Purification, Conformational Analysis, and Properties of a Family of

Aug 25, 2016 - The tigerinin family of peptides was first identified in the skin of the Indian ... Nevertheless, all the amino acids were predicted to...
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Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis Christopher M. McLaughlin,†,# Sandrina Lampis,‡,# Milena Mechkarska,†,∞ Laurent Coquet,§ Thierry Jouenne,§ Jay D. King,⊥ Maria Luisa Mangoni,∥ Miodrag L. Lukic,¶ Mariano A. Scorciapino,‡ and J. Michael Conlon*,† †

SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, U.K. Department of Chemical and Geological Sciences and Department of Biomedical Sciences, Biochemistry Unit, University of Cagliari, Cagliari, Italy § CNRS UMR 6270, PISSARO, University of Rouen, Institute for Research and Innovation in Biomedicine (IRIB), Mont-Saint-Aignan, France ⊥ Rare Species Conservatory Foundation, St. Louis, Missouri, United States ∥ Instituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy ¶ Center for Molecular Medicine, Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia

J. Nat. Prod. 2016.79:2350-2356. Downloaded from pubs.acs.org by UNIV OF LEEDS on 09/24/18. For personal use only.

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S Supporting Information *

ABSTRACT: Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 μM) and hemolytic (LC50 > 500 μM) activities but, at a concentration of 20 μg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 μg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal β-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 μM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 μM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes. Tigerinin-1R, first isolated from an extract of the skin of the Vietnamese common lowland frog Hoplobatrachus rugulosus6 in the family Dicroglossidae, is one such amphibian peptide that has excited interest as a lead compound for drug development in the treatment of patients with type 2 diabetes. Largely devoid of antimicrobial and cytotoxic properties, tigerinin-1R6 and its analogues7,8 stimulate insulin release from BRIN-BD11 clonal β-cells and the release of glucagon-like peptide-1 (GLP-1) from the GLUTag cell line9 at concentrations that are not toxic to the cells. Studies in vivo have shown that tigerinin-1R10 and its analogues8,11 enhance both insulin sensitivity and pancreatic βcell function and decrease adiposity and plasma triglycerides

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eptidomic analysis of frog skin secretions has proved to be valuable in identifying components with therapeutic potential for development into antimicrobial agents for use against drug-resistant bacteria and fungi as well as those with anticancer and antiviral properties. In addition, peptides that were first identified on the basis of their cytotoxic activities have subsequently been shown to display immunomodulatory and antidiabetic activities.1,2 Comparisons of the primary structures of the host-defense peptides in skin secretions have provided insight into the evolutionary history of species within particular families, such as the Ranidae,3 Leptodactylidae,4 and the Pipidae5 and may be used to complement data derived from the nucleotide sequences of mitochondrial and/or nuclear genes in elucidating phylogenetic relationships. © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 29, 2016 Published: August 25, 2016 2350

DOI: 10.1021/acs.jnatprod.6b00494 J. Nat. Prod. 2016, 79, 2350−2356

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tigerinin-4O (10). The values in parentheses show the approximate yields of the purified peptides in nanomoles. The amino acid sequences of the tigerinin peptides were established by automated Edman degradation, and their primary structures are shown in Figure 2. The molecular masses of the peptides, determined by MALDI-TOF mass spectrometry, were consistent with the proposed structures and demonstrate that the tigerinins were isolated in the oxidized form with a cysteine bridge and are not C-terminally αamidated. The tigerinin family of peptides was first identified in the skin of the Indian frog Hoplobatrachus tigerinus (formerly classified as Rana tigerina) in the family Dicroglossidae 18 and subsequently in the Asian frogs Hoplobatrachus rugulosus6 and Fejervarya cancrivora19 in the same family. Fejervarya is regarded as sister taxon to a clade containing Hoplobatrachus + Euphlyctis.20 As shown in Figure 3, small cyclic peptides with limited structural similarity to the tigerinins have been isolated from skin secretions of the Costa Rican frog Lithobates vaillanti in the family Ranidae21 and the East-African Mueller’s clawed frog Xenopus muelleri in the family Pipidae,22 but their evolutionary relationship to tigerinins isolated from species belonging to the Dicroglossidae is unknown. This study has led to the purification of four peptides in skin secretions of H. occipitalis whose primary structures identify them as members of the tigerinin family. In common with the vast majority of frog skin host-defense peptides, they are cationic (isolectric point pI = 8.23) and appreciably hydrophobic (Figure 2). Mass spectrometry indicated that tigerinin-4O was isolated as a dimer (observed molecular mass 2486.4 Da), but the synthetic replicate behaved as a monomer (observed molecular mass 1243.3 Da). The reason for the anomalous behavior of the naturally occurring peptide is unclear, but, as one possibility, an intermolecular disulfide bond may have been formed at some stage during the biosynthetic pathway. The genus Hoplobatrachus within the extensive subfamily Dicroglossinae (168 species) of the family Dicroglossidae comprises five species. H. crassus, H. litoralis, H. rugulosus, and H. tigerinus are distributed in Asia, with H. occipitalis being the only African species.14 Firm molecular evidence for monophyly of the Asian Hoplobatrachus has been provided, but the position of H. occipitalis was not fully clarified.23 However, the Asian and African species share many morphological and osteological features in both the adult frogs and the tadpoles. 24 Mitochondrial DNA analysis has suggested the “out of Asia” hypothesis that the genus has an oriental origin with H. occipitalis migrating to Africa in the Miocene (between 8 and 25 million years ago).24 The presence of tigerinin peptides with appreciable structural similarity to those from the Asian species in H. occipitalis skin secretions supports the proposal that all five species within the Hoplobatrachus share a common origin. Conformational Analysis. 1H and 13C resonance assignments were obtained through the analysis of a series of 2D spectra (DQF-COSY, TOCSY, NOESY, and 1 H− 13 CHSQC)25 collected from an aqueous solution of tigerinin-2O at pH 4.8 (Table S1). The presence of the disulfide bridge (Cys3−Cys11) was confirmed by the characteristic chemical shift of the cysteine residues’ Cβ, reflecting their oxidized state.26 Measured chemical shift values of 1HN, 1Hα, 1Hβ, 13 Cα, and 13Cβ for all the residues were analyzed with TALOS+ software,27 which provides predicted Φ and Ψ backbone torsion angles by comparing the experimental chemical shift values to its reference protein structure data set. Only

when administered to mice given a high-fat diet to produce obesity and insulin resistance. In addition, tigerinin-1R increases production of the anti-inflammatory cytokine interleukin-10 (IL-10) in both spleen cells from C57BL/6 mice and human peripheral blood mononuclear cells without stimulating production of the pro-inflammatory cytokines interleukin-12 (IL-12) and interleukin-23 (IL-23),12 suggesting a possible role in the treatment of sepsis. The African crowned bullfrog Hoplobatrachus occipitalis (Günther, 1858), also known as the Eastern groove-crowned bullfrog, occupies broad but disjunctive ranges in the African savannah zone with a swath in North Africa that includes portions of Morocco, Algeria, and Libya and a much larger range in sub-Saharan Africa extending from the Atlantic coast of West Africa eastward to Ethiopia, Chad, Eritrea, and Sudan and south to Angola and Mozambique.13,14 It is tolerant of a wide variety of habitats and is common over much of its range. It is listed as a Species of Least Concern by the International Union for Conservation of Nature (IUCN) Red List, although populations have been depleted in certain areas by loss of habitat and human consumption.15 The present study extends the previous work with tigerinin-1R by describing the isolation, structural characterization, conformational analysis, and biological activities of peptides belonging to the tigerinin family that are present in norepinephrine-stimulated skin secretions of H. occipitalis.

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RESULTS AND DISCUSSION Purification and Characterization of the Peptides. The pooled skin secretions from H. occipitalis, after partial purification on Sep-Pak C-18 cartridges,16 were chromatographed on a Vydac C-18 preparative reversed-phase HPLC column (Figure 1). The well-resolved peaks designated 1−4

Figure 1. Reversed-phase HPLC on a preparative Vydac C-18 column of skin secretions from H. occipitalis after partial purification on SepPak cartridges. The peaks designated 1−4 contained tigerinins and were purified further. The dashed line shows the concentration of CH3CN in the eluting solvent.

were purified to near homogeneity, as assessed by a symmetrical peak shape and a single component after mass spectrometry, by further chromatography on semipreparative Vydac C-4 and Vydac C-8 columns. Subsequent structural analysis revealed that peak 1 contained tigerinin-1O (125), peak 2 tigerinin-2O (60), peak 3 tigerinin-3O (55), and peak 4 2351

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Figure 2. Primary structure, observed molecular mass ([M + H]obs), calculated molecular mass ([M + H]calc), isoelectric point (pI), and hydrophobicity (H) of the peptides isolated from H. occipitalis skin secretions and their synthetic replicates. H is calculated using the hydrophobicity scales for amino acids of Kyte and Doolittle.17

shown in Figure 4b, a rather rigid and stretched rim-like structure, whose consistency is confirmed by a root-meansquare deviation (RMSD) of just 0.96 ± 0.38 Å, is indicated. Figure 4c represents a side view of the same 30 conformers in which side chain residues are shown for completeness and the bent conformation of the peptide backbone appears clear. The heavy atoms’ RMSD was 2.309 ± 0.705 Å. The orientations of all the residues except Arg1 appear consistent among the conformers analyzed, and an RMSD as low as 1.352 ± 0.310 Å was obtained by neglecting the first residue. Thus, while the ring portion of the peptide is somewhat rigid, it is endowed with a flexible, hydrophilic N-terminal tail. Tigerinin-2O is highly amphipathic, with complete segregation of the hydrophobic and hydrophilic residues (Figure 4d). Figure 5 shows schematically the tigerinin-2O sequence with the disulfide bridge and the experimental NOE connectivities determined in this work. Cytotoxic Activities. No tigerinin peptide showed significant hemolytic activity against mouse erythrocytes at concentrations up to 500 μM or growth inhibitory activity against the Gram-positive bacterium Bacillus megaterium and the Gram-negative bacterium Escherichia coli at concentrations up to 100 μM. The tigerinin peptides from H. tigerinus28 and F. cancrivora19 are reported to possess broad-spectrum antimicrobial activity, whereas tigerinin-1R is not active against Grampositive Staphylococcus aureus or Gram-negative E. coli at concentrations up to 500 μM.7 The reason for this discrepancy is unclear but may be a consequence of the different methodologies used to assay for antimicrobial activity. The lack of cytotoxicity of the tigerinin-O peptides is consistent with the report of Srinivasan et al.7 demonstrating a similar lack of activity for tigerinin-1R. Insulin and GLP-1 Release. The basal rate of insulin release from BRIN-BD11 glucose-responsive clonal β-cells29 in the presence of 5.6 mM glucose alone was 1.00 ± 0.03 ng/106 cells/20 min. The rate of insulin release increased to 3.05 ± 0.05 ng/106 cells/20 min (P < 0.001) by incubation with 10 mM alanine, to 3.14 ± 0.09 ng/106 cells/20 min (P < 0.001) by incubation with 1 μM GLP-1, and to 4.05 ± 0.17 ng/106 cells/ 20 min (P < 0.001) by incubation with 30 mM KCl. In common with tigerinin-1R from H. rugulosus and the more potent analogues [I10W]tigerinin-1R and [S4R]tigerinin-1R,7 the tigerinins from H. occipitalis produced a concentrationdependent stimulation of insulin release from BRIN-BD11 clonal β-cells. As shown in Figure S1, all tigerinins produced a significant (P ≤ 0.01) increase in the rate of insulin release at the maximum concentration tested (1 μM). Tigerinin-1O was

Figure 3. Comparison of the primary structures of tigerinin peptides from the Asian frogs from the Dicroglossidae (H. tigerinus, H. rugulosus, and F. cancrivora) with those from the African frog H. occipitalis. The primary structures of peptides with limited sequence similarity to the tigerinins from L. vaillanti (Ranidae) and X. muelleri (Pipidae) are also shown. aThe peptide is C-terminally α-amidated. Shading is used to indicate conserved amino acid residues. Gaps, denoted by an asterisk (*) are inserted into some sequences to maximize structural similarity.

predictions with a high consensus were used for structure calculations. Given the short sequence of tigerinin-2O, no specific secondary structure was assigned by TALOS+. Nevertheless, all the amino acids were predicted to fall within the allowed regions of the Ramachandran plot (Figure 4a) except for the N- and C-terminal residues. The analysis of NOESY spectra provided evidence for interproton throughspace dipolar interactions. On the basis of the relative intensity observed for the corresponding cross-peak, interproton distances were estimated and used as restraints within peptide structure calculations. The DYNAMO-simulated annealing scheme was applied to finally obtain 300 structures compatible with both backbone torsions and interproton distances derived from the NMR experiments. The 30 conformers with the lowest potential energy were then extracted and analyzed. As 2352

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Figure 4. (a) Ramachandran plot for tigerinin-2O. The values of the backbone torsional angles determined with TALOS+ are shown together with the corresponding uncertainty. Only the high-consensus predictions are shown. Shaded areas represent the energetically most favored regions. (b) Superposition of the backbone trace of the 30 conformers with the lowest potential energy obtained from the DYNAMO-simulated annealing scheme out of a total of 300 structures calculated. (c) Side view of the same 30 conformers. The side chains are shown in addition to the backbone trace. (d) Representative 3D structure of tigerinin-2O showing the segregation of hydrophilic (blue) and hydrophobic (yellow) residues. Atoms are represented as spheres with the radius proportional to the corresponding van der Waals radius.

release at a concentration of 1 μM (Table 1). The potency and effectiveness of tigerinin-1O were comparable to those of physiologically important incretin peptide GLP-1 under the same experimental conditions. No peptide, at concentrations up to 1 μM, produced a significant increase in the rate of release of the cytosolic enzyme LDH, indicating that the integrity of the plasma membrane had been preserved (data not shown). The mechanism of insulin-releasing action of the H. occipitalis tigerinins was not addressed in this study, but patch-clamp studies have shown that [S4R]tigerinin-1R blocks KATP channels in BRIN-BD11 cells, and the resulting depolarization indirectly increases the activity of the L-type Ca channels, leading to increase Ca2+ influx and a consequent increase in the rate of insulin secretion.8 GLUTag cells are a stable and relatively well differentiated murine enteroendocrine cell line that releases glucagon-like peptide-1 (GLP-1) in a regulated manner in response to a range of physiological and pharmacological stimulatory

Figure 5. Schematic representation of the tigerinin-2O sequence illustrating NOE connectivity. In particular, inter-residue HN−HN and HN−Hα dipolar interactions are shown.

the most potent peptide, producing an 83% increase (P < 0.001) in the rate of insulin release at a threshold concentration (minimum concentration producing a significant increase in the rate of insulin release over the basal rate in the presence of 5.6 mM glucose only) of 1 nM, and was also the most effective, producing a 395% increase (P < 0.001) in the rate of insulin 2353

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Table 1. Effects of Tigerinin Peptides on the Rate of Insulin Release from BRIN-BD11 Clonal β-Cells and GLP-1 Release from GLUTag Enteroendocrine Cellsa insulin release peptide tigerinin-1O tigerinin-2O tigerinin-3O tigerinin-4O

threshhold concentration (M) −9

10 10−7 10−7 10−6

GLP-1 release

% of basal release at 10−6 M 395 201 233 163

± ± ± ±

12*** 7*** 17** 13**

threshhold concentration (M) ND 10−7 10−6 10−8

% of basal release at 10−6 M 159 193 217.5 289.3

± ± ± ±

11 NS 17.5* 13.3* 32**

a

Basal release refers to the rate of insulin and GLP-1 release in the absence of peptide and is set at 100%. The threshold concentration is the minimum concentration of peptide producing a significant (P < 0.05) increase in the rate of release. Values shown are mean ± SEM (n = 8). *P < 0.05. **P < 0.01. ***P < 0.001. ND: not determined. NS: not significant.

agents.30 As shown in Table 1, tigerinin-2O, -3O, and -4O produced a significant (P ≤ 0.05) increase in the rate of GLP release, with tigerinin-4O being the most potent and effective peptide, producing a 289% increase in the rate of GLP-1 release at 10 nM. The effect is similar in magnitude to that produced by 10 mM glutamine (Figure S2). 31 No peptide, at concentrations up to 1 μM, produced a significant increase in the rate of release of LDH (data not shown). The ability of the tigerinins to stimulate GLP-1 release from GLUTag cells suggests that, as well as a direct stimulatory effect on insulin release by the β-cells of the pancreas, administration of the tigerinins in vivo may stimulate the L-cells of the intestine to release the potent endogenous incretin GLP-1. Consequently, the data provide further support for the contention that naturally occurring tigerinin peptides may serve as templates for the design of nontoxic and long-acting analogues for use in the treatment of patients with type 2 diabetes.1,8,11 Cytokine Production. In the first series of experiments, all four tigerinins at a concentration of 20 μg/mL produced a significant (P < 0.05) decrease in the production and release of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice (Figure 6A). Effects upon the production of IL-10 and interleukin-17 (IL-17) were not significant (data not shown).

In the second series designed to determine the dose dependency of the inhibitory effect, concentrations of tigerinin-2O and -4O as low as 1.0 μg/mL produced a significant (P < 0.05) inhibition of IFN-γ production (Figure 6B). Tigerinin1O and -3O did not produce significant inhibition of IFN-γ production at concentrations below 20 μg/mL. The effects of naturally occurring frog skin peptides on production of pro-inflammatory and anti-inflammatory cytokines by mouse cells are complex, with both inhibitory and stimulatory actions being reported.1,12 In addition, the effects of the peptides are dependent on the genetic background of the inbred strain of the mouse from which the cells are derived.12 C57BL/6 mice are regarded as prototypical Th1-biased, and Th1-type cytokines are important in producing the proinflammatory responses responsible for killing pathogenic bacteria. IFN-γ is considered to be a major Th1 cytokine, which, along with the Th17 cytokine, IL-17, is responsible for the inflammation underlying conditions such as Crohn’s disease.32 A previous study has shown that the tigerinin-like peptides tigerinin-1 M (5 μg/mL) and tigerinin-1 V (5 μg/mL) downregulated the production of IFN-γ in spleen cells from C57BL/6 mice, while the production of pro-inflammatory IL17 was not affected by either tigerinin.12 Consistent with these data, tigerinin-2O and -4O are particularly potent in inhibiting IFN-γ in mononuclear cells from these mice but do not affect IL-17 production. However, in contrast to the effect of tigerinin-1R on IL-10 production by spleen cells, effects of the four tigerinin-O peptides on IL-10 production were not significant. The role of the tigerinin-O peptides in the frog’s host defense strategy and in mediating skin functions in general and the physiological importance of the inhibitory effect on IFN-γ production in particular is unclear. Excessive cytokinemediated pro-inflammatory responses can lead to uncontrolled tissue damage, so that the tigerinins may act to attenuate the frog’s Th1 cytokine response to invasion by pathogenic microorganisms in the environment.

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EXPERIMENTAL SECTION

General Experimental Procedures. All experiments with live animals were approved by the Animal Research Ethics committee of U.A.E. University (Protocol No. A21-09) and were carried out by authorized investigators. Hoplobatrachus occipitalis frogs (n = 2; male 165 g; female 250 g) were collected at an undetermined site in the Republic of Benin, West Africa, by a United States Fish and Wildlife Service-approved importer for use in the pet trade. The animals were purchased at a pet store in the St. Louis area. The frogs were injected via the dorsal lymph sac with norepinephrine hydrochloride (40 nmol/ g body mass) and placed in a solution (100 mL) of distilled H2O for 15 min. The frogs were removed, and the collected solution was acidified by addition of trifluoroacetic acid (TFA) (1 mL) and immediately frozen. The solutions containing the secretions from both

Figure 6. Effects on the production of IFN-γ by unstimulated peritoneal cells from C57BL/6 mice by (A) 20 μg/mL of tigerinin-1O, -2O, -3O, and -4O and by (B) 1, 5, and 10 μg/mL of tigerinin-2O and -4O. *P < 0.05 compared to production in medium only. 2354

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out for 20 min at 37 °C in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 5.6 mM glucose as previously described.7 After incubation, aliquots of cell supernatant were removed for insulin radioimmunoassay.36 Control incubations were carried out in parallel with the well-established insulin stimulatory agents 10 mM alanine, 1 μM GLP-1, and 30 mM KCl. GLUTag cells were maintained in culture as previously described9 and seeded at a density of 105 cells per well. Peptides (10−10−10−6 M) were incubated with cells for 2 h at 37 °C in KRB buffer supplemented with 2 mM glucose. Control incubations were carried out in parallel with 20 mM glucose and with the well-established GLP-1 stimulatory agents glutamine (10 mM) and forskolin (5 mM).30 GLP-1 concentrations were measured by ELISA using an assay kit supplied by Abcam according to the manufacturer’s recommended protocol. In order to investigate the effects of the peptides on the integrity of the plasma membrane, BRIN-BD11 and GLUTag cells were incubated with peptides (1 nM to 1 μM; n = 3) for 20 min at 37 °C using KRB buffer supplemented with 5.6 mM glucose as previously described.7 Lactate dehydrogenase (LDH) concentrations in the cell supernatants were measured using a CytoTox96 nonradioactive cytotoxicity assay kit (Promega) according to the manufacturer’s protocol. In order to determine hemolytic activities, peptides in the concentration range 31−500 μM were incubated for 60 min at 37 °C with washed erythrocytes (2 × 107 cells) taken from male NIH Swiss mice (Harlan Ltd.) as previously described.33 The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent incubations. Activities of the tigerinins (0.39−100 μM) against the Gram-positive bacterium B. megaterium Bm11 and the Gram-negative bacterium E. coli ATCC 25922 were evaluated by a standard microbroth dilution method according to the Clinical and Laboratory Standards Institute recommended protocol.37 Minimum inhibitory concentration (MIC) producing 100% inhibition of microbial growth was determined as previously described.33 Cytokine Production. All of the animal procedures were subjected to review and approval by the Ethics Committee of the Medical Faculty, University of Kragujevac, which also complies with the National Institutes of Health (NIH) guidelines for humane treatment of laboratory animals. Experiments were performed on cells collected from the peritoneal cavity of unstimulated adult C57BL/6 mice under sterile conditions using 5 mL of cold phosphate-buffered saline according to the procedure of Ray and Dittel.38 After supplementing with 5% (v/v) fetal bovine serum (FBS; Invitrogen), the cell suspension was centrifuged (1500 rpm for 5 min). The supernatant was discarded, and the cells were resuspended in supplemented cell culture medium (RPMI 1640 containing 10% (v/ v) FBS) for further analyses. Isolated cells, representing a mixed population of macrophages, B-, T-, and NK cells, were suspended in RPMI 1640 culture medium containing 10% FBS. In the first series of experiments, peptides (20 μg/mL equivalent to approximately 14 μM) were incubated with unstimulated cells (2 × 105 cells/well) for 24 h at 37 °C in three independent incubations with 6 mice per group. After incubation, cellfree supernatants were collected and kept at −20 °C until time of analysis. In a second series, peritoneal cells were stimulated with 1, 5, and 10 μg/mL of tigerinin-1O, -2O, -3O, and -4O for 24 h at 37 °C in three independent incubations with 6 mice per group. Concentrations of IFN- γ, IL-10, and IL-17 were determined in triplicate using ELISA assay kits from R & D Systems according to the manufacturer’s recommended protocols. Statistical Analysis. 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.

frogs were pooled and partially purified on six Sep-Pak C-18 cartridges (Waters Associates) connected in series as previously described.3 The pooled skin secretions, after partial purification on Sep-Pak cartridges, were injected onto a (2.2 cm × 25 cm) Vydac 218TP1022 (C-18) reversed-phase HPLC column (Grace) equilibrated with 0.1% (v/v) TFA/H2O at a flow rate of 6.0 mL/min. The concentration of CH3CN in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm, and fractions (1 min) were collected. The peptides were purified to near homogeneity by successive chromatographies on a (1.0 cm × 25 cm) Vydac 214TP510 (C-4) column and a (1.0 cm × 25 cm) Vydac 208TP510 (C-8) column. The concentration of CH3CN in the eluting solvent was raised from 21% to 56% over 50 min, and the flow rate was 2.0 mL/min. MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument (Applied Biosystems) as previously described.3 The accuracy of mass determinations was 98%, as determined by symmetrical peak shape and absence of minor contaminants (Figure S3) and by detection of a single component by MALDI-TOF mass spectrometry (Figure 2). Conformational Analysis of Tigerinin-2O. NMR spectra were acquired at 300 K with a Varian Unity INOVA 500 high-resolution spectrometer operating at a 1H frequency of 500 MHz as previously described.33 Tigerinin-2O was dissolved in 700 μL of 10% D2O at a final concentration of 1.97 mM. The pH was adjusted to 4.8 by adding small aliquots of 0.1 M HCl. The chemical shift scale of both 1H and 13 C were referred to the methyl signal of deuterated 3-(trimethylsilyl)2,2′,3,3′-tetradeuteropropionic acid, added as internal reference at a concentration of 2 mM. 1H NMR spectra were acquired using a 6.1 s pulse (90°), 1 s delay time, 1.5 s acquisition time, and a spectral width of 6.0 kHz. 2D experiments (DQF-COSY, TOCSY, and NOESY) were recorded over the same spectral window using 2048 complex points and sampling each of the 512 increments with 64 scans. Mixing times of 80 and 260 ms were applied for the TOCSY and NOESY experiments, respectively. The 1H−13C HSQC spectra were recorded using a spectral window of 6.0 kHz for 1H and 26 kHz for 13C. Apart from the HSQC, in all other experiments a WET suppression scheme (UBURP shape centered at the water resonance with a width of 100 Hz) was applied to reduce the strong resonance from H2O.34,35 TALOS+ software was applied to analyze 1Hα, 1Hβ, 13Cα, and 13Cβ chemical shift values.27 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 structure of tigerinin-2O was obtained using a simulated annealing protocol using the Dynamo 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 crosspeaks in the NOESY spectra, and upper limits of 0.27, 0.33, and 0.50 nm, respectively, were applied. The potential energy contribution was zero below the upper limit, while a harmonic potential was applied above. Three hundred structures were calculated, and the 30 conformers with the lowest potential energy were selected for the analysis. Solvent molecules were not included in the calculations. Insulin and GLP-1 Release. BRIN-BD11 cells, maintained in culture as previously described,7 were seeded into 24-well plates and allowed to attach during overnight incubation at 37 °C. Incubations with purified synthetic tigerinins (10−12 to 10−6 M; n = 8) were carried 2355

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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.6b00494. Table S1 with 1H and 13C resonance assignments; Figure S1 showing concentration dependence of insulin release from BRIN-BD11 cells; Figure S2 showing concentration dependence of GLP-1 release from GLUTag cells; Figure S3 showing the purity of the synthetic tigerinin peptides used for biological testing (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44-7918526277. Fax: +44-2870124965. E-mail: m. [email protected]. Present Address ∞

Department of Life Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago. Author Contributions #

C. M. McLaughlin and S. Lampis contributed equally to this article. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank M. Prajeep for technical assistance in the project and Prof. P. R. Flatt, Ulster University, for providing laboratory facilities to J.M.C.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00494 J. Nat. Prod. 2016, 79, 2350−2356