Quinolones modulate ghrelin receptor signaling: Potential for a novel small molecule scaffold in the treatment of cachexia

Article abstract of DOI:10.3390/ijms19061605

Cachexia is a metabolic wasting disorder characterized by progressive weight loss, muscle atrophy, fatigue, weakness, and appetite loss. Cachexia is associated with almost all major chronic illnesses including cancer, heart failure, obstructive pulmonary disease, and kidney disease and significantly impedes treatment outcome and therapy tolerance, reducing physical function and increasing mortality. Current cachexia treatments are limited and new pharmacological strategies are needed. Agonists for the growth hormone secretagogue (GHS-R1a), or ghrelin receptor, prospectively regulate the central regulation of appetite and growth hormone secretion, and therefore have tremendous potential as cachexia therapeutics. Non-peptide GHS-R1a agonists are of particular interest, especially given the high gastrointestinal degradation of peptide-based structures, including that of the endogenous ligand, ghrelin, which has a half-life of only 30 min. However, few compounds have been reported in the literature as non-peptide GHS-R1a agonists. In this paper, we investigate the in vitro potential of quinolone compounds to modulate the GHS-R1a in both transfected human cells and mouse hypothalamic cells. These chemically synthesized compounds demonstrate a promising potential as GHS-R1a agonists, shown by an increased intracellular calcium influx. Further studies are now warranted to substantiate and exploit the potential of these novel quinolone-based compounds as orexigenic therapeutics in conditions of cachexia and other metabolic and eating disorders.

Full text of DOI:10.3390/ijms19061605

International Journal of
Molecular Sciences
Article
Quinolones Modulate Ghrelin Receptor Signaling:
Potential for a Novel Small Molecule Scaffold in the
Treatment of Cachexia
Cristina Torres-Fuentes 1 , Elena Pastor-Cavada , Rafael Cano 2 , Dalia Kandil 3,4,
,†
1,†
ID
2
5
3
2
Rachel Shanahan , Rocio Juan , Hamdy Shaban , Gerard P. McGlacken and
Harriët Schellekens 1
,3,4,
*
1
APC Microbiome Ireland, University College Cork, T12 YT20 Cork, Ireland;
ctorresfuentes@ucdavis.edu (C.T.-F.); elenapastorcavada@gmail.com (E.P.-C.)
School of Chemistry and the Analytical and Biological Chemistry Research Facility (ABCRF),
University College Cork, T12 YT20 Cork, Ireland; rafael.canom@uam.es (R.C.);
rachelshanahan91@gmail.com (R.S.); g.mcglacken@ucc.ie (G.P.M.)
Food for Health Ireland, University College Cork, T12 YT20 Cork, Ireland; dkandil@ucc.ie (D.K.);
hamdy.shaban@gmx.ch (H.S.)
Department of Anatomy and Neuroscience, University College Cork, T12 YT20 Cork, Ireland
Plant Biology and Ecology Department, Seville University, 41012 Seville, Spain; rjuan@us.es
Correspondence: h.schellekens@ucc.ie; Tel.: +353-(0)21-420-5429
2
3
4
5
*
†
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 May 2018; Accepted: 22 May 2018; Published: 30 May 2018
Abstract: Cachexia is a metabolic wasting disorder characterized by progressive weight loss,
muscle atrophy, fatigue, weakness, and appetite loss. Cachexia is associated with almost all major
chronic illnesses including cancer, heart failure, obstructive pulmonary disease, and kidney disease
and significantly impedes treatment outcome and therapy tolerance, reducing physical function and
increasing mortality. Current cachexia treatments are limited and new pharmacological strategies are
needed. Agonists for the growth hormone secretagogue (GHS-R1a), or ghrelin receptor, prospectively
regulate the central regulation of appetite and growth hormone secretion, and therefore have
tremendous potential as cachexia therapeutics. Non-peptide GHS-R1a agonists are of particular interest,
especially given the high gastrointestinal degradation of peptide-based structures, including that of
the endogenous ligand, ghrelin, which has a half-life of only 30 min. However, few compounds have
been reported in the literature as non-peptide GHS-R1a agonists. In this paper, we investigate the
in vitro potential of quinolone compounds to modulate the GHS-R1a in both transfected human cells
and mouse hypothalamic cells. These chemically synthesized compounds demonstrate a promising
potential as GHS-R1a agonists, shown by an increased intracellular calcium influx. Further studies are
now warranted to substantiate and exploit the potential of these novel quinolone-based compounds as
orexigenic therapeutics in conditions of cachexia and other metabolic and eating disorders.
Keywords: ghrelin; quinolones; cachexia; GHS-R1a
1
. Introduction
Cachexia is an advancing metabolic disorder that leads to substantial weight and muscle loss and
affects over nine million people worldwide [
major chronic illnesses such as cancer, heart failure, chronic obstructive pulmonary disease, and kidney
disease [ ]. For many years, physicians and researchers have focused their attention on the primary
illness and it took until 2006 for a formal definition for cachexia to emerge [ ]. It has been defined
1]. Cachexia is associated with the late stages of many
2
3
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as a “complex metabolic syndrome associated with underlying illness and characterized by loss of
muscle with or without loss of fat mass” [ ]. More recently, cachexia is further defined as weight loss
4
greater than 5% of body weight over 12 months or less, in the presence of chronic illness, or a body
2
mass index (BMI) lower than 20 kg/m [
5
]. The latest research suggests that cachexia is more than
a muscle disease and is related to problems in the brain’s regulation of appetite and feeding and to
inflammation and metabolic imbalances [1].
Current cachexia treatments are limited and new pharmacological strategies are urgently
needed [6]. Agonists of the ghrelin receptor (GHS-R1a) have recently received attention for
cachexia treatment. Ghrelin, which is the endogenous GHS-R1a ligand, is so far the only known
peripherally-derived hormone with orexigenic effects [7]. However, ghrelin has several other unique
functions in the regulation of energy homeostasis [8–10] and is involved in the modulation of different
physiological activities in the organism [10 11]. For example, ghrelin has been shown to be involved
,
in the central regulation of muscle metabolism [12]. As a 28-amino acid peptide hormone, it is
primarily produced in the oxyntic mucosa of the stomach as well as in other gastrointestinal tissues [13].
It acts as the endogenous ligand for GHS-R1a and causes the release of a growth hormone from the
pituitary gland [14]. Ghrelin administration increases food intake by activating GHS-R1a located
in neurons of the arcuate nucleus of the hypothalamus [10,15,16] and stimulates gastrointestinal
motility while decreasing nausea and vomiting [17]. Ghrelin has a central role in regulating the
energy balance by decreasing energy consumption via the inhibition of the sympathetic nervous
system output to brown adipose tissue, which reduces thermogenesis [18]. Therefore, activation
of ghrelin signaling may promote a positive energy balance, increased food intake, and body
weight gain [19]. However, ghrelin administration efficacy in patients is limited due to the need
for parenteral administration and a short half-life (30 min) [20]. Therefore, the development of new
GHS-R1a agonists with a longer half-life allows for once-daily administration, which could be of great
potential benefit in the treatment of cachexia [21]. Given the problematic degradation of peptidic
based structures [22,23], non-peptide GHS-R1a agonists may have higher potential as therapeutics.
However, perhaps surprisingly, very few compounds have been reported in the literature as promising
non-peptide GHS-R1a agonists. Anamorelin, (Helsinn Healthcare SA, Lugano, Switzerland), which is
a non-peptide, orally-active compound, is the only centrally-penetrant compound described to
date. It is shown to have appetite-enhancing and anabolic effects via selective agonism of the
GHS-R1a. Anamorelin is currently under development for the treatment of cancer cachexia [24–28].
Other compounds such as the polyphenol naringin, the gamma-secretase inhibitor “semagacestat,”
and biosynthetic intermediates of teaghrelins and emoghrelin have also shown a potential as GHS-R1a
agonists [29
trifluoromethyl group [32
potential in vitro and promised modulation of food intake in vivo
–
31]. Recently, we also demonstrated a novel, non-peptidic, 2-pyridone bears a crucial
33] at the 3-position, which demonstrated a selective GHS-R1a agonist
22]. However, further studies
,
[
are warranted to identify novel selective GHS-R1a-targeting small molecule agonists with increased
potency and selectivity as well as improved pharmacokinetics and bioavailability. New and improved
scaffolds are required to allow a greater diversification of the molecular structure. In this regard,
quinolone compounds (which are considered privileged molecular scaffolds) could meet some of
these requirements.
Quinolone compounds have a very diverse biological profile which includes widespread, medicinally
interesting properties ranging from antiallergenic and anticancer to antimicrobial activities [34].
Additionally, quinolones have been used in the development of numerous synthetic drugs [34].
The success of the quinolone moiety across numerous areas of biological importance is underpinned by the
ability to selectively modify its molecular framework and, therefore, grant favorable biological properties.
For example, recently, quinolones have been studied as signaling molecules for the interruption of
quorum sensing in Pseudomonas aeruginosa and other pathogens [35–39]. The interactions have shown that
exquisite specificity-of-structure is often required for biological activity, which renders the easily-decorated
quinolones as an excellent molecular base-point. Therefore, the aim of this study was to investigate the

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ability of chemically synthesized quinolone compounds to modulate the GHS-R1a knowing that the
molecular structures could be easily modified and tailored.
2
2
2
. Results
.1. Quinolones Compounds and Their Effects on GHS-R1a Signaling
.1.1. Quinolones Synthesis
The 4-Hydroxy-2-heptylquinoline and related analogs (see Figure 1) were prepared using a
procedure involving a Conrad-Limpach type cyclisation. The β-keto-ester, methyl-3-oxodecanoate was
prepared by acylation of Meldrum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) with octanoyl chloride,
which was followed by methanolysis (see Figure 1). Condensation with the corresponding aniline in
the presence of p-toluene sulfonic acid using a Dean-Stark apparatus and subsequent cyclisation of the
enamine at reflux in diphenyl ether gave the corresponding 2-heptyl-4-quinolone.
Figure 1. Quinolones synthesis. The
of Meldrum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) with octanoyl chloride, which was followed
by methanolysis. Condensation of the -keto-ester with the corresponding aniline in the presence of
β-keto-ester, methyl-3-oxodecanoate was prepared by acylation
β
p-toluene sulfonic acid using a Dean-Stark apparatus and subsequent Conrad-Limpach cyclisation of
the enamine at reflux in diphenyl ether gave the corresponding 2-heptyl-4-quinolone.
Sixteen different quinolones (Q1-Q16) were prepared and used for the investigation of their
potential to modulate the GHS-R1a. Substitution of the anthranilate ring (MeO, F, Cl, CO2Et, etc.)
focused mainly at the C6 position due to the ease of preparation (only one regioisomer is formed upon
cyclisation). However, the 7-OMe and 8-OMe compounds were also prepared and tested. Extension of
the aromatic system was also investigated along with the length of the alkyl chain (Me, n-heptyl and
n-nonyl). Lastly, substitution of the C3 position (with a Br) was also tested (see Figure 2).

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Figure 2. Chemical structure of Quinolones compounds.
2
.1.2. Quinolones Compounds Activate GHS-R1a-Mediated Intracellular Calcium Mobilization
An initial screening involving the diverse set of quinolone compounds was carried out in
order to investigate their potency to activate the GHS-R1a by measuring intracellular calcium
mobilization [22,40]. Primary signaling of the GHS-R1a is mediated via Gq/ -dependent signaling,
11
which leads to the activation of phospholipase C (PLC) and second messengers, inositol trisphosphate
IP ) and diacylglycerol (DAG). This leads to increased intracellular calcium influx from the
(
3
endoplasmic reticulum [41,42]. Exposure to quinolone compounds at 10 µM led to a small intracellular
calcium influx (<25%) in wild-type Hek293a cells. An increased calcium influx was observed in Hek
cells overexpressing the GHS-R1a (Hek293a-GHS-R1a-EGFP) (see Figure 3A). In addition, this effect
was not observed in Hek cells stably overexpressing the serotonin receptor (Hek293a-5HT2C-EGFP),
which suggests at least some selectivity of quinolones for the GHS-R1a (see Figure 3A). Although
most of the quinolone compounds displayed calcium influx levels below those generated by the
endogenous ligand ghrelin (0.3
mobilization to a similar extent to that exerted by ghrelin and were selected for further investigations
see Figure 3A). Full experimental procedures and characterization of our “hit” quinolones Q14,
µM), quinolones Q14, Q15, and Q16 facilitated intracellular calcium
(
1
13
Q15, and Q16 as well as the H and C NMR spectra are included in the Supplementary files.
The resazurin assay, which is a widely used method to analyze the viability of bacteria and mammalian
cells, demonstrated the non-toxicity of the selected quinolone compounds (see Figure S1) [43].
The Q14, Q15, and Q16 quinolones were also able to mediate intracellular calcium influx following
pre-treatment to the inverse agonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SP-analog) (1
(
(
µ
M)
see Figure 3B). Inverse agonists decrease the constitutive basal activity of G protein-coupled receptors
GPCRs), which reduces its internalization from the membrane to the cytoplasm [44]. In addition,
exposure to the SP-analog has been shown to significantly reduce GHS-R1a constitutive activity,
which increases its cell surface expression and leads to sensitization of GHS-R1a signaling [45 47].
We have also shown multiple times in our laboratory that pre-treatment with the SP-analog leads to
a significantly enhanced agonist-mediated calcium mobilization [22 40]. Additionally, SP-analogue
pre-treatment significantly enhanced (p 0.01) the endogenous ligand ghrelin-mediated calcium
influx in Hek293a-GHS-R1a-EGFP cells (see Figure 3B). A similar effect (p 0.001) was observed
–
,
≤
≤
for quinolone Q14-mediated response, which indicates high selectivity of this compound for the
GHS-R1a (see Figure 3B). Exposure to quinolones Q15 and Q16 also showed an increased intracellular
calcium mobilization upon pre-treatment with the SP-analogue but failed to reach significance
(see Figure 3B). These quinolone compounds also stimulated an intracellular calcium influx in a

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concentration-dependent manner with an efficacy of 121.2%, 94.7%, and 102.4% and EC50 of 4.5
.2 M, and 73.2 M for Q14, Q15, and Q16, respectively (see Figure 3C). Furthermore, the most active
quinolone Q15 displayed an EC , which was 25-fold higher than that reported for the endogenous
µM,
2
µ
µ
50
GHS-R1a agonist ghrelin (EC50 88 nM) and 314-fold higher when compared to the non-peptide agonist
MK-0677 (EC50 7 nM). This highlights the very promising potential of the quinolone molecular template
for the development of GHS-R1a targeting therapeutics [22].
Figure 3. Quinolone compounds (10
Hek-GHS-R1a-EGFP cells. ( ) Quinolones effects on intracellular calcium influx in wild-type Hek
cells. Hek cells stably overexpress the GHS-R1a and Hek cells stably overexpress the 5HT2C receptor.
) Quinolones induced GHS-R1a activation was enhanced following attenuation of constitutive
receptor activity by pre-treatment with the GHS-R1a inverse agonist, [D-Arg1, D-Phe5, D-Trp7,9,
Leu11]-substance P (1 M, SP-analogue), ( ) Quinolones dose-response curve on the intracellular
calcium influx in Hek cells stably overexpressing the GHS-R1a. Graphs represent the mean SEM
µM) specifically activate intracellular calcium influx in
A
(
B
µ
C
±
from at least four independent experiments with each sample performed in triplicate. The intracellular
calcium increase was depicted as a percentage of maximal calcium increase as elicited by the control
(
*
3.3% FBS). Significant differences are presented by * indicating p
** indicating p ≤ 0.001. For further statistic details, see the methods section.
≤
0.05, ** indicating p
≤
0.01 and
Intracellular calcium mobilization was also investigated with calcium imaging using confocal
fluorescent microscopy. Exposure to quinolone Q14, Q15, and Q16 led to a significant activation
(
p
≤
0.001) of calcium mobilization in both Hek cells overexpressing the GHS-R1a as well as in a
hypothalamic neuronal cell line model of an embryonic mouse. The hypothalamus cell line-N38
mHypoE-N38), which possesses endogenous levels of GHS-R1a expression [22] (see Figure 4 and live
(
imaging video clip in Supplementary files). Therefore, the addition of these quinolone compounds
showed an increased fluorescence intensity when compared to baseline with similar efficacy as
the positive control MK-0677, which is an established non-peptide agonist of the GHS-R1a [48].
In the case of Hek293a-GHS-R1a-EGFP cells, exposure to the quinolones actually resulted in a
higher intracellular calcium influx when compared to the GHS-R1a agonist MK-0677 (see Figure 4A).
Furthermore, while Q14 and Q15 showed a trend to mediate higher calcium influx (p = 0.075 and

Int. J. Mol. Sci. 2018, 19, 1605
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p = 0.085, respectively), Q16 exhibited a significant (p
compared to MK-0677 (see Figure 4A).
≤
0.001) higher calcium mobilization when
Figure 4. Quinolone compounds activate GHS-R1a receptor overexpressed in Hek cells as well as the
endogenous receptor expressed in hypothalamic neurons. Quinolone compounds (10
activate GHS-R1a receptors in transfected human embryonic kidney (Hek293a) cells (
µ
M) and MK-0677
) and embryonic
A
mouse hypothalamic cell line-N38 (B) by using cell calcium imaging through confocal microscopy (for
visualization, see Supplementary videos in Supplementary files). Significant differences compared to
the baseline and MK-0677 controls are presented with * and # respectively with * indicating p
and ***/### indicating p ≤ 0.001. For further statistic details, see the methods section.
≤
0.05
Finally, the effect of the quinolones on GHS-R1a internalization was investigated in Hek cells by
stably overexpressing the GHS-R1a-EGFP. Receptor trafficking from the membrane to the cytoplasm
was analyzed by quantifying the EGFP fluorescence intensity. As expected, exposure to the endogenous
ligand ghrelin (1
to the inverse agonist SP-analog led to a significant (p
untreated cells (see Figure 5). However, exposure to the quinolones compounds did not show any
effect on the GHS-R1a internalization process (see Figure 5).
µ
M) led to significantly (p
≤
0.01) increased GHS-R1a internalization while exposure
0.01) opposite effect when compared to
≤
Figure 5. Quinolone compounds’ effects on the GSH-R1a internalization. Quinolone compounds do not
affect GSH-R1a internalization. (
the untreated control (assay buffer), ghrelin (1
P (SP-analog) (0.5 M) and quinolones Q14, Q15 and Q16 (10
A
) Representative images are depicted following different treatments:
M), [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance
M). ( ) The graph represents the
µ
µ
µ
B
mean ± SEM of quantified fluorescence intensity (15 pictures per treatment) of the perinuclear
GHS-R1a-EGFP receptor from four independent experiments with each treatment performed in
triplicates. ** indicates p ≤ 0.01. For statistic details, see the methods section.

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3
. Discussion
Cachexia affects between 10% and 40% of patients with chronic diseases and there is no effective
therapy currently available [49]. One new approach to treat this complex metabolic syndrome is
focused on the orexigenic peptide ghrelin, which increases appetite and growth hormone secretion [50].
Indeed, several beneficial effects of ghrelin in the treatment of cachexia have been reported, such as
increased energy consumption, increased appetite and muscle tissue, reduction of adipose tissue loss,
and improved respiratory muscle strength (see Reference [50]). However, due to the necessity of
parenteral administration, ghrelin’s short half-life, and potential side-effects, its efficacy in patients
is restricted [20]. Therefore, the development of new cachexia therapeutics with the ability to mimic
ghrelin actions but with improved efficacy in patients is necessary. Quinolone compounds are
mainly known for their widespread antimicrobial properties [51] but they have also been shown
to exert different beneficial effects including antiulcer, antihistamine, or antimutagenic properties [34].
Recently, quinolones have been studied as signaling molecules for the interruption of quorum sensing
in Pseudomonas aeruginosa and other pathogens [35]. Quinolones are very versatile structures that
can be easily modified to enhance their interaction with key active sites. Different functional groups
can be incorporated to 4-(1H)-quinolones on the anthranilate backbone or at the C-2 position via
modification of the ester precursor [52]. Late stage alteration (e.g., halogenation) of the key C-3
position can also be achieved to further fine-tune the structure-activity relationship [53]. Following on
from our previous study on the use of 2-pyridones [22], we investigated the potential of quinolone
compounds synthesized in our lab to modulate appetite in our well-established screening platform by
using Hek cells stably overexpressing GHS-R1a, which is the receptor of the hunger hormone ghrelin.
A number of quinolones (Q14, Q15, Q16) displayed intracellular calcium mobilization to a similar
extent to that exerted by ghrelin in Hek cells stably overexpressing the GHS-R1a. This indicates a
significant potential to modulate the Gq/ -dependent GHS-R1a signaling pathway (see Figure 3A).
11
Exposure to quinolones did not lead to increased calcium influx in Hek293-WT cells. The quinolones
screened in this study have a long hydrophobic heptyl chain at the C2 position, which, taken together
with the more polar quinolone part of the molecule, can grant unique lipid-type qualities. Interestingly,
a longer alkyl chain at C2 has proven critical for biological activity in a number of diverse settings [33].
In this research study, the presence of the heptyl chain at the C2 position appeared to play an important
role also. For example, analog Q14 showed good intracellular calcium mobilization while Q12,
which differs only at C2 and possesses a shorter alkyl chain, did not. Remarkably, Q7 was not very
active and differed only by two carbon units from the active Q15. That said, not all quinolones with a
C2 heptyl chain were highly active. The presence of substituents at the C6 position appeared to be
well tolerated, which allowed for secondary modification. However, not all substitution at C6 gave
quinolones capable of efficient calcium mobilization. Quinolones with electron-donating groups (Q14
and Q16) were active, but those with electron withdrawing groups (Q9, Q10) were generally not with
the exception of Q15. Overall, remarkable structural specificity was observed but no clear trend could
be identified, perhaps highlighting the specific requirements for GHS-R1a activation.
The three most active quinolones Q14, Q15, and Q16 were selected for further investigations.
An increased calcium influx was also observed following exposure to these quinolones in hypothalamic
neurons (see Figure 4) with endogenous levels of GHS-R1a expression, which suggests their
potential to activate hypothalamic neurons [22]. The GHS-R1a has a very high ligand-independent
signaling or constitutive signaling at approximately 50% of its maximal Gq/11-mediated efficacy [46].
The high constitutive GHS-R1a activity serves as a desensitization mechanism, which internalizes
the receptor after signaling into endosomes and sequesters the receptor from the membrane to
the cytoplasm in an endocytosis process mediated by
shown to reduce receptor constitutive activity and to increase receptor membrane expression
and enhance ligand-mediated calcium signaling [45 47]. Additionally, Q14 showed an enhanced
β-arrestin [46]. Inverse agonists have been
,
effect on calcium signaling upon pre-treatment with an SP-analog as well as an ability to activate
the GHS-R1a in a concentration-dependent manner (see Figure 3B,C), which indicates the high

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specificity of this compound for the GHS-R1a. Q15 and Q16 also showed an increased calcium
influx following SP-analog pre-treatment but they failed to be significant. In previous studies
from our laboratory, we have also shown that the basal levels of GHS-R1a constitutive signaling
decrease upon exposure to the inverse agonist SP-analog [22,40]. However, while quinolones elicited a
significant GHS-R1a-mediated intracellular calcium mobilization, no GHS-R1a internalization was
observed (see Figure 5), which suggests that these quinolone compounds may act as functional
selective or biased GHS-R1a agonists. These compounds activate the G
but are unable to trigger -arrestin recruitment and subsequent GHS-R1a receptor internalization
and desensitization [54 56]. An example of another ghrelin receptor ligand known as JMV3018 was
also able to partially activate the G q-protein signaling pathway but unable to recruit -Arrestin [57].
αq/11-mediated pathway
β
–
α
β
In addition, recent studies including a study carried out in our laboratory [58] have demonstrated that
GHS-R1a receptor ligands display signaling bias with functional selectivity for specific downstream
signaling pathways [22,59–62]. Indeed, several different active conformations of the GHS-R1a receptor
may exist [42], with biased ligands able to induce a receptor conformation that preferentially activates
only G protein-mediated or β-arrestin-mediated downstream signaling [63].
This biased modulation of GPCRs is becoming increasingly recognized in GPCR-drug discovery,
which may allow for the discovery of more selective therapeutic compounds meant for the most
appropriate GPCRs mediated functionalities [63]. Taken together, we provide compelling evidence
for quinolone-mediated GHS-R1a activation with functional selectivity for the Gq/11 pathway.
Further studies are needed to fully understand the role of this functional selectivity in mediating the
effects of GHS-R1a receptor ligands.
In conclusion, we have identified non-peptide quinolone compounds with promising GHS-R1a
activating potential in vitro. In terms of a structure activity-relationship, the results described herein
point to the necessity of a C2 heptyl chain (a shorter Me group leads to compounds displaying a
decrease in calcium intake (see Figure 1A)). Furthermore, a comparison of the hit compound Q15
possesses a C2-heptyl chain with others such as Q7, which possesses a C2-nonyl chain. This indicates
the remarkable requirements of the chain length. Direct comparison of substitution at C6 and C7
(
(
Q11 and Q14) shows the benefit of C6 substitution. Additionally, electron withdrawing groups
Q15) and electron donating (Q14) are equally tolerated at C6, which as the bulky t-Bu group (Q16).
Such disregard for the electronics and sterics at C6 is in stark contrast to the exquisite specificity
required of the chain length at C2.
It will be interesting to see how additional variants of the quinolone scaffold modulates the
GHS-R1a receptor in vitro and how this translates to food intake and anabolic effects in vivo. The focus
will be placed on the development of quinolones with substitution at C6 in concert with fine-tuning
further hits. Certainly, further development of the quinolone molecular template is warranted
to explore its therapeutic potential in metabolic disorders including cachexia and obesity via the
modulation of the GHS-R1a receptor.
4
4
4
. Materials and Methods
.1. General Procedure for the Preparation of Substituted 2-Alkyl-4-Quinolones
.1.1. Preparation of the β-Ketoester
2
,2-Dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid) (18.7 g, 130 mmol) was dissolved in distilled
◦
dichloromethane (200 mL). The solution was cooled to 0 C under N atmosphere. Pyridine (20.5 mL,
2
2
60 mmol) and octanoyl chloride (23.8 mL, 140 mmol) were added to the cooled solution, dropwise.
◦
The solution was stirred at 0 C for 1 h and then, at room temperature, for 1 h. The mixture was
washed with 5% HCl (3
×
75 mL) and with distilled water (75 mL). The solution was then dried with
anhydrous MgSO filtered and concentrated in vacuum to yield acyl Meldrum’s acid as a brown oil,
4
which was used in the subsequent step without further purification.

Int. J. Mol. Sci. 2018, 19, 1605
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Acyl Meldrum’s acid was dissolved in MeOH (180 mL) and heated at reflux for 5 h with constant
stirring. After allowing to cool, the reaction mixture was concentrated in vacuum yielding the crude
product as an orange oil. Purification was achieved by fractional distillation, which affords the β-keto
ester as a pale yellow oil (16.7 g, 64% yield). The analytical data was consistent with previously
published studies [64].
4
.1.2. Preparation of the Quinolones
To a solution of the -ketoester (5 mmol) in hexane (10 mL) were added to the corresponding
β
aniline (5 mmol) and p-toluene sulfonic acid (0.1 mmol). The reaction mixture was heated at reflux
◦
(
>70 C) under N atmosphere overnight using a Dean-Stark system. Upon completion, the reaction
2
mixture was concentrated in vacuum to afford the crude
β
-enamino ester, which was then added
◦
drop-wise to refluxing diphenyl ether (2 mL, >260 C). Reflux was maintained for approximately 1.5 h.
After cooling to room temperature, ether (approximately 20 mL) was added to the reaction mixture
◦
and left overnight at 5 C, which allows for the quinolone product to precipitate. The quinolone
was collected by vacuum filtration, recrystallized from hot methanol (if necessary), and dried
under vacuum.
Solvents and reagents were used as obtained from commercial sources and used without
1
purification. H NMR (300 MHz) spectra were recorded on Bruker Avance 300 NMR spectrometers
in proton-coupled mode. 13C NMR (75.5 MHz) spectra were recorded on Bruker Avance 300 NMR
◦
spectrometers in proton decoupled mode at 20 C in deuterated dimethyl sulphoxide or CDCl using
3
tetramethylsilane as an internal standard. High-resolution precise mass spectra (HRMS) were recorded
on a Waters LCT Premier Tof LC-MS instrument in electrospray ionization (ESI) mode using 50%
acetonitrile-water containing 0.1% formic acid as the eluent. Samples were made up in acetonitrile.
Infrared spectra were measured as pressed potassium bromide (KBr) for solids or thin films on sodium
chloride plates for liquids on a Perkin-Elmer FT-IR spectrometer.
4
.2. Compounds Preparation
All compounds including the ghrelin receptor agonists ghrelin (Innovagen, Lund, Sweden) and
MK-0677 (Tocris, R&D Systems, Bristol, UK) as well as the inverse agonist [D-Arg1, D-102 Phe5,
D-Trp7,9, Leu11] Substance P (SP-analogue) (Tocris, R&D Systems, Abingdon, UK) and the serotonin
receptor agonist 5-hydroxytryptamine (5-HT, H9523; Sigma, St. Louis, MO, USA) were prepared in
assay buffer (1
×
Hanks balanced salt solution, HBSS, (Gibco, Waltham, MA, USA), which contained
2
0 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA)).
4
.3. Cell Culture
Human embryonic kidney cells (Hek293a) (Invitrogen, Carlsbad, CA, USA) were cultured in high
glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated
fetal bovine serum (FBS) (Sigma-Aldrich) and 1% non-essential amino acids (NEAA) (Gibco) at
◦
culture conditions (37 C and 5% CO in a humidified atmosphere). Hek293a cells were transfected
2
with a plasmid construct expressing the human GHS-R1a receptor with a C-terminal enhanced
green fluorescent protein (EGFP) tag as previously described [31] and cultured in complete DMEM
media, which contains 300 ng/
maintenance antibiotic.
µL G418 (Calbiochem, Merck KGaA, Darmstadt, Germany) as
Embryonic mouse hypothalamus cell line N38 (mHypoE-N38) (Cederlane Laboratories,
Cellutions Biosystems, Burlington, ON, Canada) was maintained in high glucose Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS)
◦
(
Sigma-Aldrich) at culture conditions (37 C and 5% CO in a humidified atmosphere).
2
Cells were grown to a confluence of >85% and subsequently split to a lower density for
continued culturing.

Int. J. Mol. Sci. 2018, 19, 1605
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4
.4. Calcium Mobilization Assay
G-protein coupled receptor-mediated changes in intracellular calcium (Ca ) were investigated
using a Flex station II multiplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA)
based on protocols described in previous studies [22 31 40]. Briefly, wild-type Hek293a cells stably
2+
,
,
expressing the GHS-R1a or the 5HT2C receptors were seeded in black 96-well microtiter plates
4
(
2.8 × 10 cells/well) and maintained for about 24 h at cultured conditions. Growth media was then
replaced by serum-free DMEM media containing 1% NEAA and the cells were incubated for another
4 h at cultured conditions. Next, cells were incubated for 90 min with assay buffer (1× Hanks balanced
salt solution, HBSS, containing 20 mM HEPES) and 1x Ca4 dye (Molecular Devices Corporation)
1:1 v/v), according to the manufacturer’s instructions. The quinolones compounds were dissolved in
2
(
assay buffer at 10
µ
M containing 0.1% DMSO (Sigma-Aldrich). Finally, compounds were automatically
◦
added by the Flexstation II during continuous fluorescent measurements for a total of 80 s at 37 C in
flex mode with an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The relative
increase in intracellular calcium [Ca2 ] was calculated as the difference between maximum and baseline
fluorescence (Vmax-Vmin) and depicted as a percentage of relative fluorescent units (RFU), which is
normalized to a maximum response (100% signal) obtained with 3.3% FBS. Background fluorescence
was obtained by cells in assay buffer alone and subtracted from RFUs. Exposure to quinolones
following a pretreatment to the inverse agonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P was
also carried out. This pretreatment was performed during the calcium dye incubation. Data were
analyzed using the GraphPad Prism software (PRISM 5.0; GraphPAD Software Inc., San Diego, CA,
USA). Sigmoidal dose-response curves were constructed using nonlinear regression analysis with
a variable slope excluding values resulting from obvious incorrect pipetting by the Flexstation II
+
(Molecular Devices Corporation).
4
.5. Calcium Imaging
Calcium imaging experiments were carried out as previously described [22]. Hek-GHSR-1a-EGFP
and mHypoE-N38 cells were seeded in poly-L-lysine coated 35 mm dishes (MatTek Corporation,
Ashland, MA, USA) at a density of 15
×
104 cells/ml and maintained for ~24 h at culture conditions
◦
◦
(
37 C and 5% CO in a humidified atmosphere). After 24 h, cells were incubated for 40 min at 37 C
2
in Fluo-4 solution (Molecular probes Incorporation., Fisher Scientific, Waltham, MA, USA)) at 4
or 8 M for Hek-GHS-R1a-EGFP and mHypoE-N38 cells, respectively. After removal of the dye,
cells were washed twice with PBS and 2 mL of assay buffer (1
µM
µ
×
Hanks balanced salt solution, HBSS,
containing 20 mM HEPES) were added to the cells, which were stored in the dark at 37 C until
◦
visualization under the microscope.
◦
Each dish plate was mounted on the stage under culture conditions (37 C and 5% CO in
2
a humidified atmosphere) for stable recording for a longer period of time using laser scanning
confocal fluorescent microscopy (FV 1000 Confocal System; Olympus, Tokyo, Japan). Fluorescence
was visualized on an inverted microscope (CKX41; Olympus) setup with a sensitive XM10 camera
(
C-BUN-F-XM10-BUNDLE) with an infrared cut filter, mercury burner (USH-103OL, Ushio America,
Cypress, CA, USA), and fluorescence condenser (CKX-RFA; Olympus). Green Fluorescence of tagged
GFP protein was monitored with an excitation wavelength of 485 nm and an emission wavelength of
5
25 nm. The same settings were used to monitor calcium mobilization signals. Image acquisition was
carried out at 500 s/pixel. Cells within a single field of view were imaged over a 1 min period in the
µ
absence of the compound (line base) with a 10-s shuttered interval between each image. When each
compound was added, the field of view was imaged over a 2 min period with a 1-s shuttered interval
between each image.

Int. J. Mol. Sci. 2018, 19, 1605
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4
.6. Internalization Assay
Receptor translocation from the membrane to the cytoplasm was carried out as previously
described [22]. Hek-GHSR1a-EGFP cells were seeded in a poly-L-lysine (Sigma-Aldrich) coated 96-well
4
microtiter plate at 3
2
×
10 cells per well and incubated for 48 h at cultured conditions. For the last
4 h of this time period, media was replaced with serum-free DMEM media. Cells were exposed to
quinolone compounds at 10 µM containing 0.1% DMSO for 1 h at 37 C. Ghrelin was used as a positive
◦
control. In addition, treatment with the inverse agonist, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance
P, was also carried out. The cells were then fixed with 4% paraformaldehyde in phosphate buffer
saline (PBS) for 20 min, washed three times with PBS, and imaged on the GE Healthcare IN Cell
Analyser 1000 (GE Healthcare, Buckinghamshire, UK) in PBS. Receptor GHS-R1a-EGFP trafficking
was quantified by analyzing the EGFP fluorescence perinuclear intensity using the INCell Analyzer
Developer Toolbox V1.6 software (GE Healthcare) as previously described [22]. Data were analyzed
and depicted using GraphPad Prism software (PRISM 5.0; GraphPAD Software Inc., La Jolla, CA,
USA).
4
.7. Statistical Analysis
Statistical analysis was performed using SPSS software (IBM SPSS statistics version 22, Armonk,
NY, USA). Normality of the data was tested by using the Shapiro-Wilk test. Normally distributed
data were analyzed by one-way ANOVA followed by the Dunnett test (when comparing against a
control group) or by the Student’s t-test (when comparing 2 groups, see Figure 3B). A non-parametric
multiple comparisons Kruskal-Wallis test was performed if data was not normal, which was followed
by, where appropriate, Mann-Whitney U tests for individual comparisons. The level of significance in
all analysis was
from N independent experiments (see figure legends for N details for each experiment). Statistical
significances are subsequently depicted as follows: * indicating p 0.05, ** indicating p 0.01 or
** indicating p ≤ 0.001.
α
= 0.05 and all tests were a two-tailed test. All graphs represent the mean
±
SEM
≤
≤
*
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/
605/s1.
1
Author Contributions: C.T.-F. analyzed and interpreted the data and wrote the manuscript with the assistance
of E.P.-C. R.C. assisted with the chemical part of the manuscript. E.P.-C. carried out the experiments with the
assistance of D.K., R.J., and H.S., R.C. synthetized the quinolones compounds with the assistance of R.S., G.P.M.
and H.S. designed the experiments, interpreted the data, and reviewed and improved the manuscript. All authors
proofread and approved the final manuscript.
Funding: The research was supported by Science Foundation Ireland (SFI/12/IP/1315 and SFI/12/RC/2275)
and the Irish Research Council for Science and Technology (IRCSET). The work was also supported by Science
Foundation Ireland in the form of a Research Center grant (SFI/12/RC/2273) to the APC Microbiome Institute.
Dr. Elena Pastor-Cavada was funded by the University of Seville, Spain.
Acknowledgments: We thank Suzanne Crotty for her technical assistance with confocal microscopy in the
BioSciences Imaging Center, University College Cork, Ireland.
Conflicts of Interest: The authors declare no conflict of interest.
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