Substantial Cellular Penetration of Fluorescent Imidazoquinoxalines

Article abstract of DOI:10.1007/s10895-020-02595-y

Fluorescent tools have revolutionized our capability to visualize, probe, study, and understand the biological cellular properties, processes and dynamics, enabling researchers to improve their knowledge for example in cancer field. In this paper, we use the peculiar properties of our Imiqualines derivatives to study their cellular penetration and distribution in a human melanoma cell line A375 using confocal microscopy. Preliminary results on colocalization with the potent protein target c-Kit of our lead are also described.

Full text of DOI:10.1007/s10895-020-02595-y

Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02595-y
ORIGINAL ARTICLE
Substantial Cellular Penetration of Fluorescent Imidazoquinoxalines
Cindy Patinote1 & Natalina Cirnat1 & Kamel Hadj-Kaddour1 & Pierre Cuq1 & Pierre-Antoine Bonnet1
&
Carine Deleuze-Masquéfa1
Received: 15 June 2020 /Accepted: 27 July 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Fluorescent tools have revolutionized our capability to visualize, probe, study, and understand the biological cellular properties,
processes and dynamics, enabling researchers to improve their knowledge for example in cancer field. In this paper, we use the
peculiar properties of our Imiqualines derivatives to study their cellular penetration and distribution in a human melanoma cell
line A375 using confocal microscopy. Preliminary results on colocalization with the potent protein target c-Kit of our lead are
also described.
.
.
.
.
.
Keywords Imidazo[1,2-a]quinoxaline Fluorescence Localization Confocal microscopy Melanoma A375 cells.
Introduction
immunomodulator marketed in cream under the name
ALDARA® effective on certain skin and urogenital cancers
such as basal cell carcinoma). From our first generation active
Imiqualines published and protected by international patents
[1–6], the chemical modulations carried out led to obtaining
the second generation [7–10], the leader of which is
EAPB02303, protected by a selection patent with internation-
al extension [11]. EAPB02303 presents an interesting and
selective profile of nanomolar cytotoxic activities on a panel
of human cancer cell lines. On our human melanoma cell line
A375 reference, the measured activities are superior to those
of vemurafenib [9]. Moreover, transcriptomic studies allowed
us to show that EAPB02303 displays an original mechanism
of action, completely independent of tubulin and different
from those of a panel of anticancer agents used in clinics
(alkylating agents, topoisomerase inhibitors, anti-metabolites,
anti-tubulin). The first in vivo results on A375 melanoma
xenografted in mice show a higher efficacy of
EAPB02303 compared to the reference docetaxel, in
the absence of anti-angiogenic or necrotic activity, a
low mitotic index and limited toxicity (unpublished re-
sults). In this context, EAPB02303 has been determined
of particularly great value as a new highly active and
selective agent for melanoma treatment [11]. The deter-
mination of its extra- or intracellular molecular pharma-
cological targets is crucial for its successful develop-
ment toward clinical steps. Actually, it is essential for
further development to determine if EAPB02303 primar-
ily binds to the cellular membrane or acts further to
internalization within the melanoma cell.
Melanoma is the most dangerous skin cancer, due to its ability
to spread throughout the body by metastases. It is thus respon-
sible for 9 deaths out of 10 linked to skin cancers. Diagnosed
and treated in the early stages, it can be cured in 90% of cases.
Protein kinase inhibitors are used to treat melanomas with a
BRAFV600 mutation: vemurafenib, dabrafenib, trametinib
and cobimetinib. However, some patients develop resistance
mechanisms. These molecular mechanisms varied but mainly
lead to reactivation of the MAPK pathway and/or activation of
the PI3K-AKT parallel signaling. On the other hand, immu-
notherapy is reserved for BRAF-negative metastatic patients
or mutated BRAF patients who have become resistant.
Patients responding to immunotherapy with monoclonal anti-
bodies (nivolumab, ipilimumab, pembrolizumab) cannot be
anticipated, and these treatments are extremely expensive.
As not all melanomas respond to these therapies, the develop-
ment of new active treatments against melanoma is a public
health concern in view of their ever increasing prevalence.
In this context, we developed original imidazoquinoxalines
series, analogues of the Imiquimod parent compound (an
Cindy Patinote and Natalina Cirnat contribute equally to this work.
* Cindy Patinote
cindy.patinote@umontpellier.fr
1
IBMM, Université de Montpellier, CNRS, ENSCM,
Montpellier, France

J Fluoresc
F
O
COOH
N
NH
N
O
F
N
N
N
SOCl2
N
NH2
NaH
N
N
N
H
N
N
H
O
NaHMDS
THF
1h 0°C, then 3h RT
reflux, 24h
DMA
O
2
reflux, 48h
3
1
4
N,N-DEA
POCl3
MW 2X15 min, 130°C
Br
N
N
N
N
N
N
N
N
NBS
CHCl3
CH3NH2
EtOH
N
Cl
NHCH3
NHCH3
reflux, 1h30
MW 20 min, 150°C
5
6
7
OCH3
HO
HO
H3CO
H3CO
(OH)2B
OCH3
BBr3
Pd(PPh3)4, Na2CO3
DME/H2O (2/1)
MW 20 min, 140°C
N
N
CH2Cl2
0°C to RT, 3h
N
N
N
NHCH3
EAPB02303
N
8
NHCH3
HNO3, H2SO4
0°C to RT, ov.
OCH3
H3CO
HO
HO
O2N
H3CO
H3CO
O2N
Br
(OH)2B
BBr3
N
N
N
O2N
N
N
N
N
N
CH2Cl2
0°C to RT, 3h
Pd(PPh3)4
Na2CO3
DME/H2O (2/1)
MW 20 min, 140°C
NHCH3
N
NHCH3
NHCH3
EAPB02303-8e
8'
7'
H2/Pd/C
THF/DMF (9/1)
HO
HO
H2N
N
N
N
NHCH3
EAPB02303-8f
Scheme 1 Synthetic pathways of EPB02303, EAPB02303-8e or EAPB02303-8 f

J Fluoresc
Fluorescence spectra of EAPB02303
UV spectra of EAPB02303
500
400
300
200
100
0
3
2
1.10^-5 M
5.10^-5 M
1.10^-4 M
5.10^-4 M
1.10^-3 M
1.10^-2 M
1
0
254
304
354
-1
Wavelength (nm)
300
400
500
600
Wavelength (nm)
Fig. 1 UV and fluorescence spectra of EAPB02303 in DMSO[22]
In vitro fluorescent imaging (FI) studies enable the de-
tection of drug-protein interactions, protein-protein inter-
actions, gene expression and cellular processes, such as
drug internalization and distribution [12–15]. The possi-
bility of studying molecular processes in detail thanks to
FI generates information related to cancer progression, as
protease activity, apoptosis, necrosis and autophagy [16,
17]. The intrinsic fluorescence of some active compounds
could be used to overcome the introduction of fluorescent
markers which could modify their biological activity. In
fact, unsaturated systems confer fluorescent properties on
heterocyclic compounds, which are consequently useful
materials for imaging. For example, autofluorescence of
anticancer drugs, as doxorubicin and ellipticine, allows
for real-time localization, accumulation, time-/dose-de-
pendent cell death studies [18, 19].
Thanks to their planar rigid π-conjugation systems, the
parent drug Imiquimod [20, 21] and our Imiqualines
derivatives[22] behave as fluorescent chromophores. We
previously recorded, analyzed and published in this journal
the unpredicted fluorescent properties of some of our elev-
en imidazo[1,2-a]quinoxaline derivatives. Differences
were observed concerning their absorbance and emission
spectra despite belonging to the same chemical family,
notably for the intensities. No auto-quenching fluorescence
was detected in the tested experimental conditions. In par-
allel of ongoing chemical biology studies using affinity
chromatography coupled with the SILAC method and as-
sociated with high performance mass spectroscopy to con-
firm specific binding with protein target(s), we investigate
in this study the cellular penetration of EAPB02303 and its
localization into human melanoma cells (A375) by using
confocal microscopy. In order to gather information and
elucidate the mechanism of action of our lead, we present
herein first the in vitro results obtained with EAPB02303,
and secondly a detailed localization study of EAPB02303-
8f, a more suitable derivative for fluorescence imaging.
Co-localization with the potent target c-Kit of our drug
was also considered.
Materials and Methods
Chemical Compounds and Prepared Solutions
EPB02303, EAPB02303-8e and EAPB02303-8f synthetic
pathways are depicted in Scheme 1. Compounds were synthe-
sized thanks to a route we previously described [11, 13, 16].
Briefly, the carbonylimidazole dimer 2 results from the conden-
sation of the 2-imidazole carboxylic acid 1 in the presence of
thionyl chloride. Addition of the o-fluoroaniline on the dimer 2
gives the intermediate 3. Cyclisation is allowed by using sodium
hydroxide in dimethylacetamide. Treatment of compound 4 with
phosphorus oxychloride and N,N-diethylaniline gives the chlori-
nated compound 5. Then, an amino group substitutes the chlo-
rine. To obtain the NHCH3 residue 6, methylamine in ethanol is
grafted under microwave assistance. The bromination of the in-
termediate 6 by N-bromosuccinimide led to compound 7. An
additional step to introduce the nitro group on position 8 is car-
ried out with nitric acid in acidic conditions and furnishes com-
pound 7’. Appropriate aryl boronic acid is introduced in position
1 via a Suzuki-Miyaura cross-coupling reactions leading to com-
pounds 8 and 8’. Intermediates were submitted to boron
tribromide to give the hydroxylated derivatives EAPB02303
and EAPB02303-8e. Hydrogenation on Pd/C gives final com-
pound EAPB02303-8 f.
Compounds were stored at 4 °C. Stock solutions at
1.10− 2 m of each compound were prepared in DMSO.
These working standard solutions were then diluted with
DMSO or Dulbecco’s Modified Eagle’s Medium (DMEM)
to obtain 1.10− 9 m to 1.10− 4 m as final concentrations for
studies described below.
Absorption and Fluorescence Spectra
Absorption spectra were recorded on an UV-visible spectro-
photometer (Cary 400, Varian) from 250 to 400 nm.
Fluorescence spectra were recorded on a fluorescence spec-
trophotometer (Cary Eclipse, Varian) from 300 to 600 nm.

J Fluoresc
Excitaꢀon wavelength
355nm
White light
Overlay
a
b
c
d
Fig. 2 Images from fluorescence microscopy experiments with fixed or
non-fixed A375 cells incubated (T) or not (NT) for 1 h at 37 °C in
medium containing 3 nM of EAPB02303. The third column represents
the overlay of direct fluorescence output (blue, first column) and
white-light reflectance (second column)

J Fluoresc
180
160
140
120
100
80
Fig. 3 UV spectra of
EAPB02303 recorded with the
same used in vitro conditions
2303 10^-5 DMEM
2303 10^-9 DMEM
weak signal
60
40
20
0
300
400
Wavelenght (nm)
500
600
Cell Culture
Cell Fluorescence Measuring
The A375 human melanoma cell line were grown in DMEM
supplemented with 10% fetal calf serum, 1% glutamine and
antibiotics and incubated at 37 °C in an atmosphere of 5%
CO2 in air. Cells were grown as attached monolayer and were
detached for experiments using short exposure to trypsin. Cell
concentrations were determined using a CASYton® counter.
The level of cellular fluorescence from fluorescence mi-
croscopy images was determined using ImageJ. The
corrected total cell fluorescence (CTCF) was calculated
by the formula:
CTCF ¼ Integrated Density–ðArea of selected cellÞ
X ðMean fluorescence of background readingsÞ:
Confocal Laser Scanning Microscopy
The confocal imaging system used was Multiphoton
Microscope Leica™ TCS SP8 equipped with a 355 nm laser
and a 63x objective (1.4 NA). Cells were grown at a density of
5.104 cells/well as a monolayer on autoclaved glass
Fluorodish® or 8-well tissue culture chambers (Sarstedt™)
at the time of drug exposure. Cells were treated with
EPB02303, EAPB02303-8e or EAPB02303-8f during vari-
ous incubation times. The cells were fixed with 4% PFA and
examined under the confocal laser scanning microscope
(Leica Microsystems™).
Results and Discussion
The efficacy of a drug relies upon one or more interac-
tion(s) with a relevant target protein implicated in the
pathological process. The detection and the characteriza-
tion of the binding of our lead to its intended target
remain a scientific challenge [23]. In order to specify
not only the mechanism of action but also the targeted
protein(s) of our lead EAPB02303 and its analogues
implicated in the treatment management of melanoma
Fig. 4 UV spectrum of
EAPB02303-8e
1.7
1.5
1.3
1.1
0.9
0.7
0.5
250
270
290
310
330
350
370
390
WAVELENGHTS (nm)

J Fluoresc
Fig. 5 Fluorescence spectra of
EAPB02303-8e
1000
900
800
700
600
500
400
300
200
100
0
excitaꢀon at 290nm
excitaꢀon at 392nm
300
400
500
600
WAVELENGHTS (nm)
disease, we decided to use the fluorescent properties of
our compounds to obtain keys for elucidating them. We
first investigated the localization, distribution and accu-
mulation of EAPB02303 and EAPB02303-8f, the most
suitable agent for confocal microscopy imaging. Even if
fluorescence is often criticized due to numerous artifacts
(ionic strength, solvent polarity, pH, temperature, natural
fluorescence of the cells) which parasitize the signal
emitted by the imaging agents, we described herein
promising results for our compound EAPB02303-8 f.
Furthermore, preliminary tests were performed here to
evaluate cKit as potent target protein, as cKit was the
only highlighted protein among a panel of screened ki-
nases (data not shown). Moreover, markers of
colocalization of the nucleus and mitochondria were
also used secondly to examine more precisely the dis-
tribution of the compound EAPB02303-8 f.
Preliminary Studies
Absorption Spectra of EAPB02303 in DMSO
The UV profile and fluorescent spectra of EAPB02303 in
DMSO were previously published [22]. Our paper showed
that UV spectra are in concordance with the presence of the
catechol moiety on the one hand and with the quinoxaline core
on the other hand (Fig. 1). Moreover, there is a quantifiable
fluorescent response with a maximum at 445 nm when excited
at 270 nm at a concentration of 1.10− 4 m.
Fig. 6 UV spectrum of
EAPB02303-8f in used
1.9
conditions
1.7
1.5
1.3
1.1
0.9
0.7
0.5
0.3
0.1
250
270
290
310
330
350
370
390
WAVELENGHT (nm)

J Fluoresc
Fig. 7 Fluorescence spectra of
EAPB02303-8f in DMSO after
exciting at 364 nm
400
350
300
250
200
150
100
50
10^-4
10^-5
10^-6
10^-7
10^-8
0
300
350
400
450
500
550
600
WAVELENGHT (nm)
Samples for Confocal Laser Scanning Microscopy
Confocal Fluorescent Microscopy
The A375 cells were plated about 24 h before the start of the
test on the 35 mm diameter glass slides (FluoroDish™) con-
taining 1.5 mL of DMEM growth medium without a phenol
red pH indicator. The number of cells seeded was adjusted to
have about 50,000 / FluoroDish™ at the time of exposure to
EAPB02303. 1.5 mL of EAPB02303 at 10− 9 m was added to
previously prepared plated cells and left for 1 h incubation at
37 °C. One FluoroDish™ was used as negative control. After
the incubation period, 1 mL of 4% paraformaldehyde (PFA)
was added (10 min in the dark, covered with a polystyrene
box). PFA was washed three times with 1.5 mL PBS during
2–5 min at RT without shaking.
Based on the significant fluorescent signal of EAPB02303,
we started confocal microscopy studies in the aim of deter-
mining the cell penetration of the compound. For the first
confocal imaging analysis, we diluted EAPB02303 stock so-
lution in DMEM without red phenol buffer until 10− 9 m as its
anticancer activity is in the nanomolar range. Even if UV
signal is low above 350 nm, the possibility to obtain a fluo-
rescence signal after excitation at 355 nm is not implausible.
To our astonishment, no fluorescence signal was detectable
after one hour of incubation time with 3 nM of EAPB02303
compared to control (Fig. 2, A compared to C, B compared to
D). Furthermore, an auto-fluorescence phenomenon of A375
Fig. 8 Fluorescence spectra of
EAPB02303-8f in DMEM after
250
exciting at 364 nm
200
10^-4
10^-5
10^-6
10^-7
10^-8
150
100
50
0
300
350
400
450
500
550
600
WAVELENGHT (nm)

J Fluoresc
Fig. 9 Superposition of
fluorescence spectra of
EAPB02303-8f at 10-4 m in
DMSO and DMEM after exciting
at 364 nm
400
350
300
250
200
150
100
50
DMEM
DMSO
0
300
350
400
450
500
550
600
WAVELENGHT (nm)
cells has been highlighted. This phenomenon is associated
with ABCG2 expression in A375 cell line as demonstrated
by Marzagalli et al.. in the same time of our study [24].
Some little round vesicles are also detected (visible as white
points in pictures of Fig. 2).
We hypothesized that the DMEM buffer may change or
interfere in the fluorescence signal of EAPB02303. In fact,
the autofluorescence of phenol-red free DMEM buffer is not
unknown, inciting manufacturers such as Thermo Fischer to
produce lower fluorescent DMEM-containing buffer [25]. In
order to eliminate the possibility that DMEM buffer impacts
and stops the fluorescent signal of EAPB02303, the absorp-
tion spectra of the compound needed to be verified in the
studied cell environment.
were further analysed for their fluorescence properties since
their biological activities are in the same nanomolar range than
the leader EAPB02303 on A375 melanoma cell line. The
advantages of EAPB02303-8e and EAPB02303-8f lie on
their electronic rich chemical groups (NO2 and NH2) grafted
on the heterocycle which are not present in the EAPB02303
chemical structure. Those chemical groups could confer use-
ful additional fluorescent properties for cellular localization.
UV spectrum of EAPB02303-8e is similar to spectra
of our quinoxaline family and is depicted in Fig. 4.
Data shows two successive waves with maxima around
260 and 292 nm respectively then a decrease until
330 nm before increasing until 400 nm. No absorbance
was detected after 400 nm in the visible region.
Considering these results, a 10− 4 m sample in DMSO is
excited at 392 nm. A very low emission response at 461 nm is
detected but not sufficient to continue the cellular localization
studies with this compound since the excitation range of the
confocal laser starts at 355 nm (Fig. 5). Out of curiosity, this
sample is excited at 290 nm, which corresponds to a peak on
its UV profile (NB: we excited at 270 nm in our previous
publication). The fluorescence response is of quality with an
emission at 453 nm (intense and wide peak) and at 580 nm
(intense and fine peak) (Fig. 5). This result shows that a com-
pound is able to absorb energy (UV profile) at certain wave-
lengths but unable to necessarily re-emit fluorescence energy.
Fluorescence Spectra of EAPB02303 in DMEM Buffer
Solutions of compound EAPB02303 between 10− 5 m and
10− 9 m in phenol-red free DMEM buffer were prepared from
the stock solution at 10− 2 m in DMSO. When excited at
355 nm, no convenient fluorescent signal was recorded, ex-
cept a very weak signal at 450 nm, which confirms us what we
observed in vitro (Fig. 3). A thin peak appears at 355 nm
corresponding to the recovering emitting light.
In vitro Localization Studies
UV and Fluorescence Spectra of EAPB02303-8e
and EAPB02303-8f in DMEM Buffer
Fig. 10 Fluorescence microscopy experiments with A375 cells incubated
„
for 15 min, 30 min, 1 and 2 h at 37 °C in medium containing 10− 5 M of
EAPB02303-8 f. Direct fluorescence observed upon irradiation with
355 nm light. The third column represents the overlay of direct
fluorescence output (blue, first column) and white-light reflectance
(second column)
As the fluorescent signal of EAPB02303 was completely lost
in the used conditions, we turned our attention to the bank of
EAPB02303 derivatives synthesized in our laboratory. Two
interesting compounds, EAPB02303-8e and EAPB02303-8f,

J Fluoresc
Excitaꢀon wavelength
364 nm
White light
Overlay
a
b
c
d
e

J Fluoresc
UV spectrum of EAPB02303-8f is depicted on Fig. 6. Data
show first maximum at 265 nm. The curve is decreasing until
a plateau around 296 nm. A secondary lower maximum is
observed around 364 nm, and fluorescence is finally lost be-
fore 400 nm. A sample at 10− 4 m in DMSO is first excited at
364 nm. The fluorescence spectrum shows a correct emission
at 455 nm. A dilution range of 10− 5 m, 10− 6 m, 10− 7 m, 10−
8 m in DMSO was prepared and the samples were excited at
364 nm. Below 10− 6 m, the signal is no longer detectable
(Fig. 7). Moreover, a dilution range of 10− 5 m, 10− 6 m, 10−
7 m, 10− 8 m of EAPB02303-8f is prepared in DMEM without
phenol red from a 10− 2 m stock solution of EAPB02303-8f in
DMSO, and same results are observed: the fluorescent signal
is lost only beyond 10− 6 m (Fig. 8). We could also observe a
fine peak corresponding to the recovering emitting light at
355 nm, which is more intense than the fluorescence answer
of the compound EAPB02303-8f at this excitation rate. The
superposition of fluorescence spectra of EAPB02303-8f in
DMEM and DMSO at 10− 4 m is depicted on Fig. 9. The
fluorescence response obtained by diluting our compound in
DMEM buffer is lower than the sample diluted in DMSO at
the corresponding concentrations. Thus, it can be deduced that
the signal loss during preliminary studies is due firstly to the
high dilution used for the incubation and secondly to the pres-
ence of the DMEM which must trap a part of or interfere with
the emitted energy. For the following results presented herein,
we used EAPB02303-8f at a 10− 5 m concentration in DMSO
in order to assure a pick-up signal. Moreover, we decided to
work on fixed cells which allow a DMEM free medium for
imaging recording.
general cytoplasmic staining (Fig. 10a). For 15 min incu-
bation (Fig. 10b), fluorescence was apparent but could not
be quantified correctly due to quick photobleaching. The
photobleaching phenomenon was observed for all incuba-
tion periods but stabilized at around 2 h. By this way, to
prevent premature light extinction, laser was off while
searching cells to be analysed and on when just starting
recording data. For 30 min incubation (Fig. 10c), some
cells seem suffering, maybe due to the high quantity of
EAPB02303-8 f. Weak increased fluorescence could be
measured after 1 h of exposure (Fig. 10d), and slightly
above background noise. Image analysis of cells ex-
posed up to 2 h revealed a 3-fold increase of fluores-
cence intensity, which is assigned to our compound
(Fig. 10e and Table 1). Here again, we noticed fluores-
cent intracellular vesical, morphologically different from
those in control cells which might suggest an endocytic
phenomenon additional to the initial passive passage of
the cellular membrane.
Co-localization Preliminary Studies
Given the results of the localization study and out of curiosity for
these fluorescent vesicles observed in cells, we tried to determine
if the target of our compound EAPB02303-8f is on the cellular
membrane using a plasmatic membrane specific dye on the one
hand and an immunofluorescent assay on the other hand. The
immunofluorescent assay was designed to determine if
EAPB02303-8f binds to the cellular receptor c-Kit.
Secondary Immunofluorescence Assay - Colocalization
of EAPB02303-8f and c-KIT
Samples for Confocal Laser Scanning Microscopy
The A375 cells were plated about 24 h before starting the test
on 35 mm diameter glass slides (FluoroDish™) containing
1.5 mL of phenol-red free DMEM growth medium. The num-
ber of seeded cells was adjusted to have about 50,000 cells/
FluoroDish™ for exposure to EAPB02303-8 f. 1.5 mL of
EAPB02303-8f at 10− 5 m in DMSO was added to previously
prepared plated cells and left in incubation at 37 °C for 15 min,
30 min, 1 and 2 h. One FluoroDish™ was used with 1.5 mL of
DSMO as negative control. After the respective incubation
periods, 1 mL of 4% paraformaldehyde (PFA) was added
and left 10 min in the dark (covered with a polystyrene box).
PFA was washed three times with 1.5 mL PBS during 2–
5 min at RT without shaking.
Samples for Confocal Laser Scanning Microscopy The A375
cells were plated about 24 h before starting the test on sterile
8-well Tissue Culture Chambers (Sarsted™) containing
0.5 mL of DMEM growth medium. The number of cells
seeded was adjusted to have about 10,000/well at the time
of exposure to EAPB02303-8 f. 0.5 mL of EAPB02303-8f
at 10− 5 m in DMSO was added to previously prepared plated
cells and left in incubation for 2 h at 37 °C. At least one well
was used as negative control. The washing buffer was
1xPBS 0.1% Tween 20 (PBST). Cells were fixed using 4%
Table 1 Corrected total cell fluorescence analyzed by confocal
fluorescent microscopy (CTCF mean values).
Results of Confocal Fluorescent Microscopy
Incubation times NT
CTCF/CTCF NT
15 min 30 min 1 h
2 h
The auto-fluorescence observed in non-treated human
melanoma cells was clearly detected in an area-
dependant way. Fluorescence was primarily observed
within cytoplasmic inclusions (such as vesicles) with a
1,0
1,3
0,92
1,1
2,9
CTCF
237,379 296,842 217,420 259,486 699,184
Images treated with ImageJ., NT = non-treated cells

J Fluoresc
T
NT
a
e
EAPB02303-8f specific
fluorescence excitaꢀon
355 nm
f
b
Alexa Fluor® 633 fluorescence
excitaꢀon
632 nm
g
c
White light reflectance
h
d
Fluorescence overlay
Fig. 11 Images from fluorescence microscopy experiments with A375
cells incubated with the primary antibody and with the secondary
antibody. T: A375 cells incubated for 2 h at 37 °C in medium
containing 10− 5 M of EAPB02303-8 f. NT: negative control A375
cells. Direct fluorescence observed upon irradiation with 355 nm light
and 632 nm. The forth column represents the overlay of direct
fluorescence outputs and white-light reflectance
PFA in PBS, pH 7.4 for 15 min at room temperature. The
permeabilization method consisted in 10 min incubation with
0.1% Triton X-100. The cells were then washed by PBS
three times. The cells were incubated for 1 h at room tem-
perature with 3% BSA in PBST to block unspecific antibod-
ies binding, and then washed three times in PBST. Cells

J Fluoresc
CellMask PM® Green
Control
CellMask PM®Green
2 h incubaꢀon with EAPB02303-8f
a
b
c
d
e
CellMask PM®Green
Fluorescence
excitaꢀon wavelength
522 nm
f
EAPB02303-8f specific
fluorescence
excitaꢀon wavelength
355 nm
g
White light
reflectance
h
Overlay
Fig. 12 Images from fluorescence microscopy experiments with A375
cells stained with CellMask® Green PM. T: A375 cells incubated for 2 h
at 37 °C in medium containing 10− 5 M of EAPB02303-8 f. NT: negative
control A375 cells. Direct fluorescence observed upon irradiation with
355 nm light and 522 nm. The forth column represents the overlay of
direct fluorescence outputs and white-light reflectance
were incubated with the diluted primary antibody (cKIT
Antibody, Abgent™) in 3% BSA PBST at room temperature
for 1 h. The solution was decanted and then washed three
times in PBST. Cells were then incubated with the secondary

J Fluoresc
antibody in 3% BSA-PBST for 1 h at room temperature in
the dark. The secondary antibody solution was decanted and
the cells were washed three times in PBST. The coverslips
were then mounted with a drop of Fluoroshield® and stored
in dark at 4 °C.
Conclusion
The development of new anticancer drugs is a scientific chal-
lenge and, presently, fluorescence is used to elucidate part of
the mechanism of cancer activity. Thanks to a first study pub-
lished in this journal, we have highlighted the peculiar fluo-
rescence properties of our Imiqualines. Among different com-
pounds, we identified EAPB02303-8f as the best one to study
the cellular penetration and localization into melanoma A375
cells. Optimization of visualization conditions were neces-
sary. By this way, we used the compound at a concentration
of 10− 5 m in DMSO for treating cells that have been then
fixed for confocal microscopy study, in order to avoid the
use and presence of DMEM which induces a loss of fluores-
cence. We were able to detect correctly our compound
EAPB02303-8f especially after 2 h of incubation, thanks to
a fluorescence 3-times higher than control. Concerning its
subcellular distribution, EAPB02303-8f is mainly internal-
ized in vesicles, without a specific accumulation in an organ-
elle or nucleus. As we presumed that EAPB02303-8f’s target
might be a cytoplasmic localized receptor cKIT, we also per-
formed preliminary tests of colocalization in A375 human
melanoma cells. This study allows us to obtain interesting
information even if EAPB02303-8f compound does not cor-
respond to a perfect fluorophore essentially due to a quick
photobleaching. In the near future, we aim to perfect the pro-
tocol of subcellular localization or, eventually, our com-
pounds by enhancing their fluorescent capacity with simple
chemical modifications. By continuing the investigation of
subcellular transport and determining the biodistribution prop-
erties at the cellular level will help us to determine the mech-
anism of action of our Imiqualines. Quantitative assessment of
the subcellular distribution of small molecules inside cells
remains a challenge and we hope that progress in this field
will continue to accelerate allowing us to obtain better results.
Results of Confocal Fluorescent Microscopy Firstly, a differ-
ence in fluorescence intensity between the A375 cells treated
with EAPB02303-8f and untreated is noticed, which is con-
sistent with previous results. We also perceived more vesicles
with higher fluorescence intensity in the treated cells
(Fig. 11a). The secondary antibody, ALEXA633® seems
more internalized and diffuse in the treated cells (Fig. 11a
and f). Nevertheless, its strong presence in untreated cells
indicates that the non-specific saturation step was not suffi-
cient (Fig. 11a and e). Interestingly, during the superposition
of the fluorescence channels, it is noticed that the vesicles
corresponding to the fluorescence of EAPB02303-8f do not
overlap with the fluorescence of the revealing antibody. As a
reminder, the incubation time with EAPB02303-8f was set at
2 h in agreement with the results of intracellular fluorescence
detection. This fluorescence was significantly greater com-
pared to the control cells during a 2 h incubation at a concen-
tration of 10− 5 m in DMEM. The incubation period with
EAPB02303-8f of 2 h is probably too long to visualize a
binding process or a plasmatic membrane target.
Fluorescence Colocalization Microscopy Analysis Using
CellMask® Green Plasmatic Membrane Fluorescent Marker
(Invitrogen™)
Samples for Confocal Laser Scanning Microscopy A375 cells
were grown on coverslips inside a tissue culture dish with the
DMEM medium and incubated with EAPB02303-8f follow-
ing the same procedure as for immunofluorescent experiment.
Cells were fixed in 4% PFA solution. The coverslips were
quickly submerged in the staining CellMask® Green PM 2X
solution (Invitrogen™) for 5 min at 37 °C. The coverslips
were rinsed in PBS three times before being mounted and
stores at 4 °C.
Acknowledgements We would like to thank Marjorie Damian from the
team Cellular Pharmacology (IBMM, Montpellier, France) directed by
Dr. Jean-Louis Banères for technical support to record UV and fluores-
cence spectra. We also thank Marie-Pierre Blanchard, head of the MRI
Imaging Platform at the Functional Genomics Institute belonging to the
Biocampus of Montpellier (France), for having recorded all the data re-
lating to confocal microscopy.
Results of Confocal Fluorescent Microscopy Figure 12 shows
the fluorescence microscopy images of plasmatic membrane
stained cells (green). Unexpectedly, the vesicles presumed to
be specific of EAPB02303-8f are more intense in non-treated
cells (Fig. 12a). Likewise, our compound does not appear to
be on the plasmatic membrane but is more likely to be diffused
in cytoplasm of A375 cells (Fig. 12f). Nevertheless, few cells
in both conditions survived the fixation step, so the compari-
son is not interpretable. Here again, one can imagine that the
incubation time with the compound EAPB02303-8f is too
long to demonstrate membrane colocalization.
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