Discovery of a novel, isothiazolonaphthoquinone-based small molecule activator of FOXO nuclear-cytoplasmic shuttling

Article abstract of DOI:10.1371/journal.pone.0167491

FOXO factors are tumour suppressor proteins commonly inactivated in human tumours by posttranslational modifications. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. Therefore, the pharmacological activation of FOXO proteins is considered as an attractive therapeutic approach to treat cancer and agerelated diseases. In order to identify agents capable of activating FOXOs, we tested a collection of small chemical compounds using image-based high content screening technology. Here, we report the discovery of LOM612 (compound 1a), a newly synthesized isothiazolonaphthoquinone as a potent FOXO relocator. Compound 1a induces nuclear translocation of a FOXO3a reporter protein as well as endogenous FOXO3a and FOXO1 in U2OS cells in a dose-dependent manner. This activity does not affect the subcellular localization of other cellular proteins including NFkB or inhibit CRM1-mediated nuclear export. Furthermore, compound 1a shows a potent antiproliferative effect in human cancer cell lines.

Full text of DOI:10.1371/journal.pone.0167491

RESEARCH ARTICLE
Discovery of a Novel,
Isothiazolonaphthoquinone-Based Small
Molecule Activator of FOXO Nuclear-
Cytoplasmic Shuttling
1
1
2
3,4
Bastien Cautain , Francisco Castillo , Loana Musso , Bibiana I. Ferreira , N1 uria de
1
1
3
Pedro , Lorena Rodriguez Quesada , Susana Machado , Francisca Vicente ,
Sabrina Dallavalle , Wolfgang Link
2
3,4
*
1
Fundacion MEDINA, Parque Tecnol o´ gico Ciencias de la Salud, Granada, Spain, 2 DeFENS Department of
Food, Environmental and Nutritional Sciences, Università di Milano, Italy, 3 Centre for Biomedical Research
CBMR), Gambelas Campus, Faro, Portugal, 4 Regenerative Medicine Program, Department of Biomedical
Sciences and Medicine, University of Algarve, Campus de Gambelas, Faro, Portugal
(
a11111
*
walink@ualg.pt
Abstract
OPEN ACCESS
FOXO factors are tumour suppressor proteins commonly inactivated in human tumours by
posttranslational modifications. Furthermore, genetic variation within the FOXO3a gene is
consistently associated with human longevity. Therefore, the pharmacological activation of
FOXO proteins is considered as an attractive therapeutic approach to treat cancer and age-
related diseases. In order to identify agents capable of activating FOXOs, we tested a col-
lection of small chemical compounds using image-based high content screening technol-
ogy. Here, we report the discovery of LOM612 (compound 1a), a newly synthesized
isothiazolonaphthoquinone as a potent FOXO relocator. Compound 1a induces nuclear
translocation of a FOXO3a reporter protein as well as endogenous FOXO3a and FOXO1 in
U2OS cells in a dose-dependent manner. This activity does not affect the subcellular locali-
zation of other cellular proteins including NFkB or inhibit CRM1-mediated nuclear export.
Furthermore, compound 1a shows a potent antiproliferative effect in human cancer cell
lines.
Citation: Cautain B, Castillo F, Musso L, Ferreira BI,
de Pedro N, Rodriguez Quesada L, et al. (2016)
Discovery of a Novel, Isothiazolonaphthoquinone-
Based Small Molecule Activator of FOXO Nuclear-
Cytoplasmic Shuttling. PLoS ONE 11(12):
e0167491. doi:10.1371/journal.pone.0167491
Editor: M a´ ria A. Deli, Hungarian Academy of
Sciences, HUNGARY
Received: June 24, 2016
Accepted: October 26, 2016
Published: December 9, 2016
Copyright: © 2016 Cautain et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
The mammalian forkhead transcription factors of the O class (FOXOs) consists of four pro-
teins, FoxO1, FoxO3, FoxO4 and FoxO6 [1,2]. FoxOs have a characteristic forkhead box DNA
binding domain and bind as monomers to their consensus DNA binding sites [3]. FOXO pro-
teins function as transcriptional regulators in the cell nucleus and activate the transcription of
genes that are involved in numerous biologically relevant processes such as metabolism, differ-
entiation, proliferation, longevity, and apoptosis [4]. FoxOs are key components of an evolu-
tionary conserved pathway downstream of insulin and insulin-like growth factor receptors.
Funding: This work was supported by Funda c¸ ão
para a Ciência e a Tecnologia (FCT) Research
Center Grant UID/BIM/04773/2013 CBMR1334. B.
I. Ferreira is the recipient of a FCT 2014 research
grant SFRH/BPD/100434/2014R. S. Machado is
the recipient of a ProRegem grant PD/BD/114258/
2016.
PLOS ONE | DOI:10.1371/journal.pone.0167491 December 9, 2016
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Discovery of a Drug-Like FOXO Activator Molecule
Competing Interests: The authors have declared
Posttranslational modifications are thought to be the main mechanism to regulate their activity
that no competing interests exist.
[5]. The Serine/Threonine kinase AKT phosphorylates FOXO proteins at three conserved
consensus sites, which leads to conformational changes that facilitate CRM-1- mediated
nuclear export abolishing FOXO-dependent gene transcription [6]. Under stress conditions
or in the absence of growth or survival factors, when the PI3K/AKT pathway is inhibited,
FOXO proteins translocate to the cell nucleus, where their transcriptional functions can be
executed [5,7]. FOXO factors have been found to be inactivated in the majority of human can-
cers, owing to the overactivation of the phosphoinositide 3-kinase (PI3K)/AKT pathway [5,7]
and have been established as bona fide tumor suppressors [8]. Accordingly, FOXOs are the
major downstream transcriptional mediators of the PI3K/AKT pathway after PI3K inhibition
[9]. Furthermore, FoxO proteins are known to determine longevity in a wide range of metazo-
ans including humans and are indispensable for lifespan extension in response to dietary
restriction regimens [10]. Recent studies have found that FOXO3A is associated with human
longevity in Japanese-Americans from Hawaii, Italians, Ashkenazi Jews, Californians, New
Englanders, Germans and Han Chinese [11]. The importance of FOXO proteins in tumor sup-
pression and aging renders them compelling targets in the quest for therapeutic agents against
cancer and to slow down the aging process. Several assays have been developed to monitor the
subcellular localization of FOXO factors [12–14]. In order to identify chemical agents with the
capacity of activating FOXO, we chose to employ a high content imaging approach to monitor
the nucleocytoplasmic translocation of a GFP-FOXO3a fusion protein in U2OS cells. In this
study, we identified and characterized a novel small FOXO relocator molecule.
Materials and Methods
Compounds
The compounds used for primary screening were obtained within the framework of COST
Actions CM1106 (Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells)
responsible for generating a compound collection for further biological evaluation. The
COST CM1106 compound collection contains synthetic molecules and natural compounds.
LY294002 and Leptomycin B were purchased from Calbiochem.
Synthesis of compounds 1a-c
General information. All reagents and solvents were reagent grade or were purified by
standard methods before use. Melting points were determined in open capillaries and are
uncorrected. Solvents were routinely distilled prior to use; dry methylene chloride was
obtained by distillation from phosphorus pentoxide. All reactions requiring anhydrous condi-
tions were performed under a positive nitrogen flow, and all glassware were oven dried. Col-
umn chromatography was carried out on flash silica gel 60 (230–400 mesh). Analytical and
preparative thin-layer chromatography (TLC) was conducted on TLC plates (silica gel 60
F254, aluminium foil) and spots were visualized by UV light and / or by means of dyeing
reagents. NMR spectra were recorded at 300 MHz. Chemical shifts (δ values) and coupling
constants (J values) are given in ppm and Hz, respectively. Compound N,N-dibenzyl-urea (2a)
was prepared according to literature procedure [15]. Compound 3a was prepared according to
literature procedure [16]. Compound 3c was prepared according to literature procedure [17].
3
-Dimethylamino-naphtho[2,3-d]isothiazole-4,9-dione (1a). To a stirred solution of
naphthoquinone (2.5 g, 16mmol) in xylene (20mL) compound 3a (777mg, 5.3mmol) was
added and the resulting mixture was heated to reflux for 2h. The solvent was removed under
reduced pressure and the residue was purified by preparative chromatography in hexane/ace-
1
tone 19:1 to afford compound 1a (314 mg, 23%): mp: 170˚C; H-NMR (300 MHz, CDCl ) δ:
3
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Discovery of a Drug-Like FOXO Activator Molecule
13
8
.31–8.11 (m, 2H), 7.89–7.77 (m, 2H), 3.12 (s, 6H). C-NMR (75 MHz, CH OH-d )) δ:
3 4
178.02, 177.49, 168.69, 167.42, 134.93, 134.87, 133.29, 132.58, 127.81, 126.88, 124.77, 41.90
(
× 2). Anal. calcd for C H N O S: C 60.45, H 3.90, N 10.85, found: C 60.37, H 3.91, N 10.87.
13
10 2 2
5-Dibenzylamino-[1,3,4]oxathiazol-2-one (3b). To a suspension of N,N-dibenzyl-urea
(1 g, 4.16mmol) in acetonitrile (12mL), chlorocarbonylsulphenyl chloride (117μL, 1.38mmol)
was added and the mixture was stirred 20 h at room temperature (RT). The solvent was
removed under reduced pressure and the residue was purified by preparative chromatography
1
in hexane/ethyl acetate 9:1 to obtain compound 3b as a pale yellow oil (254mg, 62%): H-
NMR (300 MHz, acetone-d ) δ: 7.49–7.21 (m, 10H), 4.6 (s, 4H).
6
3-(Benzyl-phenyl-amino)naphtho[2,3-d]isothiazole-4,9-dione (4). To a stirred solution
of naphthoquinone (402mg, 2.55mmol) in xylene (5mL), compound 3b (254mg, 0.85mmol)
was added and the resulting mixture was heated to reflux for 3 h. The solvent was removed
under reduced pressure, and the residue was purified by preparative chromatography in tolu-
1
ene/ethyl ether 200:1 resulting in compound 4 (219 mg, 63%): mp: 93˚C; H-NMR (300 MHz,
1
3
CDCl ) δ: 8.32–8.19 (m, 2H), 7.88–7.68 (m, 2H), 7.40–7.15 (m, 10H), 4.71 (s, 4H). C-NMR
3
(
75 MHz, CH OH-d ) δ: 177.8, 177.7, 167.8, 167.6, 137.70 (× 2), 135.0, 134.85, 133.4, 132.5,
3
4
128.4 (× 4), 128.3 (× 4), 127.9, 127.2 (× 2), 126.9, 125.27, 54.52 (× 2). Anal. calcd for
C H N O S: C 73.15, H 4.42, N 6.82, found: C 73.34, H 4.43, N 6.80.
25
18 2 2
3
-Amino-naphtho[2,3-d]isothiazole-4,9-dione (1b). To a stirred solution of compound
(20mg, 0.05mmol) in acetonitrile/H O 9:1 (2mL), cerium ammonium nitrate (133mg,
4
2
0.25mmol) was added and the resulting mixture was stirred at room temperature for 1 h. The
reaction mixture was concentrated, diluted with cold water and extracted three times with
ethyl acetate. The combined extract was washed with brine and dried on Na SO . The crude
was purified by preparative chromatography in hexane/ethyl acetate 4:1 to generate compound
2
4
1
1
b (62mg, 89%): mp: 238˚C; H-NMR (300 MHz, DMSO-d ) δ: 8.25–8.09 (m, 2H), exchange-
6
able with D2O). 13C-NMR (75 MHz, DMSO-d6) δ: 179.5, 177.7, 165.5, 164.7, 135.7, 134.7,
1
34.0, 133.9, 127.6, 127.3, 120.9. Anal. calcd for C H N O S: C 57.38 H 2.63, N 12.17, found:
11 6 2 2
C 57.42, H 2.62, N 12.19.
3
-Phenyl-naphtho[2,3-d]isothiazole-4,9-dione (1c). To a stirred solution of naphthoqui-
none (280mg, 1.77mmol) in xylene (2.5mL), compound 3c (105mg, 0.59mmol) was added and
the resulting mixture was heated to reflux for 4 h. The solvent was removed under reduced
pressure, and the residue was purified by preparative chromatography in toluene/ethyl ether
1
2
00:1 to generate compound 4 (65mg, 38%): mp: 218˚C; H-NMR (300 MHz, DMSO-d ) δ:
6
8.24–8.12 (m, 2H), 8.04–7.88 (m, 2H), 7.80–7.70 (m, 2H), 7.58–7.44 (m, 3H). 13C-NMR (75
MHz, DMSO-d6) δ: 178.6, 178.2, 169.6, 167.5, 136.0, 134.9, 134.8 (× 2), 134.0, 133.0, 130.5,
1
30.1 (× 2), 128.6 (× 2), 128.1, 127.3. Anal. calcd for C H NO S: C 70.09, H 3.11, N 4.81,
17
9
2
found: C 70.21, H 3.11, N 4.80.
Cell culture
The human osteosarcoma cell line U2OS, the breast cancer cell line MCF7, the melanoma
cell line A2058, the neuroblastoma cell line SH-SY5, the human liver cancer cell line HepG2
and the human liver epithelial cell line THLE2 were obtained from the American Type Cul-
ture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium, sup-
plemented with 10% fetal bovine serum (Sigma) and penicillin-streptomycin. Cell cultures
were maintained in a humidified incubator at 37˚C with 5% CO2 and passaged when conflu-
ent using trypsin/EDTA. Stable cell lines U2nesRELOC and U2foxRELOC cells have been
generated as described previously [13,14,18,19]. GFP-NFAT was kindly provided by L.
Gerace.
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Discovery of a Drug-Like FOXO Activator Molecule
Cytotoxic activity of the compounds
The cytotoxic activity of the different compounds was tested against four tumor cell lines in
4
the MTT colorimetric assay. Cells were seeded at a concentration of 1× 10 cells/well in 200μl
culture medium and incubated at 37˚C in 5% CO2. After 24 hours, when the monolayer
formed, the medium was replaced with a final volume of 200μl of new medium with tested
compounds or controls were added to the plates. Cells were treated with eight 2-fold serial
dilutions of each compound spanning concentrations from 50μM to 0.39μM in 1% DMSO
final. Controls are on the first and the last columns of the plates. On the first column, methyl
methanesulfonate (MMS) acts as a positive control and DMSO as a negative control. On the
last column there are four points of rotenone and doxorubicin with an initial concentration of
10mM and dilution ½. When compounds and controls were added, plates were incubated at
37˚C in 5% CO2 incubator for 72 hours. After this time, MTT solution was prepared at 5 mg/
ml in PBS 1X and then diluted at 0,5mg/ml in MEM without phenol red. The sample solution
in wells was flicked off and 100μl of MTT dye was added to each well. The plates were gently
shaken and incubated for 3 hours at 37˚C in 5% CO2 incubator. The supernatant was removed
and 100μl of DMSO 100% was added. The plates were gently shaken to solubilize the formed
formazan. The absorbance was measured using a multireader Victor™ at a wavelength of
570nm.
FOXO translocation assay
The U2foxRELOC system is a FOXO translocation assay that has been previously established
[13]. Briefly, cells were seeded at a density of 20,000 cells per well into black wall clear bottom
96-well microplates (greiner bio-one; Frickenhausen, Germany). After 12 h of incubation at
37˚C with 5% CO2, 2μl of each test compound (1mM stock) was transferred to the assay plates.
Cells were incubated with the different compounds for 30 minutes. Cells were fixed with 100%
methanol, and the nucleus stained with DAPI (Invitrogen). Cells were washed twice with 1x
phosphate-buffered saline (PBS) and stored at 4˚C before analysis. All liquid handling for com-
pound treatment, washing, fixing, and staining steps were performed by a robotic workstation
(cell culturing robot, Select T, TAP Biosystems). All experiments were performed in triplicate.
For dose response experiments cells were cultured as described above and exposed for one
hour to equal volumes of test compounds (2μl). IC50 values were calculated as being the inhib-
itor concentration that increases nuclear accumulation of the reporter protein by 50% using
Genedata Screener software (Genedata AG, Switzerland). Cells were treated with eight 2-fold
serial dilutions of each compound spanning concentrations from 50μM to 0.39μM in 1%
DMSO final.
Nuclear export assay
The U2nesRELOC assay, a previously established nuclear export assay[14] is based on the
reporter construct pRevMAPKKnesGFP. pRevMAPKKnesGFP carries the NES from MAPK
kinase (MAPKK) (or MEK) cloned between the BamHI and AgeI sites of pRev(1.4)-GFP,
sandwiched between the Rev and the green fluorescent protein (GFP) coding sequences.
Image acquisition and processing
The BD Pathway 855 High Content Bioimager (BD Biosciences; San Jose, CA) was used for
automated image acquisition. Acquired images were processed using AttoVision software (BD
Biosciences; San Jose, CA). The Bioimager was equipped with a 488/10 nm enhanced GFP
(EGFP) excitation filter, a 380/10 nm DAPI excitation filter, a 515LP nm EGFP emission filter,
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Discovery of a Drug-Like FOXO Activator Molecule
and a 435LP nm DAPI emission filter. Images were acquired in the DAPI and GFP channels of
each well using a 10 X dry objective. The plates were exposed 0.066 ms (Gain 0) to acquire
DAPI images and 0.85 ms (Gain 30) for GFP images. Cells were stained with DAPI to facilitate
microscope autofocus and to aid in the image segmentation. An image algorithm based on a
local threshold was applied to allow for the cell nucleus segmentation. Our segmentation strat-
egy assumes that the cell’s cytoplasm surrounds the nucleus. Consequently, cytoplasmic fluo-
rescence intensity is calculated from all the pixels within a circumferential ring surrounding
the nuclear ring mask. The width of the ring was defined to be small enough to avoid ambigui-
ties due to irregular cell shape. Based on the definition of cell compartments, the nuclear and
cytoplasmic levels of GFP fluorescence were quantified.
Data analysis
The BD Pathway Bioimager generates standard text files that were imported into the BD
Image Data Explorer data analysis software. The nuclear/cytoplasmic fluorescence intensity
ratios were determined by dividing the fluorescence intensity of the nucleus by the cytoplasmic
fluorescence intensity. A threshold ratio greater than 1.8 was employed to define nuclear
accumulation of fluorescent signal for each cell. Based on this procedure, we calculated the
percentage of cells per well displaying nuclear translocation or inhibition of nuclear export.
Compounds that induced a nuclear accumulation of the fluorescent signal greater than 60% of
that obtained from wells treated with 4 nM LMB (LC Laboratories, Woburn, MA, USA) were
considered to be hits. We also monitored nuclear shrinkage known to be associated with apo-
ptosis by analyzing the number of “nuclear vertices”. The term “nuclear vertices” refers to the
number of pixels that lie on the nuclear boundary. In order to estimate the quality of the HCS
assay, the Z’ factor was calculated by the equation: Z‘= 1 –[(3 × std. dev. of positive controls) +
(3 × std. dev. of negative controls) / (mean of positive controls)—(mean of negative controls)]
as previously described by Zhang and colleagues[20].
Immunofluorescence analysis by confocal microscopy
U2OS cells were cultured as described above and exposed for 30 min at 1.5μM LOM612 (com-
pound 1a). The cells were fixed with 100% methanol (5 min) and then permeablilized and
blocked for 1hour with 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBSTween.
1
The cells were then stained with FOXO3a antibody (Cell Signaling Technology #2497S),
1
FOXO1 antibody (Cell Signaling Technology #2880) or NFKB2 p100/p52 Antibody (Cell
1
1
Signaling Technology #4882) overnight at 4˚C followed by DyLight 594 donkey anti-rabbit
Abcam ab96921) secondary antibody for 1h. DAPI was used to counterstain the cell nuclei at
(
a concentration of 1.43 μM. Samples were imaged at 22˚C using 63× water immersion lenses
on a Leica SPE confocal imaging system. Images were obtained using Leica software.
qPCR analysis
mRNA from U2OS cell line was isolated using RNAeasy (QIAGEN) kit and reverse tran-
scribed using NZY First-strand cDNA Synthesis kit (NZYtech, PT). qPCR was done in techni-
cal triplicate for each sample. qPCR reaction was carried out using Lumino Ct SYBR Green
qPCR ReadyMix (Sigma). All qPCR results are representative of two separate experiments.
Data analysis was carried out using the ΔCt method. Comparisons were made using the
unpaired Student’s t test. Values represent the mean ± standard error of the mean (SEM) and
are represented as error bars. Statistical significance as indicated.
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Discovery of a Drug-Like FOXO Activator Molecule
Results
Identification of specific FOXO relocator compounds
In order to identify specific FOXO relocator compounds we designed a sophisticated screening
strategy (Fig 1). As the primary filter, we used a cellular imaging assay that follows the intracel-
lular location of FOXO proteins which we have developed previously [13] and used with suc-
cess to discover anti-cancer drug candidates, including PI3K inhibitors [21–25]. The rapid
kinetics of the assay allowed us to reduce the incubation time and minimize possible toxic
effects that might interfere with the analysis. Furthermore, this image-based high-throughput
strategy provides a filter for adequate solubility, permeability, and stability in a cellular context
and enables compounds that produce artifacts or cytotoxicity to be identified on a single cell
basis [19,26]. The U2OS cell line that stably express a green fluorescent protein (GFP)-tagged
FOXO3a were seeded in 96-well assay plates and treated with different compounds at a final
concentration of 10μM for 30 min. A total of 544 compounds were screened and their ability
to induce GFP-FOXO3a reporter protein nuclear translocation was assessed. The nuclear
accumulation of GFP induced by Leptomycin B (LMB), an inhibitor of the CMR-1-dependent
nuclear export that covalently binds to a single cysteine residue of the CRM1 protein, was used
as a reference and defined as 100% activity. 0.5% DMSO was used as a vehicle control. Primary
hits were defined as those compounds that have an activity greater than 60%. Several com-
pounds matched the criteria and were cherry-picked to confirm the activity and reproducibil-
ity. Among the confirmed hit compounds we identified compound 1a (LOM612), a newly
synthesized isothiazolonaphthoquinone. The isothiazolequinones are an extremely rare class
of compounds. To the best of our knowledge, only two natural products, pronqodine A [27]
and aulosirazole [28] and a few synthetic compounds [29,30] have been reported in the litera-
ture so far. Therefore, this skeleton can be considered as a very attractive target for biological
evaluation, offering the possibility of preparing products with several points of diversity for
decoration.
Compound 1a, together with its analogues 1b-c, were prepared according to the two-step
procedure reported in Fig 2. The 1,3,4-oxathiazol-2-ones 3a and 3b [31] were synthesized in
good yield by reaction of N,N-dimethylurea or N,N-dibenzylurea urea and chlorocarbonylsul-
phenyl chloride respectively. Then, the 1,3-dipolar cycloaddition between the nitrile sulfide
generated in situ by termal decarboxylation of 3a and 3b [32] and the naphthoquinone gave
isothiazolonaphthoquinones 1a and 4. The intermediate 4 was then treated with cerium
ammonium nitrate (CAN) in acetonitrile–water [33] at room temperature and compound 1b
was obtained in high yield. Following the same synthetic strategy isothiazolonaphthoquinones
1
c was obtained starting from 1,3,4-oxathiazol-2-ones 3c.
Interestingly, in our experiments, compound 1b (LOM621) which differs from compound
a LOM612 in the amino group by the replacement of two methyl groups by two hydrogens
1
(Fig 2) did not induce nuclear accumulation of fluorescent FOXO reporter protein at a 10μM
concentration (Fig 3A). Similarly, compound 1c, carrying a phenyl ring in place of the amino
group linked to the isothiazole scaffold also failed to produce nuclear translocation of the
reporter protein (data not shown) and was not further analyzed. These results suggest that the
mode of action of LOM612 depends on stringent structural requirements.
Dose–response analysis of FOXO translocation
We next sought to determine the half maximal effective concentration (EC ) value of
50
LOM612 compound. The FOXO reporter cells were cultured as described previously and
treated with twelve different concentrations of LOM612, LOM621 and LMB for 30 min
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Discovery of a Drug-Like FOXO Activator Molecule
Fig 1. Screening strategy to identify specific and potent FOXO translocator compounds. As a primary filter a FOXO
translocation assay has been used, followed by dose response analysis of the confirmed hit compounds. The effect of the hit
compounds on endogenous FOXO proteins has been assessed by immunohistochemistry. Then inhibitors of the general
nuclear export machinery were excluded using a nuclear export assay. Finally, the effect on other transcription factors has been
determined by immunohistochemistry using a NFKB2 specific antibody.
doi:10.1371/journal.pone.0167491.g001
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Discovery of a Drug-Like FOXO Activator Molecule
Fig 2. Synthesis of compounds 1a-c (LOM612/621/604). (a) chlorocarbonylsulphenyl chloride, N, N-dimethylurea or N,N-dibenzylurea,
acetonitrile, 2h RT for 3a: 84%, 3b: 62%; chlorocarbonylsulphenyl chloride, benzamide, toluene, 3h, reflux 3c: 70%; (b) 1,4-naftochinone,
3a, xilene, 80˚C, 3h, 23%; 1,4-naftochinone, 3b, xylene, 80˚C, 3h, 63%; (C) 1,4-naftochinone, 3c, xilene, 8h, reflux, 3h, 38%; d) CAN,
CH CN: H O 9:1, 1h, RT, 89%.
3
2
doi:10.1371/journal.pone.0167491.g002
maintaining the transferred volume constant. Fig 3B shows that LOM612 potently activates
nuclear translocation of FOXO with an EC50 value of 1.5μM. The EC50 value of the reference
compound LMB was 2.3nM (data not shown) in these experiments. In contrast, LOM621
failed to induce the accumulation of GFP-FOXO in the nucleus (Fig 3A and 3B). None of the
tested concentrations of LOM621 affected the nuclear localization of FOXO indicating that a
small difference in the chemical structure is able to abolish the effect of these quinone deriva-
tives on FOXO transcription factors. Taken together, these results show that LOM612 potently
induces FOXO translocation in a dose-dependent manner.
Analysis of the translocation of endogenous FOXO upon LOM612
exposure
As the subcellular localization of a GFP tagged FOXO reporter protein might not reflect the
nucleocytoplasmic trafficking of the endogenous protein, we performed immunohistochemi-
cal detection of FOXO3a protein in U2OS osteosarcoma cells using a specific antibody. We
analyzed if exposure to LOM612 affects also the subcellular localization of endogenous FOXO
proteins. As depicted in Fig 4A, LOM612 very efficiently induced translocation of endogenous
FOXO3a and FOXO1. As expected, exposure of U2OS cells to LOM621 didn’t show nuclear
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Discovery of a Drug-Like FOXO Activator Molecule
Fig 3. Primary screening identifies compounds capable of inducing FOXO translocation. (A) U2fox RELOC cells
were treated either with DMSO, 4nM LMB, 10μM of compound LOM 612 or compound LOM 621 for 30 min. representative
images are shown. (B) Dose-response relationship of the nuclear–cytoplasmic shuttling of FOXO following LOM 612
treatment. LOM612 induces nuclear translocation in a dose dependent manner. Represented is the percentage of cells with
more GFP fluorescence accumulation in nucleus than in cytoplasm. Results represent the mean of three independent
experiments.
doi:10.1371/journal.pone.0167491.g003
accumulation of FOXO3a and FOXO1 proteins (data not shown). These data suggests that
LOM612 can induce the nuclear translocation of FOXO factors in their physiological context.
In addition, we found that the expression of the FOXO target genes p27 and FasL were
increased upon exposure to LOM612 (S1 Fig).
Analysis of the nuclear export upon LOM612 exposure
The intracellular localization of FOXO factors is regulated by posttranslational modifications,
which can lead to their nuclear export via the nuclear export receptor CRM-1. CRM-1 is the
best studied nuclear export receptor responsible for the nuclear export of proteins that contain
a nuclear export signal (NES). Accordingly, compounds which interfere with CRM-1-mediated
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Discovery of a Drug-Like FOXO Activator Molecule
Fig 4. LOM612 specifically induces the nuclear translocation of endogenous FOXO proteins. (A) Compound LOM612 induces the nuclear
translocation of endogenous FOXO3a and FOXO1 protein detected by using a specific antibodies after 30 min of drug exposure. (B) LOM612 does not
inhibit the nuclear export. U2OS cells stably expressing nuclear export signal (NES) (Rev-NES-EGFP) reporter were treated with DMSO, LMB, LOM612
and LOM621 for 30 min. (C) LOM612 does not induce nuclear translocation of endogenous nuclear factor (NF)–κB2 protein. Representative images of the
compound-treated cells using a Leica SPE confocal imaging system. Cells were seeded automatically at appropriate density in 96-well black-wall clear-
bottom tissue culture plates and allowed to attach overnight. Cells were then treated with compounds for 30 min before paraformaldehyde (Rev-
NES-EGFP) or methanol (FOXO3a, NFKB2) fixation and DAPI staining.
doi:10.1371/journal.pone.0167491.g004
nuclear export induce the accumulation of NES-bearing proteins including FOXO factors. In
order to determine if LOM612 triggers nuclear localization of FOXO by inhibiting CRM1, we
used a high content screening approach based on mammalian cells stably expressing green
fluorescent protein (GFP)–labeled Rev protein, which contains a strong heterologous NES
[14]. We seeded the reporter cells in 96-well assay plates, incubated them over night at 37˚C
and treated with LOM612 at a final concentration of 10μM for 30 min. Upon treatment with
the nuclear export inhibitor LMB, the GFP-labeled reporter protein accumulated in the cell
nucleus (Fig 4B). In contrast, LOM621 failed to induce nuclear accumulation of fluorescence
(Fig 4B) indicating that the effect of LOM612 on subcellular FOXO localization is independent
of CRM-1.
PLOS ONE | DOI:10.1371/journal.pone.0167491 December 9, 2016
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Discovery of a Drug-Like FOXO Activator Molecule
Analysis of the NFKB2 translocation upon LOM612 exposure
The activity of many transcription factors such as FOXO, NFAT or NFkB is controlled via the
regulation of their subcellular localization. NF-κB proteins shuttle constitutively between the
cytoplasm and nucleus, and only accumulate in the nucleus and become activated upon cellu-
lar stimulation [6]. IL-1 or TNFα receptor activation induces the degradation of the NF-κB
repressor IκBα unmasking the nuclear localization signal (NLS) of NF-κB proteins and pro-
moting its nuclear translocation. To determine if LOM612 compound would lead to indis-
criminate accumulation of factors known to be regulated by subcellular localization, we
performed immunohistochemical detection of endogenous NFKB2 protein in U2OS cell line
using a specific NFKB2 p100/p52 antibody as described before [18]. We showed that LOM612
had no effect on the nuclear export of endogenous NFKB2 transcription factor in this cell line
(
Fig 4C). In addition, we exposed a cell line that stably expresses the unrelated fluorescent
reporter fusion protein nuclear factor of activated T-cells (NFAT-GFP) to 1.5μM LOM612 for
0 min. LOM612 failed to alter the subcellular localization of this protein (data not shown).
3
Taken together these results suggest that LOM612 acts on a molecular target, which specifically
regulates the subcellular localization of FOXO proteins.
Viability of human cell lines in the presence of LOM612
FOXO proteins can orchestrate several transcriptional programs including the onset of apo-
ptosis by enhancing transcription of genes encoding the apoptotic regulators and the induc-
tion of cell-cycle arrest by activation the transcription of cyclin-dependent kinase inhibitors
[
34]. In order to analyze the effect of LOM612 on the viability of human cancer cell lines we
performed dose-response analysis after 72 hours of drug exposure. Breast (MCF7), lung cancer
A2058) and glioblastoma cell lines (SHSY5Y) were seeded in 96 multiwell plates and incu-
(
bated overnight. Cells were treated with twenty different concentrations of LOM612 and
LOM621 for 72 hours. LOM612 inhibited the viability of the tested cell lines with IC50 values
in the high nanomolar or low micromolar range (Fig 5). Treatment of LOM621 also resulted
in reduced viability, but was less potent than LOM612. Interestingly, the major difference
between the two quinone derivatives was observed after the treatment of MCF7 cells which are
known to contain activating mutations within PI3K. These data suggests that LOM612 might
selectively counteract FOXO inhibition in cells with increased PI3K/AKT signaling. In order
to compare the sensitivity of cancer and non-cancer cells that share a similar biological context
to LOM612 treatment, we used well established cellular systems to test hepatcytotoxicity. We
exposed HepG2, a human liver cancer cell line commonly used as an in vitro model system for
the study of polarized human hepatocytes, and THLE2 cell line derived from primary normal
liver epithelial cells and transformed by infection with SV40 large T antigen to several different
concentrations of LOM612. Treatment of HepG2 cells with LOM612 resulted in an IC50 value
of 0.64μM. Interestingly, non-cancer THLE2 cells were five times less sensitive to LOM612
with an IC50 value of 2.76μM, suggesting that LOM612 might provide a therapeutic window
for the treatment of certain human cancers.
Discussion
Compounds capable of reactivating FOXO based on its tumor suppressor properties are con-
sidered a very attractive anti-cancer therapy [6,12,35]. A second area of interest for FOXO acti-
vating compounds might be their use to slow aging and to prevent or treat age-related diseases
[36,37]. In the present work, we report the discovery of a new compound that specifically
induces the nuclear translocation of FOXO. In an attempt to prepare derivatives of Aulosira-
zole, we identified and synthesized LOM612 –a bioactive compound. Aulosirazole was isolated
PLOS ONE | DOI:10.1371/journal.pone.0167491 December 9, 2016
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Discovery of a Drug-Like FOXO Activator Molecule
Fig 5. LOM612 compromises the viability of human cancer cell lines. (A) Breast cancer cell line MCF7,4
melanoma cell line A2058 and the neuroblastoma cell line SH-SY5 were seeded at a concentration of 1× 10
cells/well in 200 μl and treated with compounds LOM612 and LOM621 for 72 hours with eight 2-fold serial
dilutions of each compound spanning concentrations from 50μM to 0.39μM. Data is shown as mean ± SEM of
three independent experiments. ****P<0.0001 by two-way ANOVA (ns, not significant). (B) Human liver
cancer cell lines HepG2 and THLE-2 (cell line derived from primary normal liver epithelial cells) were seeded
4
at a concentration of 1× 10 cells/well in 200 μl and treated with 20 different concentrations of LOM612 from
50μM to 95pM. Data is shown as mean ± SEM of three independent experiments. ****P<0.0001 by two-way
ANOVA.
doi:10.1371/journal.pone.0167491.g005
PLOS ONE | DOI:10.1371/journal.pone.0167491 December 9, 2016
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Discovery of a Drug-Like FOXO Activator Molecule
from the blue-green alga Aulosira fertilissima and is characterized by an unusual isothiazolo-
naphthoquinone structure [28]. The authors of this work performed corbett assay to show that
Aulosirazole exhibits selective toxicity towards solid tumours. The biological activity and
unique chemical structure of aulosirazole prompted interest in the chemical synthesis of deriv-
atives which aims to identify antineoplastic agents [38]. We identified LOM612 in an image-
based high content screening assay, which monitored the subcellular localization of FOXO
proteins. Similar approaches have been used successfully to identify drug candidates for the
treatment of human tumours in which FOXO tumour suppressors are inactivated [12,13,39].
LOM612 induced FOXO nuclear translocation with an EC50 value in the high nanomolar
range indicating that the level of potency is an excellent starting point for a lead optimization
process. The observation that a minor structural change, namely the replacement of a N(Me)2
moiety with a NH (1b) or a phenyl group (1c) at position 3 renders the compound inactive in
2
the FOXO translocation assay, suggests that the dimethylamino group plays an essential role
in the interaction with the molecular target. In order to further explore the structural require-
ments, we are currently preparing a collection of chemical derivatives to perform structure
activity relationship analysis. The functional readout for these experiments is based on FOXO
subcellular translocation. However, because FOXO proteins are transcription factors that are
considered poorly druggable targets [40] it is unlikely that FOXO proteins are the direct
molecular targets of LOM612. Modern drug development is increasingly focused on targeted
strategies and as a consequence the identification of the molecular target of a bioactive com-
pound is of key importance [41]. The mode of action of Aulosirazole and its derivatives is
mostly unknown. Interestingly, the immunoregulatory enzyme indoleamine-2,3-dioxygenase
(
IDO) has been identified as a potential target of aulosirazole. Furthermore, Pronqodine A, a
structurally related natural product isolated from the soil bacterium Streptomyces sp. MK832-
5F2 has been shown to be an inhibitor of prostaglandin release [27]. Importantly, several qui-
9
nones have been identified as selective inhibitors of Cdc25 phosphatases [42–46] which are
known to dephosphorylate and in turn activate cyclin-dependent kinases (Cdks) [47]. Intrigu-
ingly, FOXO1 has been reported to be a substrate of Cdk2 [48]. In line with these findings,
Feng et al. showed that Cdc25 regulates the FOXO target gene Matrix Metalloprotein 1
(MMP1) via Cdk2 and FOXO1 [49]. However, it remains to be determined if Cdc25/Cdk2 is
also capable of regulating FOXO3a. Mode of action analysis for LOM612 should include the
determination of its inhibitory activity in an in vitro Cdc25 phosphatase assay. The subcellular
localization of FOXO proteins is regulated by a variety of upstream proteins, most prominently
serine/threonine kinases. Therefore among the primary target candidates of LOM612 are
kinases that are known to phosphorylate FOXOs at sites involved in the regulation of its intra-
cellular localization either directly, such as AKT1–3, SGK 1–3, MST-1, CK1, Cdk2, DKYR1a
and IKKβ or indirectly by activating AKT, such as PI3K and PDK1 [5,50]. Some of these
enzymes also regulate nuclear translocation of NFKB2 and hence their inhibition would also
result in the accumulation of this transcription factor [6]. Accordingly, the analysis of
LOM612 or an optimized derivative should include a screening against a selected panel of pro-
tein kinases. It is important to note that, the short drug exposure time used in the primary
translocation assay precludes any indirect mechanisms that rely on gene transcription or pro-
tein synthesis. Importantly, a more detailed study of the toxicity of LOM612 using a broader
panel of cancer cell lines would shed light on the molecular profiles that confer cellular sensi-
tivity. The FOXO-inactive analogue LOM621 is an excellent tool to identify cancer cell killing
specific to FOXO activation. It is evident that these quinone derivatives are cytotoxic indepen-
dent of FOXO activation in some cell lines. It is important to note that we observed the stron-
gest FOXO-dependent effect of LOM612 in the MCF7 breast cancer cell line, which contains
an oncogenic missense mutation in PIK3CA. This genetic alteration increases the catalytic
PLOS ONE | DOI:10.1371/journal.pone.0167491 December 9, 2016
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Discovery of a Drug-Like FOXO Activator Molecule
activity of PI3K, activating the downstream oncokinase AKT that in turn inactivates FOXO
supporting the hypothesis that LOM612 mediates part of its cytotoxic effect through FOXO.
An important caveat of the study here presented is that this FOXO-specific toxicity of
LOM612 is based on limited experimental evidence. In order to translate LOM612 into a
therapeutically useful drug candidate, a detailed characterization of its mode of action and an
analysis of the molecular profile that sensitizes cells to its cytotoxicity will be of foremost
importance. LOM612 or optimized derivatives could be developed to counteract FOXO inacti-
vation in human tumors with constitutively active PI3K/AKT signaling, or to activate cellular
defense mechanisms downstream of FOXO factors to slow age-related processes [36,37].
Supporting Information
S1 Fig. LOM612 induces the expression of FOXO target genes. U2OS cells were treated
either with DMSO or 5μM of compound LOM612 for 6 hours. qRT-PCR from U2OS cells of
FOXO target genes, p27 (A) and FasL (B) whose LOM612-induced expression is induced.
Ã
Expression relative to GAPDH. n = 2, p < 0.05.
(PPTX)
Acknowledgments
This work was supported by Funda c¸ ão para a Ciência e a Tecnologia (FCT) Research Center
Grant UID/BIM/04773/2013 CBMR1334. B. I. Ferreira is the recipient of a FCT 2014 research
grant SFRH/BPD/100434/2014R. S. Machado is the recipient of a ProRegem grant PD/BD/
114258/2016. We acknowledge COST Actions CM1106 (Chemical Approaches to Targeting
Drug Resistance in Cancer Stem Cells) for facilitating the collaboration between chemists and
biologists. We thank D. Pasarella for coordinating the COST Action CM1106. We are indebted
to N. Martinet for organizing and handling the COST Action CM1106 compound collection.
Author Contributions
Conceptualization: WL.
Formal analysis: BC FC.
Funding acquisition: WL FV SD.
Investigation: WL BC FC LM SD.
Methodology: BC FC LM BF NP LR SM.
Software: BC NP LR.
Supervision: WL FV SD.
Validation: BC NP SD.
Visualization: BC BF SM.
Writing – original draft: WL BC SD.
Writing – review & editing: WL BC BF SM.
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