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Non-Commercial Use Only Translational Medicine Reports 2019; volume 3:8142 Antibacterial and antiviral precursor, prepro-orexin, that is then potential of neuropeptides cleaved to generate the active molecules.10- Correspondence: Gianluigi Franci, 12 Orexin-A is a 33 amino acids peptide 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; Orexins; Ghrelin. Marcellino Monda, Marilena Galdiero, 28 residue neuropeptide 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 hypothalamus, where the neu- potential conflict of interest. Abstract ropeptides are generated, is responsible for eating 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 peptides are emerging as novel poten- This work is licensed under a Creative sleep-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 receptor 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. appetite 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) peptide hormone 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 gene (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 neurons, neuropeptides stood. Ghrelin is characterized by the pres- tispecific peptide synthesiser (Shimadzu act as neurotransmitters 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 hunger 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 brain and regulates food 2 equiv. of DIEA (2 M DIEA in DMF). The orexins and ghrelin. intake, body weight and glucose 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 lateral hypothalamus.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.
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