Naturally occurring FANCF–Hes1 complex inhibitors from Wrightia religiosa

Abstract

The isolation and evaluation of inhibitors of Fanconi F protein (FANCF) – hairy and enhancer of split 1 (Hes1) complex are described. A high-throughput screening assay for small-molecule inhibitors of the FANCF–Hes1 complex was realized. Successful complex formation between fluorophore (Cy3)-labeled human FANCF and immobilized rat or human HES1 on a microplate was established. Screening of our plant extract library using this system resulted in the isolation of eight natural products, including two new flavonoid glycosides (3 and 4), from Wrightia religiosa. Of these compounds, 3, 5, and 7 showed potent inhibition of the FANCF–Hes1 complex formation. Compound 7 disrupted the FANCF–Hes1 complex more efficiently than the Hes1 dimer.

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CONCISE ARTICLE
Cite this: DOI: 10.1039/
c4md00495g
Received 31st October 2014,
Accepted 21st November 2014
DOI: 10.1039/c4md00495g
www.rsc.org/medchemcomm
Naturally occurring FANCF–Hes1 complex
inhibitors from Wrightia religiosa†
Midori A. Arai,*
a
Kenji Uemura,
a
Nozomi Hamahiga,
a
Naoki Ishikawa,
a
Takashi Koyano,
b
Thaworn Kowithayakorn,
c
Tagrid Kaddar,
d
Madeleine Carreau
d
and Masami Ishibashi*
a
The isolation and evaluation of inhibitors of Fanconi F protein (FANCF) –hairy and enhancer of split 1
(Hes1) complex are described. A high-throughput screening assay for small-molecule inhibitors of the
FANCF–Hes1 complex was realized. Successful complex formation between fluorophore (Cy3)-labeled
human FANCF and immobilized rat or human HES1 on a microplate was established. Screening of our plant
extract library using this system resulted in the isolation of eight natural products, including two new
flavonoid glycosides (3and 4), from Wrightia religiosa. Of these compounds, 3,5,and7showed potent
inhibition of the FANCF–Hes1 complex formation. Compound 7disrupted the FANCF–Hes1 complex more
efficiently than the Hes1 dimer.
Introduction
Fanconi anemia (FA) is an inherited anemia associated with
bone marrow failure, progressive decline in hematopoietic
stem cells, developmental defects, and a predisposition to
cancer.
1
A common cellular phenotype is the hypersensitivity
to DNA cross-linking agents, such as mitomycin C
2
and
diepoxybutane,
3
which suggests the presence of defects in
DNA repair mechanisms. The FA core complex, which con-
sists of eight proteins (FANCA, FANCB, FANCC, FANCE,
FANCF, FANCG, FANCL, FANCM), is a key player in the DNA
cross-link repair pathway and is referred to as the FA pathway.
Mutations in any of the FA proteins cause the manifestation
of clinical features. Although several proteins that interact
with the FA core complex have been identified, such as
Fanconi anemia-association protein 24 (FAAP24),
4
FAAP100,
5
FANCM-associated histone fold protein 1 (MHF1),
6
MHF2,
6
hairy enhancer of split 1 (Hes1),
7
and C-terminal binding
protein 1 (CtBP1),
8
the details of this pathway remain
unknown. Therefore small molecules that inhibit protein–
protein interactions related to the FA pathway will not only
further our understanding of this pathway, but may also
provide novel therapeutic candidates to treat this complicated
disease.
Hes1 is a repressor type basic helix–loop–helix (bHLH)
factor that controls the fate of stem cells.
9
It was revealed
that Hes1 interacts with FANCA, FANCF, FANCG, and FANCL,
which mediates the transcriptional regulation of Hes1-
responsive genes.
7
Inhibitors of the FA protein–Hes1 complex
formation would enable the understanding and identification
of unknown FA/Hes1 cross-talk. In the present study, we
describe a rapid in vitro high-throughput screen (HTS) for
identifying inhibitors of FANCF complex formation with
Hes1. The first FANCF–Hes1 complex inhibitors isolated from
natural sources are presented here. The naturally occurring
inhibitors were isolated from Wrightia religiosa using the
screening method described herein.
Results and discussion
Glutathione-S-transferase fused human FANCF (GST–hFANCF;
full length) was expressed in Escherichia coli, then purified
with glutathione sepharose 4B. A rat Hes1 protein (3–278 aa),
with an amino acid sequence differing from human HES1 by
only one residue outside the binding region with FANCF, was
prepared as previously described.
10
To prevent GST–GST
interactions, which would result in false positives, GST-free
Hes1 protein was prepared by GST cleavage with PreScission
protease. The HTS plate assay was designed as shown in
Fig. 1. The assay was designed to use fluorophore-conjugated
FANCF to detect the FANCF–Hes1 complex using fluores-
cence intensity. Hes1 protein was immobilized on the bottom
of 96 well plates (Nunc Immobilizer™Amino Plate, Thermo).
Med. Chem. Commun.This journal is © The Royal Society of Chemistry 2014
a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana,
Chuo-ku, Chiba 260-8675, Japan. E-mail: midori_arai@chiba-u.jp,
mish@chiba-u.jp
b
Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan
c
Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand
d
Department of Pediatrics Université Laval, Cité Universitaire, Québec, Canada,
G1K 7P4, and the Centre de Recherche du CHU de Québec-CHUL, Québec, QC,
Canada G1V 4G2
†Electronic supplementary information (ESI) available: Detailed procedures for
biological experiments, isolation, activity of isolated compounds (Fig. S1 and
S2), and calculation results (Fig. S3 and S4). See DOI: 10.1039/c4md00495g
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Hes1 protein was added to the plates and incubated over
night at 4 °C. After washing with buffer, the activated spacers
remaining on the bottom of the wells were treated with
ethanolamine for 2 h at room temperature. Hes1 immobiliza-
tion was confirmed by measuring the Cy3 fluorescence inten-
sity after detection of Hes1 antibody and Cy3-conjugated
secondary antibody. It was found that 100 μl (20 μgml
−1
)of
Hes1 resulted in a sufficient amount immobilized on the
plate (data not shown). To form the FANCF–Hes1 complex,
Cy3-conjugated GST–FANCF was added to Hes1-immobilized
wells. The wells were scanned for their fluorescence intensity
after 1 h of incubation at room temperature, followed by
removal of unbound Cy3–FANCF by washing with buffer and
drying under reduced pressure. The FANCF–Hes1 complex
was detected successfully, as shown in Fig. 2 (lanes 3 and 4).
The screening assay exhibited only low levels of non-specific
binding by Cy3–GST (Fig. 2, lanes 7 and 8). As the com-
pounds were prepared in solutions containing DMSO, the
effect of the solvent on binding was investigated. Fluores-
cence intensity of the FANCF–Hes1 complex was slightly
reduced by addition of DMSO (2%) (Fig. 2, lane 4).
Our natural source extract library was then screened
using the HTS assay. Of the extracts, the MeOH extract of
Wrightia religiosa was found to contain naturally occurring
inhibitors of the FANCF–Hes1 complex. The methanol extract
of W. religiosa (28.5 g) was partitioned successively with
EtOAc, nBuOH, and water. The active nBuOH layer (8.0 g)
was subjected to ODS column chromatography and reversed-
phase HPLC. Activity-guided separation yielded eight
compounds (1–8), including two new compounds (3,4) (Fig. 3).
The isolated compounds were identified as kaempferol 3-O-α-L-
rhamnopyranosyl-IJ1→6)-β-D-glucopyranoside (1),
11
kaempferol
3-O-α-L-rhamnopyranosyl-IJ1→6)-β-D-galactopyranoside (2),
12
quercetin 4′-O-α-L-rhamnopyranosyl-3-O-α-L-rhamnopyranosyl-
IJ1→6)-β-D-glucopyranoside (5),
13
rutin (6),
14
quercetin 3-O-α-L-
rhamnopyranosyl-IJ1→6)-β-D-galactopyranoside (7),
15
wrightiadi-
one (8);
16
based on comparisons of their spectral data with
spectra in the literature. The new natural compound 3was
isolated as a yellow solid with the molecular formula
C
33
H
40
O
19
, as determined by HRAPCIMS IJm/z763.2040, calcd
for C
33
H
40
O
19
Na, [M + Na]
+
,Δ−2.2 mmu).
1
Hand
13
CNMR
analyses indicated the presence of a kaempferol structure and
three sugars (Table 1). HMBC correlations suggested the pres-
ence of glycosidic linkages between C-3 and glucose, C-4′and
Fig. 2 Human FANCF–rat Hes1 complex formation in the microplate
assay. All wells were treated with ethanolamine after Hes1/blank
immobilization, and were then incubated with Cy3-proteins followed
by washing with buffer. Excitation was 544 nm and emission was
590 nm. Error bars represent SD (n= 3). Background (each well) was
subtracted. 1, Cy3–GST–hFANCF without immobilized rHes1 (control);
2, Cy3–GST–hFANCF without immobilized rHes1 (control; DMSO
addition); almost no binding of Cy3 proteins was observed in the
wells (1and 2). 3, Cy3–GST–hFANCF with immobilized rHes1; 4,
Cy3–GST–hFANCF with immobilized rHes1 (DMSO addition); rHes1/
Cy3–GST–hFANCF complex was detected (3and 4); 5, Cy3–GST
without immobilized rHes1 (control); 6, Cy3–GST without immobilized
rHes1 (control; DMSO addition); almost no binding of Cy3 proteins
to the wells was observed (5and 6); 7, Cy3–GST with immobilized
rHes1; 8, Cy3–GST with immobilized rHes1 (DMSO addition), non-
specific interactions were minor (7and 8). Immobilization; rHes1
(5 μgml
−1
), Cy3-proteins; Cy3–GST–hFANCF (20 μgml
−1
, 0.3 μM),
Cy3–GST (7.4 μgml
−1
, 0.3 μM).
Fig. 3 Isolated natural products.
Fig. 1 (A) Schematic representation of the interaction of the FA
core complex with Hes1. (B) The HTS assay constructed to identify
inhibitors of the FANCF–Hes1 complex. In the presence of an inhibitor,
fluorescence originating from FANCF–Hes1 complex is decreased.
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rhamnose, and a 1,6-glycosidic bond between rhamnose
and glucose (Fig. 4). Compound 3was designated kaempferol
4′-O-α-rhamnopyranosyl-3-O-α-rhamnopyranosyl-IJ1→6)-β-
glucopyranoside. The new natural compound 4was isolated
as a yellow solid with the molecular formula C
33
H
40
O
20
,as
determined by HRAPCIMS IJm/z779.2004, calcd for
C
33
H
40
O
20
Na, [M + Na]
+
,Δ−0.7 mmu). Analysis of
1
Hand
13
C NMR spectra revealed the presence of a kaempferol moiety
as an aglycon and three sugars in compound 4(Table 1).
HMBC correlations suggested the presence of glycosidic link-
ages between C-3 and galactose, C-4′and rhamnose, and a 1,6-
glycosidic bond between rhamnose and galactose (Fig. 4).
Compound 4was designated kaempferol 4′-O-α-rhamnopyranosyl-
3-O-α-rhamnopyranosyl-IJ1→6)-β-galactopyranoside.
The inhibitory activities of the isolated compounds on the
human FANCF–rat Hes1 interaction were examined (see the
ESI,†Fig. S1). Of the compounds tested, 3,5, and 7produced
Table 1
1
Hand
13
C NMR data of compounds 3(A) and 4(B) (δin ppm, Jin Hz)
A.
Position
In DMSO-d
6
1
H-NMR
13
C-NMR
1
H-NMR
13
C-NMR
2 156.4 Glucose
3 133.9 1″5.27 (d, 7.2) 101.7
4 177.4 2″74.2
5 161.1 3″76.2
6 6.16 (d, 1.8) 99.2 4″70.5
7 165.4 5″75.8
8 6.38 (d, 1.8) 94.2 6″67.0
9 156.9 Rhamnose
10 103.9 1‴4.36 (s) 100.9
1′123.9 2‴70.3
2′8.11 (d, 9.2) 130.9 3‴70.0
3′7.11 (d, 9.2) 116.1 4‴71.7
4′158.0 5‴68.3
5′7.11 (d, 9.2) 116.1 6‴1.10 (d, 6.4) 18.1
6′8.11 (d, 9.2) 130.9 Rhamnose (4′)
1″″ 5.50 (s) 98.2
2″″ 70.1
3″″ 69.9
4″″ 68.4
5″″ 71.8
6″″ 1.04 (d, 6.4) 17.9
B.
Position
In DMSO-d
6
1
H-NMR
13
C-NMR
1
H-NMR
13
C-NMR
2 156.8 Glucose
3 134.0 1″5.27 (d, 7.2) 102.4
4 177.42 2″71.1
5 161.0 3″73.0
6 6.16 (d, 1.8) 99.3 4″68.1
7 165.6 5″73.7
8 6.38 (d, 1.8) 94.2 6″65.6
9 156.0 Rhamnose
10 103.7 1‴4.36 (s) 100.2
1′123.8 2‴70.6
2′8.11 (d, 9.2) 131.0 3‴70.4
3′7.11 (d, 9.2) 115.9 4‴71.9
4′158.0 5‴68.4
5′7.11 (d, 9.2) 115.9 6‴1.10 (d, 6.4) 18.1
6′8.11 (d, 9.2) 131.0 Rhamnose (4′)
1″″ 5.47 (s) 98.2
2″″ 70.3
3″″ 70.1
4″″ 71.8
5″″ 69.9
6″″ 1.04 (d, 6.4) 18.0
Fig. 4 Key HMBC and COSY correlations for compounds 3and 4.
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moderate inhibition at 50 μM. To precisely elucidate the
inhibitory activity of the isolated compounds, we subse-
quently constructed a human HES1 interaction plate assay.
Although the difference between human HES1 and rat Hes1
is only a single residue (human HES1 has 172S, which is
absent in rat Hes1), examination of human protein interac-
tions is more clinically relevant. Therefore, a human HES1
pGEX-6P-1 construct (1–280 aa, full length) was prepared and
purified using the above method. After GST removal, human
HES1 protein was immobilized on the plate. Immobilized
HES1 was detected with anti-HES1 antibody and it was
found that a concentration of 5 μgml
−1
human HES1 was
sufficient for the assay. Addition of Cy3–human FANCF to
wells containing immobilized HES1 resulted in the successful
detection of the FANCF–HES1 interaction, as shown in Fig. 5
(lane 2). A human HES1–human HES1 dimer complex was
also clearly detected (Fig. 5, lane 4). Non-specific interactions
were assessed by addition of GST protein (Fig. 5, lane 6).
Using the HTS assays described here, the inhibition of
human FANCF–human HES1 interactions by the isolated
compounds (3,5and 7) was examined (Fig. 6). The activity of
8was shown in Fig. S2 (ESI†). Wnt signal is one of the
important signals that control stem cell fate. The selectivity
in these stemness control signals would be useful. Therefore,
to check non-specific inhibition, TCF–β-catenin complex,
which is a key complex in Wnt signalling, was examined.
The TCF–β-catenin plate assay
17
was performed using the
compounds 3,5, and 7, which showed moderate inhibition of
human FANCF–rat Hes1 interaction. All compounds produced
dose-dependent inhibition of FANCF–human HES1 interaction.
Interestingly, inhibition by compounds 5and 7was improved
to 40% at 10 μM, greater than what was observed with rat
Hes1. The IC
50
values of 5and 7were 23.6 μM and 35.8 μM,
respectively. Although the inhibition levels were moderate, to
the best of our knowledge, these are the first reported inhibi-
tors of the interaction between FANCF and HES1.
To determine the specificity of the inhibitors on HES1
complexes, human HES1–human HES1 (HES1 dimer) interac-
tions were also assessed. Because the difference between the
Fig. 6 Protein interaction inhibition activity of compounds 3,5, and 7. FANCF–HES1 and HES1 dimer assay were performed on HES1
immobilized microplates. Cy3–GST–FANCF or Cy3–GST–HES1 was added to initiate complex formation. Complex formation was monitored by
determining fluorescence (Ex 544/Em 590). Immobilization; hHES1 (5 μgml
−1
), Cy3-proteins; Cy3–GST–hFANCF (20 μgml
−1
, 0.3 μM), Cy3–GST–
hHES1 (7.4 μgml
−1
, 0.3 μM). The TCF–β-catenin assay was performed on hTCF4E
1–100
immobilized microplates, with GST–mβ-catenin
128–683
(armadillo repeat; same amino acids in human β-catenin) added to initiate complex formation. Chemiluminescence was used to determine
complex formation after the addition of HRP conjugated anti-GST. Error bars represent SD (n= 3). Immobilization; hTCF4E
1–100
(5 μgml
−1
),
Cy3-proteins; Cy3–GST–mβ-catenin
128–683
(5 μgml
−1
, 60 nM).
Fig. 5 Human FANCF–human HES1 complex formation in the
microplate assay. All wells were treated with ethanolamine after
hHES1/blank immobilization, and were then incubated with Cy3-
proteins followed by washing with buffer. Excitation was 544 nm and
emission was 590 nm. Error bars represent SD (n= 3). Background
(each well) was subtracted. 1, Cy3–GST–hFANCF without immobilized
hHES1 (control with DMSO); 2, Cy3–GST–FANCF with immobilized
hHES1; hHES1/Cy3–GST–hFANCF complex was detected; 3, Cy3–GST–
hHES1 without immobilized hHES1 (control with DMSO); 4, Cy3–GST–
hHES1 with immobilized hHES1; hHES1/Cy3–GST–hHES1 complex was
detected; 5, Cy3–GST without immobilized hHES1 (control with
DMSO); 6, Cy3–GST with immobilized hHES1, non-specific interactions
was minimal. Immobilization; hHes1 (5 μgml
−1
), Cy3-proteins; Cy3–
GST–hFANCF (20 μgml
−1
, 0.3 μM), Cy3–GST (7.4 μgml
−1
, 0.3 μM).
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structures of 3and 5is only a C-3′phenolic OH, this OH
appears to enhance inhibition of both FANCF–HES1 interac-
tions and HES1 dimerization. Our results show that com-
pound 7inhibited FANCF–HES1 complex more efficiently
than HES1 dimer. The structural differences between 5and 7
are a rhamnose at the 4′position of the aglycon and a
galactose at the 3 position. The stable structures and electro-
static potential energy of isolated compounds were calculated
(see ESI,†Fig. S3). Interestingly, the internal hydrogen bonds
between rhamnose (compound 5) and between rhamnose
and OH at the 4′position (compound 7) were observed to
make the folded structures. There is a difference in the size
of cavities which were made by hydrogen bonds (Fig. S4†).
Next, we investigated the effect of compound 5on colony
formation in hematopoietic stem/progenitor cells. Briefly,
bone marrow cells were collected from femurs of wild-type
and FA-deficient mice and stem/progenitors were selected
using the StemSep negative selection procedure, according to
the manufacturer's protocol (Stemcell Technologies). Two to
5×10
3
stem/progenitor cells per ml were seeded in com-
plete methylcellulose medium (Stemcell Technologies), with
or without compound 5(50 μM), and incubated at 37 °Cin
5% CO
2
. The total number of colonies were counted and
presented as colony forming cells (CFC). Results show that
wild-type stem/progenitor cells incubated with compound 5
exhibited a 40% reduction in colony forming ability, consis-
tent with disruption of the FA pathway. FancA-deficient cells
incubated with compound 5showed reduced CFC compared
to FancA cells incubated with DMSO, whereas no effect
was observed with FancC-deficient cells (Fig. 7). The degree
of reduction in WT and FancA-deficient cells was equivalent
in the absence and presence of 5. This result indicates that
CFC inhibition by 5is not dependent on FancA. Because inhi-
bition by 5was absent in FancC-deficient cells, compound 5
might also interact with FANCC. These results suggest that
compound 5may be useful in discriminating specific func-
tions of FA proteins and/or the role of HES1 dimers in hema-
topoietic function. Indeed, the lack of effect by compound 5
in FancC-deficient cells supports previous studies suggesting
roles of FANCC in distinct pathways.
18
Conclusions
To identify FANCF–HES1 complex inhibitors derived from
natural sources, we constructed a high-throughput plate
assay using recombinant FANCF and Hes1 proteins. This
assay led to the isolation of eight compounds, including two
new flavonoid glycosides (3and 4). Compounds 3,5, and 7
showed moderate inhibition of FANCF–HES1 interactions.
We also investigated the selectivity of the inhibition using
HES1–HES1 dimer and TCF–β-catenin plate assays. Com-
pound 5exhibited selective inhibition of HES1 complexes
(FANCF–HES1 and HES1 dimer), while compound 7pro-
duced selective inhibition of the FANCF–HES1 complex, and
to a lesser extent HES1–HES1, without affecting TCF–β-
catenin complex formation. To the best of our knowledge,
this is the first report of inhibitors of FANCF–HES1 interac-
tions. The search for more active inhibitors from natural
sources continues.
Acknowledgements
We are very grateful to Prof. R. Kageyama and Prof. T. Ohtsuka
for the kind provision of plasmids and discussions. This
study was supported by a Grants-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science (JSPS), a
Grant-in-Aid for Scientific Research on Innovative Areas
‘Chemical Biology of Natural Products’from The Ministry of
Education, Culture, Sports, Science and Technology, Japan
(MEXT), the Naito Foundation, the Tokyo Biochemical
Research Foundation and a Workshop on Chirality in Chiba
University (WCCU). This work was inspired by the interna-
tional and interdisciplinary environments of the Asian Core
Program (JSPS), “Asian Chemical Biology Initiative”. This work
was also supported in parts by grants from the Canadian
Institutes of Health Research (CIHR) in partnership with the
Canadian Blood Services (CBS) and a fellowship award from
CIHR in partnership with Fanconi Canada (the Canadian
Fanconi anemia research Fund to T. K.).
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