small-molecule modulation of Wnt signaling via modulating the axin

Transcription

article
published online: 28 july 2013 | doi: 10.1038/nchembio.1309
Small-molecule modulation of Wnt signaling via
modulating the Axin-LRP5/6 interaction
© 2013 Nature America, Inc. All rights reserved.
Sheng Wang1,5, Junlin Yin2,5, Duozhi Chen2, Fen Nie1, Xiaomin Song1, Cong Fei1, Haofei Miao1,
Changbin Jing3, Wenjing Ma3, Lei Wang2, Sichun Xie1, Chen Li4, Rong Zeng4, Weijun Pan3,
Xiaojiang Hao2* & Lin Li1*
The Wnt/b-catenin signaling pathway has a crucial role in embryonic development, stem cell maintenance and human disease.
By screening a synthetic chemical library of lycorine derivatives, we identified 4-ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]
phenanthridine (HLY78) as an activator of the Wnt/b-catenin signaling pathway, which acts in a Wnt ligand–dependent manner. HLY78 targets the DIX domain of Axin and potentiates the Axin–LRP6 association, thus promoting LRP6 phosphorylation
and Wnt signaling transduction. Moreover, we identified the critical residues on Axin for HLY78 binding and showed that HLY78
may weaken the autoinhibition of Axin. In addition, HLY78 acts synergistically with Wnt in the embryonic development of
zebrafish and increases the expression of the conserved hematopoietic stem cell (HSC) markers, runx1 and cmyb, in zebrafish
embryos. Collectively, our study not only provides new insights into the regulation of the Wnt/b-catenin signaling pathway by
a Wnt-specific small molecule but also will facilitate therapeutic applications, such as HSC expansion.
T
he evolutionarily conserved Wnt/β-catenin signaling pathway,
also called the canonical Wnt signaling pathway, has important
roles in embryonic development and human diseases1. During
embryogenesis, the Wnt signaling pathway regulates cell fate specification, proliferation, differentiation and survival. Inappropriate
regulation of the Wnt signaling pathway is often linked to the progression of many human diseases, such as cancer (the pathway is
inappropriately activated), Alzheimer’s disease, familial exudative
vitreoretinopathy and bone formation disorders (the pathway is
conversely attenuated)2.
The precise control of the free β-catenin level is critical for the
Wnt/β-catenin signaling pathway. In the absence of the Wnt ligands,
cytoplasmic β-catenin is constantly phosphorylated and degraded
by the β-catenin destruction complex, which is composed of the
scaffold protein Axin, the tumor suppressor adenomatous polyposis
coli gene product (APC), casein kinase 1α, glycogen synthase kinase
3 (GSK3) and the E3 ubiquitin ligase subunit β-Trcp1. When the Wnt
ligand binds its specific receptor complex containing the seven-pass
transmembrane Frizzled receptors and the low-density lipoprotein
receptor-related protein 5/6 (LRP5/6) co-receptors, the Wnt/βcatenin signaling pathway is activated. A crucial step in transducing the Wnt signal is the recruitment of the Axin–GSK3 complex to
the receptors, which subsequently promotes GSK3-induced LRP5/6
phosphorylation and inhibits β-catenin phosphorylation, thereby
stabilizing β-catenin3. The accumulated β-catenin thus enters
the nucleus and forms complexes with lymphoid enhancer-binding
factor 1 or T-cell factors to activate the expression of the Wnt
target genes.
Axin is one of the key components in the Wnt/β-catenin
signaling pathway. The efficient assembly of distinct Axin complexes ensures the effective transduction of the Wnt/β-catenin signaling pathway4. Axin is also a concentration-limiting factor in the
Wnt/β-catenin signaling pathway5,6. However, the specific mechanism by which Wnt signaling regulates the dissociation of the Axin
protein from the β-catenin destruction complex and recruits it to
form the Axin–LRP5/6 complex has not been well defined.
Aberrant activation of the Wnt signaling pathway has been indicated in a variety of human cancers; therefore, concerted efforts
have been made to identify Wnt inhibitors, whereas little work
has been performed to discover Wnt activators, especially the
‘Wnt-specific’ Wnt activators2. To better understand the regulatory mechanism of the Wnt/β-catenin signaling pathway and to
explore new Wnt-specific pharmacologic compounds, we performed a screen on diverse synthetic chemical libraries, including a
group of lycorine derivatives. Lycorine is the main phenanthridine
Amaryllidaceae alkaloid and has antitumor activity through cytostatic, rather than cytotoxic, effects on cells7. Therefore, we included
lycorine and its various derivatives in our search. Our extensive
screening led to the discovery of HLY78 (1)8, which is a lycorine
derivative that has a synergistic effect with Wnt ligands to activate
the Wnt/β-catenin signaling pathway by promoting Axin–LRP6
complex formation. Our study investigated the use of a smallmolecule tool to modulate the differential assemblies of Axin and,
therefore, modulate Wnt signaling. Further research in this field
may present opportunities for treatment of diseases related to attenuated Wnt/β-catenin signaling.
RESULTS
HLY78 activates the Wnt/b-catenin signaling pathway
To identify small molecules that affect Wnt/β-catenin signaling
transduction, we performed a high-throughput, cell-based screen
using a number of small-molecule compound libraries, including a
library of lycorine derivatives (approximately 200 synthetic chemical compounds). Our search led to the discovery of HLY78 as an
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai, China. 2State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming, China. 3Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong
University School of Medicine, Shanghai, China. 4Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 5These authors contributed equally to this work. *e-mail: [email protected] or
[email protected]
1
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1
article
Nature chemical biology doi: 10.1038/nchembio.1309
a
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TOPFlash RLC
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Anti-SP1
Figure 1 | HLY78 activates Wnt signaling upstream of b-catenin.
(a) Chemical structure of HLY78. (b) HLY78 activates Wnt signaling in
a dose- and Wnt ligand–dependent manner. The HEK293T cells were
transfected with the TOPFlash plasmid, ΔN-β-catenin or both. The cells
were treated 18 h later for 1 h with DMSO or HLY78 followed by the
control (Ctr) conditioned medium (CM), the Wnt3a conditioned medium
or 8 mM LiCl plus the same dose of HLY78 for an additional 6 h before
the luciferase activity assays. RLC, relative luciferase count. (c) HLY78
had a synergistic effect with Wnt3a to upregulate the Wnt target genes.
The expression of the Wnt target genes, Axin2, DKK1 and NKD1, was
determined by quantitative rtPCR and normalized to GAPDH expression
(internal control). (d) HLY78 has a synergistic effect with Wnt3a to
stabilize cytosolic and nuclear β-catenin. The HEK293T cells pretreated
with HLY78 or DMSO for 1 h were incubated with the control or the Wnt3a
conditioned medium containing the same dose of HLY78 for an additional
3 h. The cells were fractionated as described in the Online Methods and
then subjected to western blot analysis. The β-actin and the SP1 served
as loading controls for the cytosolic and nuclear fractions, respectively.
The immunoblots were quantified by densitometry, and the intensity
values were normalized with those of SP1 (nuclear β-catenin) or β-actin
(cytosolic β-catenin); values are given beneath each band. Error bars
indicate the s.d. of triplicate assays in one experiment. Each experiment
was repeated at least three times.
activator of the Wnt/β-catenin signaling pathway (Fig. 1a). We show
that HLY78 has dose-dependent activating effects on the TOPFlash
luciferase reporter in the presence of the Wnt3a conditioned
medium (Fig. 1b and Supplementary Results, Supplementary
Fig. 1a,b). We also observed a similar synergistic effect when using
the recombinant Wnt3a protein (20 ng ml−1) (Supplementary
Fig. 1c). In contrast, we did not observe any apparent antiproliferative and apoptotic effect of HLY78 at concentrations up to 80 μM
(Supplementary Fig. 2). We also examined the effect of HLY78 on
endogenous Wnt target genes. HLY78 dose-dependently increased the
Wnt3a-induced expression of AXIN2 (ref. 9), DKK1 (refs. 10,11) and
NKD1 (ref. 12) (Fig. 1c). In contrast, NFAT, SRF and NFκB reporter
plasmids did not respond to HLY78 treatment at concentrations
2
that obviously activated TOPFlash (Supplementary Fig. 3a), indicating the specificity of HLY78 toward Wnt signaling.
To investigate HLY78’s synergistic effect with the Wnt protein,
we tested whether it could work synergistically with other Wnt
signaling activators, including LiCl (a GSK3β inhibitor)13 and ΔNβ-catenin (a constitutively stabilized form of β-catenin). HLY78
had no effect on the activity stimulated by LiCl treatment or ΔNβ-catenin overexpression (Fig. 1b). HLY78 also failed to activate
the TOPFlash luciferase reporter in the colon cancer cell lines
HCT116 (β-catenin mutation) and SW480 (APC deficiency),
in which the Wnt/β-catenin signaling pathway is constitutively
activated (Supplementary Fig. 3b). These findings indicated
that HLY78 acts upstream of the β-catenin degradation complex.
Consistently, HLY78 reduced the amount of phospho-β-catenin
(Ser33, Ser37 and Thr41)14 (Supplementary Fig. 4) and increased
cytosolic and nuclear β-catenin (Fig. 1d) in a dose-dependent
manner in the presence of Wnt3a. These results demonstrated that
HLY78 is an activator for the Wnt/β-catenin signaling pathway,
which acts upstream of β-catenin and functions only in the presence of the Wnt3a ligand.
HLY78 targets the Axin protein
To facilitate the identification of the cellular target (or targets) of
HLY78, we attempted to generate biotinylated HLY78 derivatives
(Supplementary Fig. 5). A series of analogs (HSS49a (2), HLY72
(3), HLY103 (4) and HLY119 (5)) were prepared, among which
only HLY119 retained the same activity as HLY78 in the synergistic
stimulation of Wnt reporter gene activity in the presence of Wnt3a
(Fig. 2a). On the basis of their structures, we suspected that the
phenethyl and N-methyl groups of HLY78 are the functional groups,
whereas the methylenedioxy group on the other side of HLY78 is
most likely not necessary for its Wnt activation function. Biotin was
attached to the dihydroxy groups at the 8,9-positions of HLY119 to
generate HLY179 (6) (Fig. 2b and Supplementary Fig. 5). HLY179
had a synergistic effect with the Wnt3a conditioned medium at
the concentration of 10 μM, whereas neither biotin alone nor
its derivative HLYC177 (7) (side chain of HLY179) was effective
(Fig. 2c). We then used HLY179 in the assays described below to
determine the potential target of HLY78 by label-free quantitative
proteomic approaches. This strategy was based on the immobilization of HLY179 or biotin to the affinity-capture cellular proteins
from the HEK293T cell lysates that were spiked with an excess
amount (80 μM) of HLY78. Of the 1,897 proteins measured with at
least two unique peptides (Supplementary Data Set 1), there were
92 proteins in the HLY179-binding group that could be quantified.
Among those proteins, Axin, which was quantified by 17 unique
peptides, specifically competed with soluble HLY78. We confirmed
the binding between HLY179 and Axin using a pull-down assay
(Fig. 2d). Meanwhile, no interaction was detected between HLY179
and other major transducers of the Wnt/β-catenin signaling pathway, including GSK3, Dvl2, LRP6 and Frizzled7 (Fig. 2e). Moreover,
the interaction between HLY179 and Axin could be effectively competed with by HLY78 (Supplementary Fig. 6a). These results substantiated our conclusion that Axin is the cellular target of HLY78.
To investigate the mechanistic details of the synergistic activation of the Wnt signaling pathway by HLY78 via its association
with Axin, we mapped the HLY78-interacting region on Axin.
We found that HLY78 binds the DIX domain of Axin (hereafter
referred to as DAX to distinguish it from the Dvl DIX domain;
Fig. 3a and Supplementary Fig. 6a). Subsequent in vitro analysis
using a purified recombinant DAX protein confirmed our mapping results and further indicated that the DAX domain of Axin
was a direct target of HLY78 (Fig. 3b). The binding between DAX
and HLY179 could be specifically competed out with HLY78
(Fig. 3b) but not with HSS49a (an inactive analog of HLY78;
Fig. 2a). Meanwhile, the DIX domain from the Dvl protein, which
nature chemical biology | Advance online publication | www.nature.com/naturechemicalbiology
article
O
3
NHMe
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O
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DAX
HLY103
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Lys780
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HLY179
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Biotin
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DMSO
Biotin
HLYC177
HLY78
HLYC60
HLY179
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Input Pulldown
+
– + –
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+ – +
Axin-Flag
GSK3β-HA
Dvl2-Flag
LRP6
Frizzled7-HA
Figure 2 | HLY78 targets the Axin protein. (a) Distinct effects of HLY78
and HLY119 (an intermediate from HLY78 to HLY179) with other analogs
on the TOPFlash reporter activity. The HEK293T cells were treated with
DMSO, HSS49a, HLY72, HLY103, HLY119 or HLY78 with the control (Ctr)
or the Wnt3a conditioned medium (CM). RLC, relative luciferase count.
(b) The chemical structure of biotinylated HLY78 (HLY179). (c) HLY179
maintains activity on Wnt signaling. The HEK293T cells were treated
with 10 μM of biotin, HLYC177, HLY78, HLYC60 or HLY179, either in the
control medium or the Wnt3a conditioned medium for the TOPFlash
reporter assays. (d) HLY179 binds the Axin protein in vivo. The HEK293T
cells treated with HLY179 or biotin were harvested and lysed, followed
by streptavidin pulldown and western blot analysis using Axin-specific
antibody (anti-Axin). (e) HLY179 specifically binds Axin. The HEK293T
cells transfected with Axin or other plasmids (as indicated) were
subjected to HLY179 or biotin treatment for 24 h, followed by streptavidin
pulldown and western blot analysis. Error bars indicate the s.d. of triplicate
assays in one experiment. Each experiment was repeated at least
three times. HA, hemagglutinin.
shares 37% homology with DAX15, failed to have detectable binding affinity for HLY179 (Supplementary Fig. 6b).
Using a previous structural study of Axin, we performed modeling for the Axin–HLY78 complex. We searched for a possible binding cavity for HLY78 on DAX using the AutoDock Vina docking
program16 and the structure of DAX (Protein Data Bank (PDB)
1WSP)17 as the receptor molecule. We analyzed the nine conformers of HLY78 outputted from Vina onto DAX and found that six
of the conformers were localized at the same cavity formed by the
juxtaposition of the residues from two neighboring protomers. We
then selected the model with the lowest estimated free energy for
binding (6.8 kcal mol−1) and modified it slightly. The modeled complex structure of Axin–HLY78 revealed that a group of residues in
Figure 3 | HLY78 directly binds the DAX domain of Axin. (a) Schematic
representation of the full-length or the truncated Axin with the indicated
affinity for HLY179. (b) HLY179 directly binds the Axin DAX domain.
Immunoblotting analysis on streptavidin-coated Sepharose after incubation
of the purified recombinant DAX with HLY179 (10 μM) or biotin in the
presence or absence of HLY78 (40 μM) or HSS49a (40 μM). (c) Cartoon
representation of HLY78 (yellow sticks) docked onto the crystal structure
of DAX (PDB 1WSP). The related two protomers of DAX are in cyan and
green, respectively. (d) The E776A, R765A or V810R mutants within the
DAX domain showed decreased binding affinity for HLY179 in vitro.
WT, wild type.
the cavity could make contacts with HLY78 through electrostatic
or hydrophobic interactions (Fig. 3c). To verify the importance of
these residues in the binding of HLY78, we generated DAX mutants
carrying R765A, E776A and V810R mutations. As predicted by
the model, these three DAX mutants showed reduced affinities for
HLY179 (Fig. 3d). In contrast, the mutation K780S, which modified
an adjacent residue pointing away from the cavity, did not affect
the binding (Fig. 3d). Next, we used surface plasmon resonance
spectroscopy to examine the binding kinetics and affinities of the
HLY179–DAX interaction through a FortéBio Octet Red system.
Wild-type DAX or the E776A mutant was captured by biosensors
and was measured for its binding affinity to different concentrations
of HLY179. HLY179 interacted directly with wild-type DAX with
a KD of 8.83 μM (Supplementary Fig. 7). In contrast, we did not
detect apparent binding between HLY179 and E776A, even at concentrations as high as 50 μM.
Our modeling analysis predicted that the inactive analogs
(HSS49a, HLY72 and HLY103) would clash with DAX when superimposed with HLY78 on DAX. HLY78 and its close derivative,
HLY72, differ only in that the phenethyl group of HLY78 is replaced
with a phenethylene group in HLY72. As shown in the model, the
phenethyl group of HLY78 could adjust its orientation and penetrate into the deep, narrow groove of the cavity, whereas the phenethylene group in HLY72 must maintain its coplanar position with
the benzene ring, which reduces its ability to fit well into the cavity
(Supplementary Fig. 8).
HLY78 enhances the Axin–LRP6 association
We investigated the mechanism by which the HLY78–Axin interaction promotes Wnt signaling transduction. Axin, a scaffold protein,
is an important component of the β-catenin destruction complex
(through its interaction with APC, GSK3 and β-catenin). Axin also
interacts with LRP6 and is required for its phosphorylation1. HLY78
enhanced the interaction of Axin, but not its E776A mutant, with
LRP6 (Fig. 4a). HLY78 also enhanced the Wnt-induced Axin–LRP6
association in a stable cell line constitutively expressing Flagtagged Axin at a similar level to the endogenous level (Fig. 4b).
Consistent with this observation, HLY78 facilitated Wnt3a-induced
Axin translocation to the cell membrane (Supplementary Fig. 9).
3
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Ctr CM + DMSO
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Figure 4 | HLY78 potentiates the Axin–LRP6 interaction. (a) The Axin bearing the E776A mutation failed to show increased binding with LRP6 under
HLY78 treatment. The HEK293T cells transfected with the indicated combinations of plasmids were treated with DMSO or HLY78 (20 μM) for 24 h
followed by immunoprecipitation (IP) using anti-hemagglutinin (HA) and western blot analysis. The immunoblots were quantified by densitometry, and
the relative intensity values are given beneath each band. (b) HLY78 facilitates the Axin–LRP6 complex formation in the Flag-tagged Axin-stable HEK293T
cell line. Ctr, control; CM, conditioned medium. (c) HLY78 enhances the Axin-LRP6 interaction in vitro. (d) Effects of HLY78 on the Wnt3a-induced LRP6
phosphorylation at Ser1490. The HEK293T cells were pretreated with DMSO or HLY78 with the indicated doses for 1 h, and the control or the Wnt3a
conditioned medium containing the same dose of HLY78 was added for an additional 30 min before harvest. Activated LRP6 was determined by western
blot analysis with anti–phospho-Ser1490 LRP6. Total LRP6 and β-actin were used as loading controls. The intensity values of the immunoblots were
normalized with those of β-actin and are given beneath each band. (e) Axin bearing the E776A mutation impairs HLY78’s activity on Wnt3a-induced
TOPFlash reporter. Cells pretreated with siRNA targeting Axin1 and Axin2 for 24 h were transiently transfected with the rescue Axin* constructs for an
additional 24 h of incubation (constructs marked with an asterisk are resistant to Axin1 and Axin2 siRNA). The TOPFlash plasmid was cotransfected for
the measurement of luciferase activity. Error bars indicate the s.d. of triplicate assays in one experiment. Each experiment was repeated at least three
times. RLC, relative luciferase count.
To determine whether HLY78 targets Axin–LRP6 directly, we
performed an in vitro LRP6–Axin interaction assay with purified
recombinant GST-LRP6C (the GST coding region was fused inframe to the entire cytoplasmic domain of LRP6 containing residues
1397–1613), CK1, GSK3 and Axin, which are expressed in mammalian cells (Fig. 4c). We found that HLY78 increased the Axin–LRP6
association directly (Fig. 4c). HLY78 had no effect on the Axin–Dvl,
a
b
Axin-HA –
Axin-∆N2-HA –
LRP6 +
GSK3β-Myc +
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Axin
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Axin–GSK3 or Axin–ΔN-β-catenin interactions (Supplementary
Fig. 10). These observations indicate that HLY78 acts by specifically
promoting the association between Axin and the LRP6 C-terminal
cytoplasmic domain.
We then examined whether HLY78 had a synergistic effect
with Wnt to induce LRP6 phosphorylation. HLY78 enhanced
LRP6 phosphorylation at Ser1490 in a dose-dependent as well as
353 437 506
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HLY78
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E776A
+
–
+
–
–
+
+
–
–
+
+
+
–
–
+
–
+
–
+
–
–
+
+
–
+
+
+
+
+
Axin-N2-Flag
Axin-∆N2-HA
‘Closed’
Hard
+
Axin
1.0 0.2 0.5 0.5
Anti-HA
+
+
–
+
+
Anti-HA
Axin-N2-Flag
Anti-Flag
–
+
–
+
+
Axin-∆N2-HA
LRP6
–
+
+
+
IP:HA
+
+
+
+
+
Anti-LRP6
LRP6
Axin
HLY78
-HA
Axin-∆N2
Axin-N2-Flag
HLY78
–
–
+
–
IP:HA
+
–
+
+
+
+
–
+
+
Axin-HA
e
d
–
+
+
+
+
Anti-Flag
Axin-N2-Flag
Anti-Flag
Axin-HA
Input
+
–
+
+
c
826
‘Open’
Axin-∆N2-HA
LRP6
Easy
+
Axin
LRP6
Axin
Figure 5 | HLY78 inhibits the interaction between Axin-DN2 and Axin-N2. (a) The Axin-DN2 interaction with LRP6 is independent of HLY78 treatment.
IP, immunoprecipitation; HA, hemagglutinin. (b) HLY78 disrupts the interaction between Axin-N2 and Axin-ΔN2. (c) LRP6 disrupts the interaction
between Axin-N2 and Axin-ΔN2. (d) The interaction between Axin-ΔN2E776A and Axin-N2 is unaffected by the treatment of HLY78. The HEK293T
cells transfected with the indicated combinations of plasmids were treated with DMSO or HLY78 (20 μM) for 24 h. The cells were then harvested for
immunoprecipitation analysis with anti-hemagglutinin (mouse) and western blot analysis using anti-hemagglutinin (mouse), anti-LRP6 (rabbit) or antiFlag (rabbit). (e) A model for the role of HLY78 in regulating Axin–LRP6 complex formation. The binding of HLY78 to the Axin C terminus disrupts its
interaction with the Axin N terminus and thus relieves the autoinhibition of Axin. Therefore, active Axin binds LRP6 more easily in situations when the Wnt
signaling pathway is activated.
4
article
Effect of HLY78 on Wnt signaling in zebrafish development
To test HLY78 activity in vivo, we extended our study to the zebrafish
model system. During zebrafish embryonic development, canonical
Wnt/β-catenin signaling patterns the dorsal-anterior structures19,20.
Ectopic activation of the Wnt pathway in ventral blastomeres (that
is, via wnt8 mRNA injection) caused dose-dependent mesodermal
defects and anterior truncations (such as eye defects) (Fig. 6a).
We postulated that HLY78 would exacerbate these phenotypes if
it functions as a positive regulator of the Wnt/β-catenin signaling
pathway during zebrafish development. Overexpression of Wnt8
resulted in eye defect phenotypes of varying severity, and treatment
with HLY78 further increased the phenotypic severity (Fig. 6a). In
contrast, dorsalization phenotypes of varying severity caused by the
wnt8 morpholino were alleviated by HLY78 (Fig. 6b).
We used in situ hybridization analysis to examine the potential
effect of HLY78 on the expression of the Wnt target gene. Injection
of the wnt8 morpholinos suppressed the expression of tbx6 and cdx4
(the ventrolateral mesodermal marker and the Wnt target genes)21,22
but not that of the no tail (ntl) gene23 (Fig. 6c). We found that the
inhibitory effect of the wnt8 morpholino on tbx6 and cdx4 expression could be significantly (P < 0.05) relieved by HLY78 treatment,
and HLY78 alone could enhance the expression of tbx6 and cdx4 but
not ntl gene expression (Fig. 6c). We also observed similar results by
quantitative real-time PCR (rtPCR) (Fig. 6d).
Wnt signaling represents a key and evolutionarily conserved signaling pathway for the generation and function of HSCs in vivo24.
WT
60
40
Small eyes
20
b
WT
d1
d2
Percentage
of total embroys
n = 49 47 43 46
500 µm
d3
WT
80
60
d1
40
20
Ctr MO
Ctr MO + HLY78 (10 µM)
wnt8 MOs
wnt8 MOs + HLY78 (5 µM)
wnt8 MOs + HLY78 (10 µM)
2.0
1.5
* **
*
*
* **
*
1.0
d3
500 µm
0
cdx4
tbx6
e
f
HLY78
XAV939
WT
60
40
Reduced
20
100
Enhanced
75 75 75 0 µM 500 µm
0 5 10 10 µM
n = 53 47 50 52 56
80
WT
60
40
Reduced
20
100
2.5
2.0
Ctr
Enhanced
80
Enhanced
75 75 75 0 µM 500 µm
0 5 10 10 µM
n = 45 42 47 45 50
80
WT
500 µm
60
40
20
0
wnt8 MOs 0
HLY78
0
0.5
Reduced
51 55 50 54
0
wnt8 MOs 0
HLY78
0
d2
75 75 75 0 µM
0 5 10 10 µM
100
n = 51
0
wnt8 MOs 0
HLY78
0
No eyes
100
0
wnt8 MOs
HLY78
WT
Percentage
of total embroys
80
0
wnt8 mRNA 1.5 1.5 1.5 0 pg
HLY78
0 5 10 10 µM
d
c
No eyes
Percentage
of total embroys
Percentage
of total embroys
100
Small eyes
Percentage
of total embroys
Finally, we investigated the mechanism through which HLY78
enhances the Axin–LRP6 association by binding the DAX domain
of Axin. A previous study revealed that the full-length Axin had
a lower affinity for LRP6 than the truncated form of Axin (residues 353–826, termed Axin-ΔN2)18. Remarkably, HLY78 could not
enhance the interaction between Axin-ΔN2 and LRP6 (Fig. 5a).
Therefore, we reasoned that Axin may assume an autoinhibitory conformation in which its N terminus folds back to interact
with other regions, making the intact Axin inaccessible for LRP6
to bind. To test this hypothesis, we generated a truncated form of
Axin, termed Axin-N2 (residues 1–353). Compared with fulllength Axin, Axin-ΔN2 showed an increased affinity for Axin-N2
(Fig. 5b). Moreover, either HLY78 or LRP6 was capable of disrupting the interaction between Axin-N2 and Axin-ΔN2 (Fig. 5b,c).
Consistently, HLY179 and HLYC60, but not HLYC177 or HSS49a,
were able to inhibit the interaction between Axin-N2 and AxinΔN2, as was HLY78 (Supplementary Fig. 14). HLY78 failed to
inhibit the association between Axin-N2 and the E776A mutant
of Axin-ΔN2 (Fig. 5d), highlighting the necessity of HLY78 binding to Axin’s DAX domain. Collectively, these results supported a
model in which HLY78 directly targets the DAX domain of Axin
to relieve its ‘autoinhibition’, hence promoting Axin–LRP6 complex
formation (Fig. 5e).
WT
n = 51 48 59 52
Gene expression
HLY78 releases Axin ‘autoinhibition’
a
Gene expression
Wnt3a-dependent manner (Fig. 4d). In addition, HLY179 and
HLYC60 (9), but not HLYC177 or HSS49a, function in a similar manner to that of HLY78 (Supplementary Fig. 11a,b). To further confirm
that Axin is the direct target of HLY78 during its regulation of the
Wnt/β-catenin signaling pathway, we applied the RNAi knockdownrescue approach, in which endogenous Axin1 and Axin2 were
silenced by siRNA and substituted by siRNA-resistant Axin1 or the
E776A mutant via transfection. We found that only Axin, but not
the E776A mutant, could successfully rescue HLY78-potentiated
Wnt3a-induced LRP6 phosphorylation (Supplementary Fig. 12).
We also observed the same results using the TOPFlash luciferase
reporter assay (Fig. 4e and Supplementary Fig. 13). These results
revealed that the Axin DAX domain may be solely responsible for
HLY78’s effect on Wnt signaling.
1.5
1.0
75 75 75 0 µM
0 5 10 10 µM
DMSO
XAV939 (5 µM)
HLY78 (80 µM)
XAV939 (5 µM)
+ HLY78 (80 µM)
DMSO (cloche mutants)
**
**
**
**
**
**
0.5
XAV939
+ HLY78
300 µm
150 µm
0
cmyb
runx1
Figure 6 | HLY78 activates Wnt signaling in vivo. (a) HLY78 exacerbates
Wnt8 overexpression phenotypes. Zebrafish embryos were classified
into three categories, wild type (WT, cyan), small eyes (green)
and no eyes (orange), on the basis of the morphological phenotype. The
number of the total embryos scored (n) under each experimental condition
is shown on top of each bar. (b) HLY78 restores the phenotype of the wnt8
morphants. Zebrafish embryos are classified into four categories, with wild
type and d3 being the most severe phenotypes. (c,d) HLY78 restores the
expression of the Wnt target genes in the ventrolateral mesoderm. Embryos
injected with the indicated morpholinos were fixed at the 60% epiboly
stage and stained for tbx6 (top), cdx4 (middle) and ntl (bottom) (c) or were
harvested for RNA extraction, followed by quantitative rtPCR on cdx4 and
tbx6 expression and normalization to the ntl (internal control) expression
level (d). (e,f) HLY78 stimulates HSC formation in an endogenous
Wnt-dependent manner. Wild-type zebrafish embryos, treated with HLY78
(80 μM), XAV939 (5 μM) or both, versus the control (Ctr) from the
three-somite stage until 36 h.p.f. were fixed for in situ hybridization with a
mixture of runx1/cmyb probe (e) and harvested for quantitative rtPCR (f).
MO, morpholino. All of the graphs show the mean ± s.d. The Student’s t-test
was applied to determine the statistical significance (**P < 0.01; *P < 0.05).
The expression of cmyb and runx1 in cloche mutants (lack of hemangioblasts)
was quantified by rtPCR to determine the non-HSC background in 36-h.p.f.
zebrafish embryos. The zebrafish cloche mutation affects both the endothelial
and hematopoietic lineages at a very early stage.
Here, we examined whether enhancing the Wnt signal by HLY78
affects the regulation of HSCs and the progenitor population by
examining the expression of two conserved HSC markers, runx1
5
article
and cmyb, at 36 h post-fertilization (h.p.f.)25. Treatment of zebrafish
embryos with HLY78 increased the expression of runx1 and cmyb
(47 embryos with increased expression out of 62 total scored),
whereas the Wnt inhibitor XAV939 (ref. 26) decreased the HSC
marker expression (30 embryos with decreased expression out of
52 total scored) and antagonized the effect of the HLY78 treatment
(30 normal embryos out of 51 embryos scored) (Fig. 6e). The
in situ hybridization results were confirmed by quantitative PCR for
runx1 and cmyb (Fig. 6f). Our observations indicate that HLY78
may regulate HSC development by enhancing Wnt signaling.
DISCUSSION
We report the discovery of the small molecule HLY78 as a new
activator of the Wnt/β-catenin signaling pathway and identify Axin
as the direct target of HLY78. Biochemical analysis and structural
modeling revealed a cavity at the dimeric interface of the Axin DAX
domain, which was critical for specific binding of HLY78. The Axin
DAX domain mediates homo- and heteropolymerization, which
may be important for its function17,27–30. Thus far, three proteins,
Axin, Dvl and Ccd1, have been found to contain a DIX domain.
A previous study suggested that Dvl may behave as a dominantnegative of Axin and regulate the latter’s function via the heterotypic interaction between Dvl-DIX and Axin-DAX27. In our view,
the binding of HLY78 has little effect on homo- or heteropolymerization of Axin. First, the residues critical for HLY78 binding
in Axin-DAX do not overlap with residues that are important for
homo- or heteropolymerization. In another previous study31, three
sites were proposed for homo- and heteropolymerization of DIX on
the basis of studies on Dvl, Axin and Ccd1. Three sites are located
at the interface formed by protomer A and B, whereas HLY78 targets the pocket formed by protomer B and C, which has attracted
little attention so far (left panel, Supplementary Fig. 15). It is very
unlikely that HLY78 will affect the interaction between protomer
A and B. Second, the critical residues for binding HLY78, such as
Arg765 and Val810, are also not involved in the interaction of protomer B with C (designated as Site IV, right panel, Supplementary
Fig. 15). Glu776 of protomer C may weakly bind Cys766 from promoter B, if at all, considering there is a distance of 3.9 Å between
them. Finally, HLY78 does not affect the interaction of Axin with
Dvl. Considering the presumption that DIX family proteins share
the same sites for homo- and heteropolymerization, it is very likely
that HLY78 will also not affect homopolymerization of Axin. Thus,
we believe that the binding of HLY78 to Axin DAX does not affect
polymerization but either directly blocks the intramolecular interaction or triggers a conformational change, leading to ‘opening’ of
the Axin structure. In fact, our model of Axin autoinhibition is not
in conflict with Axin polymerization, and both may have important
roles in regulating Axin’s function.
Previous studies have suggested that the Axin N terminus
(including the RGS domain and the linker region between the
RGS domain and the GSK-binding domain) has an inhibitory role
in Axin’s binding with its partners18,32. However, the mechanism
underlying such inhibition remains elusive. In this study, we found
that the N terminus of Axin (Axin-N2) is able to associate with the
C terminus of Axin (Axin-ΔN2) (Fig. 6b), which could be disrupted
effectively by HLY78 (Fig. 6b). Furthermore, HLY78 enhanced the
association of the full-length Axin but not that of the Axin C terminus (Axin-ΔN2) with LRP6 (Fig. 6a). These observations indicated
that an autoinhibitory conformation of Axin may be mediated by
the interactions between its N- and C-terminal domains.
It seems that activated LRP6 could also relieve the autoinhibition of Axin (Fig. 6c). Axin’s conformation may alter its function,
depending on distinct binding partners and post-translational
modifications during its differential assemblies with the ‘β-catenin
destruction complex’ and the ‘Wnt-LRP5/6 signalosome’33. The
conformational change is most likely regulated by the Wnt signaling
6
pathway and is further facilitated by HLY78. We propose a model
in which direct binding of HLY78 to the DAX domain of Axin triggers the conformational change of Axin from a ‘closed’ autoinhibitory state to an ‘open’ active state, leading to an enhancement of
the Axin–LRP6 association and the subsequent phosphorylation
and activation of LRP6 (Fig. 6e). During the review process of
this manuscript, we noted that a model of Axin autoinhibition has
recently been proposed34, strengthening the idea that autoinhibition
is a valid mechanism for Axin to regulate its function in Wnt/βcatenin signaling.
Wnt/β-catenin signaling has an important role in HSC formation and self-renewal and is essential for the HSC population
in transplantation and recovery after injury35–37. Recently, prostaglandin E2 was found to stimulate HSCs through cooperative
endogenous Wnt signaling via cAMP–PKA signaling38. In this study,
we noted that HLY78 also increased the expression of the conserved HSC transcription factors runx1 and cmyb, suggesting that
HLY78 can function as a positive regulator of HSCs by elevating
an existing Wnt signal in vertebrates. Thus, our study may inform
the clinical treatment of patients with bone marrow failure or
HSC transplantation.
Although much work has been performed to identify the antagonists of Wnt signaling, particularly with regard to cancer therapy,
little work has been performed to identify small molecules that
activate the Wnt pathway. The use of small molecules that activate
the Wnt pathway may be beneficial in the treatment of many diseases, such as osteoporosis. Several studies have linked the application of lithium (an inhibitor for both GSK3 enzymatic activity and
inositol metabolism circuits) treatment to increase bone mass in
animals with age-related osteoporosis and oophorectomy-induced
osteoporosis39,40. Unfortunately, patients have often suffered from
the side effects of the treatments41. Our study concerning the small
molecule HLY78 as a specific Wnt synergistic activator presents
another opportunity for potential clinical applications relevant to
the Wnt pathway.
Received 24 February 2013; accepted 26 June 2013;
published online 28 July 2013
Methods
Methods and any associated references are available in the online
version of the paper.
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Acknowledgments
We greatly appreciate the gift of LRP6 and MESD plasmids from X. He (Boston
Children’s Hospital), and we thank the Zebrafish Core Facility–Shanghai Institute of
Biochemistry and Cell Biology for providing zebrafish embryos. This work is supported
by the Ministry of Science and Technology of China (grant 2010CB912100 to L.L.,
2011CB966300 to X.S. and 2013CB910900 to W.P.) and the National Natural Science
Foundation of China (grants 30930052 and 31230044 to L.L. and 30830114 to X.H.).
Author contributions
S.W. designed, performed and analyzed biochemical, zebrafish and cell culture
experiments. J.Y., D.C. and L.W. designed and performed chemical synthesis under the
guidance of X.H. F.N. performed zebrafish embryo experiments. X.S. provided bioinformatics support. C.J. and W.M. performed the embryonic stem cell assays under the
guidance of W.P. C.F. provided Flag-tagged Axin-stable HEK293T cells. S.X. and H.M.
purified proteins. C.L. performed MS experiments under R.Z.’s guidance. S.W., C.F.,
W.P., X.S. and L.L. wrote the manuscript with advice from all of the authors. L.L. guided
all of the aspects of this study.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Supplementary information, chemical compound and chemical probe information are
available in the online version of the paper. Reprints and permissions information is
available online at http://www.nature.com/reprints/index.html. Correspondence and
requests for materials should be addressed to X.H. or L.L.
7
ONLINE METHODS
Chemical synthesis. The syntheses of HLY78, HLY72, HLY103, HLY119,
HLY179, HLYC177, HLYC60 and HSS49a are described in the Supplementary
Methods. The syntheses of HLY78 and HLY179 are described in Supplementary
Figure 5. HSS49a (ismine42) was isolated from Lycoris rediata.
Cell transfection and reporter gene assay. HEK293T, NIH3T3, SW480
and HCT116 cells were transfected using Lipofectamine Plus (Invitrogen)
according to the manufacturer’s instructions. For reporter gene assays, cells
were seeded in 24-well plates. Each well of HEK293T cells was transfected with
250 ng of plasmids in total, including 20 ng of TOPFlash or NFAT-Luc or 40 ng
of SRF-Luc and 25 ng of EGFP-C1. In Figure 1b, 0.25 ng of ΔN-β-catenin
was transfected. Each well of the NIH3T3 cells was transfected with 500 ng of
plasmids in total, including 25 ng of LEF-Luc, 75 ng of LEF1, 25 ng of EGFP-C1.
The LacZ plasmid was added to equalize the total amount of transfected DNA.
The cells were treated 18 h after transfection with the Wnt3a conditioned
medium or the control medium for an additional 6 h and then lysed for luciferase assays. Each well of the SW480 or the HCT116 cells was transfected with
500 ng of plasmids in total, including 100 ng of TOPFlash or FOPFlash, 100 ng
of EGFP-C1 and 300 ng of LacZ. The cells were lysed for the luciferase activity
assay 24 h after transfection. The GFP expression levels were determined for
normalization as described previously43.
Cytosol and nucleus fractionation. The HEK293T cells were grown in six-well
plates, harvested with a cell scraper into 1.5 ml of PBS and spun at 700g for
10 min. The pelleted cells were resuspended in buffer A (10 mM HEPES,
pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 10 mM NaF, 2 mM Na3VO4,
1 mM pyrophosphoric acid and CompleteTM protease inhibitors) and incubated on ice for 10 min. Cells were then passed through a 0.4-mm needlepoint
to break down the cell membrane and were then centrifuged at 700g at 4 °C for
10 min. The supernatant was collected and centrifuged at 100,000g at 4 °C for
1 h. The supernatant from the ultracentrifugation was collected as the cytosolic
fraction. For nuclear protein extraction, the pellet from the 700g centrifugation
was washed by buffer A, resuspended in buffer C (20 mM HEPES, pH 7.9,
1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 2 mM Na3VO4,
1 mM pyrophosphoric acid and CompleteTM protease inhibitors) and incubated on ice for 30 min. Nuclear extracts were recovered from the supernatants
after centrifugation at 100,000g at 4 °C for 1 h.
Quantitative rtPCR with reverse transcription. The total RNA was isolated
with a TRIzol kit (Invitrogen). Reverse transcription of purified RNA was
performed using oligo (dT) priming and Superscript III reverse transcriptase
according to the manufacturer’s instructions (Invitrogen). Quantitative rtPCR
for cdx4, tbx6, ntl, cmyb, runx1, gapdh, AXIN2, DKK1, NKD1 and GAPDH was
performed with the TaKaRa SYBR Premix Ex Taq kit on the ABI PRISM 7500
system (Applied Biosystems). The specific primers used for detecting the genes
are listed in Supplementary Table 1.
Zebrafish experiment microinjection. Embryos were produced by pair mating
of fish raised under standard conditions. The wild-type (WT) embryos were
derived from the Tuebingen strain. The antisense MO and the standard control
MO were obtained from Gene Tools (Philomath, OR). The wnt8 MOs (wnt8ORF1 MO + wnt8-ORF2 MO) have been described previously44. Embryos,
injected with the wnt8 MOs or the control MO at the one-cell stage, were
placed in water containing DMSO or HLY78 (5 μM or 10 μM, respectively) at
the sphere stage until 26 h post fertilization (h.p.f.). For sense RNA injections,
capped mRNA was synthesized using the mMessage mMachine kit (Ambion).
Embryos (one-cell stage), injected with wnt8 mRNA (1.5 pg) or control mRNA
(1.5 pg) at the one-cell stage, were placed in water containing DMSO or HLY78
(5 μM or 10 μM) at the sphere stage until 26 h.p.f.
Whole-mount in situ hybridization. Digoxigenin-UTP–labeled antisense
RNA probes were generated from linearized plasmids as templates by
in vitro transcription using the DIG RNA Labeling kit (Roche) according to
the manufacturer’s instructions. Whole-mount in situ hybridizations were
performed following the standard methods45 with minor modifications. The
embryos were photographed as previously described46. Embryos at the shield
stage and the 60% epiboly stage were shown in a lateral view with dorsal to
the right.
nature chemical biology
GST-pull down and in vitro binding assay. Recombinant proteins (GST- or
His6-tagged) were expressed and purified from Escherichia coli. For GST-pull
down kinase assays, the HEK293T cells expressing Flag-Axin were lysed by
homogenization in a hypotonic buffer containing 50 mM HEPES, pH 7.4,
1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM NaF,
1 mM Na3VO4, 0.5 mM DTT and a cocktail of protease inhibitors. The lysates
were cleared by centrifugation at 10,000g. The purified recombinant GSTLRP6C, which was bound to beads, was phosphorylated in vitro by purified
GSK3, CK1 (kinase domain) or both (New England Biolabs, 100 units per reaction) for 4 h at room temperature and washed five times before incubation with
the lysate containing Flag-Axin. Binding assays were performed at 4 °C for 6 h,
and the beads were washed five times in 1% Triton X-100 buffer before SDSPAGE and immunoblotting. For the in vitro binding assay, the recombinant
protein (His6-tagged) was purified from E. coli and then mixed in lysis buffer
for 3 h at 4 °C along with HLY179 and streptavidin beads (Thermo).
Statistical analysis. An unpaired Student’s t-test was used to evaluate the
difference between the two treatments. A value of P < 0.05 was considered
to be statistically significant, and a value of P < 0.01 was considered to be
extremely statistically significant. Statistical analyses were performed using
SPSS (Statistical Package for the Social Sciences) 13.0 statistics software.
Antibodies. Immunoblotting was performed using the following antibodies:
anti–β-catenin (610154, BD Transduction Laboratories; dilution 1:1,000);
anti–E-cadherin (610181, BD Transduction Laboratories; dilution 1:5,000);
anti-SP1 (S9809, Sigma; dilution 1:500); anti–β-actin (sc-47778, Santa
Cruz; dilution 1:5,000); anti–β-tubulin (T5168, Sigma; dilution 1:1,000);
anti–phospho-β-catenin (Ser33/Ser37/Thr41) (no. 9561P, Cell Signaling;
dilution 1:1,000); anti-LRP6 (no. 3395, Cell Signaling; dilution 1:1,000);
anti–phospho-LRP6 (Ser1490) (no. 2568, Cell Signaling; dilution 1:1,000);
anti-Axin (no. 2087, Cell Signaling; dilution 1:1,000); anti-Flag (no. 2368,
Cell Signaling; dilution 1:1,000); anti-hemagglutinin (1666606, Roche; dilution 1:1,000); anti-Flag (F3165, Sigma; dilution 1:1,000); anti-His (H1029,
Sigma; dilution 1:1,000); anti-Myc (M5546, Sigma; dilution 1:1,000); anti-GST
(G1160, Sigma; dilution 1:1,000).
Purchased chemical compounds. Lycorine with purity >98% was purchased
from Jiangsu Hanyuan Technology Ltd. D-Biotin with purity >95% was
purchased from J&K Scientific, and 2-bromoethylamine hydrobromide with
purity >98% was purchased from Sigma-Aldrich.
Plasmids. TOPFlash and FOPFlash were purchased from Millipore. Axin
(GenBank accession number NM_003502.3) was subcloned into PCMV vector to generate fusion protein with varying tags. DAX domain of Axin and
DIX domain of Dvl were subcloned into pET32a vector (Novagen, Darmstadt,
Germany) to generate a His6-tagged fusion protein. To get mutated DAX
proteins (R765A, E776A,V810R and R780S), plasmids with site-directed
mutants were constructed using the QuikChange site-directed mutagenesis kit
(Stratagene, Palo Alto, CA, USA). The specific primers used for point mutation of human Axin genes are listed in Supplementary Table 2. The bacterial
expression construct of GST-LRP6C has been described3. Axin* and AxinE776A*
(which is resistant to Axin1 and Axin2 siRNA) were generated using the
QuikChange site-directed mutagenesis kit. The specific primers used for resistant mutation of human Axin genes are listed in Supplementary Table 2. After
confirming a 100% identity match by DNA sequencing, E. coli strain BL21 was
transformed with constructs for the next step of protein purification.
Cell culture. HEK293T, HCT116 and SW480 were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS in a humidified incubator at 37 °C
and 5% CO2/95% air (v/v). NIH3T3 cells were cultured in RPMI 1640 medium
with the same supplement.
Endogenous co-immunoprecipitation. The Flag-tagged Axin stable HEK293T
cells stably expressing Flag-Axin were incubated with HLY78 (20 μM) or
DMSO for 20 h. Cells were then treated with the control or the Wnt3a conditioned medium containing the same dose of HLY78 for an additional 4 h before
harvest. Then the cells were lysed in protein lysis buffer (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 5 mM EDTA and proteinase
inhibitors) and centrifuged at 16,000g for 15 min at 4 °C. The supernatant was
doi:10.1038/nchembio.1309
incubated with anti-Flag overnight at 4 °C in addition to Protein A/G PLUS–
Agarose (Santa Cruz Biotechnology, Inc.) for the last 2 h of incubation. Mouse
IgG was used as a control. The beads were washed five times and resuspended
in 50 μL SDS loading buffer.
MS analysis. The HLY179 binding proteins (competed with HLY78) or biotin
binding proteins (competed with HLY78) were lysed by mixing them with
50 μL of solution containing 4% SDS, 100 mM Tris-HCl, pH 7.6, 0.1 M DTT
(lysis solution) and incubating them at 95 °C for 5 min. The DNA had to be
sheared by sonication to reduce the viscosity of the sample. Before starting
sample processing, the lysate had to be clarified by centrifugation at 16,000g
for 5 min. The protein samples were digested by the filter aided sample preparation (FASP) method47.
The tryptic peptides were separated by nanoflow LC and analyzed by
Q Exactive MS (Thermo Fisher Scientific) via a nanoelectrospray ion source
(Thermo Fisher Scientific)48. Briefly, mass spectra were acquired on the
Q Exactive in a data-dependent mode with an automatic switch between a full
scan and up to 20 data-dependent MS/MS scans. Target value for the full-scan
MS spectra was 3,000,000 with a maximum injection time of 30 ms and a resolution of 70,000 at m/z 200. The 20 most intense ions with a charge of two or
more from the survey scan were selected with an isolation window of 1.2 Th
and fragmented by higher-energy collisional dissociation with a normalized
collision energy of 27. The ion target value for MS/MS was set to 500,000 with
a maximum injection time of 120 ms and a resolution of 17,500 at m/z 200.
Repeat sequencing of peptides was kept to a minimum by dynamic exclusion
of the sequenced peptides for 25 s.
The acquired raw files were analyzed by MaxQuant 49 (version 1.3.0.5).
Andromeda, a probabilistic search engine incorporated into the MaxQuant
framework50, was used to search the peak lists against the Human UniProtKB
database (UniProtKB release 2012_06; 13 June 2012). Common contaminants
were added to this database. The search included cysteine carbamidomethylation as a fixed modification and N-terminal acetylation and methionine
oxidation as variable modifications. The second peptide identification option
in Andromeda was enabled. For statistical evaluation of the data obtained, the
posterior error probability and false discovery rate were used. The false discovery rate was determined by searching a reverse database. A false discovery
rate of 0.01 for proteins and peptides was required. Enzyme specificity was
set to trypsin allowing N-terminal cleavage to proline. Two missed cleavages
were allowed, and a minimum of six amino acids per identified peptide was
required. Peptide identification was based on a search with an initial mass
deviation of the precursor ion of up to 6 p.p.m., and the allowed fragment
mass deviation was set to 20 ppm.
To guarantee more reliable identification and quantification of HLY179binding proteins (competed with HLY78) or biotin-binding proteins (competed
with HLY78), proteins with only one unique peptide were removed. Totally,
1,897 proteins quantified with at least two unique peptides (Supplementary
Data Set 1). XIC (eXtracted Ion Current) of all isotopic clusters associated
with the identified peptides in the given protein group were summed up as the
protein intensity, which is the foundation of label-free quantification.
FortéBio Octet Red system assay. Samples or buffer were dispensed into 96-well
microtiter plates (Millipore, Billerica, MA) at a volume of 200 μL per well.
Operating temperature was maintained at 30 °C. Super Streptavidin Biosensors
tips (FortéBio, Inc., Menlo Park, CA) were prewetted with buffer (FortéBio)
to establish a baseline before protein immobilization. Then biotinylated
doi:10.1038/nchembio.1309
protein targets (for example, DAX or DAXE776A) were immobilized onto Super
Streptavidin Biosensors. Data were generated automatically by the Octet User
Software (version 3.1) and were subsequently analyzed from the text files using
Excel 2010. The binding profile of each sample was summarized as an ‘nm shift’
(the wavelength or spectral shift in nanometers), which represented the difference between the start and end of the 5-min sample association step51.
MTT cell viability assay. HEK293T (1 × 104) cells were seeded into each well
of 96-well plates. After being cultured for 24 h in a CO2 incubator, the cells
were treated with the dose range from 20 μM to 160 μM of HLY78 or DMSO
control for 72 h. The medium was then changed and replaced with 200 μL of
fresh growth medium with 10% FBS and 20 μL of MTT solution (Sigma). Cells
were incubated for another 4 h, and the medium was replaced by with 200 μL
of DMSO. After 15 min of incubation at 37 °C, the optical absorbance was
measured using a micro-plate reader at a 570 nm. The results are presented as
percentage of cell viability. Data collected at 0 h was set to 1 in each panel.
Assessment of apoptosis. The percentage of apoptotic cells was determined by
monitoring the translocation of phosphatidylserine to the cell surface using an
Annexin V-FITC apoptosis detection kit (Sigma, UK) according to the manufacturer’s instructions. Cells were evaluated for apoptosis using a FACSCalibur
flow cytometer (BD Biosciences) with Annexin V-FITC and PI double staining.
Fluorescence was measured with an excitation wavelength of 480 nm through
FL-1 (530 nm) and FL-2 filters (585 nm). Using flow cytometry, dot plots of
AnnexinV-FITC on the x axis against PI on the y axis were used to distinguish viable cells (which are negative for both PI and AnnexinV-FITC), early
apoptotic cells (which are Annexin V positive cells, but PI negative) and late
apoptotic or necrotic cells (which are positive for both PI and AnnexinV-FITC
staining). Nonstained cells and untreated cells were used as negative controls.
The resultant data was analyzed using CFlow plus software (Accuri C6, Flow
Cytometry System, UK).
42.Youssef, D.T. Alkaloids of the flowers of Hippeastrum vittatum. J. Nat. Prod.
64, 839–841 (2001).
43.Li, L. et al. Dishevelled proteins lead to two signaling pathways. Regulation of
LEF-1 and c-Jun N-terminal kinase in mammalian cells. J. Biol. Chem. 274,
129–134 (1999).
44.Li, Z., Nie, F., Wang, S. & Li, L. Histone H4 Lys 20 monomethylation by
histone methylase SET8 mediates Wnt target gene activation. Proc. Natl.
Acad. Sci. USA 108, 3116–3123 (2011).
45.Oxtoby, E. & Jowett, T. Cloning of the zebrafish krox-20 gene (krx-20) and its
expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095
(1993).
46.Ding, Y. et al. Caprin-2 enhances canonical Wnt signaling through regulating
LRP5/6 phosphorylation. J. Cell Biol. 182, 865–872 (2008).
47.Wiśniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample
preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).
48.Deeb, S.J., D’Souza, R.C., Cox, J., Schmidt-Supprian, M. & Mann, M.
Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes
by their protein expression profiles. Mol. Cell Proteomics 11, 77–89 (2012).
49.Cox, J. & Mann, M. MaxQuant enables high peptide identification rates,
individualized p.p.b.-range mass accuracies and proteome-wide protein
quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
50.Cox, J. et al. Andromeda: a peptide search engine integrated into the
MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
51.Abdiche, Y., Malashock, D., Pinkerton, A. & Pons, J. Determining kinetics
and affinities of protein interactions using a parallel real-time label-free
biosensor, the Octet. Anal. Biochem. 377, 209–217 (2008).
nature CHEMICAL BIOLOGY
Small Molecule Modulation of Wnt Signaling via Modulating the Axin-LRP5/6
Interaction
Sheng Wang1#, Junlin Yin2#, Duozhi Chen2, Fen Nie1, Xiaomin Song1, Cong Fei1,
Haofei Miao1, Changbin Jing3, Wenjing Ma3, Lei Wang2, Sichun Xie1, Chen Li4, Rong
Zeng4, Weijun Pan3, Xiaojiang Hao2* and Lin Li1*
1
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
2
State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
3
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences & Shanghai Jiao Tong University School of Medicine,
Shanghai 200025, China.
4
Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031, China
#
These authors contributed equally to this work.
* Correspondence: Lin Li, E-mail: [email protected]; and Xiaojiang Hao, E-mail:
[email protected].
Nature Chemical Biology: doi:10.1038/NChemBio.1309
Supplementary Table 1: Primers used for real-time quantitative PCR assays
Genes
Forward primers (5’-3’)
Reverse primers (5’-3’)
AXIN2(Homo AGGCTAGCTGAGGTGT
sapiens)
AGGCTTGGATTGGAGAA
DKK1 (Homo CTGCAAAAATGGAATATGTGT
sapiens)
CTTCTTGTCCTTTGGTGTGA
NKD1 (Homo GTCAACCACTCCCCAACATC
sapiens)
AATGGTGGTAGCAGCCAGAC
GAPDH
(Homo
sapiens)
AGGTCGGAGTCAACGGATTTG
TGTAAACCATGTAGTTGAGGTCA
cdx4 (Danio
rerio)
CTCCGGGACCAGTTTCCTAT
CTCCTTCGTTCTCGTTTTGC
tbx6 (Danio
rerio)
CAAGCTGGATTTGACTGCAA
GGGGTTTGTGAAGGCTGATA
ntl(Danio
rerio)
GAAAGTCGGTGGGATTCAGA
TCTGGGACTTCCTTGTGGTC
runnx1
(Danio rerio)
CGTCTTCACAAACCCTCCTCAA
GCTTTACTGCTTCATCCGGCT
cmyb (Danio
rerio)
GAACGGCTACGGTGGCTGG
CGTTATTTGGGCTGTTTGTGTTGC
gapdh (Danio CAGGCATAATGGTTAAAGTTGGTA CATGTAATCAAGGTCAATGAATGG
rerio)
Supplementary Table 2: Primers used for Axin mutation constructs
Mutants Primers
Forward primers (F), Reverse primers (R)
R765A
CACCCTGGTGGCGGGCCGCGCTGTCAC
GTGACAGCGCGGCCCGCCACCAGGGTG
GCCAGTTCAAGGCGCTGCTGACCAAA
E776A
TTTGGTCAGCAGCGCCTTGAACTGGC
GCTGCTGACCAGTAAGGGCAGCTACAG
K780S
CTGTAGCTGCCCTTACTGGTCAGCAGC
GAGGACGAGGCCCGCCTGCCCGTCTTTG
V810R
CAAAGACGGGCAGGCGGGCCTCGTCCTC
resistant to Axin
siRNA
CTGAAGCTGGCAAGAGCAATCTACAGGAAGTACATTC
GAATGTACTTCCTGTAGATTGCTCTTGCCAGCTTCAG
Supplementary Table 3: siRNA sequences for the targeted genes as indicated
Knockdown constructs
Target sequences (5’-3’)
Si-Axin
CGAGAGCCAUCUACCGAAATT
Si-Axin2
AGACGAUACUGGACGAUCATT
NC ( negative control)
UUCUCCGAACGUGUCACGUTT
Supplementary Table 4: Small molecule screening data
Category
Parameter
Description
Assay
Type of assay
Cell-based
Target
Wnt/β-catenin signaling pathway
Primary measurement
Detection of Firefly luciferase enzyme activities and GFP
expression
TOPFlash (Millipore) and Luciferase Assay (Roche)
Key reagents
Assay protocol
Library size
HEK293T cells were seeded in 48-well plates. Each well
of HEK293T cells was transfected with 125 ng of
plasmids in total, including 10 ng of TOPFlash and 115 ng
of LacZ plasmid. 18 hrs later, cells were treated for 1 hr
with small molecule (10 µM) followed by control
conditioned medium (CM) or Wnt3a CM plus the same
dose of small molecule for additional 6 hrs before
luciferase activity assays. The luciferase enzyme activities
were determined for Wnt signaling activity; And, GFP
expression levels were determined for normalization.
The postive small molecule only impacted luciferase
enzyme activities and did not impact GFP expression
levels.
approximately 200
Library composition
synthetic chemical compounds
Source
Additional comments
State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences
no
Format
48-well, Corning 3548
Concentration(s) tested
10 µM, 1% DMSO
Plate controls
NC043
Reagent/ compound dispensing system
Manual
Detection instrument and software
Synergy 2 (BioTek)
Assay validation/QC
NC043 inhibits Wnt signaling.
Correction factors
no
Normalization
luciferase enzyme activities were normalized by GFP
expression levels.
no
Additional comments
Library
Screen
Additional comments
Post-HTS analysis
Hit criteria
Hit rate
Additional assay(s)
TOPFlash Fluc/GFP ratio > 2 standard deviations from
the mean; or TOPFlash Fluc/GFP ratio < 0.5 standard
deviations from the mean
Approximately 0.5%
Confirmation of hit purity and structure
FOPflash reporter assay in HEK 293 Cells. TOPflash Wnt
signaling reporter assay with LiCl in HEK 293 Cells, and
immunoblotting for intra-cellular levels of the cytosolic
and nuclear β-catenin.
Compounds were verified analytically.
Additional comments
no
Supplementary References
1
Wang, W., Liu, H., Wang, S., Hao, X. & Li, L. A diterpenoid derivative
15-oxospiramilactone inhibits Wnt/beta-catenin signaling and colon cancer cell
tumorigenesis. Cell Research 21, 730-40 (2011).
Supplementary Notes
Synthesis and information of HLY78 and its analogs.
5-methyl-4-vinyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine (HLY72) -The solution of (+)-lycorine (300 mg, 1 mmol) in DMF (10 mL) was put into a
round-bottomed flask, following the CH3I (400 μL, 2 mmol) and stirred at room
temperature for 12 hrs. The reaction solution was evaporated to remove DMF and
then was charged with potassium tert-butoxide (PTB, 1.1 g, 10 mmol) and T-BuOH
(TBA, 10 mL). The mixture was heated to 90 °C and stirred for 4 hrs. The mixture
was diluted in 50 ml saturated NH4Cl and Then with EtO2 (20 mL) for twice and the
organic phase was washed with saturated NH4Cl, brine, dried over MgSO4, filtered,
and concentrated. The residue was purified by column chromatography with
petroleum ether-EtOAc (5:1) to give HLY72 as pale yellow solid (240 mg, yield 90%).
mp: 168-170 °C; 1H NMR (400 MHz, CDCl3): δ 7.58 (d, J = 7.7 Hz, 1H), 7.47 (d, J =
7.7 Hz, 1H), 7.26 (dt, J = 10.7, 7.1 Hz, 2H), 7.16 (t, J = 7.7 Hz, 1H), 6.72 (s, 1H),
5.99 (s, 2H), 5.75 (d, J = 17.8 Hz, 1H), 5.32 (d, J = 11.1 Hz, 1H), 4.03 (s, 2H), 2.51 (s,
3H); 13C NMR (100 MHz, CDCl3) : δ147.4 (C), 145.1 (C), 133.4 (CH), 133.2 (C),
129.2 (C), 126.4 (C), 125.8 (C), 124.9 (CH), 124.3 (CH), 122.7 (CH), 114.3 (CH2),
107.1 (CH), 103.6 (CH), 100.9 (CH2), 54.8 (CH2), 41.5 (CH), ESI+MS (m/z): 266
[M+H]+.
4-ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine (HLY78) -HLY72 (27 mg, 0.1 mmol) and TsNHNH2 was dissolved in THF/H2O (5 mL, 4:1) and
the solution was heated to refulx for 4 h then the mixture was cooled down and
charged with NaOAc (73.8 mg, 0.3 mmol) followed with NH4Cl (10 ml, 2 M). The
solution was extracted with Et2O (30 mL) for twice. The organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by
column chromatography with petroleum ether-EtOAc (20:1) as eluent to afford
HLY78 as a solid (25 mg, yield 95%). mp: 159-160 °C; 1H NMR (400 MHz, MeOD) :
δ7.51 (t, J = 8.4 Hz, 1H), 7.28 (d, J = 6.4 Hz, 2H), 7.20 (t, J = 7.9 Hz, 2H), 6.75 (s,
1H), 6.01 (s, 1H), 4.01 (s, 2H), 2.83 (q, J = 7.5 Hz, 2H), 2.50 (s, 3H), 1.32 (dd, J =
15.7, 8.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) : δ147.3 (C), 147.1 (C), 145.4 (C),
139.4 (C), 129.3 (C), 127.7 (CH), 126.6 (C), 126.3 (C), 124.6 (CH), 121.0 (CH),
107.2 (CH), 103.7 (CH), 100.9 (CH2), 55.2 (CH2), 41.02 (CH), 23.1 (CH2), 14.8
(CH3), HREIMS (m/z): [M]+ calcd for C17H17NO2, 267.1259; found, 267.1261.
4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diol (HLY119) -- A dry
round-bottomed flask was charged with HLY78 (55 mg, 0.2 mmol) and CH2Cl2 (10
mL). The reactor was then cooled to -78 °C and BBr3 (200 μL, 0.4 mmol) was added.
The mixture was stirred for 6 h and diluted in 50 mL saturated NaHCO3. It was
extracted with CH2Cl2 (20 mL) for twice. The organic layer was washed with brine
and concentrated. The residue
was purified by column chromatography with
chloroform-methanol (20:1) as eluent to give 5 as a pale yellow solid (35.7 mg, yield
70%). 1H NMR (400 MHz, CDCl3) : δ7.46 (d, J = 7.1 Hz, 1H), 7.27 (d, J = 2.7 Hz,
1H), 7.17-7.11 (m, 2H), 6.75 (s, 1H), 3.96 (s, 2H), 2.79 (q, J = 7.5 Hz,2H), 2.47 (s,
3H), 1.28 (dd, J = 14.7, 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) : δ143.8 (C), 143.0
(C), 139.4 (C), 129.0 (C), 127.6 (CH), 125.7 (C), 125.5 (C), 124.7 (CH), 120.9 (CH),
113.8 (CH), 113.4 (C), 110.4 (CH), 54.6 (CH2), 41.2 (CH3), 23.1 (CH2), 14.8 (CH3);
ESI+MS (m/z): 256 [M+H]+ .
5,6-Dihydrobicolorine (Hss49a) -- This compound is a alkaloid which was
isolated from lycoris rasiata. 1H NMR (CDCl3, 400 MHz) : δ7.30 (m, 1H,), 7.02 (s,
1H, H-7), 7.01 (m, 1H,), 6.85 (m, 1H), 6.76 (m, 1H), 6.69 (s, 1H, H-10), 6.01 (s,
2H, ,-OCH2O-), 4.28-4.19 (m, 2H, H-6); 13C NMR (CDCl3, 400 MHz) : δ147.6, (C)
147.5 (C), 146.6 (C), 133.9 (C), 131.1 (C), 129.9 (CH), 129.0 (CH), 118.1 (CH),
111.0 (CH), 110.9 (C), 110.2 (CH), 109.9 (CH), 101.3 (CH2, -OCH2O-), 63.8 (CH2),
30.8 (CH3); ESI+MS (m/z): 258 [M+H]+.
5,7-dihydro-4H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridine (HLY103)
-- Lycorine (28.7mg, 0.1mmol) was dissolved in DMF (2 mL) then added Burgess
regant (47.6mg, 0.2mmol). The mixture was strried at 50°C for 2 hrs under N2 then
concentrated in vacuo, and the residue was purified by column chromatography with
petroleum ether-EtOAc (100:1) to give HLY103 as a colorless solid (17.5mg, 70%).
1
H NMR (400 MHz, CDCl3) : δ7.27 (t, J = 5.5 Hz, 1H), 7.18 (d, J = 9.8 Hz, 1H,),
7.01 (d, J = 7.3 Hz, 1H), 6.77 (t, J = 7.5 Hz, 1H), 6.63 ( s, 1H), 5.96 (s, 2H), 4.06 (s,
2H), 3.32 (t, J = 7.9 Hz, 2H), 3.02 (t, J = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) :
δ149.5 (C), 147.4 (C), 146.4 (C), 128.8 (C), 128.4 (C), 126.0 (C), 123.4 (C), 123.1
(CH), 119.6 (CH), 119.5 (CH), 119.3 (CH), 107.4 (CH), 101.0 (CH2), 55.4 (CH2),
53.5 (CH2), 29.0 (CH2); ESI-MS (m/z): 250 [M-H]-.
Synthesis and information of HLY179 and its intermediates.
4-ethyl-5-methyl-8,9-bis(prop-2-tri-azole-ethylamineoxy)-5,6-dihydrophenan
thridine (HLYC60) -- To HLYC175 (9) (22 mL, 0.25 mmol) in H2O/tBuOH (2mL,
1:1) was added HLY165a (66.0 mg, 0.2 mmol), followed by CuSO4 (3.0 mg) and
sodium ascorbate solution (50 μL, 1 M solution). The solution was stirred for 15 hrs at
r.t. then was concentrated in vacuo, and the residue was purified by column
chromatography with chloroform-methanol (9:1) to give HLYC60 as a waxy stuff (60
mg, 55%).1H NMR (400 MHz, CDCl3) : δ7.78 (m, 1H), 7.51-7.41 (m, 3H), 7.15-7.08
(m, 3H), 5.23-5.16 (m, 8H), 4.52-4.43 (m, 2H), 4.42 (s, 1H), 3.50 (s, 2H), 3.45 (s, 2H),
2.78 (d, J = 8 Hz, 2H), 2.44 (s, 3H), 1.27 (t, J = 8.0 Hz, 3H); 13C NMR (150 MHz,
CDCl3) : δ147.17 (C), 144.05 (C), 139.44 (C), 135.99 (C), 134.83 (CH), 134.03 (C),
128.80 (C), 128.44 (C), 124.75 (CH), 124.51 (CH), 121.62 (CH), 121.55 (CH), 115.64
(C), 115.55 (C), 110.23 (CH), 109.84 (CH), 64.27 (CH2), 63.26 (CH2), 54.96 (CH2),
50.78 (CH2), 50.61 (CH2), 41.65 (CH3), 40.29 (CH2), 39.41 (CH2), 29.91 (CH2), 15.27
(CH3); HREIMS (m/z): [M]+ calcd for C26H33N9O2, 503.2757; found, 503.2741.
(R,S,S)-N,N'-(2,2'-(4,4'-(4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diyl
)bis(oxy)bis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1-diyl))bis(5-((
3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide)
(HLY179) -- To HLYC177 (68.6 mg, 0.22 mmol) in H2O/tBuOH (2 mL, 1:1) was
added HLY165a (10) (33.1 mg, 0.1 mmol), followed by CuSO4 (1.8 mg) and sodium
ascorbate solution (30 μL, 1 M solution). The solution was stirred for 12 hrs at 50 °C.
then was concentrated in vacuo, and the residue was purified by column
chromatography with chloroform-methanol (9:1) to give HLY179 as a colorless solid
(57.3 mg, 60%). mp: 196-197 °C; 1H NMR (400 MHz, MeOD) : δ7.96 (s, 1H), 7.95 (s,
1H), 7.58-7.51 (m, 1H), 7.46 (s, 1H), 7.19- 7.11 (m, 2H), 6.97 (s, 1H), 5.24 (s, 2H),
5.21 (s, 2H), 4.55-4.48 (m, 6H), 4.46-4.39 (m, 2H), 4.31-4.22 (m, 2H), 4.02-3.94 (m,
2H), 3.71-3.62 (m, 4H), 3.35-3.26 (m, 4H), 2.80-2.72 (m, 2H), 2.43 (s, 3H), 2.20-2.04
(m, 4H), 1.75-1.46 (m, 8H), 1.39-1.32 (m, 4H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR
(100 MHz, CDCl3) : δ176.42, 170.23, 165.98, 163.95, 153.09, 152.51, 152.04, 149.87,
149.69, 147.94, 147.35, 147.29, 144.84, 143.41, 143.20, 142.15, 138.90, 128.20,
127.50, 126.39, 126.01, 125.20, 124.26, 123.96, 120.57, 119.87, 110.56, 108.66,
92.87, 89.43, 62.94, 62.41, 61.37, 59.65, 55.06, 54.05, 41.63, 40.49, 39.58, 38.68,
34.89, 27.76, 27.51, 24.85, 22.55, 14.04, 10.24; HREIMS (m/z): [M]+ calcd for
C46H61N13O6S2, 955.4307; found, 955.4321.
4-ethyl-5-methyl-8,9-bis(prop-2-ynyloxy)-5,6-dihydrophenanthridine
(HLY165a) -- HLY119 (26 mg, 0.1 mmol) dissolved in dry THF (3 mL), following
added NaH (10 mg, 0.4 mmol) and propargyl bromide (20 μL, 0.25mmol). The
mixture was stirred at room temperature for 24 hrs and then quenched by water (50
mL) in an ice bath. The reaction solution was evaporated to remove THF and
extracted with CH2Cl2 (30 mL) for twice. The organic layer was washed with
saturated NaHCO3, brine, dried over MgSO4, filtered, and concentrated. The residue
was purified by column chromatography with petroleum ether-EtOAc (9:1) as eluent
to afford HLY165a as a solid (26.5 mg, yield 80%).1H NMR (400 MHz, CDCl3) : δ1H
NMR (400 MHz, CDCl3) δ: 7.54 (d, J = 6.9 Hz, 1H), 7.46 (s, 1H), 7.23-7.12 (m, 2H),
6.91 (s, 1H), 4.83 (s, 2H), 4.80 (s, 2H), 4.02 (s, 2H), 3.09-2.99 (m, 2H), 2.86-2.74 (m,
2H), 2.47 (s, 3H), 1.29-1.17 (m, 3H); 13C NMR (100 MHz, CDCl3) δ: 147.4 (C),
146.8 (C), 145.6 (C), 139.5 (C), 128.9 (C), 127.9 (CH), 126.8 (C), 126.3 (C), 124.6
(CH), 121.0 (CH), 113.1 (CH), 110.5 (CH), 78.6 (C), 78.4 (C), 75.9 (CH), 75.8 (CH),
57.2 (CH2), 56.9 (CH2), 54.8 (CH2), 41.31 (CH3), 23.1 (CH2), 14.8 (CH3); ESI-MS
(m/z): 330[M-H]-.
2,5-dioxopyrrolidin-1-yl-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]i
midazol-4-yl)pentanoate (HLYC176 (11)) -- A solution of D-biotin (24 mg, 0.1
mmol) in DMF (10 mL) was added with pyridine (2 mL) and DCC (41 mg, 0.2 mmol).
The mixture was stirred for 0.5 h and then charged with N-Hydroxusuccinimide (13.8
mg, 0.12 mmol). The solution was stirred for 24 hrs at r.t. then was concentrated in
vacuo, and the residue recrystallized from propanol to give HLYC176 as a colorless
solid (22 mg, 65%). mp: 204-206 °C; 1H NMR (400 MHz, [D6]DMSO) : δ6.42 (s, 1
H, 3-NH), 6.36 (s, 1 H, 1-NH), 4.27-4.32 (m, 1 H, 6a-H), 4.11-4.16 (m, 1 H, 3a-H),
3.06-3.12 (m, 1 H, SCH), 2.78-2.85 (m, 5 H, CH2CH2 (succinyl), SCH2), 2.66 (t, J =
7.3 Hz, 2 H, 2'-H), 2.57 (d, J = 11.4 Hz, 1 H, SCH2), 1.58-1.68 (m, 3 H, 3'-H, 5'H),
1.35-1.54 (m, 3 H, 4'-H, 5'-H); 13C NMR (100 MHz, [D6]DMSO) : δ170.3 (N(CO)2),
168.9 (CO2), 162.7 ((HN)2CO), 61.0 (C-3a), 59.2 (C-6a), 55.2 (SCH), 39.9 (SCH2),
30.0 (C-2'), 27.8 (C-4'), 27.6 (C-5'), 25.4(CH2CH2 (succinyl)), 24.3 (C-3'); ESI+MS
(m/z): 342[M+H]+.
N-(2-azidoethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol4-yl)pentanamide (HLYC177) -- HLYC176 (34 mg, 0.1 mmol) and HLYC175 (17
mg 0.2 mmol) was dissolved in DMF (2 mL) and then Et3N (12 mg, 1.2 mmol) was
added. The solution was stirred for 24 hrs at r.t. then was concentrated in vacuo, and
the residue was purified by column chromatography with chloroform-methanol (20:1)
to give HLYC177 as a colorless waxy stuff (23 mg, 70%). 1H NMR (400 MHz,
[D6]DMSO) δ: 8.03 (t, J = 5.3 Hz, 1 H,CONH), 6.42 (s, 1 H, 3-NH), 6.35 (s, 1 H,
1-NH), 4.26-4.32 (m, 1 H, 6a-H), 4.08-4.14 (m, 1 H, 3a-H), 3.31 (d, J = 7.6 Hz, 2 H,
CH2N3), 3.19-3.24 (m, 2H, CH2CH2N3), 3.05-3.11 (m, 1H, SCH), 2.80 (dd, J = 12.4,
5.1 Hz, 1 H, SCH2), 2.56 (d, J = 12.9 Hz, 1H, SCH2), 2.06 (t, J = 7.3 Hz, 2H, 2'-H),
1.55-1.65 (m, 1H, 5'-H), 1.39-1.55 (m, 3H, 3'-H, 5'-H),1.20-1.38 (m, 2H, 4'-H); 13C
NMR (100 MHz, [D6]DMSO) δ: 172.4 (CONH), 162.7 ((HN)2CO), 61.0 (C-3a), 59.2
(C-6a), 55.4 (SCH), 50.0 (CH2N3), 39.9 (SCH2), 38.1 (CH2CH2N3), 35.1 (C-2'), 28.2
(C-4'), 28.0 (C-5'), 25.2 (C-3'); HRESI+MS (m/z): [M+H]+ calcd for C12H20N6O2S +
H, 313.14412; found, 313.14419.
2-Azidoethylamine (HLYC175) -- 2-Bromoethylamine hydrobromide (500 mg,
2.44 mmol) and sodium azide (475.9 mg, 7.32 mmol) was dissolved in H2O (2 mL),
The mixture was heated to 75 °C and stirred for 21 hrs then was cooled down to 0 °C.
KOH (800 mg) and Et2O (2 mL) was added, the solution was extracted with Et2O
(2×10 ml), then concentrated in vacuo, and the residue was purified by column
chromatography with chloroform-methanol (20:1) to give HLYC175 as a colorless
liquid (171 mg, 82%).
1
H NMR (400 MHz, CDCl3) : δ3.30 (t, J = 5.7 Hz, 2 H,
CH2NH2), 2.80-2.84 (m, 2 H, CH2N3), 1.27 (s, 2 H, NH2); 13C NMR (100 MHz,
CDCl3) δ: 54.6 (CH2N3), 41.2 (CH2NH2); ESI+MS (m/z): 87 [M+H]+.
Supplementary Chemical Compound Information
HLY72
1
HLY78
2
HLY119
3
HSS49a
4
HLY179:
5
HLYC60
6

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