Conformational Analysis of the Host-Defense Peptides

Nov 25, 2015 - Peptides from frog skin with potential for development into agents for Type 2 ... Identification of Components in Frog Skin Secretions ...
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Conformational Analysis of the Host-Defense Peptides Pseudhymenochirin-1Pb and -2Pa and Design of Analogues with Insulin-Releasing Activities and Reduced Toxicities Giorgia Manzo,† Mariano Andrea Scorciapino,† Dinesh Srinivasan,‡ Samir Attoub,§ Maria Luisa Mangoni,⊥ Andrea C. Rinaldi,† Mariano Casu,∥ Peter R. Flatt,‡ and J. Michael Conlon*,‡ †

Department of Biomedical Sciences-Biochemistry Unit and ∥Department of Physics, University of Cagliari, I-09042 Monserrato (CA), Italy ‡ SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, U.K. § Department of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University, 17666 Al Ain, United Arab Emirates ⊥ Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, 5 00185 Rome, Italy S Supporting Information *

ABSTRACT: Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membranemimetic solvent 50% (v/v) trifluoroethanol−H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 μM. Increasing cationicity by the substitution Glu17 → L-Lys in Ps-1Pb and Glu27 → LLys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating D-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 μM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 μM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy. A previous study6 has demonstrated that Ps-1Pb and Ps-2Pa exhibit a range of properties that make them promising candidates for drug development. Both peptides show in vitro cytotoxic activity (IC50 < 12 μM) against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB231 cells, and colorectal adenocarcinoma HT-29 cells. Ps-1Pb is active (minimum inhibitory concentration MIC ≤ 20 μM) against reference strains and multi-drug-resistant clinical isolates of both Gram-positive and Gram-negative bacteria. The possibility that the peptides may have a role in cancer immunotherapy is suggested by the observation that Ps-1Pb and Ps-2Pa significantly inhibit production of IL-10, an antiinflammatory cytokine that down-regulates immune response,

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he genus Pseudhymenochirus within the family Pipidae contains the single species, Merlin’s clawed frog, Pseudhymenochirus merlini.1 Norepinephrine-stimulated skin secretions produced by this species contain multiple peptides with antibacterial and antifungal activity that are a component of the system of innate immunity that protects the animal from invasion by pathogenic microorganisms within the environment.2 Ten of these peptides show moderate sequence identity with the hymenochirins isolated from skin secretions of the sister-group species Hymenochirus boettgeri.3 However, pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) show little structural similarity to any other amphibian host-defense peptide including those previously isolated from frogs belonging to the family Pipidae.4,5 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 21, 2015

A

DOI: 10.1021/acs.jnatprod.5b00843 J. Nat. Prod. XXXX, XXX, XXX−XXX

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from mouse peritoneal macrophages while enhancing production of the pro-inflammatory cytokine IL-23. However, the therapeutic potential of the pseudhymenochirins, particularly for systemic applications, is seriously limited by their high hemolytic activity and cytotoxicity against non-neoplastic cells, such as human umbilical vein endothelial (HUVEC) cells.6 The current pandemic of type 2 diabetes constitutes a serious crisis in public health. Incretin-based therapies employing naturally occurring peptides that stimulate insulin release in response to elevated circulating glucose concentrations are receiving increasing attention for treatment of patients with this disease.7 Although regimens based upon administration of longacting analogues of glucagon-like peptide-1 (GLP-1) have met with considerable success, side effects such as nausea and vomiting in older patients and safety issues such as pancreatitis, C-cell hyperplasia, and renal failure indicate the need for new peptide-based therapeutic agents.8 Several host-defense peptides from frog skin that were first identified as a result of their antimicrobial and/or cytotoxic properties have subsequently been shown to stimulate insulin release from BRIN-BD11 clonal β-cells in vitro.9 The peptides are effective in vivo and improve islet function, glycemic control, and insulin sensitivity when administered to mice with diet-induced obesity and insulin resistance.10,11 Consequently, such peptides show potential as templates for development into agents for treatment of patients with type 2 diabetes. The initial aim of the present study was to assess the abilities of synthetic replicates of Ps-1Pb and Ps-2Pa to stimulate the rate of insulin release from BRIN-BD11 cells. In view of the high degree of toxicity of the pseudhymenochirins, it is necessary to design analogues that retain insulin-releasing activity but show reduced cytotoxicity to mammalian cells. To this end, the preferred conformations of Ps-1Pb and Ps-2Pa in the membrane-mimetic solvent 50% trifluoroethanol (TFE)− H2O were determined by NMR studies. The demonstration that both peptides adopt extended α-helical conformations led to the design of analogues containing helix-destabilizing Damino acid substitutions that display the desired properties of potent insulin-releasing activity combined with low toxicity and thus show potential as antidiabetic agents.

Figure 1. Hydropathicity plot of Ps-1Pb and Ps-2Pa. The plot was calculated using the method of Kyte and Doolittle12 with a window of three residues and a weight of 50% at the window edges. The values are normalized to 1.0.

secondary structural features. In the membrane-mimetic solvent 50% v/v H2O−TFE, application of the TALOS+ software15 to the experimental chemical shift values (Table S1) resulted in the prediction of a helical folding for both peptides. The only exceptions were the first three residues. In the case of Ps-1Pb, predicted angles for these residues received a low consensus score and were thus not included as restraints in the subsequent structural calculation. On the other hand, in the case of Ps-2Pa they were predicted with a good consensus and thus have been taken into account. Prediction of backbone angles is shown in Figure 2 on the Ramachandran plot together with the statistical uncertainty. The interproton through-space dipolar couplings obtained from the analysis of NOESY spectra are shown schematically in Figure 3. A different thickness is used for lines indicating strong, medium, and weak NOEs. A total of 96 and 101 interresidue NOEs were found for Ps-1Pb and Ps-2Pa, respectively. In both cases, short (residue i to i + 1) and medium (residue i to i + 2, 3, 4) range NOEs were found all along the sequence. The high proportion of Hα-HN NOEs with medium intensity bolsters the hypothesis of a helical conformation for both peptides. The NMR structural investigation was performed in an isotropic environment (50% v/v H2O−TFE), which is reported to shift the pre-existing folded/unfolded equilibrium toward the more structured conformation.16−19 The TFE clusters assist peptide folding by promoting interactions between the hydrophobic amino acid side chains so that the formation of backbone intramolecular hydrogen bonds is promoted.20 Both the backbone angle prediction obtained from TALOS+ (Figure 2) and the high number of sequential NOEs (Figure 3), with a high proportion of Hα-HN dipolar interactions, support this secondary structure motif. The 3D structures obtained for Ps-1Pb and Ps-2Pa are depicted schematically in Figures 4 and 5, respectively. The conformer with the lowest root-mean-square deviation (RMSD) from the average structure is shown in panel A.

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RESULTS AND DISCUSSION Conformational Analysis. The hydropathicity plots of Ps1Pb and Ps-2Pa are shown in Figure 1. Residues are ranked on the basis of their hydrophobicity,12 and the scale is normalized to 1.0. The pattern of two hydrophobic and two hydrophilic residues alternating along the sequence is evident for both peptides, suggesting the tendency to fold as stable amphipathic helices in the presence of a (phospho)lipid membrane or in a membrane-mimicking environment. For both peptides, such a pattern starts from the N-terminal end (Phe-6 for Ps-1Pb; Ile-5 for Ps-2Pa). Ps-1Pb has a marked amphipathic profile but with a relatively long C-terminal portion of hydrophobic residues, starting from residue Ala-18. In the case of Ps-2Pa, the binary alternating pattern is more extended toward the C-terminus and the average hydrophobicity is significantly higher but with a strongly hydrophilic C-terminal tail starting from residue Ser22. 1 H and 13C resonance assignments were obtained through the analysis of a series of 2D spectra (DQF-COSY, TOCSY, NOESY, and 1H−13C-HSQC)13,14 collected in H2O and 50% v/v H2O−TFE. In H2O, the spectra of both Ps-1b and Ps-2a showed poor chemical shift dispersion, indicating the lack of B

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The 3D structures resolved at the atomic level indicate a similar folding for the two peptides. Both Ps-1Pb and Ps-2Pa fold as an α-helix, incorporating a bend that separates a longer N-terminal domain with a clear segregation of hydrophobic and hydrophilic side chains on opposite sides of the helix and a shorter C-terminal tail. The Ps-2Pa conformation is more rigid than that of Ps-1Pb and is endowed with a markedly hydrophilic C-terminal domain. On the other hand, Ps-1Pb has a more pronounced amphipathicity but is characterized by a significantly higher flexibility than Ps-2Pa. The presence of a flexible bend is a feature common to many amphibian helical antimicrobial peptides such as hymenochirin-1Pa,19 esculentin1b(1−18),21 cecropin P1,22 cecropin A(1−8)−magainin 2(1− 12) hybrid,23 caerin 1.1,24 gaegurin-4,25 and maculatin 1.126 and is an important determinant of high antimicrobial potency. Insulin-Releasing Activities. Effects of the pseudhymenochirins on the rate of insulin release were studied using BRIN-BD11 rat clonal β-cells. The BRIN-BD11 cell line is a well-established model that has been widely used to study the effects of incretin agents on insulin release.27 In the first series of experiments, the basal rate of insulin release from BRINBD11 cells in the presence of 5.6 mM glucose alone was 0.97 ± 0.05 ng/106 cells/20 min. In the presence of the wellestablished insulin secretagogue 10 mM alanine, the rate of insulin release increased to 4.9 ± 0.2 ng/106 cells/20 min (p < 0.001). Ps-1Pb produced a concentration-dependent increase in the rate of insulin release with a threshold concentration [minimum concentration producing a significant (p < 0.05) increase in the rate of insulin release over the basal rate in the presence of 5.6 mM glucose only] of 0.1 nM and a mean response of 530% of the basal rate (p < 0.001) at 3 μM (Figure 6). However, at concentrations > 1 μM, Ps-1Pb produced a significant increase in the release of the cytosolic enzyme lactate dehydrogenase (LDH), indicative of permeabilization of the plasma membrane. The threshold concentration of Ps-2Pa was also 0.1 nM, and the near-maximal response to 3 μM was 700% of the basal rate (p < 0.001) (Figure 7). However, the peptide produced an even greater degree of membrane permeabilization than Ps-1Pb at concentrations > 1 μM, as indicated by a greater rate of release of LDH. Studies with a range of amphibian antimicrobial peptides have shown that increasing cationicity while maintaining amphipathicity, either naturally or by appropriate amino acid substitutions, frequently results in greater antimicrobial potency.3−6 However, increasing cationicity by the substitution Glu17 → L-Lys in Ps-1Pb and Glu27 → L-Lys in Ps-2Pa increased the threshold concentration for increasing the rate of insulin release (to 0.3 nM for [E17K]Ps-1b and to 1 nM for [E27k]Ps2Pa), and the analogues produced a similar degree of membrane permeabilization at concentrations ≥ 1 μM as the native peptides (Figure 7). While the study has shown that Ps-1Pb and Ps-2Pa stimulate insulin release from BRIN-BD11 clonal β-cells at low concentrations (≥0.1 nM), the high toxicity of the peptides precludes their use as therapeutic agents, at least for systemic applications. Consequently, in order to realize their potential as antidiabetic agents, it is imperative to design nontoxic analogues. The antimicrobial activities of cationic α-helical peptides and their ability to disrupt the plasma membrane of eukaryotic cells are dependent upon complex interactions between cationicity, hydrophobicity, conformation (helix stability), and amphipathicity.28 Studies with analogues of naturally occurring and model cationic amphipathic, α-helical

Figure 2. Ramachandran plot for (A) Ps-1Pb and (B) Ps-2Pa. Values of the backbone torsional angles were determined with TALOS+ software on the basis of the analysis of experimentally determined NMR chemical shift values. Only the high-consensus predictions are shown together with the corresponding uncertainty.

The α-helical folding is evident for both peptides, encompassing almost the entire sequence. Ps-1Pb is characterized by a bend around Ser-20. Ps-2Pa (Figure 5A) displayed a similar folding (with a significant contribution of 310 helix from residue 21 to 26, reflected by the lack of Hα-HN(i, i + 4) NOEs in this region) with the bend around residue Ile-21. Figures 4B and 5B show the amphipathic character of the two peptides. In the case of Ps-1Pb the net segregation of hydrophilic and hydrophobic residues on opposite sides of the 3D structure is clearly observed all along the sequence. In the case of Ps-2Pa, the Cterminal region is mostly hydrophilic. When the different conformers obtained with the simulating annealing were aligned using the whole backbone, Ps-2Pa showed an RMSD ranging from 0.4 to 1.8 Å with respect to the average structure, while values up to 3.2 Å were found in the case of Ps-1Pb. This was not due to poor resolution of the 3D structure but to the high mobility of the two fragments separated by the helical bend. When the two regions were separately aligned and the corresponding RMSD was recalculated, the 0.3−1.3 Å and 0.1− 0.6 Å ranges were found for the N-terminal and the C-terminal domain, respectively. C

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Figure 3. Interproton dipolar through-space interactions (NOEs) for (A) Ps-1Pb and (B) Ps-2Pa. Short- and medium-range NOEs are reported as lines connecting the two residues involved. The thickness of the lines is proportional to the relative intensity of the corresponding NOESY crosspeak, categorized into three classes: strong, medium, and weak.

Figure 4. Schematic representation of the three-demensional structure of Ps-1Pb. The structure was obtained on the basis of the NMRderived geometrical restraints. The backbone trace of the conformer with the lowest RMSD from the average structure is shown in panel A. Panel B shows the same conformer with the hydrophobic and hydrophilic residues represented as van der Waals spheres and colored in green and blue, respectively.

Figure 5. Schematic representation of the 3D structure of Ps-2Pb. Details are the same as in Figure 4.

relevant bacteria33 but shows appreciably decreased hemolytic activity. In a second series of experiments, the basal rate of insulin release from BRIN-BD11 cells in the presence of 5.6 mM glucose alone was 1.05 ± 0.03 ng/106 cells/20 min. The rate increased to 4.2 ± 0.4 ng/106 cells/20 min (p < 0.001) in the presence of 10 mM alanine and to 3.3 ± 0.2 ng/106 cells/20 min (p < 0.001) in the presence of 1 μM GLP-1(7−36)amide. Decreasing the stability of the α-helical domain of Ps-1Pb without changing cationicity by the substitution L-Arg8 → D-Arg resulted in an analogue that did not stimulate the release of LDH at either 1 or 3 μM concentration (Figure 8). The threshold concentration of [R8r]Ps-1Pb for effects on insulin release was 0.3 nM, and a concentration of 3 μM produced a 190% increase over the basal rate. The double substitution LLys8 → D-Lys and L-Lys19→ D-Lys within the α-helical domain

peptides have shown a positive correlation between hemolytic activity and hydrophobicity, stability of the helix, and hydrophobic moment (a semiquantitative measure of amphipathicity).29 A successful strategy for producing frog skin peptides with decreased cytotoxicity against human cells, such as erythrocytes, has been to incorporate D-amino acid residues into the molecule at appropriate sites in order to destabilize the helix while maintaining amphipathicity.30,31 For example, incorporation of D-lysine residues at positions 6 and 9 of hymenochirin-1B generates an analogue that retains cytotoxic potency against a range of tumor cell lines32 and clinically D

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Figure 6. Effects of Ps-1Pb on (A) insulin and (B) LDH and effects of Ps-2Pa on (C) insulin and (D) LDH release from BRIN-BD11 cells. Values are mean ± SEM with n = 8 for insulin and n = 6 for LDH. ***p < 0.001, **p < 0.01, *p < 0.05 compared to 5.6 mM glucose alone.

Figure 7. Effects of [E17K]Ps-1Pb on (A) insulin and (B) LDH and effects of [E27K]Ps-2Pa on (C) insulin and (D) LDH release from BRIN-BD11 cells. Values are mean ± SEM with n = 8 for insulin and n = 6 for LDH. ***p < 0.001, **p < 0.01, *p < 0.05 compared to 5.6 mM glucose alone.

of Ps-2Pa increased the insulin-releasing potency (threshold concentration = 0.01 nM), and the analogue produced no significant increase in the rate of LDH release at concentrations up to and including 3 μM. At this concentration, the analogue increased the rate of insulin release to 200% of the basal rate (Figure 8). The mechanism of action of the pseudhymenochirins was not investigated in this study, but work with other amphibian cationic α-helical peptides has indicated that stimulation of insulin release is mediated through membrane depolarization and activation of the KATP-dependent pathway with resulting increase in intracellular Ca2+ concentrations.34,35

Cytotoxicity Studies. Peptides of amphibian origin have been proposed as potential anticancer agents,36,37 but such compounds generally suffer from the disadvantage of lack of selectivity for tumor cells compared with non-neoplastic cells. [R8r]Ps-1b retains cytotoxic activity against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB231 cells, and colorectal adenocarcinoma HT-29 cells, but potency is decreased 3- to 4-fold compared to the native peptide (Table 1). The potency of the peptide against nonneoplastic HUVEC cells is comparable to that against MDAE

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Antimicrobial Activities. D-Amino acid substitutions have a pronounced effect on the selectivity of Ps-1Pb and Ps-2Pa for Gram-negative and Gram-positive bacteria. [R8r]Ps-1b and [K8k,K19k]Ps-2Pa displayed increased antimicrobial activity against reference strains of the clinically relevant Gram-negative bacterial pathogens Escherichia coli and Pseudomonas aeruginosa compared with the naturally occurring peptides, with [R8r]Ps1Pb 2-fold more potent than [K8k,K19k]Ps-2Pa against both microorganisms (Table 2). However, lower antimicrobial activity was exhibited by [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis.

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CONCLUSIONS The strategy of introducing D-amino acid residues into the αhelical domains of Ps-P1b and Ps-2Pa has been successful in generating analogues that retain incretin activity but are nontoxic to BRIN-BD11 cells at a concentration that produces a near-maximum response in insulin release and display low hemolytic activity. Consequently, the peptides show promise as templates for the design of long-acting analogues for use in the treatment of patients with type 2 diabetes. [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa also retain broad-spectrum antimicrobial activity against clinically relevant bacteria and thus show potential for development into new therapeutics to combat the increasing emergence of resistance to available antimicrobial drugs.

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EXPERIMENTAL SECTION

General Experimental Procedures. All peptides were supplied in crude form by EZ Biolab and were purified to near homogeneity (98%) by reversed-phase HPLC as previously described.6 All peptides showed high solubility in physiological buffers. Hydropathicity plots for both peptides were obtained using the ProtScale tool available on the ExPaSy server at http://www.expasy. org/. The values were calculated applying the method of Kyte and Doolittle12 with a window size of three residues and a relative weight of the window edges of 50%. NMR spectra were acquired at 300 K with a Varian Unity INOVA 500 high-resolution spectrometer operating at a 1H frequency of 500 MHz. Deuterated trifluoroethanol (CF3CD2OD; TFE-d3) and D2O were purchased from Sigma-Aldrich with a purity ≥ 99%. Deuterated 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid (TSP-d4) was purchased from Cambridge Isotope Laboratories with a purity of 98%. Ps-1Pb and Ps-2Pa were dissolved in 700 μL of 50% (v/v) H2O−TFEd3 at a final concentration of 2 mM. The chemical shift scales of both 1 H and 13C were referred to the methyl signal of TSP, added as internal reference at a concentration of 2 mM. 1H spectra were acquired using a 6.7 s pulse (90°), 1 s delay time, 1 s acquisition time, and a spectral width of 6.5 kHz. The WET sequence38,39 (uburp shape centered at water resonance with a width of 100 Hz) was applied to suppress the water signal. 2D experiments (1H−1H DQF-COSY, 1 H−1H TOCSY, and 1H−1H NOESY) were recorded over the same spectral window using 2048 complex points and sampling each of the 512 increments with 48 scans. Mixing times of 80 and 200 ms were applied for the TOCSY and NOESY experiments, respectively. The 1 H−13C HSQC spectra were recorded using a spectral window of 6.5 kHz for 1H and 22 kHz for 13C and sampling each of the 512 increments with 48 scans. TALOS+ software was applied to analyze 1Hα, 1Hβ, 13Cα, and 13Cβ chemical shift values.15 Briefly, the software compares the experimental values with its high-resolution structural database and provides statistical estimates of both Φ and Ψ backbone angles. Only the predictions ranked as “good” were used as restraints for structure calculation. The 3D structures of both Ps-1Pb and Ps-2Pa were obtained using a simulated annealing protocol through the Dynamo

Figure 8. Effects of [R8r]Ps-1Pb on (A) insulin and (B) LDH and effects of [K8k,K19k]Ps-2Pa on (C) insulin and (D) LDH release from BRIN-BD11 cells. Values are mean ± SEM with n = 8 for insulin and n = 6 for LDH. ***p < 0.001, **p < 0.01, *p < 0.05 compared to 5.6 mM glucose alone.

MB-231 and HT-29 cells, indicating a lack of selectivity for tumor cells. A similar decrease in cytotoxic potency (3- to 5fold) of [K8k,K19k]Ps-2Pa for MDA-MB-231 and HT-29 cells was seen, but the analogue was equipotent with Ps-2Pa against A549 cells. The peptide shows some selectivity toward lung and breast tumor cells compared with HUVEC cells but not toward HT-29 cells (Table 1). In contrast to the naturally occurring peptides and analogues containing L-amino acid substitutions, the D-amino acid-substituted peptides showed only weak hemolytic activity against mouse erythrocytes (IC50 > 100 μM). F

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Table 1. Cytotoxicities of Ps-1Pb and Ps-2Pba peptide

A549

Ps-1b [E17k]Ps-1b [R8r]Ps-1b Ps-2Pa [E27K]Ps-2a [k8k,K19k]Ps-2a

2.5 2.1 7.2 6.0 2.5 6.5

± ± ± ± ± ±

MDA-MB-231

0.2 0.7 1.5 0.6 0.5 1.1

6.6 3.6 20 6.2 4.3 17

± ± ± ± ± ±

HT-29

0.3 0.5 4 0.6 0.2 2

9.5 11 35 12 5.6 58

± ± ± ± ± ±

1.3 2 5 3 0.4 4

HUVEC 5.6 5.0 27 68 5.0 56

± ± ± ± ± ±

0.9 0.5 3 2 0.4 5

RBC 30 19 >100 7 4 >100

±5 ±4 ±2 ±1

a

A549: lung adenocarcinoma cells, MDA-MB-231: breast adenocarcinoma cells, HT-29: human colorectal adenocarcinoma cells, HUVEC: human umbilical vein endothelial cells, and RBC: mouse red blood cells. Data show mean IC50 values (μM) ± SEM. The data for Ps-1Pb and Ps-2Pa are taken from Mechkarska et al.6 in three incubations. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63 EU for animal experiments. Antimicrobial Assays. The following reference strains of microorganisms, purchased from the American Type Culture Collection, were used in the assays: the Gram-negative bacteria Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 and the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228. Minimum inhibitory concentrations (MIC) of the peptides were determined in three independent experiments by the standard microdilution method41 as previously described.42 Statistical analyses were performed using commercially available GraphPad Prism software version 5.01. Results are expressed as mean ± standard error of mean (SEM), and values were compared using two-way analysis of variance followed by Newman-Keuls post hoc test. Groups of data were considered to be significantly different if p < 0.05.

Table 2. Minimum Inhibitory Concentrations (μM) against Reference Strains of Gram-Negative and Gram-Positive Bacteriaa

a

bacteria

Ps-1Pb

[R8r]Ps-1Pb

Ps-2Pa

[K8k,K19k]Ps-2Pa

E. coli P. aeruginosa S. aureus S. epidermidis

10 20 5 2.5

2.5 10 80 40

80 80 5 5

5 20 20 40

The data for Ps-1Pb and Ps-2Pa are taken from Mechkarska et al.6

software (http://spin.niddk.nih.gov/NMRPipe/dynamo/). Unambiguous NOEs and backbone angles from TALOS+ were used as interproton distances and torsional angle restraints, respectively. In particular, NOEs were classified as strong, medium, and weak on the basis of the relative intensity of the cross-peaks in the NOESY spectra, and upper limits of 0.27, 0.33, and 0.50 nm, respectively, applied. The potential energy contribution was zero below the upper limit, while a harmonic potential was applied above. One thousand structures were calculated, and the 100 conformers with the lowest potential energy were selected for the analysis. Solvent molecules were not included in the calculations. Determination of Insulin-Releasing Activity. BRIN-BD11 cells, maintained in culture as previously described,34 were seeded into 24well plates and allowed to attach during overnight incubation at 37 °C. Incubations with purified synthetic peptides (10−11 to 3 × 10−6 M; n = 8) were carried out for 20 min at 37 °C in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 5.6 mM glucose as previously described.34,35 After incubation, aliquots of cell supernatant were removed for insulin radioimmunoassay.40 In order to investigate the effects of the peptides on the integrity of the plasma membrane, monolayers of BRIN-BD11 cells were incubated with peptides (10−11 to 3 × 10−6 M; n = 6) for 20 min at 37 °C using KRB buffer supplemented with 5.6 mM glucose as previously described.34,35 Lactate dehydrogenase concentrations in the cell supernatants were measured using a CytoTox96 nonradioactive cytotoxicity assay kit (Promega) according to the manufacturer’s protocol. Cytotoxicity Studies. Human non-small-cell lung adenocarcinoma A549 cells, human breast adenocarcinoma MDA-MB-231 cells, human colorectal adenocarcinoma HT-29 cells, and EndoGRO human umbilical vein endothelial cells were maintained at 37 °C in culture medium supplemented with antibiotics as previously described.6,32 Cells at a density of 5 × 103 cells/well were incubated for 24 h at 37 °C with peptides (10−6−10−4 M) in triplicate as described.6,32 The effect of the peptides on cell viability was determined by measurement of ATP concentrations using a CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation) and a GLOMAX Luminometer system. The IC50 value was taken as the mean concentration of peptide producing 50% cell death in a minimum of three independent experiments. The hemolytic activities of the peptides (1.6 × 10−6 to 10 × 10−6 M) against erythrocytes from male NIH male Swiss mice (Harlan Ltd.) were determined as previously described.6 The IC50 value was taken as the mean concentration of peptide producing 50% hemolysis

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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.jnatprod.5b00843. Table S1 with 1H and 13C resonance assignments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44-7918526277. Fax: +44(0)2870124965. E-mail: m. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funds from the Sardinia Regional Government 525, L.R. 7/2007, bando 2009 (grant no. CRP17385), Sapienza University of Rome (grant no. C26A14STJZ), the SAAD Contract and Trading Company, Ulster University Strategic Research Funding, and U.A.E. National Research Foundation. The authors thank K. Arafat and S. Sulaiman, U.A.E. University, for technical assistance.

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REFERENCES

(1) Frost, D. R. Amphibian Species of the World: An Online Reference, Version 6.0. Electronic Database. American Museum of Natural History: New York, 2015; http://research.amnh.org/herpetology/ amphibia/index.php (accessed September 2015). (2) Conlon, J. M.; Prajeep, M.; Mechkarska, M.; Coquet, L.; Leprince, J.; Jouenne, T.; Vaudry, H.; King, J. D. Comp. Biochem. Physiol., Part D: Genomics Proteomics 2013, 8, 352−357. (3) Mechkarska, M.; Prajeep, M.; Coquet, L.; Leprince, J.; Jouenne, T.; Vaudry, H.; King, J. D.; Conlon, J. M. Peptides 2012, 35, 269−275. (4) Conlon, J. M.; Mechkarska, M. Pharmaceuticals 2014, 7, 58−77. G

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DOI: 10.1021/acs.jnatprod.5b00843 J. Nat. Prod. XXXX, XXX, XXX−XXX