Identification of the hydantoin alkaloids parazoanthines as novel CXCR4 antagonists by computational and in vitro functional characterization

Article abstract of DOI:10.1016/j.bioorg.2020.104337

CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer metastasis and inflammatory diseases. Starting from our previously-developed pharmacophoric model, we applied a combined computational and experimental approach that led to the identification of the hydantoin alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae, as novel CXCR4 antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B was identified as the most potent CXCR4 antagonist with an IC₅₀ value of 9.3 nM, even though all the investigated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency and efficacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure–activity relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening.

Full text of DOI:10.1016/j.bioorg.2020.104337

Bioorganic Chemistry 105 (2020) 104337
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of the hydantoin alkaloids parazoanthines as novel CXCR4
antagonists by computational and in vitro functional characterization
a
,
1
b
,
1
a
,
1
b
b
Rosa Maria Vitale , Stefano Thellung , Francesco Tinto , Agnese Solari , Monica Gatti ,
a
c
c
a
Genoveffa Nuzzo , Efstathia Ioannou , Vassilios Roussis , Maria Letizia Ciavatta ,
a
,
*
b
,
d
,
*
a,*
Emiliano Manzo , Tullio Florio
, Pietro Amodeo
a
Institute of Biomolecular Chemistry, National Research Council (ICB-CNR), Via Campi Flegrei 34, 80078, Pozzuoli, (NA), Italy
Section of Pharmacology, Department of Internal Medicine, University of Genova, 16132 Genova, Italy
b
c
Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens,
Panepistimiopolis Zografou, Athens 15771, Greece
d
IRCCS Policlinico San Martino, 16132 Genova, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
CXCR4 chemokine receptor represents an attractive pharmacological target due to its key role in cancer
metastasis and inflammatory diseases. Starting from our previously-developed pharmacophoric model, we
applied a combined computational and experimental approach that led to the identification of the hydantoin
alkaloids parazoanthines, isolated from the Mediterranean Sea anemone Parazoanthus axinellae, as novel CXCR4
antagonists. Parazoanthine analogues were then synthesized to evaluate the contribution of functional groups to
the overall activity. Within the panel of synthesized natural and non-natural parazoanthines, parazoanthine-B
was identified as the most potent CXCR4 antagonist with an IC50 value of 9.3 nM, even though all the investi-
gated compounds were able to antagonize in vitro the down-stream effects of CXC12, albeit with variable potency
and efficacy. The results of our study strongly support this class of small molecules as potent CXCR4 antagonists
in tumoral pathologies characterized by an overexpression of this receptor. Furthermore, their structure–activity
relationships allowed the optimization of our pharmacophoric model, useful for large-scale in silico screening.
Hydantoin alkaloids
Parazoanthines
Natural products
CXCR4 antagonists
Molecular docking
Pharmacophoric model
1
. Introduction
Chemokines comprise a family of chemotactic cytokines, classified
agent, is a FDA-approved drug in patients with multiple myeloma and
non-Hodgkin’s lymphoma who have undergone stem cell trans-
plantation, for its mobilizing effect on hematopoietic cells [12]. Starting
from AMD3100, other molecules have been developed in an attempt to
reduce the size and improve the pharmacokinetic profile, including
AMD070 [13], GSK81239 [14], TG-0054 [15], KRH-3955 [16], and
HF5073 [17].
according to the position of the conserved N-terminal cysteine residues
into four main subfamilies: CXC, CC, CX3C and XC [1]. These small
proteins exert their homeostatic and/or inflammatory functions by
binding and activation of cognate receptors, belonging to the G-protein
coupled receptors (GPCRs) family. CXCR4, the cognate receptor of the C-
X-C chemokine CXCL12, is by far the most largely studied chemokine
receptor, due to its relevance in many pathologic processes, including
HIV infection [2], inflammatory diseases [3–5], cancer [6–9], and
WHIM syndrome [10]. In particular, CXCR4 plays a pivotal role in
cancer cell survival, proliferation, migration, invasiveness and metas-
tasis, representing a relevant therapeutic target in those diseases char-
acterized by up-regulation of CXCL12/CXCR4 signaling [11]. Currently,
the CXCR4 antagonist AMD3100, initially proposed as an anti-HIV
In a previous study [18], we developed a minimalistic pharmaco-
phoric model for CXCR4 ligands in which the essential anchoring points
on CXCR4 consisted of an aromatic and a basic functional groups,
separated by a properly-sized spacer, resulting in an upper limit of 18 Å
for the distance between the centers of mass of the two groups. This
model led to the identification of phidianidine A (Fig. 1), an alkaloid
compound of marine origin, as CXCR4 inhibitor exhibiting low micro-
molar activity. Natural compounds, representing an invaluable source of
novel chemical scaffolds endowed with a high chemical diversity, are
*
Corresponding authors.
E-mail addresses: emanzo@icb.cnr.it (E. Manzo), tullio.florio@unige.it (T. Florio), pamodeo@icb.cnr.it (P. Amodeo).
1
These authors share co-first authoriship.
https://doi.org/10.1016/j.bioorg.2020.104337
Received 12 August 2020; Received in revised form 19 September 2020; Accepted 30 September 2020
Available online 6 October 2020
0
045-2068/© 2020 Elsevier Inc. All rights reserved.

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
very useful in the search of new lead compounds, but their exploitation
in drug discovery is often hampered by their limited availability. This is
especially true for molecules from marine organisms, and/or when the
dimension/complexity of the scaffolds prevents their large-scale syn-
thesis, thus making natural compounds with a low molecular weight and
a scaffold easy to synthesize highly desirable. In this view, we included
these two latter prerequisites into a new search for naturally-occurring
molecules in our ICB collection matching the aforementioned pharma-
cophoric model. This search identified the parazoanthine (hereafter
dubbed as PARA) compound family, hydantoin alkaloids isolated from
the Mediterranean Sea anemone Parazoanthus axinellae [19,20]. The
members of this family of structurally and biologically interesting
compounds, currently synthesized at ICB laboratory, share a strongly-
basic guanidium moiety separated from a (substituted) p-phenoxy
group by a 9-bond spacer including a hydantoin ring (Fig. 1). PARAs
differ from each other either in the substitution pattern of the p-phenoxy
group, or in the saturation state of the 5,6 extracyclic bond on the
hydantoin ring. According to the latter, PARAs can be classified into
saturated (PARA-A,D,F,G,I) and unsaturated (PARA-B,C,E,H,J) com-
pounds. As for the aromatic moiety, in this work we only considered the
effects of substitution at the oxygen atom of the p-phenoxy group, and its
combinations with the 5,6 bond saturation state.
with the hydroxy group H-bonded to Thr117 (helix 3) sidechain. This
last H-bond does not occur in PARA-C due to the presence of a methoxy
group, since Thr117 acts as H-bond acceptor in PARA-A and PARA-B
ligands, being its polar hydrogen H-bonded, in turn, to His113 back-
bone CO. Instead, PARA-C methoxy group forms a H-bond with His203
(helix 5). The occurrence of a double bond in PARA-B does not affect the
binding modes since the overall arrangement essentially overlaps that of
PARA-A. However, the double bond, by conferring more rigidity to the
scaffold, could in principle favourably contribute to the binding by
reducing the entropy contribution of the ligand. The representative
poses of PARA-B and PARA-C after energy minimization, are shown in
Fig. 2A,B. A comparison with phidianidine A shows that PARAs, while
exhibiting the same overall arrangement in which the guanidium moiety
interacts with the negatively charged residues Asp97 and/or Asp187,
and the aromatic pendants are hosted in the same subpocket of the
indole moiety of phidianidine A, considerably differ in the favoured
arrangements of the central linker. The shorter distance between the
aromatic and the basic centers of mass in comparison with phidianidine
A, and the H-bonds between the hydantoin ring and the backbone of
residues in the e2 loop concur to stabilize a binding mode in which the
ligand runs parallel to the e2 loop. Instead, the linker region of phidia-
nidine A allows a greater flexibility of the ligand within the binding site,
with the amino-oxadiazole group more distant from the e2 loop and
mainly involved in H-bonds with Glu288 on helix 7, which is opposite to
the e2 loop in the receptor binding cleft [18]. Furthermore, in order to
evaluate the contribution of: a) a H-bond acceptor/donor group on the
aromatic pendant, b) the size of the aromatic group, and c) the flexibility
of the alkyl chain on ligand activity and/or potency, three derivatives
were also considered, featuring phenyl (PARA-Ph) and naphthalene
In particular, since parazoanthines A-C had already been synthe-
sized, we started our evaluation checking in silico and in vitro their ability
to act as CXCR4 ligands.
2
. Results and discussion
2
.1. Molecular docking
(
PARA-Naph) moiety, or a methoxy (correponding to the natural com-
PARA-A-C differ from each other in the presence of a hydroxy (A-B)
pound PARA-F) [20,21] group on PARA-A. PARA-F, representing the
saturated, flexible analogue of PARA-C, completes the set of possible
combinations of 5,6 bond saturations state and p-phenoxy oxygen atom
substitutions within known unbromurated PARAs. The representative
pose of PARA-Naph is shown in Fig. 2C.
or methoxy (C) group on the phenyl ring, and in the number of double
bonds in the structure: two (Δ5,6 and Δ13,14) for B-C, respectively, and
only one (Δ13,14) for A. They, in turn, differ from phidianidine A, the
previously identified CXCR4 antagonist from our ICB collection, in both
the aromatic moiety (phenyl vs indole ring) and in the length and nature
of the spacer, since they possess a hydantoin group in the place of an
oxadiazole ring, and double, in place of single, bond(s) in the carbon
chain, as well as in the distance between the two anchoring groups (~14
Å), smaller than that found in phidianidine A. Thus, to rule out possible
divergences in the binding modes in comparison to our previous lead
phidianidine A, a comprehensive molecular docking study was under-
taken. All compounds exhibited a similar arrangement into the binding
site as best pose in terms of binding energy, characterized by bidentate
ligand–protein H-bonds reinforced by ionic interactions between ligand
and Asp97 (helix 2) /Asp187 (loop e2), as well as H-bonds of the
hydantoin group with Asp187 side chain and/or carbonyl backbone and
the NH backbone atom of Arg188 (loop e2), whereas the ligand aromatic
ring is sandwiched between Arg188 and Tyr116 (helix 3) sidechains,
2.2. Synthesis
2.2.1. Synthesis of PARA-A and O-Me derivative PARA-F
The preparations of the synthetic parazoanthine A (PARA-A) and O-
methyl derivative (PARA-F) were achieved by a concise biomimetic
synthesis based on the coupling reaction of L-arginine methyl ester
dihydrochloride with isocyanate derivatives of p-coumaric acid and 4-
methoxy-cinnamic acid, respectively. The synthesis of these molecules
was performed according to the synthetic procedure of Manzo et al.
[22].
2.2.2. Synthesis of PARA-B, PARA-C and PARA-Ph
The natural alkaloids parazoanthines B and C (PARA-B and PARA-C)
Fig. 1. Structures of PARA-A, PARA-F, PARA-B, PARA-C, PARA-Ph, PARA-Naph and phidianidine A. Relevant numbering is shown on the corresponding atoms.
2

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
acetonaphthone with the hydantoinic building block (prepared ac-
cording to [23]), gave the intermediate PARA-Naph-I1; carbonyl
reduction of PARA-Naph-I1 with NaBH
4
(obtaining intermediate PARA-
◦
Naph-I2), and subsequent dehydration with p-TsOH in toluene at 110 C
/
1 h led to the intermediate PARA-Naph-I3 free from nitrogen pro-
tecting group (BOC) and with E-configuration of 13–14 double bond
(
J
13-14 = 14.8 Hz). Finally, by the guanidination of compound PARA-
Naph-I3 using the guanidinating reagent 3,5-dimethyl-1-pyrazolylfor-
maminidium nitrate in basic conditions, we obtained PARA-Naph
(
Scheme 1). The three last step led to low yield despite the different
attempts to improve it by changing reagents amounts (1–4 equivalents)
◦
and/or reaction temperature values (from r.t to 110 C). These yields
were the better we obtained.
2
.3. Biological assays
GH4C1 cells were used to test the activity of PARA compounds
against CXCR4, since this cell line was reported to express CXCR4 but
not CXCL12, whose constitutive secretion could interfere with receptor
expression [24]. In order to rule out any potential cytotoxic effects of the
tested compounds, a cell viability assay was carried out in the absence of
CXCL12 stimulation of cell proliferation. Thus, after 48 h of serum
deprivation, we treated GH4C1 cells with 1 M of PARA-A, PARA-Ph,
μ
PARA-Naph, PARA-C, PARA-B, PARA-F in FBS-free medium and in the
absence of CXCL12; control cells were treated with the same amount of
DMSO, to exclude non-specific effects of the vehicle. After 24 h of in-
cubation, we assayed cells for MTT reduction by mitochondrial en-
zymes. This test provides a direct index of cell viability, with the
obtained values being directly proportional to cell number (Fig. 3). None
of the tested molecules induced a significant variation of MTT reduction
in GH4C1 cells in comparison with vehicle, indicating that cell viability
is not affected by these treatments.
To address the possibility that PARA molecules may act as CXCR4
antagonists we evaluated their ability to antagonize the mitogenic ac-
tivity of CXCL12 in GH4C1 cells in concentration–response experiments.
For this purpose, we treated FBS-starved GH4C1 cells for 12 h with
CXCL12 (25 nM), in the absence or presence of increasing concentra-
tions (10 nM-1 M) of the molecules under study. Cell proliferation was
μ
measured using CyQuant assays that quantify the amount of nucleic
acids as a direct index of cells number.
CXCL12 induced a consistent (122 ± 1.4%) and statistically signifi-
cant (P > 0.01) proliferation of GH4C1 cells, as compared to vehicle-
treated cells. All tested PARA compounds abolished this effect in a
dose-dependent manner, although with different potency (Fig. 4).
PARA-B was the most potent CXCR4 antagonist displaying an IC50 value
of 9.3 nM, followed by PARA-A (18.7 nM), PARA-Naph (70.7 nM),
PARA-Ph (116 nM), and PARA-F (269 nM). It was not possible to
calculate the IC50 value for PARA-C, since the compound showed a
biphasic curve, with a strong inhibition at low concentrations (10–50
nM) and a loss of efficacy at high concentrations. As far as efficacy is
concerned, all compounds were able to completely abolish CXCL12
proliferative effects, with the exception of PARA-Ph that, even at the
Fig. 2. Ribbon representation of CXCR4 complex with PARA-B (A), PARA-C (B)
and PARA-Naph (C). PARA-B, PARA-C and PARA-Naph are shown in ball&stick
representation and coloured in cyan, light green and plum, respectively. The
residues within 4 Å from ligand are shown in stick representation. Nitrogen,
oxygen, and sulphur atoms are painted white, blue, red and yellow, respec-
tively. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
highest concentration tested (1
μ
M), was not able to bring proliferation
M PARA-F caused a reduction of
back to the control level. Moreover, 1
μ
GH4C1 DNA content below control levels, as did PARA-C at low con-
centrations (10–50 nM), raising the possibility of an inverse agonist
activity of these compounds.
and the derivative 18-deoxy-parazoanthine B (PARA-Ph) were realized
according to the procedure described by Tinto et al. [23].
Linear concentration-dependent activities of PARA-A, PARA-Naph,
PARA-Ph, and PARA-B were also demonstrated by measuring the mod-
ulation of DNA neosynthesis induced by CXCL12 in GH4C1 cells (Suppl.
Fig. 1). By using BrDU incorporation assays we observed that CXCL12-
induced GH4C1 cell proliferation was antagonized by these PARA
compounds, with similar efficacies to those observed using the CYQuant
assay. These results indicate that all tested compounds are able to affect
CXCR4 activation concering CXCL12 proliferation signaling, which
represents one of the main pathways involved in tumor development,
2
.2.3. Synthesis of PARA-Naph
The synthesis of PARA-Naph was inspired by Tinto et al. procedure
[
23]; we prepared the intermediate
α
-bromo-1-acetonaphthone by
α
-bromination of 1-acetonaphthone with bromine in acetic acid
(
Scheme 1).
The succeeding coupling reaction (Scheme 1) of
α-bromo-1-
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R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
Scheme 1. Synthetic procedure for the preparation of PARA-Naph.
including pituitary adenomas [25,26].
One of the main functions of CXCR4 in normal and tumor cells is the
activation of intracellular pathways related to cell motility. To further
investigate the efficacy of PARA compounds as CXCR4 antagonists, we
evaluated their ability in antagonizing CXCL12-induced GH4C1 cell
migration. To this end, we measured the number of cells that permeate
through a porous membrane under CXCL12 driving chemotactic force.
In our experimental protocol, fluorescence dye-stained GH4C1 cells
were seeded into transwell upper chambers, while CXCL12 was added
into the lower chambers; the number of chemoattracted cells through
the transwell membrane was quantified by counting cells under epi-
fluorescence microscopy after 6 h of migration. Cells were treated with
PARA compounds before their staining and plating in transwell upper
chambers (Fig. 5). Vehicle-treated cells displayed a significant sensi-
tivity to CXCL12-dependent migration, as the number of migrating cells
increased by 44% in comparison to untreated cells (Fig. 5A). CXCR4
blockade by all PARA molecules, used at 1 M, significantly reduced the
μ
number of migrating cells. Among the tested compounds, PARA-A and
PARA-Ph showed lower efficacy, reaching a reduction of CXCL12-
induced migration of about 30%, while the remaining showed a
greater effect causing about 50% of inhibition (Fig. 5C).
Fig. 3. PARA compounds are not toxic for GH4 C1 cells. GH4C1 cell were
treated with all the studied compounds (1 µM) and cell viability tested using
MTT reduction assay.
ERK1/2 activation is one of the main intracellular signals by which
CXCR4 transduces intracellularly external cues [27]. Thus, to confirm
4

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
Fig. 4. PARA compounds inhibit GH4C1 proliferation induced by CXCL12. Cells, treated with CXCL12, increased proliferative activity by 22%, an effect
concentration-dependently inhibited by all the tested compounds, although with different potency and efficacy. Each panel represent the concentration-response
curve (10–1000 nM) of the PARAs on CXCL12 (25 nM)-induced cell proliferation. EC50 values are reported within the respective graphs. ◦◦ = p < 0.01 vs. con-
trol values; * and ** = p > 0.05 and 0.01 vs. CXCL12.
Fig. 5. Effect of PARA compounds on CXCL12-dependent GH4C1 cell migration. A) Cell migration was evaluated using a trans-well approach in which CXCL12 was
added to the lower chamber as chemoattractor. Cell migration was monitored taking advantage of the vital Vybrant™ CFDA SE fluorescent dye labelling of GH4C1
cells and measuring fluorescence on the lower side of the trans-well (B) using a fluorescence microscope and the ImageJ software (C). CXCL12 increased GH4C1 cell
migration by 44% (A), an effect that was abolished by all the PARA compounds (B, C). * and ** = p > 0.05 and 0.01 vs. CXCL12.
the specificity of the observed biological effects we checked whether the
tested compounds were able to interfere with CXCL12-dependent
phosphorylation/activation of ERK1/2 MAP kinase in GHC1 cells. In
particular, we evaluated by immunoblotting ERK1/2 phosphorylation
levels after 15 min of treatment with CXCL12 (25 nM), while PARA
compounds (50 nM) were added to the cells 30 min before CXCL12
pretreatment with all PARA compounds reduced ERK1/2 phosphoryla-
tion induced by CXCL12. Among highly statistically significant results
(P < 0.01 vs. CXCL12 treatment), PARA-B and PARA-C induced stronger
effects, while PARA-Naph elicited a moderate response. The other
compounds produced lower effects, with still statistically significant
results, although reaching a lower significance (P < 0.05) (Fig. 6B).
Finally, we decided to down-regulate CXCR4 expression in GH4C1
cells to demonstrate that the tested PARA compounds act through an
antagonistic activity at this chemokine receptor. We used CRISPR-CAS9)
(
Fig. 6A). CXCL12 increased ERK1/2 activation by two-fold in respect to
basal values, a response that was higher than that elicited by FBS, used
as positive control (+113% vs. + 61% over basal value, Fig. 6B). Cell
5

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
Fig. 6. Effect of PARA s on ERK1/2 activation. ERK1/2 activity in GH4C1 cells evaluated in Western blot using phopsho-specific antibody (A) and quantification by
◦
◦
densitometric analysis (B). CXCL12-induced increase of ERK1/2 phosphorylation was significantly reduced by all the PARA compounds tested.
= p < 0.01 vs.
control values; * and ** = p < 0.05 and 0.01 vs. CXCL12.
approach to interfere with CXCR4 expression. From gene interference,
we selected several clones, 9 of which were expanded and tested for
CXCX4 mRNA content by RT-PCR, in comparison to GH4C1 cells (Suppl
Fig. 2A). From this screening, we chose the clone A11 that showed the
most significant reduction in CXCR4 mRNA (named GH4A11). CXCR4
down-regulation was then confirmed by immunoblotting analysis
(Suppl Fig. 2B). Comparing growth ability of GH4A11 and parental
GH4C1 cells, using the MTT reduction assay, we did not observe dif-
ferences in the proliferation rate (data not shown), thus we used this cell
line to test the proliferative and migratory activities induced by CXCL12
(25 nM) and the reversal by the PARA compounds (50 nM-1
μM).
GH4A11 proliferation rate, evaluated using CyQuant assay, was neither
Fig. 7. PARA compounds were unable to modulate GH4A11 cell proliferation, measured by CyQuant evaluation of DNA content, in both basal conditions and in the
presence of CXCL12.
6

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
increased by CXCL12 nor reduced in the presence of the PARA com-
pounds, which did not also affect basal growth in the absence of CXCL12
3. Conclusions
(
Fig. 7). The only significant difference was a slight paradoxical increase
of proliferation induced by PARA-B in the presence of CXCL12 (P <
.05).
Similar results were also obtained evaluating GH4A11 cell migra-
In conclusion, through a combined computational and experimental
approach, we identified parazoanthines as potent CXCR4 antagonists.
The validation of the in silico results was obtained by assessing the ability
of PARA derivatives to antagonize CXCL12-dependent proliferation,
migration and phosphorylation/activation of ERK1/2 MAP kinase in rat
pituitary adenoma cell line GH4C1 cells. All tested compounds were able
to antagonize the down-stream effects of CXC12, albeit with variable
potency and efficacy, in the low nanomolar-micromolar range. More-
over, these results allowed the development of a more stringent phar-
macophoric model, useful for large-scale in silico screening, and
candidate PARA derivatives as potentially useful therapeutics tools to
negatively modulate the CXCL12/CXCR4 axis in tumoral pathologies
characterized by a overexpression of this receptor.
0
tion, which was neither increased by treatment with CXCL12 nor
reduced by all PARA compounds in the presence or absence of CXCL12
(
Fig. 8). These results clearly confirmed that the all tested compounds
act via CXCR4 antagonism to revert CXCL12 induction of GH4C1 pro-
liferation and migration.
2
.4. Structure-activity relationships and pharmacophoric model
optimization
The activity of PARA compounds as CXCR4 antagonists has been
evaluated looking at their ability to counteract the effects of CXCL12/
CXCR4 signalling on proliferation, migration and phosphorylation/
activation of ERK1/2 MAP kinase (pERK). In all assays, PARA-B was
identified as the most active and potent compound, highlighting the
significance of both the double bond on the alkyl spacer and the
occurrence of the hydroxy group on the phenyl moiety. In fact, both
PARA-A and PARA-Ph were less active than PARA-B, with a more dra-
matic effect for PARA-Ph. The bulkier naphthalene group performed
better than the phenyl substituent on proliferation and migration, but
not at pERK assay, suggestive of a biased antagonism effect induced by
this substituent, a behaviour already observed by other CXCR4 ligands
4. Experimental part
4.1. Computational methods
Docking studies were performed with AutoDock 4.2[29]. Both the
crystallographic structure of CXCR4 (PDB entry: 3OE0) and the inves-
tigated ligands were processed with AutoDock Tools (ADT) package to
merge non-polar hydrogens, calculate Gasteiger charges and desolvation
parameters, and select all rotatable ligand bonds as flexible. Grids for
docking evaluation with a spacing of 0.375 Å and 70x70x50 points,
centered in the ligand binding pocket, were generated using the program
AutoGrid 4.2 included in Autodock 4.2 distribution. Lamarckian Genetic
Algorithm (LGA) was adopted to perform 100 docking runs with the
following parameters: 100 individuals in a population with a maximum
of 15 million energy evaluations and a maximum of 37,000 generations,
followed by 300 iterations of Solis and Wets local search. PARA-CXCR4
complexes were selected on the basis of binding energy and cluster
population. The x-ray structure was cleaned of the fusion protein at the
intracellular loop 3 (IL3) and completed in its not-resolved loop regions
using MODELLER program version 9.15[30] and the resulting com-
plexes, after addition of all hydrogen atoms, underwent energy mini-
mization with SANDER module of Amber16 package[31] using the
ff14SB version of AMBER force field for the protein and GAFF for the
ligand. Ligand atomic charges were obtained with the RESP methodol-
ogy[32], according to the prescription for molecule parametrization in
the GAFF force field. This latter requires an ab initio full geometry
optimization at the Hartree-Fock level using the STO-3G basis set, fol-
lowed by a single-point energy calculations on the optimized molecule
at the RHF/6-31G* level. Ab initio calculations were performed with
GAMESS program[33].
[
28]. The methylation of the hydroxy group is well tolerated in the
migration and pERK assays, even increasing the activity of PARA-F in
comparison to PARA-A in the migration assay. Instead, in the prolifer-
ation assay the methoxy group induces inverse agonism. However, the
biphasic curve observed for PARA-C is suggestive of off-target effects at
higher concentrations that balance the CXCR4-mediated anti-
proliferative activity. The comparison between the phidianidine A and
parazoanthines B-C scaffolds and the greater potency as CXCR4 antag-
onists of the latter, suggest that a shorter distance (~14 Å instead of 18
Å) between the guanidine group and the aromatic moiety improves the
activity. It is reasonable to speculate that the shorter linker, as well as
the two double bonds on the alkyl chain, reduce the flexibility of the
ligands, causing in turn a reduction of the unfavourable entropy
contribution upon binding. The occurrence of a bulky aromatic group
such as the indole group in the phidianidine A or naphthalene in PARA-
Naph, is not mandatory for the antagonist activity, but the phenyl group
has to compensate the reduced size featuring an hydroxy or methoxy
group to engage H-bonds with the polar residues lying in the subpocket
hosting the aromatic moiety. Furthermore, in both phidianidine A and
parazoanthines, the alkyl linker features a five-membered ring able to
form H-bonds with the receptor. Thus, the previous minimalistic phar-
macophoric model can be refined as shown in Fig. 9.
4
.2. Synthesis (synthetic procedures for PARA-Naph)
NMR spectra of the synthetic compounds were recorded on a Bruker
Fig. 8. Lack of PARA compound effects on cell migration in presence of CXCR4 downregulation. In trans well experiments GH4A11 cells showed no responses to both
stimulatory effect of CXCL12 and the inhibitory activity of PARA compounds.
7

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
Fig. 9. Optimized pharmacophoric model.
+
Avance-400 (400.13 MHz) in CDCl
3
and CD
3
OD (δ values are referred to
(2H, bq, J = 6.1 Hz); HRMS (ESI) m/z 308.1325 [M + H] (calcd for
1
CHCl
3
and CD
3
OD at 7.26 and 3.34 for H NMR and at 77.0 and 49.0
C
18
H
17
N
3
O
2
, 307.1321).
ppm for 13C NMR respectively). HR-MS spectra were acquired by a Q-
Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Sci-
entific). TLC plates (Kieselgel 60 F254) and silica gel powder (Kieselgel
PARA-Naph: Compound PARA-Naph-I3 (8.6 mg, 0.02 mmol) was
dissolved in anhydrous DMF (1.0 mL) with DIPEA (6 µL, 0.03 mmol) and
3,5-dimethyl-1-pyrazolylformaminidium nitrate (6 mg, 0.03 mmol); the
mixture was stirred overnight at room temperature under argon atmo-
sphere, evaporated, fractioned by silica gel chromatography using a
gradient of chloroform and methanol and further purified by HPLC RP-
6
0, 0.063–0.200 mm) were from Merck.
All the reagents were purchased from Sigma-Aldrich and used
without any further purification.
PARA-Naph-I1: hydantoinic building block (150 mg, 0.587 mmol)
was dissolved in anhydrous DMF (6.0 mL); potassium carbonate (121
mg, 0.876 mmol) and TBAB (14 mg) were added; the mixture was
phase (H
2
O-TFA 0.1%/MeOH, from 60/40 to 50/50 in 20 min, RP-
amide analytical column, flow rate 1 mL/min) to give PARA-Naph (3
1
mg, 0.008 mmol, 40%); H NMR (CD
3
OD): δ 8.34 (1H, d, J = 14.2 Hz),
◦
heated at 50 C under argon atmosphere with molecular sieves and
8.13 (1H, d, J = 8.5 Hz), 7.92 (1H, d, J = 6.6 Hz), 7.86 (1H, d, J = 8.3
Hz), 7.68 (1H, d, J = 7.0 Hz), 7.61 – 7.44 (3H, m), 7.22 (1H, d, J = 14.6
Hz), 5.89 (1H, bt, J = 7.8 Hz), 3.42 (2H, bt, J = 6.8 Hz), 2.60 (2H, bq, J
α
-bromo-1-acetonaphthone (176 mg, 0.416 mmol) was added after
1
5–30 min; after 2 h the reaction was evaporated and purified by silica
1
3
gel chromatography using a gradient of chloroform and methanol to
give compound PARA-Naph-I1 (58 mg, 0.137 mmol, 33%); 1H NMR
= 7.0); C NMR (CD OD): δ 162.5 (C), 157.2 (C = NH), 153.8 (C), 133.7
3
(C), 133.5 (C), 133.3 (C), 129.4 (CH), 128.9 (CH), 126.7 (CH), 124.5
(
CD
3
OD): δ 8.89 (1H, bs, NH), 8.67 (1H, d, J = 8.37 Hz), 8.04 (1H, d, J =
(CH), 124.0 (CH), 119.9 (CH), 118.3 (CH), 110.1 (CH), 40.9 (CH2), 27.3
+
8
.2 Hz), 7.99 (1H, d, J = 7.27 Hz), 7.88 (1H, d, J = 7.95), 7.60–7.50 (2H,
(CH2); HRMS (ESI) m/z 350.3277 [M + H] (calcd for C19
19 5 2
H N O ,
m, overlapped), 5.91 (1H, t, J = 8.23 Hz) 5.06 (2H, s), 3.22 (2H, m), 2.44
349.1539).
(
(
(
2H, m) 1.44 (9H, s, Boc methyls); 13C NMR (CD
C), 156.2 (C, Boc), 153.8 (C), 132.7 (CH), 127.6 (CH), 127.1 (CH) 124.9
CH), 109.9 (CH), 80.2 (C, Boc), 45.1 (CH ), 38.7 (CH ), 26.8 (CH ),
, Boc methyls); HRMS (ESI) m/z 423.1798 [M +
, 423.1794).
3
OD): δ 194.4 (C), 162.4
4.3. Biological assays
4.3.1. Cell cultures
2
2
2
2
9.4, 29.0, 28.6 (CH
+
3
Na] (calcd for C23
H
25
N
3
O
5
GH4C1 rat pituitary adenoma cells were cultured in 50:50 DMEM:
F12 medium containing penicillin/streptomycin and supplemented with
PARA-Naph-I2: PARA-Naph-I1 (58 mg, 0.137 mmol) was dissolved
in ethanol (4 mL) and sodium borohydride (6 mg, 0.156 mmol) was
added; the mixture was stirred for 2 h at room temperature and after
addition of some drops of water was partitioned between water (20 mL)
L-glutamine and 10% of fetal bovine serum (FBS) and maintained in 5%
CO2 in humidify air at 37 ◦C [24].
and ethylacetate (20 mL); the organic phase was evaporated to give
4.3.2. Generation of CXCR4 knockout GH4 cells with CRISPR-Cas9
The CRISPR-Cas9 and green fluorescent protein (GFP) fusion protein
expression vector U6RNA-Cas9-2A-GFP expressing gRNA targeting
CXCR4 was purchased from Sigma-Aldrich. GH4C1 cells were plated in a
6-well plate in complete growth medium to achieve about 80% conflu-
ence on the day of transfection. Cells were transfected with Lipofect-
amine™ 3000 Transfection Reagent (ThermoFisher Scientific)
according to the manufacturer’s guidelines. The co-expression of Cas9
and GFP from the same mRNA allowed the possibility of tracking of
transfection efficiency and enriching cultures with CXCR4-knockout
cells by fluorescence activated cell sorting (FACS) 24 h post-
transfection. Cells were collected, diluted and seeded individually into
96-well plates, for isolating single colonies. Each colony was harvested
and transferred to a 24-well plate. The cells were allowed to proliferate
until checked for CXCR4 expression by RT-PCR and Western blot, and
used for functional assays. Due to the lower residual expression of
CXCR4 GH4C1 A11 clone was selected for the experiments.
1
PARA-Naph-I2 (39 mg, 0.091 mmol, 66%); H NMR (CDCl
3
): δ 8.25
(
1H,m), 8.06 (1H, m), 7.88 – 7.70 (2H, m, overlapped), 7.58–7.32 (3H,
m, overlapped), 5.86 (1H, m, overlapped) 5.79 (1H, m, overlapped,
epimer,), 5.62 (1H, m, overlapped, epimer), 3.98 – 3.64 (4H, m, over-
lapped) 1.44 (9H, s, Boc methyls); 13C NMR (CD
OD): δ 170.7 (C), 164.7
3
(
C), 155.3 (C, Boc), 127.7 (CH), 127.0 (CH) 124.7 (CH), 122.1 (CH),
1
10.1 (CH), 80.1 (C, Boc), 70.3 (CH, epimer), 68.2 (CH, epimer) 59.1
(
CH
2
), 47.6 (CH
2
), 44. (CH
2
), 27.7 (CH3, Boc methyls); HRMS (ESI) m/z
, 425.1951).
+
4
25.1948 [M + Na] (calcd for C23
27 3 5
H N O
PARA-Naph-I3: Compound PARA-Naph-I2 (39 mg, 0.091 mmol)
was dissolved in toluene (5 mL) with monohydrated p-toluensulfonic
◦
acid (70 mg, 0.37 mmol); the mixture was heated at 110 C for 1 h,
evaporated and partitioned between a saturated sodium bicarbonate
water solution (100 mL) and chloroform (100 mL); chloroform was dried
with sodium sulfate and purified further with silica gel chromatography
using a gradient of chloroform and methanol to give PARA-Naph-I3 (6
1
mg, 0.02 mmol, 22%); H NMR (CDCl
3
): δ 8.33 (1H, d, J = 14.8 Hz), 8.14
(
1H, d, J = 8.6 Hz), 7.90 (1H, d, J = 6.6 Hz), 7.90 (1H, d, J = 8.3 Hz),
4.3.3. Proliferation and viability assays
7
7
.83 (1H, d, J = 7.0 Hz), 7.76 (1H, d, J = 7.9 Hz), 7.65 – 7.41 (3H, m),
.18 (1H, d, J = 14.5 Hz), 5.79 (1H, bt, J = 6.8 Hz), 2.87 (2H, m), 2.44
4
.3.3.1. Cell proliferation assay CyQUANT®. DNA synthesis, as index of
8

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
cell proliferation, was determined by fluorescence kit CyQUANT® (Life
Technologies, USA). After treatments, GH4C1 and GH4A11 cells were
incubated with CyQUANT® for 1 hr, as reported [34]. The dye-DNA
complexes were read using a microplate fluorescence reader (Tecan®
Infinite 200 Pro, Switzerland) ex/em 485/530 nm.
4.3.3.6. Antibodies. P42/44 ERK1/2 and phospho-p44/42 (ERK1/2)
rabbit polyclonal antibodies (Cell signaling). CXCR4, monoclonal mouse
antibody (Abcam).
α-tubulin and β-actin, monoclonal mouse antibodies
(Sigma Aldrich). HRP-conjugated anti-rabbit IgG goat antiserum (Bio-
Rad Laboratories).
4
.3.3.2. Cell proliferation ELISA BrdU kit (Roche). Modulation of GH4C1
4.3.3.7. Statistical analysis. All data are presented as mean ± SEM of at
least three experiments performed in triplicate. Statistical significance
was established at p-value < 0.05. Prism version 5.02 (GraphPad, San
Diego, CA) software was used to analyze the results.
cell proliferation induced by CXCL12 was evaluated by measuring 5-
′
bromo-2 -deoxyuridine (BrdU) incorporation in newly synthesized
cellular DNA [35]. Briefly, GH4C1 cells were seeded into 48 cluster
4
plates at the density of 2.5x10 cells/well and treated for with CXCL12
(
25 nM) in the absence or presence of tested compounds. After 20 h, 10
Declaration of Competing Interest
µmol/l of BrdU was added to cell cultures and further incubated for 4 h
at 37 C. subsequently cells were fixed and incubated with peroxidase-
◦
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
conjugated anti-BrdU antibodies. The amount of immunocomplexes
was detected by adding peroxidase substrate tetramethyl benzidine.
2 4
Reaction was stopped with 1 M H SO . Absorbance was measured
spectrophotometrically at 450 nm using microplate reader BioTek
ELx800 (Winooski VT, USA).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104337.
4
.3.3.3. MTT reduction assay. Mitochondrial function, as index of cell
viability, was evaluated by measuring intracellular reduction of 3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,
Sigma-Aldrich) to water insoluble purple formazan by mitochondrial
dehydrogenase [36]. Briefly, 0.25 mg/ml MTT was added to culture
References
[
1] A. Zlotnik, O. Yoshie, The Chemokine Superfamily Revisited, Immunity 36 (2012)
05–716, https://doi.org/10.1016/j.immuni.2012.05.008.
[2] C.A. Didigu, R.W. Doms, Novel Approaches to Inhibit HIV Entry, Viruses 4 (2012)
◦
medium and incubated at 37 C for 3 hrs; medium was then removed,
7
and formazan crystals were dissolved in dimethylsulfoxide. Absorbance
was measured spectrophotometrically at 570 nm using microplate
reader BioTek ELx800 (Winooski VT, USA).
3
09–324, https://doi.org/10.3390/v4020309.
[
[
[
[
3] P.A. Hernandez, R.J. Gorlin, J.N. Lukens, S. Taniuchi, J. Bohinjec, F. Francois, M.
E. Klotman, G.A. Diaz, Mutations in the chemokine receptor gene CXCR4 are
associated with WHIM syndrome, a combined immunodeficiency disease, Nat.
Genet. 34 (2003) 70–74, https://doi.org/10.1038/ng1149.
4
.3.3.4. Migration assay. Modulation of CXCL12-induced chemo-
4] J.S. Song, C.M. Kang, H.H. Kang, H.K. Yoon, Y.K. Kim, K.H. Kim, H.S. Moon, S.
H. Park, Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on
bleomycin induced murine pulmonary fibrosis, Exp. Mol. Med. 42 (2010) 465,
https://doi.org/10.3858/emm.2010.42.6.048.
attraction of GH4C1 and GH4A11 cells was assayed measuring cell
migration through microporous membranes [37]. Cells were plated into
4
6
well clusters at a density of 30x10 cells/well, serum deprived for 48 h
5] A. Wang, A.-M. Fairhurst, K. Tus, S. Subramanian, Y. Liu, F. Lin, P. Igarashi, X.
J. Zhou, F. Batteux, D. Wong, E.K. Wakeland, C. Mohan, CXCR4/CXCL12
Hyperexpression Plays a Pivotal Role in the Pathogenesis of Lupus, J. Immunol.
182 (2009) 4448–4458, https://doi.org/10.4049/jimmunol.0801920.
6] A. Bajetto, F. Barbieri, A. Pattarozzi, A. Dorcaratto, C. Porcile, J.L. Ravetti, G. Zona,
R. Spaziante, G. Schettini, T. Florio, CXCR4 and SDF1 expression in human
meningiomas: a proliferative role in tumoral meningothelial cells in vitro, Neuro.
Oncol. 9 (2007) 3–11. http://www.hubmed.org/display.cgi?uids=17108064.
7] B.A. Teicher, S.P. Fricker, CXCL12 (SDF-1)/CXCR4 pathway in cancer, Clin. Cancer
Res. 16 (2010) 2927–2931. http://www.hubmed.org/display.cgi?uids=20484021.
8] X. Sun, G. Cheng, M. Hao, J. Zheng, X. Zhou, J. Zhang, R.S. Taichman, K.J. Pienta,
J. Wang, CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression,
Cancer Metastasis Rev. 29 (2010) 709–722. http://www.hubmed.org/display.cgi?
uids=20839032.
and treated with the different compounds at the concentration of 1 µM.
After 12 h of treatment cells were stained with fluorescent dye
◦
Vybrant™ CFDA SE (ThermoFisher) 10 µM for 15 min at 37 C, gently
harvested in serum free medium and plated in the upper chambers of
FluoroBlok™ HTS 96 multiwell (8 µm pore, Corning) at the concentra-
tion of 1.5x10 per chamber. CXCL12 (25 nM) was added in the lower
4
[
[
compartment and migration was evaluated after six hours by counting
the number of the cells that have migrated in the lower chambers with
confocal microscope (BioRad MRC 1024 ES) (3 microscopic fields for
each condition). The medium in upper and lower chamber was devoid of
fetal bovine serum throughout the experiment to avoid unspecific
chemoattraction.
[
9] H. Maganti, A. Visram, R. Shorr, J. Fulcher, M. Sabloff, D.S. Allan, Plerixafor in
combination with chemotherapy and/or hematopoietic cell transplantation to treat
acute leukemia: A systematic review and metanalysis of preclinical and clinical
studies, Leuk. Res. 97 (2020), 106442, https://doi.org/10.1016/j.
4
.3.3.5. Immunoblotting. Cells were plated in 60 mm petri dishes (2 ×
5
leukres.2020.106442.
[10] S. Milanesi, M. Locati, E.M. Borroni, Aberrant CXCR4 Signaling at Crossroad of
1
0 / dish), serum deprived for 48 h and treated with SDF-1
α
in the
WHIM Syndrome and Waldenstrom’s Macroglobulinemia, Int. J. Mol. Sci. 21
absence or the presence of PARA compounds. After 15 min of treatment
cells were lysed in Tris-HCl 20 mM pH 8 NaCl 137 mM, EDTA 2 mm,
glycerol 10%, NP-40 1%, phenylmethylsulfonyl fluoride 1 mM and
cOmplete Mini protease inhibitor cocktail (Roche)[38]. After centrifu-
gation (10 min, 500 × g) to remove nuclei, concentration of protein in
cytoplasmic fractions was assessed by Bradford assay. 30 µg of proteins
from each sample were boiled in Laemmli buffer and size-fractionated
by 12% SDS-PAGE. Resolved proteins were electroblotted onto 0.2 µm
polyvinylidene difluoride membrane (Bio-Rad Laboratories) and probed
with primary antibodies. Immunoreactive bands were visualized adding
HRP-labeled secondary antibodies followed by Clarity TM Western ECL
(
2020) 5696, https://doi.org/10.3390/ijms21165696.
[
[
[
11] R.J. Miller, G. Banisadr, B.J. Bhattacharyya, CXCR4 signaling in the regulation of
stem cell migration and development, J. Neuroimmunol. 198 (2008) 31–38,
https://doi.org/10.1016/j.jneuroim.2008.04.008.
12] E. De Clercq, The AMD3100 story: the path to the discovery of a stem cell mobilizer
(
Mozobil), Biochem. Pharmacol. 77 (2009) 1655–1664, https://doi.org/10.1016/j.
bcp.2008.12.014.
13] N.D. Stone, S.B. Dunaway, C. Flexner, C. Tierney, G.B. Calandra, S. Becker, Y.-
J. Cao, I.P. Wiggins, J. Conley, R.T. MacFarland, J.-G. Park, C. Lalama, S. Snyder,
B. Kallungal, K.L. Klingman, C.W. Hendrix, Multiple-Dose Escalation Study of the
Safety, Pharmacokinetics, and Biologic Activity of Oral AMD070, a Selective
CXCR4 Receptor Inhibitor, in Human Subjects, Antimicrob. Agents Chemother. 51
(
2007) 2351–2358, https://doi.org/10.1128/AAC.00013-07.
[
[
14] S. Jenkinson, M. Thomson, D. McCoy, M. Edelstein, S. Danehower, W. Lawrence,
P. Wheelan, A. Spaltenstein, K. Gudmundsson, Blockade of X4-Tropic HIV-1
Cellular Entry by GSK812397, a Potent Noncompetitive CXCR4 Receptor
Antagonist, Antimicrob. Agents Chemother. 54 (2010) 817–824, https://doi.org/
(
Bio-Rad Laboratories) and quantified using ChemiDoc imaging system,
equipped with Image lab 4.0.1 software (Bio-Rad Laboratories). Quan-
tification of the signal was performed by densitometry and measured as
1
0.1128/AAC.01293-09.
ratios of housekeeping (
content.
α-tubulin or β-actin) used to normalize protein
15] W.-T. Hsu, H.-Y. Jui, Y.-H. Huang, M.-Y.-M. Su, Y.-W. Wu, W.-Y.-I. Tseng, M.-
C. Hsu, B.-L. Chiang, K.K. Wu, C.-M. Lee, CXCR4 Antagonist TG-0054 Mobilizes
Mesenchymal Stem Cells, Attenuates Inflammation, and Preserves Cardiac Systolic
9

R.M. Vitale et al.
Bioorganic Chemistry 105 (2020) 104337
Function in a Porcine Model of Myocardial Infarction, Cell Transplant. 24 (2015)
313–1328, https://doi.org/10.3727/096368914X681739.
the tumor microenvironment, Front. Cell. Neurosci. 8 (2014), 144, https://doi.org/
10.3389/fncel.2014.00144.
1
[
16] T. Murakami, S. Kumakura, T. Yamazaki, R. Tanaka, M. Hamatake, K. Okuma,
W. Huang, J. Toma, J. Komano, M. Yanaka, Y. Tanaka, N. Yamamoto, The Novel
CXCR4 Antagonist KRH-3955 Is an Orally Bioavailable and Extremely Potent
Inhibitor of Human Immunodeficiency Virus Type 1 Infection: Comparative Studies
with AMD3100, Antimicrob. Agents Chemother. 53 (2009) 2940–2948, https://
doi.org/10.1128/AAC.01727-08.
[28] B. Hitchinson, J.M. Eby, X. Gao, F. Guite-Vinet, J.J. Ziarek, H. Abdelkarim, Y. Lee,
Y. Okamoto, S. Shikano, M. Majetschak, N. Heveker, B.F. Volkman, N.I. Tarasova,
V. Gaponenko, Biased antagonism of CXCR4 avoids antagonist tolerance, Sci.
Signal. 11 (2018) eaat2214, https://doi.org/10.1126/scisignal.aat2214.
[29] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.
J. Olson, AutoDock4 and AutoDockTools4: Automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, https://doi.org/
10.1002/jcc.21256.
[30] N. Eswar, B. Webb, M.A. Marti-Renom, M.S. Madhusudhan, D. Eramian, M.-Y.
Shen, U. Pieper, A. Sali, Comparative Protein Structure Modeling Using Modeller,
in: Curr. Protoc. Bioinforma., John Wiley & Sons, Inc., Hoboken, NJ, USA, 2006:
pp. 5.6.1-5.6.30. doi:10.1002/0471250953.bi0506s15.
[31] H.G. D.A. Case, R.M. Betz, D.S. Cerutti, T.E. Cheatham, III, T.A. Darden, R.E. Duke,
T.J. Giese, C. A.W. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, A. Kovalenko,
T.S. Lee, S. LeGrand, P. Li, I. Lin, T. Luchko, R. Luo, B. Madej, D. Mermelstein, K.M.
Merz, G. Monard, H. Nguyen, H.T. Nguyen, J.S. Omelyan, A. Onufriev, D.R. Roe, A.
Roitberg, C. Sagui, C.L. Simmerling, W.M. Botello-Smith, L.X. and P.A.K. R.C.
Walker, J. Wang, R.M. Wolf, X. Wu, AMBER 2016, (2016).
[32] T. Fox, P.A. Kollman, Application of the RESP Methodology in the Parametrization
of Organic Solvents, J. Phys. Chem. B. 102 (1998) 8070–8079, https://doi.org/
10.1021/jp9717655.
[
[
17] X. Fang, Q. Meng, H. Zhang, B. Liang, S. Zhu, J. Wang, C. Zhang, L.S. Huang,
X. Zhang, R.T. Schooley, J. An, Y. Xu, Z. Huang, Design, synthesis, and biological
characterization of a new class of symmetrical polyamine-based small molecule
CXCR4 antagonists, Eur. J. Med. Chem. 200 (2020), 112410, https://doi.org/
1
0.1016/j.ejmech.2020.112410.
18] R.M. Vitale, M. Gatti, M. Carbone, F. Barbieri, V. Felicit `a , M. Gavagnin, T. Florio,
P. Amodeo, Minimalist Hybrid Ligand/Receptor-Based Pharmacophore Model for
CXCR4 Applied to a Small-Library of Marine Natural Products Led to the
Identification of Phidianidine A as a New CXCR4 Ligand Exhibiting Antagonist
Activity, ACS Chem. Biol. 8 (2013) 2762–2770, https://doi.org/10.1021/
cb400521b.
19] N. Cachet, G. Genta-Jouve, E.L. Regalado, R. Mokrini, P. Amade, G. Culioli, O.
P. Thomas, Parazoanthines Aꢀ E, Hydantoin Alkaloids from the Mediterranean Sea
Anemone Parazoanthus axinellae, J. Nat. Prod. 72 (2009) 1612–1615, https://doi.
org/10.1021/np900437y.
[
[
20] C. Audoin, V. Cocandeau, O. Thomas, A. Bruschini, S. Holderith, G. Genta-Jouve,
Metabolome Consistency: Additional Parazoanthines from the Mediterranean
Zoanthid Parazoanthus Axinellae, Metabolites. 4 (2014) 421–432, https://doi.org/
[33] M.S. Gordon, M.W. Schmidt, Advances in electronic structure theory, in: C.E.
Dykstra, G. Frenking, K.S. Kim, G.E. Scuseria (Eds.), Theory Appl. Comput. Chem.,
Elsevier, Amsterdam, 2005: pp. 1167–1189. doi:10.1016/B978-044451719-7/
50084-6.
1
0.3390/metabo4020421.
[
[
21] J. Wefer, T. Lindel, Total Synthesis of the Marine Natural Product Parazoanthine F
by Copper-Mediated C-N Coupling, European J. Org. Chem. 2015 (2015)
[34] V. Villa, S. Thellung, A. Corsaro, F. Novelli, B. Tasso, L. Colucci-D’Amato, E. Gatta,
M. Tonelli, T. Florio, Celecoxib Inhibits Prion Protein 90–231-Mediated Pro-
inflammatory Responses in Microglial Cells, Mol. Neurobiol. 53 (2016) 57–72,
https://doi.org/10.1007/s12035-014-8982-4.
[35] A. Pattarozzi, E. Carra, R.E. Favoni, R. Würth, D. Marubbi, R.A. Filiberti, L. Mutti,
T. Florio, F. Barbieri, A. Daga, The inhibition of FGF receptor 1 activity mediates
sorafenib antiproliferative effects in human malignant pleural mesothelioma
tumor-initiating cells, Stem Cell Res. Ther. 8 (2017) 119, https://doi.org/10.1186/
s13287-017-0573-7.
[36] F. Barbieri, R. Würth, A. Pattarozzi, I. Verduci, C. Mazzola, M.G. Cattaneo,
M. Tonelli, A. Solari, A. Bajetto, A. Daga, L.M. Vicentini, M. Mazzanti, T. Florio,
Inhibition of Chloride Intracellular Channel 1 (CLIC1) as Biguanide Class-Effect to
Impair Human Glioblastoma Stem Cell Viability, Front. Pharmacol. 9 (2018), 899,
https://doi.org/10.3389/fphar.2018.00899.
6
370–6381, https://doi.org/10.1002/ejoc.201500823.
22] E. Manzo, D. Pagano, G. Nuzzo, M. Gavagnin, M.L. Ciavatta, First synthesis of
parazoanthine-A and its O-Me derivative, Tetrahedron Lett. 53 (2012) 7083–7084,
https://doi.org/10.1016/j.tetlet.2012.10.066.
[
[
23] F. Tinto, D. Pagano, E. Manzo, Synthesis of parazoanthine B and analogs,
Tetrahedron. 71 (2015) 4379–4384, https://doi.org/10.1016/j.tet.2015.04.040.
24] T. Florio, S. Casagrande, F. Diana, A. Bajetto, C. Porcile, G. Zona, S. Thellung,
S. Arena, A. Pattarozzi, A. Corsaro, R. Spaziante, M. Robello, G. Schettini,
Chemokine Stromal Cell-Derived Factor 1 Induces Proliferation and Growth
α
Hormone Release in GH4C1 Rat Pituitary Adenoma Cell Line through Multiple
Intracellular Signals, Mol. Pharmacol. 69 (2006) 539–546, https://doi.org/
1
0.1124/mol.105.015255.
[
[
[
25] F. Barbieri, A. Bajetto, R. Stumm, A. Pattarozzi, C. Porcile, G. Zona, A. Dorcaratto,
J.-L. Ravetti, F. Minuto, R. Spaziante, G. Schettini, D. Ferone, T. Florio,
Overexpression of Stromal Cell-Derived Factor 1 and Its Receptor CXCR4 Induces
Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas, Clin. Cancer
Res. 14 (2008) 5022–5032, https://doi.org/10.1158/1078-0432.CCR-07-4717.
26] R. Würth, F. Barbieri, A. Pattarozzi, G. Gaudenzi, F. Gatto, P. Fiaschi, J.-L. Ravetti,
G. Zona, A. Daga, L. Persani, D. Ferone, G. Vitale, T. Florio, Phenotypical and
Pharmacological Characterization of Stem-Like Cells in Human Pituitary
Adenomas, Mol. Neurobiol. 54 (2017) 4879–4895, https://doi.org/10.1007/
s12035-016-0025-x.
[37] M. Gatti, A. Solari, A. Pattarozzi, C. Campanella, S. Thellung, L. Maniscalco, R. De
Maria, R. Würth, A. Corsaro, A. Bajetto, A. Ratto, A. Ferrari, A. Daga, F. Barbieri,
T. Florio, In vitro and in vivo characterization of stem-like cells from canine
osteosarcoma and assessment of drug sensitivity, Exp. Cell Res. 363 (2018) 48–64,
https://doi.org/10.1016/j.yexcr.2018.01.002.
[38] A. Bajetto, A. Pattarozzi, A. Corsaro, F. Barbieri, A. Daga, A. Bosio, M. Gatti,
V. Pisaturo, R. Sirito, T. Florio, Different Effects of Human Umbilical Cord
Mesenchymal Stem Cells on Glioblastoma Stem Cells by Direct Cell Interaction or
Via Released Soluble Factors, Front. Cell. Neurosci. 11 (2017), 312, https://doi.
org/10.3389/fncel.2017.00312.
27] R. Würth, A. Bajetto, J.K. Harrison, F. Barbieri, T. Florio, CXCL12 modulation of
CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of
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