prevent fast renal clearance leading to prolonged half-life.57,58 The plasma protein binding of 1 was determined by equilibrium dialysis using mouse (CD-1) plasma (see Supporting Information) and estimated to be 99%, which means that 1% of 1 is unbound. Tissular Distribution of [3H]1 in Mice. On the basis of the in vivo blood PK analyses showing that distribution of circulating 1 from the circulation to the systemic tissues of mice was almost complete 20−30 min after iv injection, three postdose kinetic points were chosen for the biodistribution study: 4 h (determining the selectivity of drug uptake by target tissues), 30 h, and 48 h (determining the stability of drug uptake by target tissues). 8225



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Figure 5. Intravenous dosing. Representative mapping of mouse target organs after iv injection of [3H]1. (A) Emission of radioactivity of midsagittal and lateral sagittal sections of mouse whole body 4, 30, and 48 h after iv injection of 5 μCi/100 μg of [3H]1 was acquired using the high-resolution β-radiometric imager. Top picture, scan of one section (4 h post dose); three lower pictures, β-imager analysis of the corresponding section at 4, 30 and 48 h postadministration, respectively. The 20-μm sections were exposed for 40 h. Highest level of radioactivity (red area) is seen in the renal tissue. (B) Quantitative profile of radioactivity in various mouse tissues at 4, 30, and 48 h postadministration of 5 μCi/100 μg [3H]1. Assessment of quantitative regional differences was performed with computer-assisted image analysis using the β-vision program. The number of β-particles emitted per area is expressed as counts/(mm2/40 h acquisition); the tissue detection threshold, determined within muscle areas, is represented as a red line (around 200 cpm/(mm2/40 h)). Bars represent mean ± SD of 2−6 determinations.

Figure 6. Intranasal dosing. Representative mapping of mouse target organs after intranasal injection of [3H]-1 .(A) Emission of radioactivity of midsagittal and lateral sagittal sections of mouse whole body 4 and 48 h after intranasal injection of 5 μCi/20 μg of [3H]1 was acquired with the high-resolution β-radiometric imager. Top picture, scan of one section (4 h post dose); two lower pictures, β-imager analysis of the corresponding sections at 4 and 48 h postadministration, respectively. The 20-μm sections were exposed for 40 h. Red areas correspond to the highest uptake of radioactivity. Highest levels of radioactivity (red area) are detected in the renal, pulmonary, and naso-pharyngeal tissues at 4 h postdose. (B) Quantitative profile of radioactivity in various mouse tissues at 4, 30, and 48 h postadministration of 5 μCi/20 μg [3H]1. Assessment of quantitative regional differences was performed with computer-assisted image analyses using the β-vision program. The number of β-particles emitted per area is expressed as counts/(mm2/40 h acquisition). The red line shows the tissue detection threshold (about 200 cpm/(mm2/40 h)) determined within muscle areas. Bars represent mean ± SD of 2−6 determinations. 8226



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Figure 7. Liver and kidney distribution of biotinylated 1. Five hours after iv injection of 100 μg of 4, kidneys and liver were excised and fixed. Tissue sections were labeled with avidin-peroxidase or with HE. Brown color shows positive signal. Control tissues from naive mice were incubated in the same conditions. A signal was detected in the cortex and deep medulla of the kidney (B). Compound 4 was more specifically localized in epithelial cells of some cortical convoluted tubules (C) and in a high proportion of epithelial cells from the deep medulla (D). No signal was detected in cortical glomeruli (black stars, C). In the liver, the signal was diffuse and granular (F), predominantly localized in small cells lining sinusoids (Kupffer cells; black arrowheads) (G). Control animals did not display any signal in the kidney or in the liver (A, E). C, cortex; M, medulla; P, papilla. Data are representative of at least three mice.

To characterize at the cellular level the fixation of 1 in the kidneys and liver, we injected iv a biotinylated version of 1 (sequence 4 in Figure 1). The bactericidal activity of 4 on B. anthracis germinated spores was slightly weaker than that of 1 (64.8% ± 6.1% killing at 5 μg/mL (SEM, n = 18)). Four hours postdose, 4 was detected in the kidney cortex and deep medulla (Figure 7), more specifically in epithelial cells of multifocal convoluted tubules in the cortex and of most collecting tubules in the deep medulla. No signal was detected in cortical glomeruli. In the liver, the signal was diffuse and more pronounced in small cells lining sinusoids, probably Kupffer cells. Proof of Concept of in Vivo Potential Therapeutic Activity of 1. The bactericidal and PK properties of 1 were thus compatible with an application of oligourea foldamers to in vivo therapeutics. We focused on two of the main routes of bacterial infection in humans, the inhalational and cutaneous routes. The B. anthracis model of infection is highly informative because the infectious process can develop through natural routes in normocompetent mice, without any immune manipulation.47 Spores of the encapsulated nontoxinogenic 9602P strain were thus inoculated in mice treated or not with 1.47,59,60 In the cutaneous model of lethal infection, a significant increase in time of survival, as well as partial survival, was observed compared with the control animals (Figure 8A, left panel; p < 0.001). A similar delay in time to death was also observed in inhalational infection (Figure 8A, right panel; p < 0.001). Dimerization of 1 Increases Its Bactericidal Activity. We addressed the possibility whether multimerization of 1 could increase its efficiency. We previously reported dimerization to strongly potentiate gene transfection efficacy of an amphipathic cationic foldamer designed to deliver nucleic acids into cells.54 Indeed, 6, a covalent dimer of 1 with a thioether linkage55 (Figure 1), showed a significantly higher bactericidal efficiency, with an EC50 of 0.2 μg/mL for germinated spores

In the β-imager radiograms of whole mouse body sagittal sections, 4 h after iv injection of [3H]1, a dense (red areas) and distinct accumulation of radioactivity was apparent in the kidney and, to a lesser extent, in the liver (blue areas) (Figure 5A). In these organs, the tissue uptake was stable for at least 48 h. In the kidney, the radioactivity was localized within the cortical area (Figure 5A). More radioactivity (10−50-fold) was present in these locations than in muscle areas as assessed by direct quantitative regional distribution using computer-assisted image analysis (Figure 5B). No radioactive signal was detected within the lung, brain, or intestine tissues. This overestimation of the anatomic plasma spaces may reflect the presence of sequestration sites within these tissues. The [3H]1 gross anatomic distribution and quantification at 4 and 48 h after intranasal inoculation is shown in Figure 6. The upper airway (nasopharyngeal duct opening into the tubeshaped respiratory pharynx) sequestered a significant amount of the radioactivity at 4 h (Figures 6A and S1). A large amount of radioactivity was transiently sequestered within the lung, quantifiable up to 4 h postdose. Using this delivery route, we show that nasally administered 1 also gains primarily access to the kidney cortical area. The renal tissue uptake was stable for at least 48 h. The maximal measured radioactivity in the kidney was approximatively 70% of that measure from iv dose, which indicates good bioavailability of 1. Taken together, our data provide evidence of a major, selective, and stable uptake of the native 1 by the cortical area of the kidney. Meanwhile, a low elimination of [3H]1 in urine (2−4% over 6 h) was observed. To determine whether the state of the radioactive species detected in the organs (native molecule vs degradation products), acid homogenates from kidney 4 and 30 h postdose were analyzed after methanol extraction by RP-HPLC. The radioactivity was predominantly recovered in the peak corresponding to the chromatographic characteristics of native 1 even after 30 h post-iv-injection: 89% ± 10% (Figure S2). 8227



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However, as soon as germination was triggered, the nascent bacterium became susceptible to the bactericidal activity of 1. Furthermore, the presence of the PDGA capsule did not hinder the bactericidal activity on the bacilli. Cathelicidin bactericidal activity is also not impeded by the PDGA capsule.8 The EC50 values were equivalent for germinated spores and encapsulated bacilli (3.3 μg/mL, that is, 2.4 μM), while 2-fold higher for unencapsulated bacilli. The negatively charged capsule may thus favor interaction with the positively charged 1 and increase the local concentration of these molecules. The EC 50 determined for 1, that is, in the micromolar range, is equivalent to what is reported for cathelicidin8 and defensins.56,62,63 The bactericidal effect was extremely rapid, as 50% of the bacteria were killed in less than 60 s. We also report that the bactericidal activity of 1 in vitro is significantly amplified by covalent dimerization. Owing to the amphiphilic cationic nature of 1, one may hypothesize that the mechanisms leading to bacterial death resemble what has been reported for positively charged defensins and cathelicidins, that is, interaction with the negatively charged phospholipid bacterial membrane bilayer, mainly composed of phosphatidylglycerol.56,62,63 This mode of action is consistent with previous findings showing that 1 interacts strongly with, and disrupts, negatively charged phospholipid membranes.36,64 It is noteworthy that the γpeptide 3, despite strong structural homology with 1, has only residual activity on the different forms of B. anthracis. These data are in line with a previous study demonstrating the weak antibacterial activity of 3 against S. aureus and E. coli and further support a key role of the urea backbone for bactericidal activity.37 The origins of this difference are not yet fully understood but could be related to subtle differences in polarity and geometry between the two helical backbones. Alternatively it has recently been shown that aliphatic oligoureas are ideally preorganized to interact with anionic guests in a site specific manner at the positive end of the helix dipole.65,66 This property may suggest a complementary mechanism to account for the interaction properties of antimicrobial oligourea helices with model phospholipid membranes as well as bacterial membranes. Synthetic foldamers with backbones consisting of nonstandard amino acid residues, or even without amino acid units, manifest intrinsic differences with the natural α-peptides they intend to mimic. Properties such as binding to the target, in vitro proteolytic stability or membrane translocation are commonly investigated.23,25,31,54,67−69 However, in vivo behavior of artificial backbones (PK, potency) remains largely overlooked.40,69,70 Our study thus intended to gain specific information on these original parameters with this amphiphilic cationic oligourea foldamer 1. PK parameters of 1 in the mouse bloodstream demonstrate its rapid distribution (half-time distribution 3−5 min) to the body tissue compartments. Its half-time β-elimination from the bloodstream was evaluated at 99−103 min. By analogy, corresponding values for the 99mTc-labeled ubiquicidin (UBI), a small bactericidal cationic 6654 Da protein, and for corresponding synthetic antimicrobial peptide fragments (6− 18 residue long) were between 17 and 142 min.71 Using the radioactive 1 and the biotinylated 4, we show accumulation of the oligourea in the kidney and the liver through iv administration. Interestingly, tissular biodistribution was similar after delivery by the inhalational route, with further localization in the nasopharynx and the lung. This suggests that intranasal

Figure 8. In vivo protective efficiency of 1 and 6 treatment during cutaneous and inhalational B. anthracis infection. Mice were infected in the ear pinna (left panels) or intranasally (right panels) with spores of the 9602P B. anthracis strain as described in the Materials and Methods section and treated with oligoureas 1 (A) and 6 (B). Oligoureas (20 μg) were administered 15 min, 6 h, 24 h, and 48 h after spore inoculation either in the ear pinna (left panels) or intranasally (right panels). Survival was monitored for 15 days. Survival curve (closed symbols) significance against nontreated mice (open symbols) was calculated with the Kaplan−Meier statistical test using the Graphpad software; ***p < 0.0001; **p < 0.001. Histological analysis of the kidneys did not show any lesion at day 10 of infection with treatment.

and 0.5 μg/mL for encapsulated bacilli, that is, 17- and 7-fold lower, respectively, compared with the values for the cognate 1 monomer (Figure 2C). The toxic dose of 6 inducing 50% cell lethality (RAW 264.7 macrophage cell line) was 19 ± 0.6 μg/ mL (mean ± SEM, n = 3) giving a selectivity ratio of 95 for B. anthracis germinated spores, higher than that of 1. A very similar anthracidal activity was measured for cognate dimer 7 bearing a disulfide linkage (EC50 = 0.25 μg/mL, see Figure S3) indicating that the nature of the linker has little influence on the activity in vitro. Local in vivo treatment with 6 led to similar control of B. anthracis infection as the monomer 1, by both cutaneous (Figure 8B, left panel) and inhalational routes (Figure 8B, right panel), with a similar delay in time to death (p = 0.006 and 95% and lyophilized. Tritiation of 1. Tritiated water (high specific radioactivity) was prepared by reduction of palladium oxide (26 mg, freshly activated at 110 °C under reduced pressure) under an atmosphere of 99% tritium gas (RC-TRITEC, typically 10−15 Ci at 2 bar) for 2 h at room temperature. When pressure was stable (typically 0.6 bar), the remaining gas was eliminated by pumping and trapped on a La/Ni/ Mn alloy device. Then the tritiated water was transferred by simple freeze-drying and trapping into a solution of 1 (2 mg, 1.4 μmol) in pure TFA (150 μL). The mixture was stirred for 2 h at room temperature and then transferred into a pear-shape flask. The tritiated water and exchangeable tritium atoms were eliminated by repeated dissolution/evaporation cycles (at least 3 times, 3 × 10 mL) of a 50:50 mixture of methanol in water to a final volume of about 0.1 mL. This solution was purified by analytical RP-HPLC (Waters Atlantis dC18 column 100 Å, 3 μm, 2.1 × 150 mm2) using a linear gradient A/B from 100:0 to 0:100 in 30 min at 1 mL·min−1 (A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile). The volume of the combined HPLC fractions containing the tritiated compound was reduced in vacuum. [3H]1 (0.73 mCi) was dissolved in water (1.15 mL) to a final concentration of 0.63 mCi/mL. The specific radioactivity was estimated to 40−45 Ci·mmo1−1 by UV titration (285 nm, ε = 11400 M−1·cm−1) of the final solution. PK Analyses of Radiolabeled 1 in the Mouse Blood. In vivo time course of blood distribution and elimination of the [3H]1 was investigated in conscious BALB/c mice after an iv injection of 0.5 × 106 cpm/(100 μg/5.8 × 10−2 μmol) [3H]1 in 50 μL of PBS. Blood (10−20 μL) was withdrawn 0.6, 2, 3.5, 5.5, 8.7, 15, and 240 min after compound administration via the retro-orbital sinus. The urinary bladder content was collected at the last selected point (240 min). The radioactivity content of the samples was determined using a liquid scintillation Wallac β-radiometer. Biodistribution of Radiolabeled 1 in Whole Mouse Body. Each BALB/c mouse received either 100 μg (5 μCi) [3H]1/50 μL PBS by iv administration or 20 μg (5 μCi) [3H]1 by intranasal administration. Two minutes prior to the assigned time (4, 30, and 48 h postdose) the halothane anesthetized mouse was restrained into a flat box in lateral position, and at selected time was immediately immersed in a −80 °C mixture of isopentane and dry ice for 30 min.

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CONCLUSION By virtue of their modified backbones, foldamer mimics of natural peptides are likely to manifest altered properties including improved proteolytic stability but also different PK profiles and as a result different activities in vivo. The in vivo PK properties of an amphiphilic cationic oligourea foldamer (1) described here, which reveal high tissue uptake, slow elimination (low renal clearance), and high stability of the modified backbone are consistent with two studies conducted earlier on β-peptides52 and peptoids51 and pave the way for future development of bioactive urea-based foldamers. Oligourea 1, which was originally designed to structurally mimic helical AMPs by segregating cationic and hydrophobic side chains at the oligourea helix surface, exhibits remarkable in vitro bactericidal activities against the different differentiation forms of B. anthracis, the causative agent of anthrax. These results and the finding that this membrane active foldamer showed some protection against B. anthracis in a murine model of infection suggest that it could be equally useful in vivo against multiresistant Gram-positive bacteria such as MRSA. Various chemical approaches (i.e., covalent dimerization (this work), isosteric backbone modifications,75 or fused peptide/oligourea chimeras76) are available to further tune the physical properties of 1 and optimize bactericidal activities against Gram-negative bacteria or mycobacteria, both of public health concern, and for biodefense control. 8229



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Mice were then placed in a self-sealing plastic bag and kept at −80 °C during 24 h and then stored at −30 °C during at least 48 h until their embedding. The frozen body was rapidly shaved off and its tail and hind leg were cut. Then, the body was blocked in mounting medium (4% carboxy-methyl cellulose gel and OCT-tissue compound) at −70 °C using the Leica embedding sample-holder system (120 × 250). The entire system was then stored at −25 °C. Whole body sagittal sections (20 μm) were made at −25 °C using the cryo-macrotome CM3600 (Leica, France). Each section adhering to Scotch tape (Neschen, Benelux) was successively maintained on a metallic framework and left in the cryo-macrotome chamber for at least 7 days to ensure complete drying. Selected whole mouse body sections were exposed on a surface detector of 20 × 20 cm2 and placed in the gaseous detector of βparticles (the real-time β-imager 2200; Biospace Lab, France) for at least 40 h. Mapping and quantification of [3H]1 bound in organs or compartments within the whole mouse body section were recorded and analyzed by computer-assisted image analysis (β vision program). The linearity of this method of detection allows measurement of the specific binding of the [3H]-compound within various structures of the same section. Background or nonspecific binding was evaluated within cardiac or muscle area of each section. Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) of Tissue Extracts. A C18-bonded silica nonpolar stationary phase (C18-ACE column, 5 μm 100 Å, 150 × 4.6 mm2; Phenomenex, France) with A/B elution mobile phase (A, water, 0.1% TFA; B, acetonitrile, 0.1% TFA) was used. The RP-HPLC system consisted of the Surveyor plus MPUMP system (ThermoScientific, France) equipped with a temperature-controlled autosampler, a UV detector precalibrated at 224 and 254 nM and a Radiomatic 150TR detector (PerkinElmer, France). The elution gradient conditions were as following: after 10 min under isocratic solvent conditions in 20% B, a 15 min linear solvent gradient from 20% to 100% B was applied to elute sample components under the control of the ChromQuest driver software. Each acid (HCl 1 N) homogenized tissue was submitted to methanol/0.1% TFA extraction (4:1, vol/vol sample) followed by 48 h lyophilization and pyrolyzed water resuspension before being eluted on the C18 RP-HPLC column. Cytotoxic Assay. Cells of the RAW 264.7 macrophage cell line (2 × 105 cells per well) were incubated with increasing concentrations of 1 and 6 in 96-well plates for 4 h. Viability was then assessed through the MTT assay.79 Histology. Five hours after intravenous injection of 100 μg of 4, the biotinylated analogue of 1, in mice, kidneys and liver were removed and fixed in 10% neutral buffered formalin. Four micrometer sections were cut and labeled with a streptavidin−peroxidase polymer (S9420, Sigma, USA) or stained with hematoxylin−eosin. Sections from naive mice were used as controls. In Vivo Infection and Oligourea Treatment. Mice were infected with spores of the 9602P strain, six mice per group, either in the cutaneous tissue of the ear pinna (3.2 log10 ± 0.4 spores) or by intranasal route (5.7 log10 ± 0.2 spores).59 Cutaneous infections were performed under light anesthesia by injecting 10 μL of the spore suspension in PBS into the ear pinna. Intranasal inoculation was performed in lightly anesthetized mice by deposing the spore inoculum in 20 μL of PBS upon inhalation into the right nostril. Oligoureas (20 μg in 10 μL) were administered 15 min, 6 h, 24 h and 48 h after spore injection at the same site either in the ear pinna or intranasally. In vivo experiments were performed at least twice. Statistical Analysis. Statistical analysis and graphing was performed using GraphPad Prism 4 software (GraphPad Software Inc.) and unpaired two-tailed Student t tests. Significance was considered at p ≤ 0.05.

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Detailed synthetic procedures, detailed characterization of new oligomers, and supplementary Tables S1−S5 and Figures S1−S10 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: +33 1 45 68 83 12. E-mail: [email protected]. *Phone: +33 5 40 00 30 20. E-mail: [email protected]. Author Contributions

E.T. and J.-P.C contributed equally. P.L.G. and G.G. share senior coauthorship. Notes

The authors declare the following competing financial interest(s): G.G. is co-inventor on a patent application covering oligourea derivatives with antibacterial activities.

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ACKNOWLEDGMENTS We dedicate this work to the memory of Ana Cardona, who directed the first pharmacokinetic studies of 1. She will remain in our hearts. We greatly thank Alain Cosson and Maria de Moura for their expert technical assistance and Scott Nichols and Taylor Feeley for their participation in the preliminary experiments during their research period in the laboratory. This work was supported by ANR and DGA (Project ANR-12ASTR-0024). E.T. was funded by Fondation pour la Recherche Médicale, FRM No. DEA20090616230. Predoctoral fellowships from DGA and Conseil Régional d’Aquitaine (to S.A.), and from ImmuPharma France and ANRT (to P.C.) are gratefully acknowledged.

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ABBREVIATIONS AMPs, antimicrobial peptides; CBRN, chemical, biological, radiological, and nuclear; CFU, colony forming unit; MRSA, methicillin-resistant S. aureus; PDGA, poly(γ-D-glutamic acid); RP-HPLC, reversed-phase high pressure liquid chromatography; SEM, standard error of the mean; TFA, trifluoroacetic acid; UBI, ubiquicidin

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00144. 8230



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