In Vitro and in Vivo Characterization of Novel Stable Peptidic Ghrelin Analogs: Beneficial Effects in the Settings of LPS-Induced Anorexia in Mice

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In vitro and in vivo characterization of novel stable peptidic ghrelin analogs: beneficial effects in the settings of LPS-induced anorexia in mice

Martina Holubová, Miroslava Blechová, Anna Kákonová, Jaroslav Kuneš, Blanka Železná, and Lenka Maletínská

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague,

Czech Republic (MH, MB, AK, JK, BŽ, LM)

Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic (JK) Downloaded from

jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Running title: Orexigenic and anti-inflammatory effects of ghrelin analogs

Corresponding author:

Dr. Lenka Maletínská

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,

Flemingovo náměstí 2, 166 10 Prague 6, Czech Republic e-mail: [email protected], tel: +420 220183567, fax: +420 220183585

Text pages: 38 Downloaded from

Tables: 2

Figures: 7 jpet.aspetjournals.org References: 51

Abstract: 250 words

Introduction: 750 words at ASPET Journals on September 26, 2021 Discussion: 1498 words

List of non-standard abbreviations: Ada, 1-adamantaneacetyl; AgRP, agouti-related peptide; ANOVA, analysis of variance; Ala-OtBu, tert-butyl-alanine; bla, beta lactamase; bromooct, 8-bromooctanoyl; Cha, cyclohexylalanine; dec, decanoyl; decen, decenoyl; Dpr, diaminopropionic acid; ELISA, enzyme-linked immuno sorbent assay; FRET, fluorescence resonance energy transfer; GAPHD, glyceraldehyde 3-phosphate dehydrogenase; GH; growth hormone; GHRP, growth hormone releasing peptide; GHS-R1a, growth hormone secretagogue receptor 1a; HPLC, high-performance liquid chromatography; IL, interleukin; i.p., intraperitoneal; i.v., intravenous; Kd, dissociation constant; Ki, inhibition constant; LPS, lipopolysaccharide; MS, mass spectrometry; myr, myristoyl; Nal, naphthylalanine; NPY, neuropeptide Y; oct, octanoyl; palm, palmitoyl; PheCl2, dichlorophenylalanine; PheNO2, nitrophenylalanine; Q-TOF, quadrupole time-of-flight; Sar, sarcosine; s.c., subcutaneous;

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S.E.M., standard error of the mean; TNF-α, tumor necrosis factor alpha; undecyn, 10- undecynoyl.

Recommended section assignment: Drug Discovery and Translational Medicine

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ABSTRACT

Ghrelin, the only known orexigenic gut hormone produced primarily in the stomach, has lately gained attention as a potential treatment for anorexia and cachexia. However, its biological stability is highly limited, therefore a number of both peptide and non-peptide ghrelin analogs have been synthesized. In this study, we provide in vitro and in vivo characterization of a series of novel peptide growth hormone secretagogue receptor

(GHS-R1a) agonists, both under non-pathological conditions and in the context of Downloaded from lipopolysaccharide (LPS)-induced anorexia. These analogs were based on our previous series modified by replacing the Ser3 with diaminopropionic acid (Dpr), the N-terminal Gly with

4 sarcosine and Phe with various non-coded amino acids. New analogs were further modified jpet.aspetjournals.org by replacing the n-octanoyl bound to Dpr3 with longer or unsaturated fatty acid residues, by incorporation of the second fatty acid residue into the molecule, or by shortening the peptide chain. These modifications preserved the ability of ghrelin analogs to bind to the membranes at ASPET Journals on September 26, 2021 of cells transfected with GHS-R1a, as well as the GHS-R1a signaling activation. The selected analogs exhibited long-lasting and potent orexigenic effects after a single subcutaneous (s.c.) administration in mice. The stability of new ghrelin analogs in mice after s.c. administration was significantly higher when compared to ghrelin and [Dpr3]ghrelin, with half-lives of approximately 2 hours. A single s.c. injection of the selected ghrelin analogs in mice with

LPS-induced anorexia significantly increased food intake via the activation of orexigenic pathways and normalized blood levels of proinflammatory cytokines, demonstrating the anti- inflammatory potential of the analogs.

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INTRODUCTION

Cachexia is a multifactorial wasting condition characterized by ongoing loss of muscle mass with/without loss of fat mass. This condition, which is a result of catabolic/anabolic imbalance driven by a combination of reduced food intake and altered metabolism, accompanies advanced cancer or chronic progressive diseases where cachexia and anorexia often co-exist and induce malnutrition (Evans et al., 2008; Argilés et al., 2017). The major mechanisms underlying the development of cachexia include chronic inflammation, abnormal inflammatory cytokines release and increased activity of the sympathetic nervous system, Downloaded from which contribute to systemic hypermetabolism and a negative energy balance (Colldén et al.,

2017). Cachexia is correlated with a worsened prognosis and increased mortality rate of jpet.aspetjournals.org certain diseases (DeBoer, 2011). Current therapeutic interventions are limited (reviewed in

(Argilés et al., 2017)). Lately, ghrelin and other agonists of growth hormone secretagogue receptor (GHS-R1a) have gained attention as potential anti-cachectic treatments (Molfino et at ASPET Journals on September 26, 2021 al., 2014).

Ghrelin, the only known orexigenic gut hormone and endogenous ligand of GHS-R1a, is secreted primarily from the stomach. This 28-amino-acid peptide contains a serine esterified by n-octanoic acid, a unique modification necessary for its biological activity

(Kojima et al., 1999). Structure-function studies have revealed that the N-terminal tetrapeptide octanoylated at Ser3 preserves most of the in vitro biological activity of the full- length ghrelin molecule (Matsumoto et al., 2001). The N-terminal positive charge and Phe4 are essential for the biological activity of ghrelin (Van Craenenbroeck et al., 2004). The ester bond in Ser3-O-octanoyl is an easy target for hydrolysis; only 10 to 20% of circulating ghrelin is octanoylated, with half-life in blood lasting only a few minutes (Hosoda and Kangawa,

2012).

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Ghrelin maintains the positive energy balance of the organism and has a key role in increasing appetite, food intake and body weight, facilitating adipose tissue accummulation and regulating energy metabolism (Delporte, 2013). These effects, together with the anti- inflammatory effects mediated by peripherally distributed GHS-R1a, make ghrelin administration a promising anti-cachectic therapeutic strategy (Zhou et al., 2017). Ghrelin administration to cachectic rodents increased appetite and body weight (DeBoer et al., 2007;

Tsubouchi et al., 2014), prevented cisplatin-induced hyperalgesia, lipid catabolism and weight loss (Garcia et al., 2008; Garcia et al., 2013), prevented tumor- and cisplatin-induced muscle Downloaded from wasting and decreased inflammation (Chen et al., 2015). Clinical trials showed that intravenous (i.v.) administration of synthetic ghrelin to cachectic patients is safe, it effectively jpet.aspetjournals.org increases appetite and improves nausea and exertional capacity (Strasser et al., 2008; Hiura et al., 2012; Miki et al., 2013).

However, because of the short lifetime of native ghrelin, its usefulness as a therapeutic at ASPET Journals on September 26, 2021 agent is limited (Borner et al., 2016). Therefore a number of peptide and non-peptide

GHS-R1a agonists have been tested. BIM-28125 and BIM-28131 induced weight gain and decreased muscle atrophy in rat heart failure models (Palus et al., 2011; Lenk et al., 2013).

The peptides GHRP-1, GHRP-2, GHRP-6 and hexarelin were tested in rats with cardiac cachexia (Xu et al., 2005). HM01 attenuated anorexia-cachexia syndrome in tumor-bearing rats (Borner et al., 2016). Hexarelin and JMV2894 counteracted anorexia and preserved muscle function in rats with cisplatin-induced cachexia (Conte et al., 2017). Z-505 ameliorated chemotherapy- and cancer-induced cachexia-anorexia in rodents (Shiomi et al.,

2017). Anamorelin was successfully employed in clinical trials in cancer patients (Garcia et al., 2015; Temel et al., 2016; Currow et al., 2017). JMV1843 (macimorelin) is currently in an ongoing phase II trial for treatment of cancer-induced cachexia (clinicaltrials.gov;

NCT01614990).

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Based on the structure-activity studies, we designed ghrelin analogs with enhanced stability. Ghrelin was modified by replacing the Ser3 with diaminopropionic acid (Dpr) to protect the molecule from hydrolysis by esterases, according to Bednarek (Bednarek et al.,

2000). Gly1 was replaced with sarcosine (Sar) and Phe4 with β-cyclohexylalanine (Cha) or

L-1-naphthylalanine (Nal) to protect the analogs against the activity of aminopeptidases or chymotrypsin-like proteinases, respectively. These designed analogs possess similar affinities as ghrelin for cell membranes with transfected GHS-R1a, and they show potent orexigenic effect and high stability in mouse plasma (Maletínská et al., 2012). Downloaded from

The aim of this study was to design a new series of potent long-acting stable GHS-R1a agonists, which were modified by replacing the n-octanoyl bound to Dpr3 with another fatty jpet.aspetjournals.org acid residue (longer-chain, unsaturated, or substituted), by incorporation of the second fatty acid residue, by modification of amino acid sequention or by shortening the peptide chain.

These analogs were characterized concerning their binding affinity to GHS-R1a, signaling at ASPET Journals on September 26, 2021 activation, orexigenic effect, growth hormone release, pharmacokinetics in mice and effects in the mouse model of lipopolysaccharide (LPS)-induced sepsis/anorexia, a model for early steps in cachexia.

MATERIALS AND METHODS

Peptide synthesis and iodination

Rat ghrelin [Gly-Ser-Ser(O-octanoyl)-Phe-Leu-Ser-Pro-Glu-His-Gln-His-Gln-Lys-Ala-

Gln-Gln-ArgLys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg], [diaminopropionic acid3]ghrelin ([Dpr(N-octanoyl)3]ghrelin) and their analogs were assembled in a solid-phase

ABI 433A synthesizer (Applied Biosystems, Foster City, CA, USA) by the stepwise coupling of the corresponding Fmoc amino acids to the growing chain on Wang resin

(1% divinylbenzene, 100–200 mesh, 0.22 mmol/g) (IRIS Biotech GmbH, Marktredwitz,

Germany). Lipidization with the corresponding fatty acid was performed on fully protected

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USA). The structures of all peptides are shown in Tab. 1.

Ghrelin was iodinated at His9 with Na125I (Izotop, Budapest, Hungary) using Iodo-Gen

(Pierce, Rockford, IL, USA) according to the protocol recommended by the manufacturer and purified as described previously (Maletínská et al., 2012). The specific activity of the [125I]ghr was approximately 2000 Ci/mmol. Aliquots of the purified radiolabeled peptides were dried Downloaded from by evaporation and kept at -20 °C. The radiolabeled peptides were used for binding studies within 1 month. jpet.aspetjournals.org Competitive binding studies

Competitive binding studies were performed according to a previously described procedure (Demange et al., 2007; Maletínská et al., 2012). Briefly, isolated plasma at ASPET Journals on September 26, 2021 membranes from LLC-PK1 cells with transfected human GHS-R1a receptor (a kind gift from

Prof. Martinez, IBMM, Montpellier, France) were used. Cell membranes (3 µg of protein/tube) were incubated with 0.05 nM 125I-ghrelin and nonradioactive ghrelin/ghrelin analogs at concentrations ranging from 1 pM to 10 µM for 45 min at 25 °C in 250 µL of binding buffer composed of 50 mM Tris pH 7.4 (Sigma-Aldrich, St. Louis, MO, USA, cat. no. T6066), 5 mM MgCl2 (Sigma-Aldrich, St. Louis, MO, USA, cat. no. M2393), 2.5 mM

EDTA (Sigma-Aldrich, St. Louis, MO, USA, cat. no. EDS), 0.1% bovine serum albumin

(Serva GmbH, Heidelberg, Germany, cat. no. 11920), 0.1 mg/mL bovine pancreatic trypsin inhibitor (Sigma-Aldrich, St. Louis, MO, USA, cat. no. T0256). The binding reaction was terminated by the addition of ice-cold washing buffer composed of 20 mM Tris pH 7.4,

10 mM MgCl2, 2.5 mM EDTA and 0.015% Triton X-100 (Sigma-Aldrich, St. Louis, MO,

USA, cat. no. X100) followed by rapid filtration over GF/C filters (Whatman, Clifton, NJ,

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USA, cat. no. 1822-915) presoaked with 0.5% polyethylenimine (Sigma-Aldrich, St. Louis,

MO, USA, cat. no. P3143) in binding buffer using a Brandel cell harvester (Brandel Inc.,

Gaithersburg, MD, USA).

Bound radioactivity was determined by gamma counting (Wizard 1470 Automatic

Gamma Counter; PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).

Experiments were carried out in duplicate at least three times. Non-specific binding defined using an excess of ghrelin (10 µM) was always less than 15% of the total binding.

Beta-lactamase-dependent fluorescence resonance energy transfer (FRET) assay Downloaded from

The T-RexTM TangoTM GHSR-bla (GHSR-beta lactamase) U2OS cell line, overexpressing GHS-R1a and containing a beta-lactamase reporter gene under the control of a jpet.aspetjournals.org UAS response element, was obtained from Thermo Fisher Scientific (Waltham, MA, USA, cat. no. K1819). Cells were grown in McCoy´s 5A medium (Thermo Fisher Scientific,

Waltham, MA, USA, cat. no. 16600-082) supplemented with 10% dialyzed fetal bovine at ASPET Journals on September 26, 2021 serum (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 26400-036), 0.1 mM non- essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 11140-050),

25 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 15630-080),

1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 11360-070) and antibiotics according to manufacturer´s instructions – penicillin-streptomycin (Biosera,

Nuaille, France, cat. no. LM-A4118), zeocin (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. R250-01), hygromycin (Thermo Fisher Scientific, Waltham, MA, USA, cat. no.

10687-010), geneticin (Biosera, Nuaille, France, cat. no. LM-A4115). The cells were maintained at 37 °C in a humified atmosphere containing 5% CO2 and passaged twice a week at 80 to 90% confluence. Cell viability was determined using the trypan blue dye (Sigma-

Aldrich, St. Louis, MO, USA, cat. no. T8154); cells were counted manually using the Bürker counting chamber.

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Cells were plated at 40,000 cells/well in FreestyleTM Expression Medium (Thermo

Fisher Scientific, Waltham, MA, USA, cat. no. 12338-018) with 1.25 ng/mL doxycycline

(MP Biomedicals, Santa Ana, CA, USA, cat. no. 195044) in a 96-well plate (Cellvis,

Mountain View, CA, USA, cat. no. P96-1.5H-N) and incubated for 20 h at 37 °C/5% CO2.

The assay was performed according to the manufacturer´s protocol (GeneBLAzer® GPCR

Cell-based assays, Thermo Fisher Scientific, Waltham, MA, USA, cat. no. K1096 and

K1156), and the tested compounds were added at concentrations ranging from 1 pM to 0.1

µM. The fluorescent plate reader Tecan Infinite® M1000 (Tecan Group Ltd., Mannedorf, Downloaded from

Switzerland) was operated with a 405-nm excitation wavelength and a 460-nm or 530-nm emission wavelength via bottom read. Experiments were carried out in duplicate at least three jpet.aspetjournals.org times.

Effect of ghrelin analogs on acute food intake in mice

All of the animal experiments described in this study were performed according to the at ASPET Journals on September 26, 2021 ethical guidelines for animal experiments and Czech Republic law 246/1992 and were approved by the Committee for experiments with laboratory animals of the Academy of

Sciences of the Czech Republic (the decision no. 38/2013 was issued on 8/4/2013).

Male C57BL/6 mice (Charles River, Sulzfeld, Germany) were housed at a temperature of 22 ± 2 °C under a daily cycle of 12/12 h light/dark (light from 6:00 a.m.) with free access to water and a standard chow diet Ssniff® R/M-H (Spezialdiäten GmbH, Soest, Germany, cat. no. V1534) which contained 58%, 9% and 33% of calories from carbohydrate, fat and protein, respectively.

Mice were placed into separate cages for one week before the experiment, and they had free access to water and food pellets. Just before the experiment, the food pellets were removed from the cages. At 8:00 a.m., the mice were injected subcutaneously (s.c.) with 0.2 mL of saline, ghrelin or ghrelin analogs (dissolved in saline) at a dose of 5 mg/kg of body

JPET Fast Forward. Published on June 18, 2018 as DOI: 10.1124/jpet.118.249086 This article has not been copyedited and formatted. The final version may differ from this version. JPET #249086 weight (n=5-8). The effect of the new analogs was compared with the effect of octanoylated ghrelin analogs [Sar1, (Dpr-N-oct)3]ghrelin, [Sar1, (Dpr-N-oct)3, Nal4]ghrelin and [Sar1, (Dpr-

N-oct)3, Cha4]ghrelin which were previously published by Maletínská et al. (Maletínská et al.,

2012). 15 min after the injection, the mice were given preweighed food pellets. Food intake was monitored at 30-min intervals for at least 8 hours. The mice had free access to water during the experiment. After the initial screening, analogs, which were shown to be more effective than ghrelin or [Dpr3]ghrelin, were tested in 2 separate experiments.

The selected ghrelin analogs 4 and 10 were injected s.c. into mice at doses of 0.2 to Downloaded from

5 mg/kg (0.2 mL/mouse) and food intake was monitored at 30-min intervals for at least 8 hours to evaluate the dose-response relationship. jpet.aspetjournals.org Pharmacokinetics of selected ghrelin analogs in mice

C57BL/6 male mice were s.c. injected with ghrelin, [Dpr3]ghrelin or one of the selected ghrelin analogs no. 4, 7, 10 and 20 at a dose of 5 mg/kg of body weight (0.2 mL/mouse, n=4). at ASPET Journals on September 26, 2021

Blood was collected before the injection and 0.5, 1, 2, 4, 8 and 16 hours after the injection.

Plasma was separated and stored at -20 °C. The concentration of ghrelin analogs in the samples was determined using a Rat/Mouse Ghrelin (Total) enzyme-linked immuno sorbent assay (ELISA) kit (Merck-Millipore, St. Charles, MO, USA, cat. no. EZRGRT-91K) according to the manufacturer´s instructions, and the corresponding ghrelin analog was used as a calibration standard. The samples were diluted appropriately to fall within the range of the assay (0.04-10 ng/mL). The lowest ghrelin level detectable by the assay was 0.04 ng/mL.

The specifity of the assay was as follows: rat/mouse ghrelin (active) 85%, des-octanoyl rat/mouse ghrelin 100%, human ghrelin (active) 115%, des-octanoyl human ghrelin 249%, canine ghrelin (active) 71%, porcine ghrelin (active) 48%.

Growth hormone (GH) release after s.c. administration of selected analogs to mice

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The effect of selected ghrelin analogs on GH release was determined in 3-month-old male C57BL/6 mice. Mice were s.c. injected with saline, ghrelin, [Dpr3]ghrelin and selected ghrelin analogs [Sar1, (Dpr-N-oct)3, Nal4]ghrelin and analogs no. 4 and 7 at a dose of 5 mg/kg of body weight (0.2 mL/mouse, n=6). 10 min after the injection, blood was collected, plasma was prepared and stored at -20 °C until use. GH levels in the plasma samples were determined by Rat/Mouse Growth Hormone ELISA kit (Merck-Millipore, St. Charles, MO, USA, cat. no.

EZRMGH-45K) according to the manufacturer´s instructions. The samples were diluted appropriately to fall within the range of the assay (0.07-50 ng/mL). The limit of sensitivity of Downloaded from the assay was 0.07 ng/mL. The specifity of the assay was as follows: rat growth hormone

100%, may cross react with hamster, canine, and primate growth hormone. jpet.aspetjournals.org Effects of selected ghrelin analogs in a mouse model of LPS-induced anorexia

One week before the experiment, male C57BL/6 mice were placed into separated cages and habituated to an automatic system for food intake monitoring (Developmental workshop at ASPET Journals on September 26, 2021

IOCB, Prague, Czech Republic). The animals were randomized into two groups, the LPS group and the control group. At 5:00 p.m., the LPS group was injected intraperitoneally (i.p.) with 100 µg/kg of body weight LPS (Escherichia coli 055:B5 serotype, Sigma-Aldrich, St.

Louis, MO, USA, cat. no. L2880) in 0.2 mL of saline. The control group received an i.p. injection with an equal volume of saline. Food intake was monitored automatically with the food intake monitoring system. At 7:00 a.m. on the following day, the mice of both groups were s.c. injected with 0.2 mL of either saline or ghrelin analog 10 (n=4-5 mice per group).

Food intake was monitored for another 10 hours.

At the end of the experiment, the mice were sacrificed by decapitation, blood was collected, and plasma was prepared and stored at -20 °C. Hypothalami were dissected on top of a Petri dish filled with ice, using scissors and curved forceps. Immediately after dissection,

Proinflammatory cytokine levels in the blood plasma were determined using the

Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel (Merck-Millipore, St.

Charles, MO, USA, cat. no. RECYMAG65K27PMX). The manufacturer claims that there is no or minimum cross-reactivity between the antibodies and any other analytes in the panel.

Standard curve ranges of the particular analytes are as follows: IL-1α and IL-2 12.2 – 50,000 pg/mL, IL 4 4.9 – 20,000 pg/mL, IL-6 73.2 – 300,000 pg/mL, IL-10 7.3 – 30,000 pg/mL, Downloaded from interferon γ 14.6 – 60,000 pg/mL, TNF-α 2.4 – 10,000 pg/mL.

Samples of hypothalami were homogenized, total RNA was extracted and RNA jpet.aspetjournals.org concentration was determined as previously described (Holubová et al., 2014). Determination of the mRNA expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) was performed using an at ASPET Journals on September 26, 2021

ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to compensate for variations in the amount of input RNA and the efficiency of reverse transcription, and the modified formula 2-ΔCt was used to calculate the relative gene expression.

After this pilot study, the experiment was repeated with LPS-treated mice only, testing the orexigenic effect of ghrelin and analogs 4, 7 and 10 (n=4-8 mice).

Data analysis and statistics

Data are presented as the means ± S.E.M. The competitive binding curves were plotted using GraphPad Prism software (San Diego, CA, USA) while comparing the best fit for single binding sites models. IC50 values were obtained from nonlinear regression analysis, and inhibition constants (Ki values) were calculated from the IC50 values using the Cheng-Prusoff

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equation (Cheng and Prusoff, 1973). The dissociation constant of the radioligand (Kd) value obtained from saturation binding experiments was 0.18 nM.

The results of the beta-lactamase assay were analyzed by the nonlinear regression of log agonist versus response using GraphPad Prism software. EC50 was calculated as the concentration of the peptide that yielded 50% of the maximal response.

In some cases, the data from the food intake experiments and the model of LPS-induced anorexia are expressed as % of the respective control (as described in the Tables and Figure legends) to enable comparison of the data from multiple experiments. Data from all in vivo Downloaded from experiments were analyzed using GraphPad Prism software. T-test, one-way ANOVA followed by Dunnett post hoc test, repeated measures ANOVA or two-way ANOVA followed jpet.aspetjournals.org by Bonferroni post hoc test were used where appropriate, as described in the Tables and

Figure legends. P < 0.05 was considered statistically significant.

RESULTS at ASPET Journals on September 26, 2021

Design and synthesis of ghrelin analogs

The structures of ghrelin and its analogs used in this study are shown in Table 1.

The peptide sequences were assembled on a solid phase support, as described in the Materials and methods section. The identity and purity of the peptides was verified by HPLC and MS.

The purity of peptides was higher than 95%.

The original serine in position 3 of ghrelin was replaced with Dpr, which can form a stable amide bond with octanoic acid [Dpr(N-octanoyl)] instead of the original ester bond

[Ser(O-oct)] which can be easily hydrolyzed by esterases (Bednarek et al., 2000). To further increase the stability of the ghrelin analogs, glycine at the N-terminal position was replaced with sarcosine (all analogs), and phenylalanine at position 4 was replaced with one of the following noncoded amino acids: naphthylalanine (Nal, analogs 3-5 and 15), cyclohexylalanine (Cha, analogs 6-8 and 16), dichlorophenylalanine (PheCl2, analogs 9-10),

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nitrophenylalanine (PheNO2, analogs 11-12) or tert-butyl-alanine (Ala-OtBu, analog 13).

Some analogs were then further modified by replacing the n-octanoyl bound to Dpr in position 3 with one of the following: A. a longer-chain fatty acid residue – decanoyl (analogs

1, 3, 6, 17, 19, 21), myristoyl (analogs 2, 4, 7, 10, 12, 18, 20, 22) or palmitoyl (analogs 5 and

8); B. unsaturated fatty acid residue – 9-decenoyl (analog 23) or N-10-undecynoyl (analog

24); C. other residues 1-adamantaneacetyl (analog 25) or 8-bromooctanoyl (analog 26). Other analogs were modified by incorporation of the second fatty acid residue into the structure, namely, palmitoyl bound to the secondary aminogroup of Lys in C-terminal part of the Downloaded from molecule (analogs 14-18 and 20-21), or by shortening of the peptide chain to 18 amino acids from the N-terminus (analogs 19-22). jpet.aspetjournals.org Competitive binding of ghrelin analogs to GHS-R1a and beta-lactamase-dependent

FRET assay

Rat ghrelin, [Dpr3]ghrelin and the novel ghrelin analogs competed with rat 125I-ghrelin at ASPET Journals on September 26, 2021 for binding to plasma membranes from LLC-PK1 cells with transfected human GHS-R1a receptor with a Ki mostly in the nanomolar range, or even lower, as shown in Table 2, with the exception of analogs 10, 11, 12, 15, 16, 18 and 22 which had Ki values in the range of

10-8-10-7 M. Activation of the beta-lactamase reporter gene in the U2OS cell line overexpressing GHS-R1a by ghrelin or selected ghrelin analogs revealed agonistic

-10 -8 characteristics of the ghrelin agonists with EC50 in the range of 10 to 10 , with most of the analogs activating the receptor comparably to ghrelin (Table 2).

Effect of ghrelin analogs on acute food intake in mice

The effects of s.c. administered ghrelin analogs on acute food intake were tested in freely fed mice. The results of the acute food intake experiments are shown in Table 2 and

Fig. 1 and 2. Most of the tested analogs significantly increased food intake in comparison with the saline-treated group, as shown by the food intake at 495 min after administration of the

JPET Fast Forward. Published on June 18, 2018 as DOI: 10.1124/jpet.118.249086 This article has not been copyedited and formatted. The final version may differ from this version. JPET #249086 peptide. Moreover, the orexigenic effect of analogs 1-10 and 18-22 was significantly higher than the effect of ghrelin at that time point (Table 2).

Fig. 1 shows the impact of fatty acid length on the orexigenic effect of the analogs.

Previously published (Maletínská et al., 2012) octanoylated ghrelin analogs [Sar1, (Dpr-N- oct)3]ghrelin, [Sar1, (Dpr-N-oct)3, Nal4]ghrelin and [Sar1, (Dpr-N-oct)3, Cha4]ghrelin were modified by replacing the octanoyl with decanoyl, myristoyl or palmitoyl. We found that although the elongation of the fatty acid chain slightly deteriorates the affinity of the analogs for the GHS-R1a receptor (Table 2), the elongation significantly improves the orexigenic Downloaded from effect of the compounds. This amelioration is especially pronounced when decanoyl or myristoyl is used; further elongation does not seem to contribute to further increase in food jpet.aspetjournals.org intake but still preserves the higher orexigenic effect relative to that of ghrelin or

[Dpr3]ghrelin. The effect of the tested analogs was long-term, lasting at least 8 hours after administration (Fig. 1 and Table 2). at ASPET Journals on September 26, 2021

Fig. 2 shows the dose-dependent orexigenic effect of [Sar1, (Dpr-N-myr)3, Nal4]ghrelin

1 3 4 (analog 4) and [Sar , (Dpr-N-myr) , PheCl2 ]ghrelin (analog 10), which significantly increased food intake at a dose of 5 mg/kg and 1 mg/kg, respectively.

Pharmacokinetics of selected ghrelin analogs in mice

As expected, ghrelin and [Dpr3]ghrelin were eliminated rapidly from mouse blood plasma, reaching zero concentration approximately 1 hour after administration. In contrast, all tested ghrelin analogs exhibited significantly prolonged stability in mouse blood, with half- lives of approximately 2 hours. Interestingly, all of the analogs persisted in the blood at a significant amount even 8 hours after administration (Fig. 3).

Growth hormone release after s.c. administration of selected ghrelin analogs to mice

Ghrelin, [Dpr3]ghrelin and the octanoylated ghrelin analog [Sar1, (Dpr-N-oct)3,

Nal4]ghrelin were shown to cause a significant increase in the blood levels of growth hormone

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10 min after the injection in 3-month-old mice. However, this effect on growth hormone release was not observed after the administration of myristoylated ghrelin analogs 4 and 7

(Fig. 4).

Effects of selected ghrelin analogs in mouse model of LPS-induced anorexia

Administration of bacterial lipopolysaccharide significantly decreased food intake in mice; during the initial 10 hours after LPS administration, the animals did not consume any food, whereas the control mice consumed approximately 3 g overnight, on average (Fig. 5A).

A single s.c. injection of myristoylated ghrelin analog 10 ([Sar1, (Dpr-N-myr)3, Downloaded from

4 PheCl2 ]ghrelin) significantly increased food intake in both the control mice (Fig. 5B) and

LPS-treated mice (Fig. 5C). Similar results were observed when myristoylated ghrelin analogs jpet.aspetjournals.org 4 and 7 were used to reverse the effect of LPS treatment (Fig. 5D).

Administration of analog 10 normalized blood levels of proinflammatory cytokines IL-2

(Fig. 6A) and TNF-α (Fig. 6B) which were increased by the administration of LPS. The at ASPET Journals on September 26, 2021 treatment with analog 10 also tended to decrease the levels of IL-1α and IL-4 but the result did not reach significance (data not shown). The levels of other cytokines measured by

Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel (e.g., IL-6, IL-10, interferon γ) were not significantly affected by the administration of analog 10 (data not shown).

Treatment with analog 10 significantly increased the expression of mRNA for orexigenic neuropeptides AgRP (Fig. 7A) and NPY (Fig. 7B) in the hypothalamus after the s.c. administration to LPS-treated mice, thereby activating orexigenic pathways. The mRNA expression of IL-1β and TNF-α in the hypothalamus was non-significantly decreased by the administration of analog 10 (data not shown).

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DISCUSSION

In this study, we provide in vitro and in vivo characterization of a series of novel peptidic GHS-R1a agonists, both under non-pathological conditions and in the context of

LPS-induced anorexia.

Peptides have the following advantages over other compounds targeting G protein- coupled receptors: they are less toxic than most of the small-molecule ligands and less immunogenic than large antibody-based drugs, they exhibit high target affinity and specificity, and they do not tend to interact with other drugs. Nevertheless, the usefullness of Downloaded from peptides as therapeutic agents is still limited by their low metabolic stability, poor bioavailability and fast body clearance (Ahrens et al., 2012). Similarly to many other jpet.aspetjournals.org regulatory peptides, ghrelin lacks in vivo stability; to achieve this stability, the peptide requires modifications, such as amino acid substitution, to resist in vivo degradation and preserve affinity for the receptor. Charron at al. described a stable fluorine-bearing at ASPET Journals on September 26, 2021 ghrelin(1-8) analog with positions 1, 3, 4 and 8 modified with isonipecotic acid, Dpr, Nal and

Thr, respectively, stating that the addition of naphthyl groups into the molecule structure makes the peptides structurally related to the GHS family of peptidomimetics (e.g., ipamorelin), where multiple hydrophobic pockets are accessed by the ligand (Charron et al.,

2017). Protection of the octanoyl group on Ser3 against the activity of esterases by replacing

Ser3 with Dpr was previously described by Bednarek at al.; this modification did not affect the binding or signaling properties of [Dpr3]ghrelin (Bednarek et al., 2000). In our previous study

(Maletínská et al., 2012), we described a series of novel peptidic GHS-R1a agonists based on

[Dpr3]ghrelin, which were further modified with noncoded amino acids (Sar in position 1 and noncoded bulky hydrophobic phenylalanine derivatives, e.g., Nal or Cha, in position 4). These ghrelin analogs were found to be highly stable with potent and long-lasting orexigenic effect.

Here, we describe another series of ghrelin analogs based on our previous study.

JPET Fast Forward. Published on June 18, 2018 as DOI: 10.1124/jpet.118.249086 This article has not been copyedited and formatted. The final version may differ from this version. JPET #249086

It was shown previously that when Ser3 is acylated with a large aliphatic group, the binding affinity is as effective as that of natural ghrelin; however, when small, less hydrophobic acids are used, the binding affinity is lost (Bednarek et al., 2000). That finding demonstrated that ghrelin is able to tolerate large lipophilic groups in position 3; therefore, in this study, we describe a series of analogs carrying various saturated, unsaturated or substituted fatty acids. Lipidization of peptides is a common tool to overcome the inability to cross the blood-brain barrier (Zhang and Bulaj, 2012); here we have shown that lipidization of ghrelin analogs with various fatty acids not only preserves their biological effects and ability Downloaded from to act centrally after peripheral administration but also renders most of the analogs more biologically effective than those that are octanoylated. jpet.aspetjournals.org In the in vitro studies, we confirmed that the modifications made to ghrelin analogs did not deteriorate their ability to bind to cell membranes with transfected GHS-R1a. With the exception of analogs 11 and 12 (with nitrophenylalanine in position 4), which had Ki values in at ASPET Journals on September 26, 2021

-7 the 10 M range, all other analogs had Ki values comparable with that of ghrelin or

[Dpr3]ghrelin, mostly in the 10-10 to 10-9 M range. In certain subseries of analogs, we observed that when a longer fatty acid was used, the affinity was one order of magnitude lower than in the analog with the same modifications of the peptide chain but with a shorter fatty acid (e.g. analog 1 vs. 2, 3 vs. 4 and 5, 6 vs. 7 and 8, 9 vs. 10, 17 vs. 18, 21 vs. 22).

However, this slight deterioration in binding affinities did not seem to reduce the in vivo effects of these analogs, suggesting that better stability of the compounds likely compensates for decreased binding capacities. All tested ghrelin analogs, again with the exception of analogs 11 and 12, also activated GHS-R1a signaling to an extent comparable to ghrelin.

The effect of ghrelin analogs on food intake persisted long-term, lasting up to 10 hours after administration, depending on the dose. This phenomenon, as was shown by pharmacokinetics measurements, was likely caused by enhanced resistance of the analogs

JPET Fast Forward. Published on June 18, 2018 as DOI: 10.1124/jpet.118.249086 This article has not been copyedited and formatted. The final version may differ from this version. JPET #249086 against degradation by proteases enabled by the incorporation of the specific alternative fatty acid and non-coded amino acids (besides an amide-bond attachment of fatty acid to Dpr3).

Analogs modified in position 4 (with Nal, Cha and PheCl2) were found to be especially effective and to increase food intake in mice significantly more compared with ghrelin, particularly when decanoyl or myristoyl was used; further elongation of the fatty acid chain did not seem to contribute to further increase in food intake. The 18-amino-acid shortened analogs also exhibited highly significant orexigenic effect.

The stability of the new ghrelin analogs in vivo in mice after s.c. administration was Downloaded from significantly higher when compared to ghrelin and [Dpr3]ghrelin, which were eliminated rapidly from mouse blood plasma. In contrast, all tested ghrelin analogs showed significantly jpet.aspetjournals.org prolonged stability in mouse blood, with half-lives of approximately 2 hours. The half-life extension of peptides by lipidization is generally achieved by an increased binding to serum albumin, which leads to a gradual peptide release and prolonged circulation time (Mäde et al., at ASPET Journals on September 26, 2021

2014), enabling a longer time of action of the peptide. This might also be the case for the tested ghrelin analogs, considering the double peak in concentration-time profiles following the administration of a single dose.

In our previous study, we reported that ghrelin, [Dpr3]ghrelin and octanoylated ghrelin analogs [Sar1, (Dpr-N-oct)3, Nal4]ghrelin and [Sar1, (Dpr-N-oct)3, Cha4]ghrelin significantly increased the release of growth hormone after a single injection (Maletínská et al., 2012).

However, this effect on GH release was not observed after the administration of myristoylated ghrelin analogs 4 and 7. This lack of GH release could actually be an advantage for the potential use of the analogs in the treatment of anorexia/cachexia syndromes, because there is still a possibility of association between GH and tumor growth in adults. There is strong evidence from animal and cell experiments suggesting the involvement of GH in the

As ghrelin has been proposed as a potential therapeutic target for anorexia/cachexia, we tested several selected potent analogs in the mouse LPS-induced anorexia model. Treatment with LPS, a major constituent of the cell wall of gram-negative bacteria, is commonly used to induce an acute phase response associated with pronounced hypophagia and wasting syndrome (Borner et al., 2012). A single LPS injection causes a transient and unsustained response, with the weight decrease and muscle degradation most obvious at 24 hours after Downloaded from administration (Duan et al., 2014). In our study, a single subcutaneous administration of

1 3 4 analog 10 ([Sar , (Dpr-N-myr) , PheCl2 ]ghrelin) effectively counteracted LPS-induced jpet.aspetjournals.org hypophagia and significantly increased food intake; similar results were observed after the administration of other myristoylated analogs 4 and 7.

The lack of appetite observed in inflammatory states is believed to be mediated by at ASPET Journals on September 26, 2021 elevated circulating levels of proinflammatory cytokines (particularly IL-1β, TNF-α and IL-6) which are among the most potent endogenous anorectic factors. LPS is a potent inducer of proinflammatory cytokines from macrophages and microglia; however, the neural pathways responsible for LPS-induced anorexia are not fully understood (Buchanan and Johnson, 2007;

Liu et al., 2016). Although it has been postulated that inflammatory anorexia might be caused by the inhibition of AgRP neurons by proinflammatory factors, no clear evidence for the involvement of arcuate nucleus neurons in inflammatory anorexia was demonstrated (Gautron et al., 2005; Liu et al., 2016). Duan et al. reported decreased AgRP but increased NPY mRNA expression 24 hours after LPS administration to rats (Duan et al., 2014; Duan et al., 2015).

Similarly, in our study, we did not observe any significant difference in the expression of neuropeptides after LPS administration. However, as expected, administration of ghrelin agonist 10 to LPS-treated mice significantly increased the expression of orexigenic

JPET Fast Forward. Published on June 18, 2018 as DOI: 10.1124/jpet.118.249086 This article has not been copyedited and formatted. The final version may differ from this version. JPET #249086 neuropeptides AgRP and NPY, as did ghrelin in rats with LPS-induced hypophagia (Rivas et al., 2017), consistent with the increased food intake.

Administration of analog 10 normalized the blood levels of proinflammatory cytokines

IL-2 and TNF-α which were increased by the administration of LPS. This finding is in agreement with multiple previous in vitro and in vivo studies which demonstrated the anti- inflammatory effects of ghrelin. Systemic administration of LPS was shown to suppress plasma ghrelin in rats (reviewed in (Bando et al., 2017)), likely via the effects of IL-1β

(Bando et al., 2017). Although endogenous ghrelin might not be produced in sufficient Downloaded from amounts to neutralize the LPS-induced cytokine production, an exogenously administered

GHS-R1a agonist may provide significant neutralizing capability. Ghrelin inhibited the jpet.aspetjournals.org production of IL-1β and TNF-α in LPS-stimulated murine macrophages (Waseem et al.,

2008) and the LPS-induced IL-6 release from mouse dopaminergic neurons (Beynon et al.,

2013), and decreased serum proinflammatory cytokines levels during endotoxemia in rats at ASPET Journals on September 26, 2021

(Wang et al., 2009) and mice (Chorny et al., 2008). GHRP-2 attenuated the concentrations of

TNF-α and IL-6 and repressed the activation of the transcription factor nuclear factor kappa B in LPS-induced acute lung injury in rats (Li et al., 2010).

Conclusions

Taken together, the described novel peptidic GHS-R1a agonists exhibited a high binding affinity to cell membranes with transfected GHS-R1a and a high ability to induce

GHS-R1a signaling activation, comparable to native ghrelin. Selected analogs showed high in vivo stability and long-lasting and potent orexigenic effects after a single s.c. administration to mice. A single s.c. injection of the selected ghrelin analogs to mice with LPS-induced anorexia significantly increased food intake via the activation of orexigenic pathways and normalized blood levels of proinflammatory cytokines, indicating the potential of these analogs for anti-cachectic treatment.

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Acknowledgements

The authors would like to thank H. Vysušilová and T. Elbert (IOCB CAS, Prague,

Czech Republic) for the technical assistance and peptide iodination, respectively. We also thank Z. Lacinová (First Faculty of Medicine, Charles University in Prague and General

University Hospital, Prague, Czech Republic) for mRNA expression and cytokines measurement, and J.A. Fehrentz and J. Martinez (IBMM, Montpellier, France) for providing the plasma membranes with transfected human GHS-R1a receptor. Downloaded from Authorship Contributions

Participated in research design: Holubová, Železná, and Maletínská.

Conducted experiments: Holubová and Kákonová. jpet.aspetjournals.org

Contributed new reagents or analytic tools: Blechová.

Performed data analysis: Holubová, Kákonová, and Maletínská.

Wrote or contributed to the writing of the manuscript: Holubová, Kuneš, Železná, and at ASPET Journals on September 26, 2021

Maletínská.

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Footnotes

This work was supported by the Czech Academy of Sciences [grants RVO:61388963 and

RVO:67985823].

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Legends for Figures

Fig. 1: Effect of ghrelin analogs on cumulative food intake after s.c. administration to fed mice. All peptides were administered s.c. at a dose of 5 mg/kg and food intake was monitored for 8 hours. A. Effect of analogs 1 [Sar1, (Dpr-N-dec)3]ghrelin and 2 [Sar1, (Dpr-N- myr)3]ghrelin compared with the octanoylated analog [Sar1, (Dpr-N-oct)3]ghrelin. B. Effect of analogs 3 [Sar1, (Dpr-N-dec)3, Nal4]ghrelin, 4 [Sar1, (Dpr-N-myr)3, Nal4]ghrelin and 5 [Sar1,

(Dpr-N-palm)3, Nal4]ghrelin compared with the octanoylated analog [Sar1, (Dpr-N-oct)3,

Nal4]ghrelin. C. Effect of analogs 6 [Sar1, (Dpr-N-dec)3, Cha4]ghrelin, 7 [Sar1, (Dpr-N-myr)3, Downloaded from

Cha4]ghrelin and 8 [Sar1, (Dpr-N-palm)3, Cha4]ghrelin compared with the octanoylated [Sar1,

(Dpr-N-oct)3, Cha4]ghrelin. The curves are representative of the results obtained from at least jpet.aspetjournals.org 2 independent experiments. The data are presented as means ± S.E.M. and were analyzed by repeated measures ANOVA followed by Bonferroni post hoc test. Significance is * P < 0.05,

** P < 0.01, *** P < 0.001 vs. saline-treated group (n=5-8). at ASPET Journals on September 26, 2021

Fig. 2: Dose-dependent orexigenic effect of ghrelin analogs 4 and 10 after s.c. administration to fed mice. Ghrelin analogs 4 [Sar1, (Dpr-N-myr)3, Nal4]ghrelin (A) and 10

1 3 4 [Sar , (Dpr-N-myr) , PheCl2 ]ghrelin (B) were injected s.c. at doses of 0.2, 1 and 5 mg/kg and food intake was monitored for 8 hours. The data are presented as means ± S.E.M. and were analyzed by repeated measures ANOVA followed by Bonferroni post hoc test. Significance is

* P < 0.05, ** P < 0.01, *** P < 0.001 vs. saline-treated group (n=5-6).

Fig. 3: A. Pharmacokinetics of selected ghrelin analogs in vivo after s.c. administration to mice. Mice were s.c. injected with ghrelin and selected ghrelin analogs at a dose of

5 mg/kg. Blood was collected before the injection (time 0) and 0.5, 1, 2, 4, 8 and 16 hours after injection and the concentration of ghrelin analogs was measured by commercially

B. Detail of initial 2 hours after analogs administration. Data are presented as means ±

S.E.M., n=4.

Fig. 4: Effect of selected ghrelin analogs on growth hormone release in mice. 10 min after the s.c. administration of ghrelin or ghrelin analogs (5 mg/kg) to 3-month-old mice (n=6), blood was collected and GH was measured in blood plasma using commercially available

ELISA kit. Data are presented as means ± S.E.M. and were evaluated using the unpaired Downloaded from t-test. Significance is * P < 0.05, ** P < 0.01 vs. saline-treated group.

jpet.aspetjournals.org Fig. 5: Effect of ghrelin analog 10 on food intake in a mouse model of LPS-induced anorexia. At 5:00 p.m., the LPS group was i.p. injected with LPS (100 µg/kg) in 0.2 mL of saline, the controls received an equal volume of saline. Overnight food intake was monitored at ASPET Journals on September 26, 2021 automatically with the food intake monitoring system. At 7:00 a.m. on the following day, the mice of both groups were s.c. injected with 0.2 mL of saline or ghrelin analog 10, [Sar1, (Dpr-

3 4 N-myr) , PheCl2 ]ghrelin. Food intake was monitored for another 10 hours. A. Cumulative food intake during the whole experiment. B, C. Cummulative food intake from the ghrelin analog injection to the end of the experiment. D. Comparison of orexigenic effect of ghrelin and selected ghrelin analogs in LPS-treated mice. Data are presented as means ± S.E.M. and were evaluated using the one-way ANOVA folowed by Bonferroni post hoc test (A) or unpaired t-test vs. the corresponding saline-treated group (B, C, D). Significance is * P <

0.05, ** P < 0.01, *** P < 0.001 (A.-C. n=4-5, D. n=4-8).

Fig. 6: Effect of ghrelin analog 10 on IL-2 (A) and TNF-α (B) levels in a mouse model of

LPS-induced anorexia. Blood samples were collected 24 hours after LPS administration and

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10 hours after analog 10 administration. Data are presented as means ± S.E.M. and were evaluated using the two-way ANOVA folowed followed by Bonferroni post hoc test.

Significance is * P < 0.05, *** P < 0.001 (n=4-5).

Fig. 7: Effect of ghrelin analog 10 on hypothalamic mRNA expression of orexigenic neuropeptides AgRP and NPY in a mouse model of LPS-induced anorexia. Hypothalami were dissected 24 hours after LPS administration and 10 hours after analog 10 administration.

Gene expressions were measured by RT-PCR, using GAPDH as control. Data are presented Downloaded from as means ± S.E.M. and were evaluated using the two-way ANOVA folowed followed by

Bonferroni post hoc test. Significance is *** P < 0.001 (n=4-5). jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Tables jpet.aspetjournals.org Table 1: Structures of ghrelin analogs

Aaa-Ser-Bbb-Ccc-Leu-Ser-Pro-Glu-His-Gln-His-Gln-Lys-Ala-Gln-Gln-Arg-Ddd-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Eee-Leu-Gln-Pro-Arg.

No. Compound No. aa Position 1 (Aaa) 3 (Bbb) 4 (Ccc) at ASPET Journals on September 26, 2021 16 (Ddd) 24 (Eee) Ghrelin 28 Gly Ser(O-oct) Phe Lys Lys [Dpr3]ghr 28 Gly Dpr(N-oct) Phe Lys Lys 1 [Sar1, (Dpr-N-dec)3]ghr 28 Sar Dpr(N-dec) Phe Lys Lys 2 [Sar1, (Dpr-N-myr)3]ghr 28 Sar Dpr(N-myr) Phe Lys Lys 3 [Sar1, (Dpr-N-dec)3, Nal4]ghr 28 Sar Dpr(N-dec) Nal Lys Lys 4 [Sar1, (Dpr-N-myr)3, Nal4]ghr 28 Sar Dpr(N-myr) Nal Lys Lys 5 [Sar1, (Dpr-N-palm)3, Nal4]ghr 28 Sar Dpr(N-palm) Nal Lys Lys 6 [Sar1, (Dpr-N-dec)3, Cha4]ghr 28 Sar Dpr(N-dec) Cha Lys Lys 7 [Sar1, (Dpr-N-myr)3, Cha4]ghr 28 Sar Dpr(N-myr) Cha Lys Lys 8 [Sar1, (Dpr-N-palm)3, Cha4]ghr 28 Sar Dpr(N-palm) Cha Lys Lys 1 3 4 9 [Sar , (Dpr-N-oct) , PheCl2 ]ghr 28 Sar Dpr(N-oct) PheCl2 Lys Lys 1 3 4 10 [Sar , (Dpr-N-myr) , PheCl2 ]ghr 28 Sar Dpr(N-myr) PheCl2 Lys Lys 1 3 4 11 [Sar , (Dpr-N-oct) , PheNO2 ]ghr 28 Sar Dpr(N-oct) PheNO2 Lys Lys 1 3 4 12 [Sar , (Dpr-N-myr) , PheNO2 ]ghr 28 Sar Dpr(N-myr) PheNO2 Lys Lys 13 [Sar1, (Dpr-N-oct)3, Ala-OtBu4]ghr 28 Sar Dpr(N-oct) Ala-OtBu Lys Lys 14 [Sar1, (Dpr-N-oct)3, Lys(N-palm)24]ghr 28 Sar Dpr(N-oct) Phe Lys (Lys-N-palm) 15 [Sar1, (Dpr-N-oct)3, Nal4, Lys(N-palm)24]ghr 28 Sar Dpr(N-oct) Nal Lys (Lys-N-palm) 16 [Sar1, (Dpr-N-oct)3, Cha4, Lys(N-palm)24]ghr 28 Sar Dpr(N-oct) Cha Lys (Lys-N-palm) 17 [Sar1, (Dpr-N-dec)3, Lys(N-palm)24]ghr 28 Sar Dpr(N-dec) Phe Lys (Lys-N-palm) 18 [Sar1, (Dpr-N-myr)3, Lys(N-palm)24]ghr 28 Sar Dpr(N-myr) Phe Lys (Lys-N-palm)

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19 [Sar1, (Dpr-N-dec)3]ghr(1-18) 18 Sar Dpr(N-dec) Phe Lys Lys 20 [Sar1, (Dpr-N-myr)3]ghr(1-18) 18 Sar Dpr(N-myr) Phe jpet.aspetjournals.org Lys Lys 21 [Sar1, (Dpr-N-dec)3, Lys(N-palm)16]ghr(1-18) 18 Sar Dpr(N-dec) Phe (Lys-N-palm) Lys 22 [Sar1, (Dpr-N-myr)3, Lys(N-palm)16]ghr(1-18) 18 Sar Dpr(N-myr) Phe (Lys-N-palm) Lys 23 [Sar1, (Dpr-N-decen)3]ghr 28 Sar Dpr-N-decen Phe Lys Lys 1 3

24 [Sar , (Dpr-N-undecyn) ]ghr 28 Sar Dpr-N-undecyn Phe at ASPET Journals on September 26, 2021 Lys Lys 25 [Sar1, (Dpr-N-ada)3]ghr 28 Sar Dpr-N-ada Phe Lys Lys 26 [Sar1, (Dpr-N-bromooct)3]ghr 28 Sar Dpr-N-bromooct Phe Lys Lys

Ada, 1-adamantaneacetyl; Ala-OtBu, tert-butylalanine; bromooct, 8-bromooctanoyl; Cha, cyclohexylalanine; dec, decanoyl; decen, decenoyl;

Dpr, diaminopropionic acid; Nal, naphthylalanine; myr, myristoyl; oct, octanoyl; palm, palmitoyl; PheCl2, dichlorophenylalanine; PheNO2, nitrophenylalanine; undecyn, 10-undecynoyl.

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Table 2: Biological properties of ghrelin analogs – competitive binding studies, receptor activation assay and orexigenic effect

Analog 125I-ghrelin binding to Activation of bla Food intake in fasted mice human GHS-R1a reporter gene (5 mg/kg s.c.) (LLC-PK1 (GHS-R1a at 495 min membranes) overexpressing cells)

Ki [nM] EC50 [nM] % of saline- significance vs.: treated group saline ghrelin Ghrelin 0.69±0.05 0.59±0.33 167.9±19.4 ** - 3

[Dpr ]ghr 0.64±0,03 NT 223.4±32.5 ** NS Downloaded from 1 0.63±0.03 0.91±0.13 416.6±73.2 *** ** 2 2.11±0.16 2.14±1.05 418.8±45.4 *** *** 3 0.65±0.07 2.39±0.23 425.2±73.0 *** **

4 2.02±1.11 4.19±1.54 425.5±49.5 *** *** jpet.aspetjournals.org 5 4.25±0.64 4.42±0.58 303.8±40.5 *** ** 6 1.04±0.06 0.49±0.08 421.1±75.2 *** ** 7 6.42±0.51 1.01±0.19 437.8±66.8 *** ** 8 5.90±0.38 0.99±0.13 270.5±38.3 *** *

9 2.45±0.37 1.29±0.71 667.6±126 *** *** at ASPET Journals on September 26, 2021 10 16.8±1.81 9.50±9.30 836.8±176 *** *** 11 230±25.6 170±103 184.7±82.7 NS NS 12 126±7.18 45.8±23.9 201.4±64.0 NS NS 13 4.72±0.45 NT 144.8±17.1 NS NS 14 6.10±0.39 NT 172.7±21.4 * NS 15 23.4±5.34 NT 148.8±12.1 * NS 16 20.8±4.32 NT 174.2±17.2 ** NS 17 4.27±0.45 2.94±0.44 373.9±209 NS NS 18 14.9±2.94 4.38±1.90 394.4±81.8 ** *** 19 3.71±0.78 NT 280.9±48.9 ** * 20 3.38±0.90 NT 353.6±66.4 *** * 21 5.99±0.79 1.51±0.30 297.1±51.9 * ** 22 50.9±8.65 3.79±2.67 333.4±59.4 ** ** 23 1.52±0.43 NT 128.6±39.8 NS NS 24 0.83±0.02 NT 192.0±20.2 ** NS 25 0.27±0.12 NT 181.3±21.9 ** NS 26 0.79±0.08 NT 195.6±28.9 ** NS

NS, not significant; NT, not tested. Data from in vitro studies are presented as the means ±

S.E.M. of at least three separate experiments. In the competitive binding studies, Ki was

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calculated using the Cheng-Prusoff equation. EC50 is the concentration of peptide that yielded 50% of the maximal effect. Food intake was analyzed at 495 min after the s.c. administration of tested peptides and is presented as percentage of the saline-treated group consumption to compare data from multiple experiments. Data were evaluated by unpaired t-test (n=5-8 mice in at least 2 experiments). Significance is * P < 0.05, ** P < 0.01, *** P < 0.001.

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Figures

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Fig. 2 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Fig. 3

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Fig. 4 Downloaded from

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at ASPET Journals on September 26, 2021

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Fig. 5 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Fig. 6 Downloaded from jpet.aspetjournals.org

at ASPET Journals on September 26, 2021

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Fig. 7 Downloaded from jpet.aspetjournals.org

at ASPET Journals on September 26, 2021