Article Cite This: J. Med. Chem. 2019, 62, 6276−6286
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Positive Charge Patterning and Hydrophobicity of MembraneActive Antimicrobial Peptides as Determinants of Activity, Toxicity, and Pharmacokinetic Stability Tracy A. Stone,‡ Gregory B. Cole,† Dorna Ravamehr-Lake,† Huong Q. Nguyen,† Farheen Khan,† Simon Sharpe,† and Charles M. Deber*,† †
Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4 Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, M5S 1A8 Ontario, Canada
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‡
S Supporting Information *
ABSTRACT: Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH2). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.
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into antimicrobial therapeutics.3,9 While some natural CAPs have been reported to exhibit low toxicity to zwitterionic mammalian membranes,4 many are capable of inducing lysis of erythrocytes or mammalian cellsa feature that is often accompanied by relatively high peptide hydrophobicity and amphipathicity.10−19 This phenomenon is especially prominent in studies of de novo synthetic CAPs and synthetic variants of natural CAPs, where it is far easier to control for variations in peptide length, composition, and charge than in comparisons across sequences of natural CAPs. This observed toxicity, coupled with poor pharmacokinetics, is largely responsible for the failure of many CAPs in the clinic.3,9 Approaches to increased stability and safety of these compounds thus remain in high demand. To address these objectives, our lab had earlier designed a synthetic novel CAP, 6K-F17 (sequence KKKKKKAAFAAWAAFAA-NH2).20 Unlike the naturally occurring CAPs that typically have an amphipathic design with positively charged residues positioned on one face of the helix, the six Lys residues in 6K-F17 are clustered at the N-terminus of the sequence. This unique sequence design endows 6K-F17 with high antimicrobial activity against a range of bacteria yet with
INTRODUCTION The emergence of widespread antibiotic resistance to many common and often “last resort” antibiotics necessitates the need for development of novel antimicrobial agents.1,2 Antimicrobial peptides represent an excellent and underutilized source of potential new therapeutics.3 These naturally occurring peptides are ubiquitous in nature, occurring in all three kingdoms of life, and exhibit a broad range in activity, primary sequence, secondary structure, and mechanism of action.4,5 Among the natural CAPs, we have focused on the linear, helical, cationic antimicrobial peptides (CAPs), most commonly isolated from the skin of Anura (tailless amphibians).6 Natural CAPs are typically enriched in positively charged (Lys, Arg) and hydrophobic residues and can range in length from as few as nine amino acids up to 140, with an average length of 31 amino acids.6 Generally, the primary sequences of these helical peptides are amphipathic, with positively charged residues distributed along a single face of the folded helix.4,5 The overall net positive charge of CAPs is responsible for their selective targeting to negatively charged bacterial membranes over zwitterionic eukaryotic membranes, where ostensibly the peptides disrupt the integrity of the bacterial membrane, resulting in cell death.5,7 Despite the discovery of antimicrobial peptides dating back to the late 1930s,8 very few have successfully been developed © 2019 American Chemical Society
Received: April 18, 2019 Published: June 13, 2019 6276
DOI: 10.1021/acs.jmedchem.9b00657 J. Med. Chem. 2019, 62, 6276−6286
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Table 1. Natural CAPs with Cationic Charge Clustersabcdefg
a
Peptide name and Antimicrobial Peptide Database (APD) identification number.6 bPeptide sequences. Positively charged residues (Lys, Arg) are indicated in red. Positively charged clusters of at least four or more Lys/Arg in the primary sequence are underlined. cSource of the peptide and name of the species from which the peptide was isolated. dSequence hydrophobicity reported as ΔG (kcal/mol) from water to the bilayer, based on the Octanol-Interface scale32 and calculated using membrane protein explorer (MPEx).33 eCorresponding hydrophobic moments are reported in parentheses, calculated from the vector sum of side chain hydrophobicity along a helical wheel plot using MPEx.33 fReported toxicity from primary sources. “Yes” indicates a high level of toxicity to erythrocytes. “No” indicates low or no toxicity to erythrocytes. “Untested” indicates the lack of experimental data to accurately classify it as toxic or nontoxic. gReference(s) for the initial discovery of the peptide and toxicity measurements.
virtually no toxicity to mammalian cells.7,20−24 We have previously shown the impact of positioning the charge cluster on the C-terminus7 and on both termini23 of this peptide, with the peptide having N-terminal positioning of the charge cluster, exerting the greatest antimicrobial activity. In the present work, we have now expanded our studies to (1) establish the extent of occurrence of charge clustering in a collection of nearly 400 natural CAPs and (2) address systematically the impacts of charge patterning within a series of designed peptides. We show that the clustering of charged residues to the peptide terminus can offer improved selectivity for bacterial membranes, reduced toxicity to host cells, and importantly, increased resistance to nonspecific proteolysis.
as charge-clustered, none of these peptides have complete segregation of charged residues to a terminus, as they all contain at least one additional charged residue in the remainder of the peptide. For this reason, the amphipathicity, as measured by hydrophobic moment, remains quite high for some of the peptides (Table 1). In assessing the toxicity of seven naturally charge-clustered peptides, three of them (BM moricin, misgurin, and lactococcin G-a) have “low” toxicity, as reported in the literature;25−27 two remain untested (lactoferricin and MS moricin), although the high sequence similarity between MS moricin and the nontoxic BM moricin suggests low toxicity for both peptides;25 and two are toxic (melittin and ponericin W1).28−31 The high toxicity of melittin has been attributed to its ability to form pores in membranes,28 while ponericin W1, having a similar design to melittin and also originating from an insect venom, has been proposed to function through a similar mechanism.29 Notably, the average hydrophobicity of these two toxic peptides is higher than that of the nontoxic peptides (Table 1). Peptide Design. Given that the clustering of positive charges within 6K-F17 imparts excellent selectivity for bacterial over mammalian membranes, we undertook to determine the properties that would be imparted to this peptide when its residues were arranged in the traditional amphipathic design charges dispersed throughout the sequence and segregated to one face of a helixas is predominant in naturally occurring CAPs. In this context, we modulated the amphipathic character
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RESULTS Charge Clustering is Rare in Naturally Occurring Antimicrobial Peptides. In undertaking this work, it was immediately of interest to determine if high charge density or “charge-clustering”, occurs in natural CAPs. Using the online Antimicrobial Peptide Database (APD),6 we generated a set of α-helical CAPs that displayed activity against bacteria (Gramnegative and Gram-positive, 395 sequences in total). Synthetic, chimeric, and nonribosomally synthesized sequences were removed from the dataset (23 sequences). Of the remaining 372 peptide sequences, only seven (1.9%) contained four or more consecutive Lys/Arg residues in the primary sequence or “clustered” (Table 1), establishing that charge clustering is a rare event in natural CAP sequences. Despite being classified 6277
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Table 2. Sequences of 6K-F17 and 6K-F17-4L Peptides and Their Amphipathic Variants, Studied in this Workabc
a
Peptide sequences. Positively charged (Lys) residues are shown in red. 4-Leu peptide sequences are depicted beneath the corresponding Ala parent sequence. bRetention time (min) on a C-18 column, with a mobile phase composition of either 80% solvent A/20% solvent B for 0-Leu peptides or 65% A/35% B for 4-Leu peptides (see the Experimental Section for mobile phase compositions). Error is reported as standard deviation in parentheses. cHydrophobic moment as calculated from the vector sum of side chain hydrophobicity along a helical wheel plot using MPEx33 and the Octanol-Interface scale.32
of the charge-clustered parent 6K-F17 peptide by moving Lys residues from the N-terminus into the hydrophobic core of the sequence in a stepwise manner (Table 2). Lys residues were positioned within the hydrophobic core to maximize the observed hydrophobic moment and hence the presence of two distinct helical faces: one containing the Lys residues and the other containing the hydrophobic residues, including bulky, aromatic residues (Phe, Trp) (Figure 1). Hydrophobic momentsa measure of the vector sum of side chain
hydrophobicity perpendicular to the helix axiswere used to assess the degree of amphipathicity within the sequence, viz., the larger the hydrophobic moment, the more amphipathic the sequence.34 A second peptide series, in which four Ala residues were substituted for four Leu residues, was created to test the effects of amphipathicity in peptides with an average hydrophobicity closer to most natural CAPs (Table 2). Leu residues were positioned to emphasize the distinction between charged and hydrophobic faces. To assess changes in local hydrophobicity as a function of sequence patterning, we measured peptide retention times on a C18 HPLC column. For both peptide series (0-Leu and 4Leu), charge-clustered sequences were retained the longest on the column and therefore construed as the most hydrophobic, whereas amphipathic sequences eluted the earliest (Table 2; 6K-F17 at 18.4 min vs 1Kamp at 4.2 min). Overall, chargeclustered peptides “read out” as relatively more hydrophobic than their amphipathic counterparts. Charge Clustering Decreases Peptide Toxicity to Human Red Blood Cells. The 6K-F17 and amphipathic variants were virtually nontoxic to human red blood cells up to 320−640 μM (50
6K-F17-4L 5Kamp-4L 4Kamp-4L 3Kamp-4L 2Kamp-4L 1Kamp-4L
12.5 3.1 12.5 3.1 1.6 1.6
>50 12.5 >50 3.1 3.1 3.1
50 6.3 >50 3.1 3.1 3.1
PA287 (μM)
Hemolysis at 40 μMc
PA330 (μM)
PA380 (μM)
3.1 50 >50 >50 50 >50
25 50 >50 >50 >50 >50
12.5 50 50 >50 >50 >50
0% 0% 0% 0% 0% 0%
25 6.3 >50 3.1 3.1 3.1
>50 12.5 >50 6.3 6.3 6.3
>50 >50 >50 3.1 1.5 6.3
33% (± 22) 49% (± 22) 8% (± 2.0) 100% (± 4.5) 89% (± 7.3) 81% (± 9.4)
0-Leu
4-Leu
Minimum inhibitory concentrations (MICs, μM) against E. coli K12 bacteria. bMinimum inhibitory concentrations (μM) against P. aeruginosa bacteria, lab strain (PAO1) and persister strains (PA) isolated from cystic fibrosis (CF) patients at the Hospital for Sick Children. c% hemolysis at 40 μM, standard deviation is reported in parentheses. One-way analysis of variance comparison of the mean of 6K-F17-4L to the means of the amphipathic peptides showed significantly higher hemolysis for 3Kamp-4L (p = 0.002), 2Kamp-4L (p = 0.0063), and 1Kamp-4L (p = 0.0124). a
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Figure 3. N-terminal clustering of Lys increases the resistance to proteolytic degradation. (A) % peptide remaining over time (minutes) after exposure to proteinase K. Data represent the average of at least two independent experiments. Error is reported as standard deviation. Peptides are listed in the legend in the order of decreasing % peptide remaining. To the right, representative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of peptides treated with ProtK over 1 h. (B) Top panel, % 5-carboxytetramethylrhodamine (TAMRA)-labeled peptides remaining over time (hours) after incubation in blood plasma (6K-F17 and a positive control with the six Lys residues in the 6K-F17 sequence “scrambled”; see the Experimental Section). (C) Experiments similar to those shown in (B), using lung sputum taken from CF patients. Data represent the average of at least two independent experiments. Error is reported as standard deviation.
Figure 4. CD spectra of 6K-F17 and peptide variants in bacterial and mammalian model membranes. (A) 6K-F17 and amphipathic variants (25 μM) in E. coli lipid bilayers (2 mg/mL) and (B) POPC lipid bilayers (2 mg/mL). (C) 6K-F17-4L and amphipathic variants (25 μM) in E. coli lipid bilayers and (D) POPC lipid bilayers. All spectra represent the average of at least three independent experiments. Spectra are listed in the legends in the order of increasing helicity at 222 nm.
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Figure 5. Tryptophan quenching in bacterial and mammalian model membranes. (A) 6K-F17 and amphipathic variants in E. coli lipid bilayers. (B) 6K-F17-4L and amphipathic variants in E. coli lipid bilayers. (C) 6K-F17-4L and amphipathic variants in POPC lipid bilayers. Trp was quenched through addition of increasing amounts of acrylamide. (D) 6K-F17-4L and amphipathic variants in POPC lipid bilayers supplemented with increasing amounts of dibrominated lipid 1-palmitoyl-2-(11,12-dibromo)stearoyl-sn-glycero-3-phosphocholine (Br-PC 11,12). In all cases, peptide concentration was 10 μM whereas lipid concentration was 0.8 mg/mL. Data points represent the average of at least two independent experiments. Error is reported as standard deviation. Peptides are listed in the legend in the order of decreasing quenching.
amphipathic variants showed varying interactions with bacterial membranes, as indicated by a range in helical character (CD spectroscopy, Figure 4A) and blue shifts (Trp fluorescence, Figure S2). The more amphipathic sequences displayed the most helical character and largest blue shifts, suggesting that amphipathic sequences may be more buried in bacterial membranes than charge-clustered sequences, and therefore may be exerting their antimicrobial activity through an alternative mechanism of action. In contrast, in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranesa mimic of mammalian membranes (zwitterionic, net neutral charge)the peptides all fail to adopt secondary structures (Figure 4B), supporting the observed low toxicity of these peptides toward human red blood cells (Table 3). The 4-Leu peptides in E. coli lipid bilayers gave similar results to the 0-Leu peptides, with amphipathic sequences adopting the most helical structures (Figure 4C). In the presence of POPC lipid bilayers, the 4-Leu peptides, having displayed high hemolytic activity against human erythrocytes (Figure 2), exhibit a dramatic increase in helical character (CD spectroscopy; Figure 4D) and blue shifts (Trp fluorescence; Figure S3) relative to their lower hydrophobicity counterparts (Figure 4B). Amphipathic sequences again adopted the most helical character and largest blue shifts. Application of a water-soluble Trp quencher (acrylamide) revealed that despite the increased helical character of the amphipathic 1Kamp peptide relative to 6K-F17, the Trp from both peptides was similarly protected from the aqueous quencher in E. coli membranes (Figure 5A). However, the 4Leu peptides revealed 1Kamp-4L to be significantly less exposed to the aqueous quencher than 6K-F17-4L, implying that the Trp from 1Kamp-4L was buried more deeply into the bilayer (Figure 5B). Further Trp quenching experiments revealed the more hemolytic 4-Leu amphipathic peptide, 1Kamp-4L, to be the
most deeply buried in POPC membranes (Figure 5C). Studies using bilayers substituted with dibrominated lipid (11,12BrPC) quenchers confirmed that 1Kamp-4L has its Trp residue positioned closest to the core of the bilayer (∼6.5 Å from the core) relative to its charge-clustered counterpart 6KF17-4L (Figure 5D). Altogether, these data further suggest that charge-clustered sequences interact with bilayers in a different manner than the amphipathic sequences. Charge-Clustered Sequences are More Disruptive to Bacterial Membranes in Vitro than Amphipathic Sequences. Membrane disruption assays using terbium(III)loaded bacterial lipid vesicles and external dipicolinic acid (DPA) (see the Experimental Section) indicate that chargeclustered sequences are significantly more disruptive to anionic lipid bilayers than traditional amphipathic sequences, regardless of average hydrophobicity (Figure 6). Thus, while amphipathic sequences show minimal liposome disruption (∼8% for 1Kamp) after 20 min, charge-clustered 6K-F17 induces ∼35% disruption (Figure 6A). The 4-Leu peptides, having a higher average hydrophobicity, induce more lysis overall yet the trend remains the same, with charge-clustered sequences inducing the most lysis (∼80%) and amphipathic sequences the least (∼40%) (Figure 6B). In comparisons of the slopes for 0-Leu- and 4-Leu-induced lysis, the peptides with increased hydrophobic character exerted their liposome disruption activity much more rapidly (Figure 6A,B). Disruption of POPC liposomes (mimic of mammalian membranes) was tested for 4-Leu peptides (Figure 6C) and, analogous to the hemolysis results (Figure 2; Table 3), the amphipathic variants were the most disruptive to the mammalian bilayers (∼25−70%) relative to the chargeclustered variants (∼8−4%). Peptides with low average hydrophobicity (no Leu substitutions) were not tested as they showed minimal interactions with POPC lipid bilayers through CD experiments (Figure 4B) and were nontoxic to human erythrocytes up to 640 μM (Table 3). 6281
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why charge clusteringa sequence design that appears optimal for selective targeting to bacterial membranesoccurs only infrequently in naturally occurring CAPs. Several reasons may be suggested. First, despite the demonstrable high therapeutic potential of a charge-clustered sequence (vide infra), amphipathic sequence designs may have several benefits over charge clustering in nature. For example, ribosomal synthesis of multiple consecutive Lys or Arg sequences has been shown to stall ribosome translation and recruit proteases to the stalled ribosome complex.40 Thus, the concentration of positive charge from multiple consecutive Lys/Arg residues likely enables the formation of favorable electrostatic interactions with the negatively charged ribosomal exit tunnel, thereby stalling translation.41,42 Therefore, the synthesis of chargeclustered sequences in vivo may be hindered at the translational level. This may explain why charge clusters in natural CAPs are never more than four positively charged residues in a row and are generally located on the C-terminus, or end, of the sequence (Table 1). In a further example, amphipathic character in natural CAPs may be important for functions beyond antibacterial activity. Many of the natural CAPs in our database have reported activity against organisms other than bacteria, including fungi, viruses, cancer cells, mammalian cells, parasites, and insects.6 These broad ranges in activity may favor an amphipathic design over the clustering of positively charged residues together, particularly when required for the targeting and disruption of varying membrane types (neutral/zwitterionic and negatively charged). Additionally, protease susceptibility may be a desirable trait in natural CAPs. Most characterized CAPs have been isolated from the skin of Anura (tailless amphibians), where they are released from granular (venom) glands in a prepro-protein form that in itself is nontoxic.43 Proteolytic cleavage by secreted enzymes is therefore required to “activate” these peptides. These activated forms of natural CAPs, having high average hydrophobicity and an amphipathic design, are often toxic to host cells, making prolonged exposure potentially dangerous. As observed in our protease degradation studies (Figure 3), amphipathic sequences are significantly more susceptible than charge-clustered peptides to protease degradation, a feature that appears to be required for initial activation of natural CAPs and subsequently for clearing from susceptible host cells. Charge Clustering in CAPs: Improved Target Specificity, Host Toxicity, and Protease Resistance. Here, we have demonstrated how charge clustering in the antimicrobial peptide 6K-F17 has uncoupled antibacterial activity from hemolytic activity. The ability of 6K-F17 to maintain high antimicrobial activity and low toxicity to erythrocytes may manifest from an increase in local hydrophobicity imparted by the charge-clustered designwhich incidentally has both a charge cluster and an uninterrupted hydrophobic clusteras observed through increased retention time on a C18 column (Table 2). This increase in local hydrophobicity, while the average hydrophobicity is kept low, may ensure that only once the charge cluster provides electrostatic attraction to anionic membranes does the hydrophobic domain induce membrane rupture. Amphipathic variants lacking the hydrophobic cluster found in 6K-F17 are therefore not as effective at rupturing bacterial membranes. This effect is emphasized in liposome disruption assays, where charge-clustered sequences (6K-F17 and 6K-F17-4L) are always better at inducing leakage in
Figure 6. Peptide-induced disruption of membranes. % liposome disruption to E. coli lipid bilayers upon addition of (A) 6K-F17 and amphipathic variants or (B) 6K-F17-4L and amphipathic variants. (C) % liposome disruption to POPC lipid bilayers upon addition of 6KF17-4L and amphipathic variants. Liposomes were loaded with Tb3+ and suspended in a buffer containing dipicolinic acid (DPA). Then, 10 μM peptide was added and Tb3+ fluorescence was monitored over time. All samples were normalized to 100% liposome disruption with Triton. Fluorescence traces represent the average of at least three independent experiments.
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DISCUSSION Implications of Charge Clustering and Amphipathicity in Natural CAPs. The data reported in Table 1 clearly establish that not only is charge clustering rare in natural CAPs, it appears that it is explicitly avoided in nature. Yet, as shown in the present work, we observed that the clustering of positive charges to the terminus of 6K-F17 has two clear advantages over a corresponding amphipathic sequence pattern: (1) increased antimicrobial activity, accompanied by low toxicity to host cells when the peptide overall average hydrophobicity is low and (2) increased resistance to rapid/ complete degradation by proteases. One may thus inquire as to 6282
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four or more positively charged (Lys/Arg) residues occurring consecutively in the primary sequence. Bacterial Strains, Blood Cells and Plasma, and Lung Sputum. Bacterial strains tested include E. coli K12 BW25113 strain (Dharmacon Inc., CO), P. aeruginosa PAO1 lab strain (Dharmacon Inc., CO), and four clinical isolates of persister strains of P. aeruginosa (214, 287, 330, 380), obtained from the sputum of cystic fibrosis patients at the Hospital for Sick Children that had persisting infections post-treatment with inhaled tobramycin. Human blood samples (red blood cells and plasma) were drawn fresh from consenting donors in the lab. Clinical isolates of P. aeruginosa and lung sputum were obtained with informed consent from CF patients with chronic infection followed at the Hospital for Sick Children (Toronto, Canada). The consent was obtained from a parent or legal guardian if not of age. All methods were performed in accordance with the relevant guidelines and regulations for research involving human subjects at the Hospital for Sick Children. Antimicrobial susceptibility testing was performed as per the Clinical Laboratory Standards Institute.48 Peptide Quantification. Peptides were purchased from SynPeptide Co., Ltd. (Shanghai, China) with ≥98% purity, confirmed by mass spectrometry and analytical reverse-phase high-performance liquid chromatography. Peptides were obtained as lyophilized powders that were dissolved in water and quantified by absorbance at 280 nm using the extinction coefficient 5690 M−1 cm−1. Peptides were stored as frozen aliquots until use. Then, 5-carboxytetramethylrhodamine (TAMRA; Novabiochem, Massachusetts) labeled 6KF17 and 3K-F17scr (a scrambled positive control for degradation, sequence: KKKAAFKAAKWKAAFAA-NH2) were synthesized inhouse using standard Fmoc solid phase synthesis methods and fluorescently labeled on the N-terminus, as previously described.49 Briefly, 86 mg of TAMRA and 76 mg 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate were added to 100 mg of crude peptide resin dissolved in 2 mL of dimethylformamide with 37.6 μL of diisopropylethylamine. The reaction was protected from light and nutated for 3 h, before being washed, and the labeling was repeated. Labeled peptides were cleaved and purified using standard protocols.49 Fluorescently labeled peptides were quantitated by absorbance of TAMRA at 565 nm, and the corresponding extinction coefficient is 91 000 M−1 cm−1. Liposome Preparation. Total E. coli lipid extract, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2(11,12-dibromo)stearoyl-sn-glycero-3-phosphocholine (Br-PC 11,12) were purchased from Avanti Lipids Inc. (Alabama) as chloroform stocks. Appropriate amounts of lipid were dried into thin films, lyophilized overnight, and brought up in 1 mL of water, vortexed, frozen, and lyophilized again. The resulting lyophilized lipid powder was brought up in the appropriate buffer and freeze-thawed five times (dry ice/50 °C water bath). Samples for CD and Trp fluorescence were passed through a 0.2 μm sized filter seven times and left to equilibrate overnight. Samples for the liposome disruption assay were passed through a 0.4 μm filter 10 times and immediately buffer exchanged on a size exclusion column (Superdex 200 10/300, GE Healthcare Life Sciences). Liposomes were diluted appropriately prior to use (2 mg/mL for CD, 0.8 mg/mL for fluorescence, and 2.5% (v/ v) for liposome disruption assays). Peptides in CD and fluorescence experiments were added to preformed liposomes and allowed to equilibrate overnight prior to recording spectra. Reverse-Phase High-Performance Liquid Chromatography Retention Times. Peptides dissolved in water (25 μM) were injected onto an XBridge BEH130 C18 column (Waters) with an isocratic mobile phase 80% A (90% H2O, 10% acetonitrile, 0.1% trifluoroacetic acid (TFA)) and 20% B (90% acetonitrile, 10% H2O, 0.1% TFA) (for the 0-Leu peptide series) or 65% A/35% B (for the 4Leu peptide series). Peptide elution times were monitored by absorbance at 214 nm. The void elution of uracil was used to correct for day-to-day differences in elution. Hemolysis Assay. Toxicity against mammalian membranes was tested by measuring the peptide concentration at which red blood cells begin to undergo hemolysis. Fresh human red blood cells were
negatively charged E. coli membranes (Figure 6A,B) but not in neutral POPC membranes (Figure 6C). Interestingly, the increased liposome disruption of E. coli lipid bilayers by charge-clustered sequences does not correlate with antibacterial assays for peptides of raised hydrophobicity (4-Leu). This apparent contradiction may arise from differences in the solubility of the charge-clustered sequences vs amphipathic sequences in the presence of extracellular components of bacteria, such as LPS antigen chains and secreted sugars,44 which are absent in the terbium(III) liposome disruption assay. Furthermore, charge-clustered sequences differ from amphipathic sequences in how they interact with lipid bilayers, as evidenced through variations in the secondary structure and Trp burial. An amphipathic peptide, having a hydrophobic face positioned along the helical axis opposite to a positively charged face, can lie atop the membrane perpendicular to the bilayer normal. In contrast, a charge-clustered sequence, containing both an uninterrupted positive charge cluster and a hydrophobic stretch, may interact with the bilayer in a more dynamic manner, reminiscent of a detergent. Considering the naturally charge-clustered peptides listed in Table 2, the two toxic CAPs, melittin and ponericin W1, function through formation of a pore.28,29 However, 6K-F17, being relatively less hydrophobic (ΔG = 18.6 kcal/mol [from water to bilayer]33) vs melittin and ponericin W1; ΔG = 12.1 kcal/mol and ΔG = 15.8 kcal/mol, respectively,33 and much shorter (17 amino acids in total, 11 hydrophobic amino acid stretches) than both melittin and ponericin W1 (26 and 25 amino acids respectively), is unlikely to assemble into a melittin-like pore.45 The observed resistance of charge-clustered 6K-F17 to proteolytic degradation by Proteinase K, and to endogenous proteases in blood plasma and CF patient lung sputum, identifies a straightforward approach to improving the pharmacokinetic properties of peptide therapeutics without the use of D-isomeric or nonproteogenic amino acids, which can be expensive to incorporate, are not amenable to production by molecular biology techniques and can be potentially toxic to cells.46,47 The ability of the charge cluster to prevent proteolytic degradation can most plausibly be attributed to an electrostatic repulsion of the cluster away from the enzyme active site and/or binding pocket.
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CONCLUSIONS The unique design of clustering positive charges to one terminus, in concert with a relatively lower core average hydrophobicity, endows antimicrobial peptide 6K-F17 with remarkable selectivity for bacterial over mammalian membranesa feature that could be used as a template for the optimization of natural CAPs of similar length and hydrophobicity. The increased resistance of 6K-F17 to proteolytic degradation vs amphipathic counterparts evokes a general strategy for the de novo design of novel peptide therapeutics with high resistance to nonspecific proteolysis in vivo.
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EXPERIMENTAL SECTION
Database Construction. A database of 395 helical natural cationic antimicrobial peptides was generated using the online Antimicrobial Peptide database.6 Selected sequences were required to have a helical structure (verified by NMR, X-ray crystallography, or CD) and exhibit antibacterial activity (Gram-negative and/or Grampositive bacteria). Synthetic, chimeric, and nonribosomally synthesized peptides were removed from the dataset (23 sequences). The remaining 372 sequences were then screened for those containing 6283
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Tryptophan Fluorescence and Quenching. Fluorescence spectra were recorded on a Photon Technology International fluorimeter using a 1 cm path length quartz cuvette. Tryptophan was excited at 280 nm, and emission spectra were recorded between 300 and 400 nm with a step size of 1 nm. Peptide concentrations were 10 μM in 10 mM Tris buffer 10 mM NaCl pH 7.4, supplemented with either 5 mM SDS or 0.8 mg/mL total E. coli lipid extract or POPC. Samples were background subtracted, and the wavelength of maximal fluorescence emission intensity was recorded. For acrylamide quenching, tryptophan was excited at 295 nm (rather than 280 nm to avoid absorbance from acrylamide itself) and emission spectra were recorded between 310 and 400 nm with a step size of 1 nm. For quenching within the membrane, dibrominated lipids were introduced into POPC samples at 10, 20, 30, and 40 mol %. Tryptophan was excited at 280 nm, and emission was read between 310 and 400 nm with a step size of 1 nm. Stern−Volmer plots were generated by plotting F0/F vs concentration of the quencher. Larger Stern−Volmer slopes indicate increased quenching of Trp to the given quencher. Liposome Disruption Assay. Terbium (Tb3+)-loaded liposomes were diluted with a buffer containing dipicolinic acid (DPA), and Tb3+ fluorescence was measured using a Photon Technology International fluorimeter and a 1 cm path length quartz cuvette. Tb3+ was excited at 314 nm (2 nm slit width), and the emission was recorded over time at 544 nm (5 nm slit width). Peptides were added to a stirring solution of Tb3+-loaded liposomes in DPA buffer, and fluorescence was recorded for 10 min. Fluorescence of liposomes alone was subtracted from those with added peptide. All samples were normalized to the fluorescence of a 0.1% triton-lysed control.
isolated through centrifugation. Red blood cells were washed in phosphate-buffered saline (PBS) and resuspended to a final concentration of 4% (v/v) in PBS. Two-fold serial dilutions of peptides ranging from 0 to 640 μM were incubated for 1 h at 37 °C with 100 μL of red blood cells for a total volume of 200 μL per well. Nonlysed cells were spun out, and the supernatant was measured for absorbance at 540 nm. Blood cells treated with PBS alone represented the negative control (0% hemolysis), and blood cells treated with 1% triton represented the positive control (100% hemolysis). % hemolysis is reported as % hemolysis = (Abs540 sample − Abs540 negative control) × 100% (Abs540 positive control − Abs540 negative control) Proteolytic Degradation by Proteinase K, Blood Plasma, and Lung Sputum. Peptides (20 μM) were incubated with purified proteinase K (isolated from the fungus Engyodontium album, formerly Tritirachium album, Invitrogen, California) (100 μg/mL) in buffer (10 mM Tris-HCl, 10 mM NaCl, pH 7.4) at 37 °C for up to an hour. At each time point, 5 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol was added to inhibit protease function. For each replicate peptide sample, a corresponding control sample was made with “dead” or preinhibited enzyme (ProtK pretreated with PMSF prior to addition to peptide) to ensure proper inhibition of enzyme activity. Samples were diluted with 4X SDS-PAGE sample-loading buffer and stored at −20 °C. Samples were boiled at 70 °C for 10 min prior to being run on a 12% Bis-Tris NuPAGE gel, 200 V for approximately 35 min. Gels were stained with Coomassie Blue stain for visualization. Percentage peptide remaining was quantitated by measuring the density of bands using ImageJ software.50 Bands were all background subtracted and normalized to the intensity of the band at T0 (no protease present). TAMRA-labeled 6K-F17 and a scrambled variant, 3K-F17scr (control peptide for degradation, sequence: KKKAAFKAAKWKAAFAA-NH2), were used to measure peptide degradation in human blood plasma and in CF patient lung sputum. Blood plasma was separated from red blood cells through centrifugation and immediately frozen and stored at −80 °C until use. Plasma was thawed on ice and diluted with buffer (10 mM Tris-HCl, 10 mM NaCl, pH 7.4) and 40 μM peptide to a final volume of 280 μL (9% v/ v blood plasma). Lung sputum was pooled from various CF patients, filtered to remove bacteria and frozen at −80 °C until use. Sputum was thawed on ice and diluted with buffer (10 mM Tris-HCl, 10 mM NaCl, pH 7.4) and 40 μM peptide to a final volume of 280 μL (70% v/v sputum). Both plasma and sputum samples were incubated at 37 °C with shaking (200 rpm) and protected from light for 24 h. Time points were removed, and protease activity inhibited through addition of 5 mM PMSF. Samples were diluted with SDS-PAGE loading buffer and run on gels as described above. Peptide bands were visualized by exciting the fluorescent TAMRA label. Minimum Inhibitory Concentration (MIC) Assay. Antimicrobial activity was measured following standard microtiter dilution protocols in Mueller-Hinton Broth (MHB). Briefly, 2-fold serial dilutions of peptides ranging from 0 to 50 μM were incubated for 20 h at 37 °C with 50 000 cfu/well of an overnight culture of bacteria grown in MHB. Minimum inhibitory concentrations were recorded as the peptide concentration at which bacterial growth is inhibited, as measured by the OD600. All recorded OD600 readings were background subtracted. Circular Dichroism Spectroscopy. Secondary structure determination was carried out using a Jasco J-720 spectropolarimeter. Peptide concentrations were 25 μM in 10 mM Tris buffer 10 mM NaCl pH 7.4, supplemented with either 2 mg/mL total E. coli lipid extract or 2 mg/mL POPC. Samples were read using a 0.1 cm cuvette path length with five accumulations per run. Spectra represent the average of at least three independent replicates. All spectra were background subtracted and converted to mean residue molar ellipticity using standard formulas.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00657. Table of MIC and hemolysis of Lys-tag shortened peptides (e.g. 5K-F16, 4K-F15, and 3K-F14); Figure of Lys-tag shortened peptide proteinase K degradation; tryptophan blue shifts in the presence of bilayers made from the E. coli lipid extract and POPC lipids (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Charles M. Deber: 0000-0003-4024-4993 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported, in part, by grants to C.M.D. from the Cystic Fibrosis Foundation (United States) (Grant #1610) and from the Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery Grant #05577) and to S.S. from NSERC (Discovery Grant #06146). D.R.-L. was the recipient of a RESTRACOMP Research Studentship Award from the Hospital for Sick Children.
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ABBREVIATIONS 11,12- Br-PC, 1-palmitoyl-2-(11,12-dibromo)stearoyl-sn-glycero-3-phosphocholine; APD, antimicrobial peptide database; CAP, cationic antimicrobial peptide; DPA, dipicolinic acid; HFIP, 1,1,1,3,3,3-hexafluoro2-propanol; POPC, 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine; MHB, mueller-Hinton 6284
DOI: 10.1021/acs.jmedchem.9b00657 J. Med. Chem. 2019, 62, 6276−6286
Journal of Medicinal Chemistry
Article
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broth; TAMRA, 5-carboxytetramethylrhodamine; TFE, 2,2,2,trifluoroethanol
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