Translational Medicine Reports 2019; volume 3:8142

Antibacterial and antiviral precursor, prepro-, that is then potential of cleaved to generate the active molecules.10- Correspondence: Gianluigi Franci, 12 Orexin-A is a 33 amino acids Department of Experimental Medicine, including 4 cysteines linked by two intra- University of Campania Luigi Vanvitelli, Carla Zannella, Debora Stelitano, chain disulfide bonds.13,14 Post-translational Via Costantinopoli 16, 80138 Naples, Italy. E-mail: [email protected] Veronica Folliero, Luciana Palomba, modifications of this peptide include a Tiziana Francesca Bovier, Roberta pyroglutamyl cyclization at the N-terminal Key words: Antibacterial; Antiviral; Astorri, Annalisa Chianese, and a C-terminal amidation. Orexin-B is a Neuropeptides; ; . Marcellino Monda, Marilena Galdiero, 28 residue and like orexin-A Gianluigi Franci is characterized by a C-terminal amida- Contributions: CZ and DS contributed equal- 10,14 ly; VF, LP, TFB, RA, AC, MM, MG, data col- Department of Experimental Medicine, tion. The two neuropeptides share 46% 10 lecting and manuscript reviewing and refer- University of Campania Luigi Vanvitelli, of sequence homology. Two-dimensional NMR spectroscopy analysis of soluble ence search; GF, data collecting, figures Naples, Italy preparation and manuscript writing. orexin-B revealed that its structure consists 10 of two α–helices linked via a flexible loop. Conflict of interest: the authors declare no The lateral , where the neu- potential conflict of interest. Abstract ropeptides are generated, is responsible for behavior and energy homeosta- Funding: VALEREplus Program. The emergence of multidrug resistant sis.6,15,16 Indeed, orexins play a key role in bacteria is a global health threat and the dis- feeding behavior and body weight regula- Received for publication: 28 February 2019. covery of new antimicrobial agents is an Accepted for publication: 13 March 2019. tion, sympathetic activation, control of absolute priority. In this context endoge- metabolic status and energy expenditure, nous are emerging as novel poten- This work is licensed under a Creative -wake cycles,17 and regulation of glu- tial candidates. In this work, we assessed Commons Attribution 4.0 License (by-nc 4.0). cose level in the blood.6,18,19 These neu- the antimicrobial effects of orexins and ropeptides fulfill their functions interacting ©Copyright C. Zannella et al., 2019 ghrelin neuropeptides against gram-nega- only with two G-protein-coupled receptors Licensee PAGEPress, Italy tive (Escherichia coli, Salmonella (orexin 1 and 2).6 Recently, orexin- Translational Medicine Reports 2019; 3:8142 typhimurium, Klebsiella pneumoniae) and B has been reported having bactericidal doi:10.4081/tmr.8142 gram-positive (Staphylococcus aureus) bac- 20 teria. Orexin-B and ghrelin showed a potent effects. use bactericidal effect at concentration equal to Another neuropeptide suggested to pos- sess some antimicrobial activity is the We investigated the antimicrobial activ- or greater than 25 µg/ml. No antimicrobial 21 activity has been observed for orexin-A. peptide ghrelin. Ghrelin, also ity of orexin-A, orexin-B and ghrelin Furthermore, we investigated the antiviral known as lenomorelin, is 28-amino-acid against Escherichia coli, Salmonella proprieties of the three peptides against her- (3,3kDa) that was first iso- typhimurium, Klebsiella pneumoniae and lated and identified from rat stomach Staphylococcus aureus. Moreover, we eval- pes simplex virus 1 (HSV-1). We found that 22 orexin-B, but not orexin-A is effective for endocrine cells. It is produced mainly uated the antiviral proprieties of the same from the enteroendocrine cells in the peptides against herpes simplex virus 1 HSV-1 infectivity inhibition. 23 mucosal layer of the stomach, but it was (HSV-1). found also in the pancreas, kidney, small and large intestines24 and in salivary 25 Introduction glands. The human Ghrelin (GHRL) Materials and Methods Nowadays, antibiotics resistance is a encodes a 117 residue precursor (pre- major public health concern. The identifica- proghrelin), that is cleaved by endoproteas- tion of new antimicrobial compounds is a es to produce the mature ghrelin peptide. Peptide synthesis top priority to face this situation.Non-commercial In this sce- The ghrelin peptide exists in two forms acy- Peptides were prepared by standard 9- nario neuropeptides are emerging as novel lated ghrelin and desacylated ghrelin, the fluorenylmethoxycarbonyl polyamine promising antimicrobial agents. functions of the latter are poorly under- solid-phase synthesis, using a PSSM8 mul- Synthesized in , neuropeptides stood. Ghrelin is characterized by the pres- tispecific peptide synthesiser (Shimadzu act as in the central and ence of an n-octanoyl group on the serine in Corporation Biotechnology Instruments peripheral nervous system. These peptides third position (in some species on the threo- Department, Kyoto, Japan). The TGA resin -1 have a key role in the control of several bio- nine) that increases the hydrophobicity of (substitution 0.3 mmol g ) was used as the logical processes included immune and the ghrelin molecule26 and it is important for solid-phase support, and syntheses were inflammatory responses.1-3 In the recent the activity of the peptide. Ghrelin has been performed on a scale of 100 mmol. All past, some neuropeptides, such as the discovered as a natural ligand of the growth amino acids, 4 equiv. relative to resin load- vasoactive intestinal polypeptide and neu- hormone secretagogue receptor.27 ing, were coupled according to the ropeptide Y, have been reported having Increasing evidence supports a role for TBTU/HOBT/DIEA method: 1 equiv. of antimicrobial activities.4,5 ghrelin in the regulation of and Fmoc-amino acid, 1 equiv. of TBTU, 1 In this work we explored both the metabolism.28 Ghrelin induces orexigenic equiv. of HOBT (1 M HOBT in DMF) and antimicrobial and antiviral proprieties of signaling in the and regulates food 2 equiv. of DIEA (2 M DIEA in DMF). The orexins and ghrelin. intake, body weight and Fmoc protecting group was removed with Orexin-A and orexin-B are neuropep- metabolism.29 Moreover, this peptide boosts 30% piperidine in DMF (v/v). Peptides tides produced in the .6- up the immune response20 and inhibits the were fully deprotected and cleaved from the 9 The two peptides are synthetized from a production of proinflammatory cytokines.30 resin by hydrofluoric acid treatment: 89%

[Translational Medicine Reports 2019; 3:8142] [page 1] Article trifluoroacetic acid (TFA) solution contain- colonies of each strain grown overnight on was calculated with respect to no-peptide ing 5.5% thioanisole, 3.3% ethandithiol and MHA plates were resuspended in MHB and control experiments. 2.2% anisole as scavengers; the crude pep- incubated at 37°C until visibly turbid. This tides were precipitated with ice-cold ethyl log-phase inoculum was resuspended in Cytotoxicity against eukaryotic cells ether, filtered, re-dissolved in water and 0.9% sterile saline to reach an appropriate Peptide cytotoxicity was measured by a lyophilised. The crude peptides were puri- optical density at 600 nm (OD600) (with a lactate dehydrogenase (LDH) assay and fied to homogeneity by preparative reverse- Bio-Rad microplate reader; Bio-Rad was carried out according to manufacturer’s phase high-pressure liquid chromatography Laboratories, Hercules, CA) corresponding instructions using a cytotoxicity detection (HPLC) on a Waters Delta Prep 3000 chro- to a concentration of about 1×108 CFU/ml. kit (Roche Diagnostic SpA, Milan, Italy). matographic system, equipped with an UV This standardized inoculum was diluted Release of LDH from the cytosol to culture Lambda Max Mod. 481 detector. The sam- 1:10 in MHB, and the inoculum size was is a marker of cell death. The increase of ples were injected on a Jupiter confirmed by colony counting. LDH activity in the supernatant was related with the percentage of necrotic cells. Vero (Phenomenex) C18 column (21.20 mm×25 Antimicrobial-activity assay cells were cultured in a 96-well plate at a cm, 15 μm) eluted with a H2O/0.1% TFA Susceptibility testing was performed density of 3×104 cells/well for 24 h, fol- (A) and CH3CN/0.1% TFA (B) solvent mix- using the broth microdilution method out- lowed by treatment with investigated con- ture. A linear gradient from 20 to 75% of B lined by the Clinical and Laboratory centrations of each peptides for 24h. over 55 min at a flow rate of 20 mL min-1 Standards Institute using sterile 96-well Maximal LDH release was obtained after was employed. The collected fractions were microtiter plates (Falcon, NJ). Serial 2-fold the treatment of control cells with 1% solu- lyophilised to dryness and analysed by ana- dilutions (from 100 to 0,78 μg/ml) of each tion of Triton X-100 (Sigma Chemical lytical reverse-phase HPLC on a Shimadzu peptide were prepared in cation-adjusted Company) for 10 min at room temperature. class-LC10 equipped with a diode array MHB at a volume of 100 μl/well. Each well One hundred microliter of supernatant from detector SPD-M10AV using a Phenomenex was inoculated with 5 μl of the standardized the top of all the wells was mixed with the C18 analytical column (10×250 mm, 10 bacterial inoculum, corresponding to a final prepared detection kit reagent. After 30 min μm); a linear gradient from 20 to 75% of B 5 -1 test concentration of about 5×10 CFU/ml. incubation, the absorbance (Abs) was mea- over 55 min at a flow rate of 1 mL min was Antimicrobial activities were expressed as suredonly at 490 nm by Tecan spectrophotome- used. The identity of purified peptides was the % reduction of microbial growth ter plate. For each experiment, at least confirmed by Maldi spectrometry. Their observed after 24 h of incubation at 37°C. three replicate wells were examined. sequences, molecular weight and net Each assay was performed in triplicate, and charges are indicated in Table 1. All purified the mean antimicrobial activity is reported.use peptides were obtained with high yields Hemolytic assay (50-60%). Virus entry assays The hemolytic activity of the peptides Peptides were dissolved in DMEM was determined using fresh human erythro- Cells and viruses without serum and used at serial 2-fold dilu- cytes from healthy donors. Blood was cen- African green monkey kidney cells tions (from 100 to 0,78 μg/ml). Experiments trifuged and the erythrocytes were washed (Vero) (ATCC CCL-81) were grown in were conducted in parallel with scrambled three times with 0.9% NaCl. Peptides were Dulbecco’s modified Eagle’s medium peptides and no-peptide controls. To assess added to the erythrocyte suspension (5%, (DMEM) supplemented with 10% fetal the effect of peptides on inhibition of HSV vol/vol) at a final concentration ranging bovine serum. HSV-1 (strain SC16), carry- infectivity, a co-treatment assay was per- from 0.78 to 100 μg/ml in a final volume of ing a lacZ gene driven by the CMV IE-1 formed. The cells were incubated the pep- 100 μl. Samples were incubated with agita- promoter to express β-galactosidase, was tides at each dilution in the presence of viral tion at 37°C for 60 min. The release of propagated on Vero cells monolayers. The inoculum for 45 min at 37°C. After inacti- hemoglobin was monitored by measuring following bacterial strains were included in vation of nonpenetrated viruses by citrate the Abs of the supernatant at 540 nm. The our study: the Gram-negative bacteria E. buffer (pH 3.0), cell monolayers were incu- control for zero hemolysis (blank) consisted coli ATCC 11219, Pseudomonas aerugi- bated for 48 h at 37°C in DMEM supple- of erythrocytes suspended in the presence nosa ATCC 13388, Salmonella enterica mented with carboxymethylcellulose. of peptide solvent. Hypotonically lysed ery- serovar typhimurium ATCC 14028,Non-commercial and K. Finally, monolayers were fixed, stained throcytes were used as a standard for 100% pneumoniae ATCC 10031 and the Gram- with X-gal (5-bromo-4-chloro-3-indolyl-b- hemolysis. The percent hemolysis was cal- positive bacterium S. aureus ATCC 6538. D-galactopyranoside), and plaque numbers culated using the following equation: To standardize the bacterial cell suspension were scored. Experiments were performed [(Abssample − Absblank)/(Abstotal lysis − for assay of antibacterial activity, some in triplicate and the percentage of inhibition Absblank)] ×100.

Table 1. Sequences, molecular weights and net charges of the peptides. Name Sequence MW (as TFA salt) Net charge (pH 7) Ghrelin Gly-Ser-Ser(n-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser- 3370.9 5.1 Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-OH Orexin-B Arg-Ser-Gly-Pro-Pro-Gly-Leu-Gln-Gly-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Ala-Ser-Gly-Asn-His- 2899.36 4.1 Ala-Ala-Gly-Ile-Leu-Thr-Met-NH2 Orexin-A Gln-Pro-Leu-Pro-Asp-Cys-Cys-Arg-Gln-Lys-Thr-Cys-Ser-Cys-Arg-Leu-Tyr-Glu-Leu-Leu-His-Gly- 3561.12 1.9 Ala-Gly-Asn-His-Ala-Ala-Gly-Ile-Leu-Thr-Leu-NH2 MW, molecular weights; TFA, trifluoroacetic acid.

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Results

Antimicrobial-activity of orexins and ghrelin We assessed the antimicrobial activity of the synthesized peptides orexin-A, orex- in-B and ghrelin on the bacterial strains E. coli, S. enterica serovar typhimurium, K. pneumoniae and S. aureus, following the broth microdilution method outlined by the Clinical and Laboratory Standards Institute. As shown in the Figure 1, a dose-dependent effect has been evidenced for orexin-B and ghrelin from concentrations equal to 12.5 μg/mL, or greater. In particular, at 25 μg/ml or higher concentrations of orexin-B or ghrelin the relative % of viable bacteria was minimal, revealing a potent antimicrobial effect. Moreover, the results obtained for orexin-B and ghrelin are in accordance with what has been reported previously reported for E. coli,20 whereas S. aureus, tested with the broth microdilution method, resulted sensitive. In contrast, no bacterial killing only effect has been observed for orexin-A, test- ed on the four bacterial strains. Figure 1. Histogram of antibacterial activities of orexin-A, orexin-B and ghrelin expressed Anti-viral proprieties of orexin-B in %. use The ability of the peptides to reduce HSV-1 infectivity has been evaluated by a co-treatment assay. As negative control we included the medium in which the peptides have been dissolved (vehicle) and as posi- tive control the cytolytic peptide mellitin. As shown in Figure 2, only for orexin-B a significative, dose-dependent antiviral effect has been found. The effect started at 25 μg/mL, exceeding the IC 50 value at 100 μg/mL. No antiviral effect has been detect- ed for orexin-A and ghrelin at the tested concentrations. Cytotoxicity of orexins and ghrelin As shown in Table 2, orexin-A, orexin- Figure 2. Histogram of relative % of viral inhibition of herpes simplex virus 1 infection. B and ghrelin are not cytotoxic.Non-commercial However,

Table 2. Cell viability and hemolysis caused by exposure to different concentration of peptides orexin-A, orexin-B and ghrelin. Cell via- bility is expressed as percentage of lactate dehydrogenase released compared to a positive control (Vero cells treated with a 1% solution of Triton X-100 for 10 min at room temperature). Hemolysis is expressed as percentage of released hemoglobin respect to a positive control made of hypotonically lysed erythrocytes. All experiments were conducted in triplicate. Peptide concentrations (µg/ml) Cell viability (%) Hemolysis (%) Orexin-A Orexin-B Ghrelin Orexin-A Orexin-B Ghrelin 100 92 89 91 16 8 15 50 94 96 95 11 2 9 25 100 100 97 1 0 2 12,5 100 100 100 0 0 0 6,25 100 100 100 0 0 0 3,125 100 100 100 0 0 0 1,56 100 100 100 0 0 0 0,78 100 100 100 0 0 0

[Translational Medicine Reports 2019; 3:8142] [page 3] Article we observed a slight reduction of cell via- Caro M, Gonzalez-Rey E. of hypothalamic neuropeptides and G bility in Vero cells at the highest concentra- Neuropeptides as pleiotropic modula- protein-coupled receptors that regulate tions tested (50-100 μM). The difference tors of the immune response. feeding behavior. Cell 1998;92:573-85. between the cytotoxic effects of the three Neuroendocrinology 2011;94:89-100. 15. Nakazato M, Murakami N, Date Y, et al. peptides was mild, and slightly more 2. Delgado M, Ganea D. Anti-inflammato- A role for ghrelin in the central regula- marked only at 100 μM, where orexin-B ry neuropeptides: a new class of tion of feeding. Nature 2001;409:194-8. was found to be the most endowed with a endogenous immunoregulatory agents. 16. Tolle V, Bassant MH, Zizzari P, et al. mild cytotoxic effect. Brain Behav Immun 2008;22:1146-51. Ultradian rhythmicity of ghrelin secre- The results of the hemolityc assays are 3. Gonzalez-Rey E, Delgado M. Anti- tion in relation with GH, feeding behav- reported in Table 2. The peptides do not inflammatory neuropeptide receptors: ior, and sleep-wake patterns in rats. induce hemolysis at concentrations less new therapeutic targets for immune dis- Endocrinology 2002;143:1353-61. than 25μM. A modest hemolityc effect orders? Trends Pharmacol Sci 17. Hoyer D, Jacobson LH. Orexin in sleep, appears at 50 and 100μM concentrations for 2007;28:482-91. addiction and more: is the perfect all three peptides. 4. El Karim IA, Linden GJ, Orr DF, Lundy insomnia drug at hand? Neuropeptides FT. Antimicrobial activity of neuropep- 2013;47:477-88. tides against a range of micro-organ- 18. Messina G, Viggiano A, De Luca V, et isms from skin, oral, respiratory and al. Hormonal changes in menopause Discussion and Conclusions gastrointestinal tract sites. J and orexin-a action. Obstet Gynecol Int Neuroimmunol 2008;200:11-6. 2013;2013:209812. In the last years several endogenous 5. Brogden KA, Guthmiller JM, Salzet M, 19. Rani M, Kumar R, Krishan P. Role of peptides have been reported having antimi- Zasloff M. The nervous system and orexins in the central and peripheral crobial activity, and among these some neu- innate immunity: the neuropeptide con- regulation of glucose homeostasis: ropeptides. It has been suggested that these nection. Nat Immunol 2005;6:558-64. Evidences & mechanisms. cationic peptides bind to the negatively 6. Messina G, Dalia C, Tafuri D, et al. Neuropeptides 2018;68:1-6. charged bacterial cell wall exerting their Orexin-A controls sympathetic activity 20. Ohta K, Kajiya M, Zhu T, et al. Additive antimicrobial effect. In line with this, orex- only and eating behavior. Front Psychol effects of orexin B and vasoactive ins sequence is rich in positive charged 2014;5:997. intestinal polypeptide on LL-37-medi- residues.20 7.Burdakov D, Gonzalez JA. ated antimicrobial activities. J In this study we assessed the antimicro- Physiological functions of glucose- Neuroimmunol 2011;233:37-45. bial activity of orexin-A and orexin-B inhibited neurones. Acta Physiol (Oxf)use21. Wilcox T, Hirshkowitz A, Hawkins L, against the gram-negative E. coli, S. thy- 2009;195:71-8. Boas DA. The effect of color priming phimurium and K. pneumoniae and the 8. Cowley MA, Smith RG, Diano S, et al. on infant brain and behavior. gram-positive S. aureus. The distribution and mechanism of Neuroimage 2014;85:302-13. Orexins share 46% of sequence homol- action of ghrelin in the CNS demon- 22. Kojima M, Hosoda H, Date Y. Ghrelin ogy, despite this only orexin-B showed strates a novel hypothalamic circuit reg- is a growth-hormone-releasing acylated antimicrobial proprieties. ulating energy homeostasis. peptide from stomach. Nature We found that orexin-B was effective 2003;37:649-61. 1999;402:656-60. against all the strains tested. Our results are 9. Boss C, Roch C. Recent trends in orexin 23. Date Y, Kojima M, Hosoda H, et al. consistent with the results of Ohta et al. research-2010 to 2015. Bioorg Med Ghrelin, a novel - reporting bactericidal activity of orexin-B 20 Chem Lett 2015;25:2875-87. releasing acylated peptide, is synthe- against E. coli and P. aeruginosa. 10. Lee JH, Bang E, Chae KJ, et al. sized in a distinct endocrine cell type in Furthermore, we assessed the antiviral Solution structure of a new hypothalam- the gastrointestinal tracts of rats and activity of these neuropeptides against ic neuropeptide, human hypocretin- humans. Endocrinology HSV-1. Orexin-B showed a dose-response 2/orexin-B. Eur J Biochem 1999;266: 2000;141:4255-61. antiviral effect, exceeding the IC 50 value at 831-9. 24. Sakata I, Sakai T. Ghrelin cells in the 100 μg/mL. On the other hand, ghrelin did gastrointestinal tract. Int J Pept not show antiviral activity at the assessedNon-commercial11. Lang M, Soll RM, Durrenberger F, et al. 2010;2010. concentrations. However, it was effective Structure-activity studies of orexin a 25. Groschl M, Topf HG, Bohlender J, et al. against all the gram positive and gram neg- and orexin B at the human orexin 1 and ative tested bacteria. Ghrelin, like orexin-B, orexin 2 receptors led to orexin 2 recep- Identification of ghrelin in human sali- is a cationic peptide and shows a similar tor selective and orexin 1 receptor pre- va: production by the salivary glands isoelectric point and it has been suggested ferring ligands. J Med Chem and potential role in proliferation of oral that it could share the same bactericidal 2004;47:1153-60. keratinocytes. Clin Chem 2005;51:997- mechanism.21 Further studies are required to 12. Chieffi S, Carotenuto M, Monda V, et 1006. investigate the mechanism underlying the al. Orexin system: the key for a healthy 26. Bednarek MA, Feighner SD, Pong SS, anti-viral activity of orexin-B. However, life. Front Physiol 2017;8:357. et al. Structure-function studies on the taken together our results indicate that neu- 13. Okumura T, Takeuchi S, Motomura W, new growth hormone-releasing peptide, ropeptides represent promising candidates et al. Requirement of intact disulfide ghrelin: minimal sequence of ghrelin as both antiviral and anti-bacterial agents. bonds in orexin-A-induced stimulation necessary for activation of growth hor- of gastric acid secretion that is mediated mone secretagogue receptor 1a. J Med by OX1 receptor activation. Biochem Chem 2000;43:4370-6. Biophys Res Commun 2001;280:976- 27. Kojima M, Hosoda H, Matsuo H, References 81. Kangawa K. Ghrelin: discovery of the 14. Sakurai T, Amemiya A, Ishii M, et al. natural endogenous ligand for the 1. Souza-Moreira L, Campos-Salinas J, Orexins and orexin receptors: a family growth hormone secretagogue receptor.

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Trends Endocrinol Metab 2001;12:118- 29. Chabot F, Caron A, Laplante M, St- 30. Dixit VD, Schaffer EM, Pyle RS, et al. 22. Pierre DH. Interrelationships between Ghrelin inhibits - and activation- 28. Muller TD, Nogueiras R, Andermann ghrelin, and glucose homeosta- induced proinflammatory cytokine ML, et al. Ghrelin. Mol Metab sis: Physiological relevance. World J expression by human monocytes and T 2015;4:437-60. Diabetes 2014;5:328-41. cells. J Clin Invest 2004;114:57-66.

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