Novel drug-like somatostatin receptor 4 agonists are potential analgesics for neuropathic pain

Article abstract of DOI:10.3390/ijms20246245

Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via the somatostatin sst₄ receptor without endocrine actions. Therefore, sst₄ is considered to be a novel target for d

Full text of DOI:10.3390/ijms20246245

International Journal of
Molecular Sciences
Article
Novel Drug-Like Somatostatin Receptor 4 Agonists
are Potential Analgesics for Neuropathic Pain
Boglárka Kántás 1,2,†, Rita Börzsei 3,†, Éva Szo˝ke 1,2, Péter Bánhegyi 4, Ádám Horváth 1,2
Ágnes Hunyady 1,2, Éva Borbély 1,2, Csaba Hetényi 1, Erika Pintér 1,2,† and
,
Zsuzsanna Helyes 1,2, ,†
*
1
Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Szigeti str. 12,
H-7624 Pécs, Hungary
Szentágothai Research Centre and Centre for Neuroscience, University of Pécs, Ifjúság str. 20,
H-7624 Pécs, Hungary
2
3
4
Department of Pharmacology, Faculty of Pharmacy, University of P
Avicor Ltd., Herman Ottó str. 15, H-1022 Budapest, Hungary
Correspondence: zsuzsanna.helyes@aok.pte.hu
é
cs, Szigeti str. 12, H-7624 P
é
cs, Hungary
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 14 October 2019; Accepted: 9 December 2019; Published: 11 December 2019
Abstract: Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and
anti-inflammatory effects via the somatostatin sst4 receptor without endocrine actions. Therefore,
sst4 is considered to be a novel target for drug development in pain including chronic neuropathy,
which is an emerging unmet medical need. Here, we examined the in silico binding, the sst4-linked
G-protein activation on stable receptor expressing cells (1 nM to 10 µM), and the effects of our
novel pyrrolo-pyrimidine molecules in mouse inflammatory and neuropathic pain models. All four
of the tested compounds (C1–C4) bind to the same binding site of the sst4 receptor with similar
interaction energy to high-affinity reference sst4 agonists, and they all induce G-protein activation.
C1 is the more efficacious (
In vivo testing of the actions of orally administered C1 and C2 (500
γ
-GTP-binding: 218.2%
±
36.5%) and most potent (EC50: 37 nM) ligand.
g/kg) showed that only
µ
C1 decreased the resiniferatoxin-induced acute neurogenic inflammatory thermal allodynia and
mechanical hyperalgesia significantly. Meanwhile, both of them remarkably reduced partial sciatic
nerve ligation-induced chronic neuropathic mechanical hyperalgesia after a single oral administration
of the 500 µg/kg dose. These orally active novel sst4 agonists exert potent anti-hyperalgesic effect in a
chronic neuropathy model, and therefore, they can open promising drug developmental perspectives.
Keywords: sst4 receptor; anti-hyperalgesic; docking; molecular modeling; G-protein activation;
neurogenic inflammation; resiniferatoxin; inflammatory pain; neuropathic pain
1. Introduction
Our group made the discovery more than 2 decades ago that somatostatin released from the
activated capsaicin-sensitive peptidergic sensory nerve terminals exerts potent anti-inflammatory and
antinociceptive effects [
pointing out that a sensory nerve-derived peptide is able to induce systemic hormonal (endocrine-like)
effects by getting into the bloodstream and reaching distant parts of the body [ ]. Besides the
1–3]. These results established the proof-of-concept of “sensocrine” regulation [4]
2,3
peripheral effects, somatostatin is also an important neurotransmitter in the central nervous system
involved in a broad range of functions including pain transmission, motor and mood coordination and
endocrine regulation [5–9]. Although somatostatin could be potentially useful for the treatment of
several diseases including different pain conditions, the therapeutic application of the native peptide is
Int. J. Mol. Sci. 2019, 20, 6245; doi:10.3390/ijms20246245
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 6245
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strongly limited by its diverse effects and rapid degradation and consequently short elimination half-life
(<3 min) [9]. However, stable and potent synthetic analogs could be potential analgesic candidates.
A wide range of somatostatin effects are mediated via five inhibitory G-protein-coupled receptor
subtypes (GPCRs) [10,11] which have seven transmembrane domains (TMDs). They are divided into
two classes on the basis of their phylogeny, structural homologies and pharmacological properties.
The somatotropin release-inhibiting factor 1 (SRIF1) receptor class involves sst2, sst3 and sst5 mediating
important endocrine actions of somatostatin (e.g., inhibition of growth hormone, insulin, glucagon
secretion), and the SRIF2 class includes sst1 and sst4 [10]. It is well known that sst4 receptor is
present in the dorsal root ganglia cells and spinal cord dorsal horn, and can also mediate analgesic
effects along with the
anti-inflammatory, antinociceptive and anti-hyperalgesic effects of somatostatin are mediated by the
sst4 receptor without influencing endocrine functions [ 14 17]. Therefore, the sst4 receptor has
δ-opioid receptor [12,13]. We provided several lines of evidence that the broad
1,4, –
become a well-established novel drug target and the development of sst4 agonists has recently been
included in the scope of several pharmaceutical companies [13,18–23].
Several hepta- and octapeptide somatostatin analogs, such as TT-232 [24
,25], were shown
to induce anti-inflammatory and antinociceptive effects [13 26 29], predominantly via sst4
,
–
activation [13,28,30]. Despite the great effectivity of the peptide agonists in preclinical models, they
are not appropriate for oral application that would be preferred for chronic treatment. Therefore,
small molecule nonpeptide analogs were synthesized for drug developmental purposes. J-2156 is
a 1-naphthalenesulfonylamino-peptidomimetic, which is a sst4 “superagonist” [31], having potent
anti-inflammatory, analgesic and antidepressant actions [15,32–35].
NNC26-9100 and L-803,087 are compounds of other structurally distinct classes of highly selective
small molecule sst4 agonists [36
studies, NNC26-9100 and L-803,087 were effective in models of different neurological diseases,
such as Alzheimer’s disease and epilepsy [38 39], but they are not suitable for oral administration,
,37], and they are widely used as reference materials [9]. In previous
,
which presumably contributed to them not being candidates for drug development.
Here, we report the sst4 receptor binding and activation of novel small molecule pyrrolo-pyrimidine
molecules (Compound 1 = C1, Compound 2 = C2, Compound 3 = C3, Compound4 = C4) (Figure 1) [40],
as well as their effects in an acute neurogenic inflammatory hyperalgesia model mimicking peripheral
and central sensitization mechanisms and in a translationally relevant chronic neuropathic pain model.
O
O
HN
HN
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
Compound 2
Compound 4
Compound 1
Compound 3
Figure 1. Lewisstructuresofthetestedpyrrolo-pyrimidineligands. C1andC2areethylenelinker-containing
compounds formerly patented [40]; C3 and C4 are structurally very similar, but are methylene
linker-containing molecules.

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2. Results
2.1. Binding of the Novel Pyrrolo-Pyrimidine Compounds to the sst4 Target In Silico: Structural Calculations
The docking calculations resulted in the representative atomic resolution model of four
pyrrolo-pyrimidine ligand structures bound to the sst4 receptor and the corresponding target–ligand
interaction energies. The structures of the docked complexes were analyzed, and all target residues
were collected with a closest heavy atom distance of 3.5 Å to the docked ligand representatives (Table 1).
Table 1. Target residues interacting with representative docked ligand structures within 3.5 Å marked
with a cross.
Residues
Compound 1
Compound 2
Compound 3
Compound 4
Trp207
Ser208
Ala209
Val212
Val213
Phe216
Tyr276
Gln277
Lue280
Asn282
Leu283
Asp289
Ala290
Thr291
His294
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
All ligands bind to sst4 with a similar interaction energy of
−
8.24
±
0.41 kcal/mol (Table 2).
Since more than 70% of interacting residues are identical for all ligands, they share a common binding
site, which was expected due to their similar structures. All ligands satisfy the criteria of drug-likeness
characterized by the Lipinksi’s rule of five (RO5) (Table 2).
Table 2. Target–ligand interaction energies and Lipinski’s rule of five descriptors, i.e., molecular weight
(MW), logarithm of octanol/water partition coefficient (mlogP) [41], numbers (N) of H-donor and
H-acceptor atoms.
Compound 1
Compound 2
Compound 3
Compound 4
Einter (kcal/mol)
MW
mlogP
NH-donor
NH-acceptor
−8.54
328.4
3.60
1
−7.64
386.5
3.68
1
−8.31
314.4
3.38
1
−8.46
372.5
3.47
1
2
3
2
3
A closer inspection of Figure 2 underlines the above similarities between representative binding
modes of the ligands. All molecules are stabilized by an H-bond with Gln279 (residue numbering
is according to the UniProt database entry No. P31391). The secondary amine group of compounds
containing ethylene linker forms contact with the side-chain oxo group of Gln279, while the secondary
amine group of ligands with methylene linker forms H-binding with the backbone oxo group of
Gln279. Furthermore the 7H-pyrrolo[2,3-d]pyrimidine core and the ethylphenyl or the methylphenyl
moiety of the ligands fit into the hydrophobic cavity defined by Val212, Val213, Phe216, Leu280 and
Leu283. Furthermore, the benzyl ring of C2 is involved in aromatic–aromatic (π-stacking) interactions
with His294.

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Figure 2. (A) Ligand pairs having ethylene (red: C1, blue: C2) or methylene (green: C3, magenta: C4)
intramolecular linker (Figure 1) share a common binding pocket. Target residues interacting with docked
representatives within 3.5 Å are indicated with thin lines. For comparison of the binding modes, ligand
pairs with the same intramolecular linker are shown in the same panel; (B) 7H-pyrrolo[2,3-d]pyrimidine
core and the ethylphenyl moiety of C1 and C2 fit into the hydrophobic cavity defined by Val212, Val213,
Phe216, Leu280 and Leu283. Both molecules are stabilized by an H-bond with Gln279. The benzyl ring
of C2 is involved in aromatic–aromatic (π-stacking) interactions with His294.
2.2. Somatostatin-Receptor-4-Linked G-Protein Activation by the Novel Pyrrolo-Pyrimidine Compounds on
Stable Receptor-Expressing Cells
All four of the compounds induced concentration-dependent sst4 activation on sst4-expressing
CHO cells as shown by the [35S]GTP
γS binding assay. The EC50 values demonstrating the potency of
the ligands were 37, 66, 149 and 70 nM in the cases of C1, C2, C3 and C4, respectively. The maximal
activation values over the basal activities of the receptor showing the efficacy of the compounds were
218.2%
±
36.5%, 203%
±
30.8%, 189%
±
36.3% and 177.3%
±
32.9% for C1, C2, C3 and C4, respectively.
C1 and C2 were the most potent and efficacious sst4 receptor agonists, while C3 and C4 were weaker
agonists (Figure 3), therefore further in vivo tests were performed with the C1 and C2 compounds.

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Figure 3. Effect of Compounds 1–4 on sst4 receptor-linked G-protein activation. [35S]GTP
γ
S binding
S binding
induced by the compound in sst4-expressing CHO cells. The ligand-stimulated [35S] GTP
γ
reflects the GDP–GTP exchange reaction on
α-subunits of G-proteins by receptor agonists. Increasing
concentrations of all compounds result in similar concentration-dependent stimulations of [35S]GTP
γ
S
binding. Each data point represents the mean
EC50 values.
±
SEM of n = 3 experiments; dashed lines indicate the
2.3. C1 Compound Decreases RTX-Induced Inflammatory Thermal Allodynia and Mechanical Hyperalgesia
The ethylene linker-containing, patented pyrrolo-pyrimidine sst4 agonists (C1 and C2) as described
by the in silico and in vitro results above were tested in vivo as well in pain models. Intraplantar RTX
injection (20
and 41.05
µ
L, 0.1
µ
g/mL) decreased the heat threshold from 46.06
±
0.36 to 34.65
4.1%) after 10, 20 and 30 min,
0.05 to 5.11
0.42, 6.64 ± 0.29
31.38% 3.55% mechanical
± 1.51, 41.16 ± 2.25
◦
±
1.74 C (
−
24.6% 3.5%, 10.4% 5.2% and 10.7%
±
−
±
−
±
respectively, and decreased the mechanonociceptive threshold from 9.70
and 6.65 0.34 g, which means 47.25% 4.42%, 31.52% 2.88%,
±
±
±
−
±
−
±
−
±
hyperalgesia following 30, 60 and 90 min, respectively. Oral pretreatment with C1 (500 mg/kg), but not
with C2, significantly decreased the acute neurogenic inflammatory heat hyperalgesia at 10 and 30 min
and mechanical hyperalgesia at 30 min (Figure 4).

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Figure 4. Effect of a single oral treatment with our novel compounds in 500 µg/kg on RTX-induced
drop of the (
well as the (
(M) vehicle-treated mice served as controls. Data points represent the means
per group (* p < 0.5, ** p < 0.01, vs. respective pretreatment self-control values, two-way ANOVA,
Bonferroni0s multiple comparison test for comparison).
A
C
) heat thermonociceptive and (
) mechanonociciceptive thresholds of the untreated left hindpaws. The methylcellulose
SEM of n = 5–12 mice
B) mechanonociceptive thresholds of the right (treated),
±
2.4. C1 and C2 Compounds Reduce Chronic Neuropathic Mechanical Hyperalgesia
Seven days after sciatic nerve ligation the mechanonociceptive threshold of the operated
limbs decreased from 9.58
mechanical hyperalgesia, while the mechanosensitivity of the contralateral paws did not change.
Oral administration of the methylcellulose vehicle did not alter the mechanosensitivity of the paws
±
0.06 to 6.61
±
0.14 g in all groups, representing approximately 30%
60 min later (pretreatment hyperalgesia: 7.80
7.46 0.22 g (23.06% 2.59%)). Oral pretreatment with both C1 and C2 (500
neuropathic mechanical hyperalgesia 1 h later from 6.51 0.18 g (31.22%
(9.98% 3.92%) and from 6.53 0.30 g (31.39% 3.39%) to 8.04 0.31 g (15.70%
(Figure 5).
±
0.26 g (28.99%
±
2.28%), post-treatment hyperalgesia:
g/kg) significantly reduced
2.11%) to 8.53 ± 0.37 g
2.84%), respectively
±
±
µ
±
±
±
±
±
±
±

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Figure 5. Effect of a single oral treatment with C1 and C2 compounds (500 µg/kg) on neuropathic
mechanical hyperalgesia 7 days after partial tight ligation of the right sciatic nerve. Triplets of the
columns represent mechanonociceptive thresholds on the ( ) operated ipsilateral and ( ) unoperated
A
B
contralateral limbs (in grams) before and after the operation, before and 60 min after treatment with the
respective test compound or the vehicle (methylcellulose = M). Results are expressed as means ± SEM
of the mechanonociceptive thresholds (n = 8 mice per group, * p < 0.5, ** p < 0.01, *** p < 0.001,
**** p < 0.0001 vs. respective pretreatment self-control values, two-way ANOVA, Bonferroni0s multiple
comparison test for comparison).
2.5. Selectivity Profile of Compound 1
Specific binding, enzymatic activity and agonistic/antagonistic effect of Compound 1 were
investigated in 1 µM concentration in this assay, as usually done in such tests. This is 10 times higher
than its EC50 value determined in the G-protein activation assay. The results revealed that this high
concentration of Compound 1 had no remarkable effect on voltage-gated K+ and Ca2+ channels, COX-2,
PDEs, dopamine or opioid receptors, but some agonistic effect on CB1 and CB2 cannabinoid receptors

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(30.8% and 58.9%), respectively. Actions under 50% are not functionally relevant (Table 3). See in
Supplementary Materials.
Table 3. Compound 1 inhibitory activity at 1 µM.
Target
Specific Binding (%) Enzymatic Activity (%)
Agonist/Antagonist Effect (%)
K+ channel hERG
Ca2+ channel
COX-2
23.6
19.4
−10.4
−15.0
−0.6
2.7
PDE3A
PDE4D2
MAOA
CB1
CB2
30.8/−1.5
58.9/7.7
D1
D2S
Delta (DOP)
Kappa (KOP)
Mu (MOP)
3.3/−10
17.2/−11.5
−0.3/−1.4
−1.5/23.6
5.5/20.2
3. Discussion
In the present paper we provide the first in silico, in vitro and in vivo data that novel
4-phenetylamino-7H-pyrrolo[2,3-d]pyrimidine derivatives designed and patented by us are sst4
receptor agonists with effective analgesic properties. Our ethylene linker-containing compounds
significantly inhibit chronic neuropathic hyperalgesia after single oral administration.
Evaluation on the basis of the Lipinski’s RO5 clearly showed the drug-likeness of these compounds.
RO5 was originally designed to predict the aqueous solubility and intestinal permeability of new, orally
available molecules using four simple physicochemical parameters: higher absorption or permeation
are more likely if the molecular weight, the Moriguchi logP (mlogP) and the numbers of H-donors
and H-acceptors are under 500, 4.15, 5 and 10, respectively [42]. Since then, several simple properties
to complex calculations were published that may explain the relationship between the structure of
the candidates and their general kinetic profile or drug-likeness properties. A comprehensive review
concluded that the usefulness of molecular size and lipophilicity are limited as predictors of general
drug-likeness of a molecule [43]. However, these properties may have statistical success in special cases
of nervous system or dermatological diseases [43]. We found that all of the four pyrrolo-pyrimidine
ligands of the present study satisfy the RO5 criteria (Table 2). These compounds target the nervous
system, and an oral administration route would be preferred. Based on the structures and the Lipinski’s
RO5 results, the investigated compounds are likely to cross the blood–brain barrier and have good
pharmacokinetics and bioavailability. Molecular modeling studies were performed to investigate
the binding mode of our novel, patented ethylene linker-containing compounds (C1 and C2) and for
comparison with two structurally similar, methylene linker-containing ligands (C3 and C4). Docking
calculations and analyses of the residues interacting with the docked representatives within 3.5 Å
show that all molecules bind to the same site with similar interaction energy to the sst4 receptor.
J-2156, a high-affinity, selective sst4 agonist [31
,44,45], binds to a region called the high-affinity binding
pocket [44 46]. Our novel structures bind near this high-affinity binding pocket, and their binding mode
,
is almost the same as described in case of other high-affinity ligands involved [44]. It is hypothesized
that ligand interaction with the conserved aspartic acid in TM3 of somatostatin receptors is necessary
for agonist binding, however, Liu et al. suggested that hydrogen bonding with Gln279 might be an
alternative key interaction between the ligand and the receptor [44]. All molecules of the present study
are stabilized by an H-bond with Gln279.
After in silico demonstration of the sst4-binding of the four compounds, their receptor-activating
abilities were examined in the γGTP binding assay on sst4-receptor-expressing cells. All the compounds

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elicited G-protein activation; therefore, they are considered to be sst4 agonists, but C1 proved to have
the highest potency and efficacy. Since the structurally similar methylene linker-containing C3 and C4
investigated for comparison in this assay were not stronger, but weaker agonists, we investigated only
our novel molecules, C1 and C2, in vivo.
C1, but not C2, inhibited the RTX-induced acute neurogenic inflammatory thermal allodynia
and mechanical hyperalgesia. However, both compounds remarkably alleviated the traumatic
nerve-injury-provoked neuropathic mechanical hyperalgesia; C1 induced approximately 70%, while C2
evoked 50% anti-hyperalgesic effects.
Intraplantar injection of RTX, a selective and ultrapotent agonist of the transient receptor potential
vanilloid 1 (TRPV1) capsaicin receptor, evokes acute neurogenic inflammatory reaction with rapidly
developing, relatively short thermal allodynia, which is mediated predominantly by peripheral
sensitization mechanisms. RTX releases proinflammatory neuropeptides, such as substance P and
calcitonin-gene-related peptide in the innervated area, which trigger a local inflammation cascade
and induce peripheral sensitization of the nociceptive nerve endings [47]. Certain inflammatory
mediators, e.g., bradykinin, and prostaglandins activate/sensitize the TRPV1 receptor on the sensory
nerve terminals via protein kinases C and A [48–50]. The heat allodynia is followed by mechanical
hyperalgesia mediated not only by peripheral mechanisms, but central sensitization processes in the
spinal cord and different pain-processing brain regions as well [51]. The antiallodynic effect of our C1
compound is consistent with our previous data showing similar effect of the heptapeptide somatostatin
analog TT-232 with sst1/sst4 activating ability [52]. Based on its physicochemical properties and the
results of the Lipinsky’s RO5 being appropriate to estimate the kinetic parameters of drugs acting
on the central nervous system, C1 is likely to cross the blood–brain barrier. Therefore, the inhibitory
action of C1 on mechanical hyperalgesia is suggested to be due not only to peripheral mechanisms,
but also to diminished central pain sensitization. This is supported by the localization of sst4 receptors
on primary sensory neurons, in the dorsal horn of the spinal cord as well as in brain regions playing an
important role in pain processing, like the amygdala, hippocampus and somatosensory cortex [53,54].
An explanation for the ineffectiveness of the weaker sst4 agonist C2 in this model can be other,
currently not identified mechanisms of action of C1, such as sst1 or opioid receptor agonism, and/or
kinase inhibition. A limitation of this study is the lack of data for the selectivity of our compounds.
Partial ligation of the sciatic nerve [55] is a reliable and widely used disease model of
traumatic neuropathic pain in rodents. As a result of the operation, significant damage develops
in the thinly myelinated and unmyelinated fibers leading to abnormal sensory functions, such as
hyperalgesia, without disabling motor functions [55,56]. Single oral pretreatment with both C1 and
C2 resulted in a 60%–70% analgesic effect in this model. This is in agreement with earlier data
showing that both the heptapeptide sst4 agonist TT-232 and the nonpeptide superagonist J-2156 exert
potent and dose-dependent analgesic effects (10–100 µg/kg i.p.), TT-232 even reversed mechanical
hyperalgesia [15,27]. The importance of these results is highlighted by the fact that neuropathic pain
is very resistant to conventional analgesics: the effect of opioids and NSAIDs is almost absent in
neuropathic conditions [27,57–59]. Adjuvant analgesics (e.g., antiepileptics, antidepressants) might
have limited effects in certain cases, but they could exert several serious adverse effects [60].
Specific binding, enzymatic activity and agonistic/antagonistic effects were also investigated in
case of Compound 1. It should be noted that Compound 1 was applied in 1 µM concentration in this
assay, as usually done in such tests, which is 10 times higher than its EC50 value determined in the
G-protein activation assay. The results revealed that this high concentration of Compound 1 had no
remarkable effect on voltage-gated K+ and Ca2+ channels, COX-2, PDEs, dopamine or opioid receptors,
but some agonistic effect on CB1 and CB2 cannabinoid receptors (30.8% and 58.9%, respectively).
Of course, it cannot be excluded that CB1/CB2 receptor agonism of Compound 1 is involved in its
anti-inflammatory and analgesic effects, but further investigations are needed to clarify these issues.
Determining the exact selectivity and, particularly, the safety of these compounds was beyond the
scope of this study. Here we aimed to provide state-of-the-art evidence for these small molecule

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compounds to bind to and activate the sst4 somatostatin receptor and, most importantly, to exert
anti-hyperalgesic effect after oral administration.
In conclusion, here we demonstrate that our new pyrrolo-pyrimidine ligands are drug-like sst4
agonists and provide proof-of-concept for their effectiveness to inhibit chronic neuropathic pain.
Therefore, they could open promising perspectives for the development of a novel type of analgesics
appropriate for the treatment of this unmet medical need.
4. Materials and Methods
4.1. In Silico Molecula Modeling Studies (Structural Calculations)
4.1.1. Preparation of Ligand and Target Structures.
All ligand structures were built in Maestro (Shrödinger, LLC New York, NY, USA) [61].
The semiempirical quantum chemistry program package MOPAC (Stewart Computational Chemistry,
Colorado Spings, CO, USA) [62] was used to minimize the raw structures with a PM7
parametrization [63]. The gradient norm was set to 0.001. Force calculations were applied on the
energy-minimized structures and the force constant matrices were positive definite. The structure
of sst4 receptor was created by homology modeling and energy-minimized by GROMACS 5.0.2
(GROMACS Development teams at the Royal Institute of Technology and Uppsala University, Uppsala,
Sweden) [64] as described in our previous study [46]. Gasteiger–Marsilli partial charges were assigned
to both the ligand and target atoms in AutoDock Tools (The Scripps Research Institute, La Jolla, CA,
USA) [65], and united atom representation was applied for nonpolar moieties. These energy-minimized
structures were converted to Protein Databank (PDB) format and forwarded to docking calculations.
4.1.2. Grid Calculation and Docking.
Docking of all ligands was performed with AutoDock 4.2.6 (The Scripps Research Institute,
La Jolla, CA, USA) [65], focusing on the extracellular region of the sst4 target. In order to reduce false
positive conformations, the transmembrane and intracellular target regions were not included in the
docking search. Flexibility was allowed at all active torsions of the ligand, but the target was treated
rigidly. The docking box was centered on the extracellular region of sst4 including 80
×
80
×
80 grid
points at a 0.375 Å spacing by AutoGrid 4 (The Scripps Research Institute, La Jolla, CA, USA) [65].
Lamarckian genetic algorithm was used for global search. After 10 docking runs, ligand conformations
were ranked by the corresponding calculated interaction energy values and subsequently clustered
using a root-mean-square deviation (RMSD) tolerance of 3.5 Å between cluster members. Rank 1 was
analyzed and selected as representative structure for each ligand.
Ligand-based descriptors including the molecular weight (MW), the logarithm of octanol/water
partition coefficient (mlogPo/w) and the numbers of H-donor and H-acceptor atoms in the molecules
were calculated from the ligand PDB files using SwissADME (Swiss Institute of Bioinformatics,
Lausanne, Switzerland), a free webserver (http://www.swissadme.ch) [66].
4.2. Somatostatin-Receptor-4-Linked G-Protein Activation Assay
Membrane fractions were prepared from CHO cells, which express the sst4 receptor,
in Tris–ethylene glycol-bis(2-aminoethyl)tetraacetic acid (Tris–EGTA) buffer (50 mM Tris–HCl (Sigma,
St. Louis, MO, USA), 1 mM EGTA (Sigma, St. Louis, MO, USA), 3 mM MgCl2 (Sigma, St. Louis,
MO, USA), 100 mM NaCl (Sigma, St. Louis, MO, USA), pH 7.4, 10 µg of protein/sample). The fractions
were incubated in the buffer containing 0.05 nM guanosine triphosphate (GTP) (BioChemica
International Inc., Melbourne, Florida, USA), labeled on the gamma phosphate group with 35S
([35S]GTP
γ
S) (Isotop Institute, Budapest, Hungary) and increasing concentrations (1 nM to 10
C1, C2, C3 and C4 compounds for 60 min at 30 ◦C, in the presence of 30
M guanosine diphosphate
(GDP) (Sigma, St. Louis, MO, USA). We determined the nonspecific binding in the presence of 10
µM) of
µ
µM

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unlabeled GTPγS and total binding in the absence of test compounds. Samples were filtrated through
Whatman GF/B glass fiber filters using 48-well Slot Blot Manifold from Cleaver Scientific (Cleaver
Scientific Ltd., Rugby, Warwickshire, United Kingdom). Filters were washed with ice-cold 50 mM
Tris–HCl buffer (pH 7.4) and radioactivity was measured in a β-counter (PerkinElmer Inc., Waltham,
MA, USA). The test-compound-induced G-protein activation was given as percentage over the specific
[35S]GTPγS binding detected in the absence of agonists [56,67].
4.3. Acute Neurogenic Inflammatory Thermal Allodynia and Mechanical Hyperalgesia
The investigated compounds (500 µg/kg p.o.) or the vehicle were administered orally 1 h before
the induction of the acute neurogenic inflammation by intraplantar RTX (Sigma, St. Louis, MO, USA)
injection (20 µL, 0.1 µg/mL) into the right hindpaw. RTX evokes an acute neurogenic inflammatory
reaction with the development of a rapid thermal allodynia, mainly due to peripheral sensitization
mechanisms, and a later developing mechanical hyperalgesia due also to central sensitization [68].
The nociceptive heat threshold was determined before (control) and 10, 20 and 30 min after RTX
administration with an increasing-temperature hot plate (IITC Life Science, Woodland Hills, CA, USA).
Mice were placed on a plate which was then heated up from 25 ◦C at a rate of 12 ◦C/min until the
animal showed nocifensive behavior (licking, lifting or shaking of the hindpaw); that temperature
was considered as the noxious heat threshold. The mechanonociceptive threshold was measured with
an electronic von Frey device (dynamic plantar aesthesiometer (DPA), Ugo Basile, Comerio, Italy)
prior to RTX injection and 30, 60 and 90 min afterwards. The measurement at 30 min was performed
immediately after the last heat threshold measurement. The experiment consisted of two separate
series on consecutive days and the number of animals in the control group was six per day. Therefore,
the control group contained 12 mice, and six and five mice were used in the C1- and C2-treated
groups, respectively.
4.4. Chronic Traumatic Neuropathic Pain Model
After conditioning and two control mechanonociceptive measurements on 2 consecutive days,
mice were anaesthetized by the combination of ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.).
Traumatic mononeuropathy was achieved by one-third to one-half part ligation of the right sciatic
nerve [55]. Significant decrease in the mechanical threshold develops 7 days after operation [56,69,70].
The mechanonociceptive threshold of the plantar surface of the hindpaws was measured by DPA on
the 7th postoperative day. The paw withdrawal threshold was obtained in grams (the maximal value
was 10 g; ramp time of 4 s). The compounds (500 µg/kg) or the vehicle were administered orally on
day 7, one hour before the repeated measurement. The experiment consisted of separate series on
3 consecutive days, and there were 2–3 vehicle-treated control animals in every series. Therefore,
each group contained 8 mice.
4.5. Selectivity Profile Determination
The evaluation was performed by Eurofins Cerep (France). Compound binding was calculated as
a % inhibition of the binding of a radioactively labeled ligand specific for each target. Enzyme inhibition
effect was calculated as a % inhibition of control enzyme activity. Cellular agonist effect was calculated
as a % of control response to a known reference agonist for each target, and cellular antagonist
effect was calculated as a % inhibition of control reference agonist response for each target. In each
experiment the respective reference compound was tested concurrently with Compound 1 [71
See in Supplementary Materials.
–78].
4.6. Synthesis of the Compounds
The starting 4-chloro pyrrolo-pyrimidines (R1 = H or methyl) were obtained from commercial
sources. After N-benzylation, they were coupled with the appropriate phenylethylamines, which yielded

Int. J. Mol. Sci. 2019, 20, 6245
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our patented [40] ethylene linker-containing molecules, or benzylamines, which resulted in structurally
similar, methylene linker-containing compounds (Figure 6). See in Supplementary Materials.
Figure 6. Preparation route. (i) NaH, DMF, BnBr; (ii) R2-amine, DMSO, 100 ◦C.
4.7. Solution Preparation
In the sst4 receptor activation assay, all the compounds were dissolved in dimethyl sulfoxide
(DMSO) (Szkarabeusz Ltd., Pécs, Hungary). The concentration of the stock solutions was 10 mM and
was diluted with distilled water to reach the final concentrations. For the in vivo experiments, 1 mg of the
compounds was rubbed dry in a braying mortar, suspended thoroughly in 1 mL 1.25% methylcellulose
(MC) (Pharmacy of University of P
water to get a 1000 g/mL stock solution freshly every experimental day. Most microsuspensions
looked opalescent; they were shaken properly, sonicated, and further diluted with 1.25% MC to obtain
the 25 g/mL solution for oral administrations (0.2 mL/10 g body weight for the 500 g/kg dose).
écs, Pécs, Hungary) solution and then dissolved in sterile bidistilled
µ
µ
µ
The solutions were shaken and sonicated again directly before use. The vehicle was always 1.25% MC
dissolved in sterile bidistilled water.
4.8. Animals and Ethics
Male NMRI mice (8–12 weeks old) bred in the Laboratory Animal House of the Department of
Pharmacology and Pharmacotherapy of the University of Pecs were kept in standard plastic cages at
24–25 ◦C, under a 12–12 h light–dark cycle and provided with standard rodent chow and water ad
libitum. All experimental procedures complied with the recommendations of the 1998/XXVIII Act of
the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of
Animal Experiments (63/2010) and were approved by the Ethics Committee on Animal Research of
Pecs University according to the Ethical Codex of Animal Experiments; license was given (license No.
BA1/35/55-50/2017). We made all efforts to minimize the number and suffering of the animals used in
this study.
4.9. Statistical Analysis
In the in vivo experiments, all data were expressed as means
±
SEM of n = 5–21 mice per group
and analyzed with two-way ANOVA followed by Bonferroni0s Multiple Comparison test. In all cases
p < 0.05 was considered to be statistically significant.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/24/6245/s1.
Author Contributions: Conceptualization, R.B., C.H., E.P. and Z.H.; Data curation,
Á
.H. (
gnes Hunyady) and Z.H.; Funding acquisition, .S., C.H. and Z.H.;
m Horv th); Methodology, B.K. and .H. ( m Horv th); Project
Ágnes Hunyady)
and
É
.B.; Formal analysis, B.K.,
.S. and
Á
Á
.H. (
.H. (
Á
Á
É
á
Investigation, B.K., R.B.,
É
dá
á
Á
Á
d
á
administration, E.P. and Z.H.; Resources, C.H. and Z.H.; Supervision, C.H. and Z.H.; Visualization, B.K., R.B. and
.S.; Writing—original draft, B.K., R.B., .S., P.B., C.H. and Z.H.; Writing—review and editing, .S., C.H. and E.P.
É
É
É

Int. J. Mol. Sci. 2019, 20, 6245
13 of 17
Funding: This work was funded by the Hungarian National Research, Development and Innovation Office
(K123836), 2017-1.2.1-NKP-2017-00002 (NAP-2; Chronic Pain Research Group), EFOP-3.6.1.-16-2016-0004 and
GINOP 2.3.2-15-2016-00050 “PEPSYS”.
of the Hungarian Academy of Sciences. We acknowledge the grant of computer time from the Governmental
Information Technology Development Agency, Hungary. The University of P cs is acknowledged for a support
by the 17886-4/23018/FEKUTSTRAT excellence grant, and by PTE OK-KA No:2019/KA-2019-31. C.H.’s work
was supported by a grant co-financed by Hungary and the European Union (EFOP-3.6.2-16-2017-00008) and the
NKP-19-4 New National Excellence Program of the Ministry for Innovation and Technology. B.K’s work was
É.B. É.S. and C.H. were supported by the János Bolyai Research Scholarship
é
Á
Ú
supported by
ÚNKP-19-3 New National Excellence Program of the Ministry for Innovation and Technology and
Gedeon Richter’s Talentum Foundation.
˝
Acknowledgments: The authors thank László Orfi and Tamás Szu˝ts for the collaboration in the synthesis of the
compounds and D
dear colleagues, J nos Szolcs
were essential in the present project.
ó
ra Ömböli for expert technical assistance. We dedicate this work to our highly appreciated
á
á
nyi and György K ri, who sadly passed away recently, and whose contributions
é
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
GPCR
TMD
SRIF1
RO5
G-protein coupled receptor
transmembrane domain
somatotropin release-inhibiting factor
Lipinksi’s rule of five
MW
molecular weight
mlogP
RTX
logarithm of octanol/water partition coefficient according to Morigucchi
resiniferatoxin
TRPV1
PDB
transient receptor potential vanilloid 1
Protein Databank
RMSD
GTP
root mean square deviation
guanosine triphosphate
GDP
DPA
DMSO
MC
guanosine diphosphate
dynamic plantar aesthesiometer
dimethyl sulfoxide
methylcellulose
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