Sialyltransferase Inhibitors Suppress Breast Cancer Metastasis

Article abstract of DOI:10.1021/acs.jmedchem.0c01477

We report the synthesis and evaluation of a series of cell-permeable and N- versus O-selective sialyltransferase inhibitors. Inhibitor design entailed the functionalization of lithocholic acid at C(3) and at the cyclopentane ring side chain. Among the series, FCW34 and FCW66 were shown to inhibit MDA-MB-231 cell migration as effectively as ST3GALIII-gene knockdown did. FCW34 was shown to inhibit tumor growth, reduce angiogenesis, and delay cancer cell metastasis in animal models. Furthermore, FCW34 inhibited vessel development and suppressed angiogenic activity in transgenic zebrafish models. Our results provide clear evidence that FCW34-induced sialyltransferase inhibition reduces cancer cell metastasis by decreasing N-glycan sialylation, thus altering the regulation of talin/integrin/FAK/paxillin and integrin/NFκB signaling pathways.

Full text of DOI:10.1021/acs.jmedchem.0c01477

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Sialyltransferase Inhibitors Suppress Breast Cancer Metastasis
Chih-Wei Fu, Han-En Tsai, Wei-Sheng Chen, Tzu-Ting Chang, Chia-Ling Chen, Pei-Wen Hsiao,
and Wen-Shan Li*
Cite This: J. Med. Chem. 2021, 64, 527−542
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ABSTRACT: We report the synthesis and evaluation of a series of cell-permeable and N- versus
O-selective sialyltransferase inhibitors. Inhibitor design entailed the functionalization of
lithocholic acid at C(3) and at the cyclopentane ring side chain. Among the series, FCW34
and FCW66 were shown to inhibit MDA-MB-231 cell migration as effectively as ST3GALIII-
gene knockdown did. FCW34 was shown to inhibit tumor growth, reduce angiogenesis, and
delay cancer cell metastasis in animal models. Furthermore, FCW34 inhibited vessel
development and suppressed angiogenic activity in transgenic zebrafish models. Our results
provide clear evidence that FCW34-induced sialyltransferase inhibition reduces cancer cell
metastasis by decreasing N-glycan sialylation, thus altering the regulation of talin/integrin/FAK/
paxillin and integrin/NFκB signaling pathways.
application in in vitro studies or for in vivo use to repress
cancer metastasis. With few exceptions,26−29 inhibitor designs
have concentrated on creating tight-binding substrate ana-
logues.30−39 Because most of these inhibitors are not cell
permeable, their use in biological and clinical applications is
restricted. To overcome the availability of STs, recent advances
in the design of various expression systems for recombinant
enzyme production open up the opportunity to screen
compounds against a wider panel of ST enzymes.40 In
addition, inhibitors that can preferentially act on ST isozymes
have not yet been reported. Thus, the discovery of cell-
permeable inhibitors that can target specific ST isozymes in
vivo, and thus, minimize potential side effects, is essential for
the development of effective chemical therapeutics to prevent
metastasis.
INTRODUCTION
■
Metastasis is the primary cause of death in cancer patients. The
multistep process by which cancer cells migrate from the
tumor site to reach and proliferate in secondary organs is
known as the metastatic cascade. During this process, cancer
cells detach from the tumor, move through basement
membrane barriers and the extracellular matrix (ECM), enter
the vascular system, and adhere to a new site where they
proliferate.1−3 Inhibitors that can block this process are
urgently needed.
The known correlation between metastasis and abnormally
high levels of N- and O-sialylation of cancer-cell surface
glycoproteins4−8 directed our attention to the development of
chemical compounds to inhibit glycoprotein sialylation, which
is catalyzed by members of the sialyltransferase (ST) family.9
The ST family comprises more than 20 glycoprotein-specific
and glycolipid-specific, α2,3-, α2,6-, and α2,8-linkage trans-
ferring enzymes.10,11 The ST family is known to play an
important role in tumor cell progression, invasion and
metastasis,12−15 especially in breast cancer.16−18 Previous
studies have shown that overexpression of genes encoding
the sialyltransferases ST6GALI and ST3GALIV protects tumor
cells against hypoxia, modulates tumor progression, adhesion
and migration in pancreatic cancer cell lines, and enhances the
metastatic potential of cancer cells in vivo.19−23 Furthermore,
the sialyltransferase ST6GALNACV is known to mediate
breast cancer metastasis to the brain.24
RESULTS AND DISCUSSION
■
Design of Isozyme-Specific and Cell-Permeable ST
Inhibitors by Employing the “LCA-Extension Strategy”.
In an earlier work, we discovered that the lithocholic acid
(LCA)41 component of the natural product soyasaponin I
serves as an ideal platform for the development of cell-
permeable ST inhibitors.42−44 Specifically, the LCA adducts
Lith-O-Asp and AL10 depicted in Figure 1 were shown to
Received: August 24, 2020
Published: December 28, 2020
Despite ample documentation of the importance of post-
translational sialylation of glycoproteins and glycolipids to the
regulation of immune cells25 or cell−cell adhesion and to the
regulation of tumor cell metastasis and invasion, an inhibitor
with ST isozyme preference has yet to be identified for
https://dx.doi.org/10.1021/acs.jmedchem.0c01477
© 2020 American Chemical Society
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Figure 1. Illustration of the lead ST inhibitors discovered in previous work (left panel) and two second-generation inhibitors examined in the
current work. The blue line defines the lead-inhibitor-binding site of the ST, whereas the red line depicts a hypothetical subsite to be filled by the
PEG moiety of the second-generation inhibitors. NBD is the abbreviation for nitrobenzoxadiazole.
Table 1. Inhibitory Potency and Specificity of Sialyltransferase Inhibitors
Cpd
IC50 (μM)
selectivity ratio
g
g
g
α2,3-N-ST3GALIII
α2,6-N-ST6GALI
α2,3-O-ST3GALI
α2,3-O-/α2,3-N-
α2,3-O-/α2,6-N-
a
d
2aA
2bA
2cA (FCW34)
2dA (FCW66)
2eA
3aB
3bB
3cB
3dB
3eB
4aC
4bC
4cC
4dC
4eC
Lith-O-Asp
AL10
LCAG
Soyasaponin I
0.8 0.1
4.2 0.3
4.33 0.04
3.6 0.4
4.90 0.08
5.0 0.2
7.2 0.1
6.7 0.3
5.4 0.5
5.7 0.4
7.5 0.4
5.6 0.2
5.1 0.1
4.6 0.4
5.0 0.4
5.7 0.4
15.8 0.1
1.5 0.5
0%
>625
>119
>116
>139
>102
>99
>70
>74
>94
>88
>67
>89
>97
>109
>101
>88
3
a
b
b
b
d
e
0.77 0.02
1.74 0.09
1.01 0.07
1.0 0.1
c
0% (74%)
>649 (<1299)
a
d
e
0% (60%)
>287 (<575)
a
d
e
0% (50%)
>495 (≤990)
a
d
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
39
>516
a
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
a
a
a
a
a
a
a
a
a
12
0.9 0.1
c
1
3
15
13.2 0.6
15% (41%)
59% (83%)
9
f
a
b
b
0%
c
f
a
68%
a
b
c
d
Inhibition percentage was measured at 500 μM. Inhibition percentage was measured at 1 mM. Not determined. The selectivity ratio, in
parentheses, is expressed as the value of 500 μM/individual IC50 from α2,3-N-ST3GALIII without correction for the contribution of IC50 from
e
α2,3-O-ST3GALI. The selectivity ratio, in parentheses, is expressed as the value of 1000 μM/individual IC50 from α2,3-N-ST3GALIII without
f
g
correction for the contribution of IC50 from α2,3-O-ST3GALI. Inhibition percentage was measured at 100 μM. The selected ST isozymes, rat
α2,3-N-ST3GALIII (CalBiochem #566218), human α2,6-N-ST6GALI (R&D systems #7620-GT), and rat α2,3-O-ST3GALI (CalBiochem
#566227), were used in this study.
exhibit low micromolar inhibitory activity against several
sialyltransferases, ST3GALIII, ST6GALI, and ST3GALI
(Table 1; the IC50 range is 12−39 and 0.9−13.2 μM,
respectively), as well as to display significant antimetastatic
properties.42−44 To increase the affinity for a target protein,
chemically designed compounds will be used to probe
biologically relevant chemical space and improve the potency
against different isoforms of enzymes based on a structure-
based design approach. From structural minimization and
visualization of AL10 and the designed ST inhibitors, we
observed an extended space with hydrophobic and hydrophilic
amino acid residues, such as P320(B), P259(B), V261(A),
W302(B), H370(B), S322(B), S189(B), C353(B), G324(B),
A190(B), and N212(B) in two asymmetric polypeptide chains
(A/B) of ST6GALI (PDB 6QVT), where the polyethylene
glycol (PEG) moiety of designed inhibitors might develop
additional interactions (see the Molecular Docking section and
Supporting Information). In the present study, the lead
inhibitor Lith-O-Asp was modified to extend the cyclopentane
ring side chain with one or more ethylene glycol units and
elaborate the C(3) aspartate ester moiety, as is illustrated in
Figure 1 with the Lith-O-Asp analogues FCW34 and FCW66.
By employing this “LCA-extension strategy”, we set out to
generate isozyme-specific, cell-permeable ST inhibitors. The
three series of compounds prepared are shown in Scheme 1.
The “2A” series, which includes FCW34 and FCW66,
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a
Scheme 1. Synthesis of ST Inhibitors
a
Reagents and conditions: (a) (i) Fmoc-L-Asp(O-tBu)-OH, DCC, DMAP, CH2Cl2, RT, 8 h, 50%; (ii) 1a−1e N3 linker, DCC, DMAP, CH2Cl2,
RT, 3 h, (10a 88%, 10b 86%, 10c 80%, 10d 71%, 10e 75%); (b) (i) DBU, CH2Cl2, RT, 2 h; (ii) NBD-Cl, NaHCO3, THF-EtOH, RT, 2 d or p-
nitrobenzoic acid, 2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetic acid, HBTU, DIPEA, CH2Cl2, RT, 3 h (two steps: 12aA 45%, 12bA 32%, 12cA
34%, 12dA 31%, 12eA 32%, 12aB 91%, 12bB 87%, 12cB 72%, 12dB 73%, 12eB 70%, 12aC 88%, 12bC 83%, 12cC 75%, 12dC 75%, 12eC 68%);
(c) TFA, H2O, CH2Cl2, RT, 2 h (2aA 85%, 2bA 82%, 2cA(FCW34) 91%, 2dA(FCW66) 88%, 2eA 85%, 3aB 80%, 3bB 82%, 3cB 90%, 3dB 82%,
3eB 84%, 4aC 90%, 4bC 82%, 4cC 85%, 4dC 80%, 4eC 82%).
possesses the C(3) N-nitrobenzoxadiazole (NBD)-L-aspartate
ester moiety, whereas the “3B” series possesses a N-p-
nitrophenyl (NP)-L-aspartate ester moiety, and the “4C” series
a N-7-methoxycoumarin (MC)-L-aspartate ester moiety. With-
in each of the three series, the annotations a−d correspond to
the number of ethylene glycol (EC) units that make up the
PEG moiety. These three moieties, NBD, NP, and MC, were
uncovered through the initial screening of SAR study using the
drug-like substructure. The inclusion of NBD moiety is based
on the substructure of benzoxadiazole group from Isradipine,
approved by the FDA for medical use in 1989.
ST3GALI with compounds Lith-O-Asp, 2aA−2eA, 3aB−
3eB, and 4aC−4eC was monitored using the UPLC-based
inhibition assay. The IC50 values determined for the tight-
binding ST isozyme−inhibitor pairs and the % inhibition
determined for the weak-binding ST isozyme−inhibitor pairs
are reported in Table 1 (lithocholylglycine, LCAG, and
soyasaponin I as the positive control). The series 2aA−2eA
displayed an order of magnitude tighter binding to α2,3-N-
ST3GALIII (IC50 range is 0.8−1.7 μM) compared to Lith-O-
Asp (IC50 = 12 μM). In contrast, while the IC50 value
determined for Lith-O-Asp inhibition of α2,3-O-ST3GALI is
39 μM, the IC50 values measured for 2aA−2dA are ∼1 mM
and for 2eA IC50 > 1 mM. Likewise, whereas 2aA−2eA, 3aB−
3eB, and 4aC−2eC display ∼2−3-fold tighter binding to α2,6-
N-ST6GALI (IC50 values: 4−7 μM) than that observed with
Lith-O-Asp (IC50 value: 16 μM), they do not show inhibition
of α2,3-O-ST3GALI when tested at a concentration of 1 mM,
in contrast to IC50 = 39 μM measured for Lith-O-Asp. These
results indicate that 2aA−2eA, 3aB−3eB, and 4aC−2eC can
be used to inhibit the two N-glycoprotein sialyltransferases
α2,3-N-ST3GALIII and α2,6-N-ST6GALI without impacting
the activity of the O-glycoprotein sialyltransferase α2,3-O-
ST3GALI.
Synthesis of ST Inhibitors. Steps taken toward the
chemical synthesis of 2aA−2eA, 3aB−3eB, and 4aC−4eC are
summarized in Schemes 1, S1 and S2 (for chemical structures:
see Supporting Information Chart S1). Synthetic intermediates
10a−10e were prepared via dicyclohexylcarbodiimide (DCC)-
promoted esterification of LCA with Fmoc-Asp(t-Bu)-OH and
with PEG-N3 linkers of varying lengths (1a−1e, see the
Supporting Information). Removal of the Fmoc-protecting
group from 10a−10e with 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to generate the free amine was followed by amination
with NBD-Cl under basic conditions to yield 12aA−12eA, or
by amide coupling with p-nitrobenzoic acid or 2-(7-methoxy-2-
oxo-2H-chromen-4-yl)acetic acid, using HBTU as the coupling
reagent, to furnish 12aB−12eB or 12aC−12eC. Removal of
the t-Bu-protecting group with TFA provided the final
products 2aA−2eA, 3aB−3eB, and 4aC−4eC.
The design ideas were inspired by the results of previous
inhibitory potency studies of LCA and its analogues toward ST
isozymes.10 Further employing the “LCA-extension strategy
leads to the discovery of target compounds, FCW34 and
FCW66. The complete structure−activity relationship and
optimization are depicted in Figure S1. Our initial studies
focused on the effect of C(3) amino acid moiety and observed
Activity and Isozyme Selectivity of ST Inhibitors In
Vitro. The inhibition of selected ST isozymes rat α2,3-N-
ST3GALIII, human α2,6-N-ST6GALI, and rat α2,3-O-
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Figure 2. A comparison of the effects of siRNA-induced ST3GALIII gene knockdown with those of FCW34- and FCW66-induced ST3GALIII
inhibition on the migration ability of MDA-MB-231 cells in vitro. (A) Images of cells transfected with three ST3GALII siRNA A, B, and C
(STEALTH siRNAs, HSS143938, HSS143939, and HSS143940 from Thermo Fisher with different target sequences), respectively, for 72 h or
exposed to 10 or 20 μM FCW34 or FCW66 and allowed to migrate for 16 h (see the Supporting Information). (B) A plot of results from (A) in the
chart format. All data were expressed as mean SEM of three experiments (*P < 0.05, **P < 0.01).
LCA with a specific amino acid sequence, such as L-Asp-LCA,
L-Glu-LCA, D-Glu-LCA, L-Asp-L-Asp-LCA, and L-Asp-L-Asp-
Gly-Gly-LCA, displaying potent inhibitory activity against
human STs, α2,3-N-ST, and α2,6-N-ST, respectively.28,29
Building on the optimal structure of L-Asp-LCA, we extended
the additional groups in the N-terminal of Asp and found the
heterocyclic moiety (NBD, NP, and MC) exhibiting greater
inhibitory activities against α2,3-N-ST, α2,3-O-ST, and α2,6-
N-ST compared to that of L-Asp-LCA (Lith-O-Asp),
suggesting that perhaps a heterocyclic moiety at the N-
terminal position could occupy an interactive region in the
active site pocket of ST isozyme. For example, AL10,
possessing an NBD group at the N-terminal position, displayed
improved inhibitory potency against α2,3-N-ST, α2,3-O-ST,
and α2,6-N-ST with respective IC50 values of 0.9, 13.2, and 1.5
μM (Table 1), which are with 3−13-fold increased potency
over L-Asp-LCA (Lith-O-Asp). Besides redesigning the potent
ST inhibitors based on previous results, the approach to solely
tune the steroidal core structure (Figure S1), the B- and C-
ring-modified homolactones and homolactams,26 was accom-
plished to uncover several improved ST inhibitors, as
compared to the parent compound LCA, indicating that the
modified homolactones and homolactams are located inside
the binding pocket and the B- and C-rings are proximal to the
hydrophilic region in the active site of the ST isozyme. As we
knew, the structure of LCA belongs to the “tadpole scaffold”
and relatively few positions are available where additional
substituents can be added. To explore the target-binding site
for further binding regions to achieve additional binding
interactions, we use the “LCA-extension strategy” to extend the
cyclopentane ring side chain with one or more ethylene glycol
units (n = 1−5). This action leads to the discovery of ST
inhibitors with isozyme preference, FCW34 and FCW66
(Figure 1 and Table 1). According to the SAR results in
(Figure S1), the cyclopentane ring side chain with one or more
ethylene glycol units play an important role in ST isozyme
selectivity. Perhaps the extension of the cyclopentane ring side
chain is allowed in the active sites of α2,3-N-ST and α2,6-N-
ST; however, this action cannot be accommodated by α2,3-O-
ST.
ST Inhibitors Inhibit the Migration Ability of Breast
Cancer Cells via Suppression of α2,3-N-ST3GALIII-
Catalyzed N-Glycoprotein Sialylation. The patients of
triple-negative breast cancer (TNBC) respond poorly to the
treatment of both endocrine therapy and HER2-targeted
therapy. The treatments available against TNBC are mainly
surgery, radiation, and chemotherapy in clinic practice. To
overcome the limited treatment options, we plan to accelerate
the development of an innovative ST inhibitor to gain new
targeted therapies for TNBC patients. The MDA-MB-231 cell
line is used in this study as it represents one of the important
cell lines of TNBC. Our next goal was to determine if the
second-generation ST inhibitors 2aA−2eA also inhibit breast
cancer cell motility. Before that, the antiproliferative effect of
compounds 2aA−2eA, 3aB−3eB, and 4aC−4eC was tested
on MDA-MB-231 cells. All of them did not cause an inhibitory
effect on cell growth (>90% survival) at the tested
concentrations (>60 μM). For this purpose, the Boyden
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Figure 3. Effects of FCW34 and positive control, Taxol, on tumor progression and angiogenesis of MDA-MB-231 cells in vivo. Mice bearing the
established MDA-MB-231/Luc tumors (100 mm3) were treated with FCW34 (10 mg/kg/mouse/3 d) or Taxol (10 mg/kg/mouse/3 d) as a
positive control via intraperitoneal injection. (A) Bioluminescence images taken after 8 weeks and (B) graph showing a comparison of the
quantification of photo units between the mice treated with FCW34 after 60 d versus the mice treated with Taxol after 50 d (n = 6 per group).
(C,D) After the tumors had reached the size of ∼100 mm3, the mice were treated with vehicle, FCW34, or Taxol, and subsequently, monitored for
tumor growth. After the mice were sacrificed, the tumors were harvested for further analysis and the blood vessel network in breast cancer tissues
was examined by CD31 immunostaining analysis (E). The number of vessels in the breast cancer tissue counted using the CAST system and
expressed as mean SEM (n = 6 per group, right panel). (F) The proliferation activity examined by Ki-67 immunostaining and quantification of
proliferation index, Ki-67+ cells, in tumor tissues (n = 6 per group, right panel). The asterisks indicate statistical significance (*p < 0.05, **p <
0.01).
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Figure 4. The effects of FCW34 and FCW66 on talin-1 and integrin interaction in cultured MDA-MB-231 cells. (A,B) Colocalization of p-talin-1
with integrin β1 or integrin β3 in MDA-MB-231 cells observed by immunofluorescence analysis. After treatment with DMSO (control), 20 μM
FCW34, or 20 μM FCW66 for 48 h, the cells were permeabilized with Triton X-100 and stained with anti-p-talin-1 (red) and with (A) anti-integrin
β1 (green) or with (B) integrin β3 antibodies (green). (C) Western blots of p-talin-1 immunoprecipitate from MDA-MB-231 cells treated with
DMSO, FCW34, or FCW66 (10 and 20 μM). p-Talin-1 and integrins β1 and β3 were detected using the corresponding antibodies. (D) The
relative amounts of p-talin-1 and integrins β1 and β3 observed in the western blots and reported as mean SEM for triplicate experiments.
Chamber assay was used to measure the impact of exposure of
MDA-MB-231 cells to (20 μM) inhibitor. Based on the results
from this preliminary screen (see Figure S2), 2cA and 2dA
(hereafter referred to as FCW34 and FCW66, respectively)
were identified as having the greatest potency as cancer cell
migration inhibitors. To determine the IC50 values for FCW34
and FCW66, cell motility was monitored over a 16 h period as
a function of their concentration, ranging from 2 to 30 μM.
The results obtained (see Figures S3 and S4) demonstrate that
both inhibitors abrogate serum-induced cell migration in a
concentration-dependent manner and define FCW34 IC50
10.64 0.09 μM and FCW66 IC50 = 12.0 0.1 μM
=
(lithocholylglycine, LCAG, and soyasaponin I as positive
control).
To explore a possible link between FCW34 and FCW66
acting as inhibitors of ST3GALIII and cancer cell motility, we
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examined the effect of α2,3-N-ST3GALIII knockdown by
α2,3-N-ST3GALIII siRNA on cell motility (Figure S5). As
shown in Figure 2A,B, compared to the control, knockdown of
the α2,3-N-ST3GALIII gene significantly inhibited the
migration activity in MDA-MB-231 cells, similar to the
inhibitory effects observed for FCW34 and FCW66 exposure.
Taken together, these findings suggest that FCW34 and
FCW66 might retard cancer cell migration via inhibition of
α2,3-N-ST3GALIII-catalyzed N-glycoprotein sialylation.
ST Inhibitors Inhibit the Migration Ability of Breast
Cancer Cells via Suppression of Integrin Sialylation. Our
next step was to determine if inhibition of integrin sialylation
might be the cause of inhibited mobility in cancer cells exposed
to FCW34 and FCW66. This possibility was suggested by
increasing evidence that protein sialylation plays a critical role
in tumor metastasis, coupled with the knowledge that aberrant
N-glycosylation of integrin, the major cellular receptor of the
extracellular matrix in cell migration, is associated with cancer
cell behavior.45−48 Until now, studies designed to elucidate the
biological effects of integrin sialylation have been restricted by
the limited availability of cell-permeable ST inhibitors. In the
present study, western blot analysis of MDA-MB-231 cell
extracts was carried out using biotinylated Maackia amurensis
Lectin II (MAL II) as the probe for (α-2,3)-linked sialic acid
residues. We observed that FCW34 and FCW66 significantly
suppressed the sialylation of integrin β subunits β1, β3, β4, and
β5 (Figure S6).
FCW34 Exhibits In Vivo Spontaneous Metastasis
Inhibition and Reduces Tumor Growth. Because Taxol
is one of the clinically effective drugs for the treatment of
breast cancer, it is used to treat patients with metastatic breast
cancer. In order to ensure the accuracy and efficacy of animal
experiments, Taxol was selected as the positive control in this
experiment. To determine if FCW34 inhibits tumor growth
and metastasis in vivo, a BALB/c mice model of MDA-MB-
231/Luc was used. As shown in Figure 3A−D, the FCW34- or
Taxol-treated mice displayed profound tumor suppression and
delayed lung metastasis. The results of histological analysis
(Figure S7A) show a significantly smaller number of metastatic
foci in the lungs of FCW34- or Taxol-treated mice than in the
control mice. In addition, analysis of H&E-stained adjacent
lymph node sections revealed fewer metastatic nodules in the
FCW34- or Taxol-treated mice than in the control mice
(Figure S7B). Finally, the results of blood tests and body
weight assessments indicate that treatment of mice with
FCW34 or Taxol is not detrimental to their health (Figure S8).
Taken together, these findings suggest that FCW34 is an
excellent candidate for evaluation as a lead for an
antimetastatic drug for cancer treatment.
marker, Ki-67 nuclear protein.51,52 The synchronized images
and the corresponding proliferation indexes shown in Figure
3F reveal that the accumulation of Ki-67-positive cells in the
FCW34- or Taxol-treated mice is significantly less than that
observed for the control mice. These results indicate that
FCW34 inhibits proliferation and neovascularization, and thus,
tumor progression.
FCW34 and FCW66 Suppress Cancer Metastasis
through Disruption of Talin-Driven Integrin Activation
by Reducing N-Glycan Sialylation and Talin Phosphor-
ylation. We next directed our attention to determining if
FCW34 and FWC66 affect the talin−integrin signaling
pathways. Integrins mediate cell adhesion to the extracellular
matrix, and therefore, play an important role in cancer. Talin is
a key regulator of integrin affinity and avidity.53−55 In order to
maintain integrin in an activated state, the talin N-terminal
head domain must interact with the β integrin cytoplasmic
tail.56,57 In order to determine if N-glycan sialyation affects
integrin-dependent signaling, we used FCW34 and FCW66 to
inhibit the sialyltransferases and immunofluorescence and
coimmunoprecipitation (Co-IP) assays to monitor the
integrin−talin association events. The results obtained and
reported in Figure 4A,B indicates that FCW34 and FCW66
significantly reduce colocalization of integrins β1 and β3 and
phospho-talin-1 (p-talin-1) in MDA-MB-231 cells. Figure
4C,D shows that FCW34 and FCW66 decrease p-talin-1
association with integrins β1 and β3 in a dose-dependent
manner. We interpret these findings to support the proposal
that FCW34 and FCW66 induce the reduction of N-glycan
sialylation and talin phosphorylation and that this might be
critical for the disruption of talin-driven integrin activation and
clustering, and thus, for the suppression of cancer metastasis.
FCW34 and FCW66 Decrease Cell Migration Ability
through Inhibition of Talin/Integrin/FAK/Paxillin and
Integrin/NFκB Signaling Pathways. In light of the
importance of ECM/integrin-directed cell behavior,58−60 we
investigated whether the inhibitor induced the decrease in the
migration ability of cells, and therefore, the reduction of tumor
progression and angiogenesis in mice resulted from the
suppression of talin/integrin/FAK/paxillin signaling or integ-
rin/NFκB signaling.61−64 As shown in the images reported in
Figure S9A, the exposure of MDA-MB-231 cells to FCW34
and FCW66 (0, 10, and 20 μM) results in significant decreases
in the expression levels of integrin isoforms (αν, α5, β1, β3, β4,
and β5). Tyrosine (i.e., Tyr397, Tyr576, Tyr577, and Tyr925)-
phosphorylated focal adhesion kinase (p-FAK) (Tyr397,
Tyr576, Tyr577, and Tyr925) and Tyr118-phosphorylated
paxillin (p-paxillin) were found to be reduced in a dose-
dependent manner by treatment of cells with FCW34 or
FCW66 (Figure S9B). In addition, FCW34 and FCW66
effectively inhibited phosphorylated p-IKKα/β, p-IκBα, and p-
NF-κB (Figure S10), demonstrating that FCW34 and FCW66
regulate the talin/integrin/FAK/paxillin and integrin/NFκB
signaling pathways and suppress breast cancer growth and
angiogenesis in vivo.
Having established that FCW34 treatment suppresses tumor
growth and metastasis in the mouse model, we carried out
further histological analysis of collected tumor tissues to study
the molecular mechanisms of FCW34-induced suppression.
The cluster of differentiation 31 (CD31) adhesion molecule,
also known as platelet-endothelial cell adhesion molecule-1
(PECAM-1), was used as a marker of angiogenesis49,50 to
obtain the immunostaining images shown in Figure 3E. The
images reveal a significantly smaller number of vessels
surrounding tumor nodules in the FCW34-treated mice than
in the control mice. Quantification analysis of vessels
confirmed that angiogenesis was significantly reduced in
FCW34- or Taxol-treated mice. Next, the extent of tumor
proliferation was assessed using the cellular proliferation
In order to evaluate the efficiency of the compound
permeability into cells, a parallel artificial membrane
permeability assay (PAMPA) model was performed.65,66
FCW34 and FCW66 were tested by the PAMPA method
(pH = 7.4) in line with reference compounds, chloramphenicol
(high permeability), diclofenac (medium permeability), and
theophylline (low permeability), at 200 μM concentrations.
Apparent permeability (Papp) values were 8.8 0.2 × 10−6, 7.1
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Figure 5. Effect of Fcw34 on vessel development in transgenic zebrafish models. The influence of Fcw34 (20 μM) application on blood vessel
development in Tg(kdrl:mCherry)ci5 × Tg(fli1a:negfp)y7 zebrafish embryos was analyzed at various time intervals. (A) Bright-field images of Fcw34-
treated embryos; (B) fluorescence microscopy analysis of Fcw34-treated embryos at 30 h postfertilization (hpf). Upper panels show the anatomical
locations of ISV and CVP for observation; middle panel shows the measurements of ISV length (corresponding to the boxed area in the upper
panel) in control and Fcw34-treated embryos. The sprouting length of ISV in embryos (n = 10 per group) was analyzed at 30 hpf. Data were mean
SEM of triplicate experiments; bottom panels highlight the morphology and neovascularization in CVP morphology, as indicated by arrows.
0.5 × 10−6, 1.4 0.1 × 10−6, 3.6 0.3 × 10−6, and 9.0 0.4
× 10−6 cm/s for chloramphenicol, diclofenac, theophylline,
FCW34, and FCW66, respectively (Figure S11). In general,
compounds with Papp values between 1.1 × 10−6 and 1.0 ×
10−5 are mostly considered to have better pharmacokinetic
characteristics. Obviously, the Papp values of FCW34 and
FCW66 are within this range and FCW66 exhibited a 2−3-fold
permeability potency increase over FCW34, indicating that
FCW 66, possessing one more ethylene glycol unit of the
cyclopentane ring side chain, facilitates the passive perme-
ability.
FCW34 Is Associated with Antiangiogenesis Pro-
gression in a Zebrafish Model. To further validate the
antiangiogenic activities of FCW34, we employed Tg-
(kdrl:mCherry)ci5 × Tg(fli1a:negfp)y7 zebrafish embryos for
monitoring the neovascularization status in the intersegmental
vessels (ISV) and caudal vein plexus (CVP).67 Treatment with
FCW34 did not elicit a noticeable defect in the gross
morphology of zebrafish embryos (Figure 5A). However,
FCW34 treatment clearly perturbed the ISV and CVP
formations (Figure 5B). These results further demonstrate an
underlying relationship between FCW34-induced ST inhib-
ition and reduced angiogenic activity in vivo.
observation explains why a different cell line is associated
with a distinctive level of endogenous STs. In MDA-MB231
cells, the designed ST inhibitors, FCW34 and FCW66, are able
to interact with endogenous STs, which could be occasionally
forced overexpression under oxidative pressure, cellular
signaling, and other factors.
Physicochemical Analysis of Designed ST Inhibitors.
For two decades, the rule of five (Ro5) has been widely used as
the concept of drug-like properties to predict and categorize
the small molecule candidates compared to oral drugs. The
physicochemical parameters most commonly used are
molecular weight (MW), the calculated logarithm of the
water/octanol partition coefficient (clog P), number of
hydrogen bond acceptors (HBA), number of hydrogen bond
donors (HBD), topological polar surface area (TPSA)/
molecular polar surface area (MPSA), and number of rotatable
bonds (RotB). Because some FDA-approved oral drugs do not
strictly obey the rule of five (Ro5), Doak and co-workers
proposed reasonable outer limits for physicochemical param-
eters of a possible molecule to be orally absorbed.68,69 The
predicted molecular descriptors of the outer limit include MW
≤ 1000, −2 ≤ clog P ≤ 10, HBA ≤ 15, HBD ≤ 6, TPSA ≤
250, and RotB ≤ 20.
Endogenous Expression of ST6GALI, ST3GALIII,
ST3GALI, and ST6GALNACI Proteins in Different Cell
Lines. To indirectly evaluate the designed ST inhibitor-
targeted sialyltransferases in cancer cells, we examined the
existence of endogenous ST6GALI, ST3GALIII, ST3GALI,
and ST6GALNACI proteins. As shown in Figure S12, the
majority of cancer cell lines, MCF7, Hs578T, BT549, MDA-
MB-468, MDA-MB-231, and HeLa cells, have endogenous
ST6GALI, ST3GALIII, ST3GALI, and ST6GALNACI pro-
teins. It is interesting to note that MCF7 and MDA-MB468
cells showed high levels of immunostaining for ST6GALI and
ST3GALIII, respectively. In contrast, most of the cancer cell
lines exhibited abundant endogenous ST3GALI and ST6GAL-
NACI. It is important to note that the human normal epithelial
cell line M10 has no detectable endogenous ST3GALIII
protein but displayed higher level of immunostaining for
ST6GALI compared to other cancer cell lines. This
The selected compounds, 2aA−2eA, 2aB−2eB, and 2aC−
2eC, and Taxol used in this study with the calculated
parameter distributions are depicted in Supporting Information
Table S1. The MW range of the selected compounds alters
from 709 to 953, which is higher than that (MW ≤ 500) of the
Ro5 hypothesis. However, the high MW compounds have
reached the market recently, such as Taxol. The clog P value of
the selected compounds varies from 8.5 to 10.2 compared to
clog P ≤ 5 of Ro5 and 4.7 of Taxol, indicating that lipophilicity
is too high, and a subsequent modification of the steroidal ring
to reduce the lipophilic character is essential. The predicted
HBA ranges from 7 to 14, and the Ro5 rule requires HBA ≤
10. All the selected compounds satisfy the Ro5 hypothesis,
HBD ≤ 5. The calculated TPSA/MPSA values fall within the
range of 203−273. The RotB range of selected compounds
vary from 16 to 29, which is higher than that (14) of Taxol,
suggesting that the number of rotatable bonds from the PEG
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Article
linker and the steroidal aliphatic side chain should be reduced
in order to provide an oral druggable space.
progression and angiogenesis in vivo. This represents an
important step forward in ST-targeted breast cancer treat-
ments. Furthermore, our findings suggest that the sensitization
of tumors to FCW34 through manipulation of N-glycan
sialylation, followed by regulation of the talin/integrin/FAK/
paxillin and integrin/NFκB signaling pathways (Figure S14)
could be a feasible therapeutic approach.
Molecular Docking. To further study the binding mode of
the designed ST inhibitors, such as 2aA−2eA, 3cB−3dB, and
4cC−4dC with respect to that of the reference inhibitors
AL10, LCAG, and Lith-O-ASP in the ST6GALI structure
(PDB 6QVT),70 we built a conformation of two asymmetric
polypeptide chains (A/B) of human ST6GALI structure for
molecular docking, minimization, and binding energy calcu-
lations. Except ST6GALI, the crystal structure of porcine
ST3GALI (2WNF) was also used to construct the donor−
acceptor conformation, but it was not successful during the
minimization process.71 Simulated annealing process shows
that all the protein−inhibitor complexes are stable at 700 K.
On this basis, we found that the designed inhibitor complexes,
2aA−2eA, 3cB−3dB, and 4cC−4dC, showed lower binding
energy (−21.42 to −35.48 kcal/mol) than AL10 (−9.37 kcal/
mol), LCAG (−14.73 kcal/mol), and Lith-O-ASP (−9.58
kcal/mol) in Table S2. This finding is consistent with the
observation that 2aA−2eA, 3cB−3dB, and 4cC−4dC display
higher inhibitory potency than LCAG and Lith-O-ASP but not
AL10, because there are some unfavorable bumps and
unfavorable negative−negative interactions in the protein−
AL10 complex. Compared with the protein−ligand structure
between AL10 and 2aA/2bA/2cA/2dA/2eA, we observed an
extended space with hydrophobic and hydrophilic amino acid
residues, such as P320(B), P259(B), V261(A), W302(B),
H370(B), S322(B), S189(B), C353(B), G324(B), A190(B),
and N212(B) in two asymmetric polypeptide chains (A/B) of
ST6GALI (PDB 6QVT), where the PEG moiety of designed
inhibitors might develop additional interactions. As can be
seen in Figure S13, PEG moiety-containing inhibitor−protein
complexes show extra key interactions involving several
hydrophilic amino acid residues, S322(B), H370(B),
C353(B), S189(B), D274(B), and Q235(B), indicating that
these key interactions include the formation of conventional
hydrogen bonding and electrostatic attraction. In addition, the
shorter or longer alkyl chain (PEG linker) of the ST inhibitor
seems to occupy at the similar binding pocket.
We also observed the important hydrogen-bonding inter-
action and a salt-bridge formation between the nitro group or
nitrogen atom of NBD and Pro/Ser/Lys residues (P371(A),
S323(A), S322(A), and K268(B)). Furthermore, the steroidal
skeleton of 2aA−2eA, 3cB−3dB, 4cC−4dC, AL10, LCAG,
and Lith-O-ASP is well adapted to the hydrophobic cavity of
ST6GALI, in which the cavity is mostly surrounded by
nonpolar residues, such as P259(A), P259(B), V261(A),
V261(B), Trp302(A), Trp302(B), P320(A), and P320(B).
Interestingly, the azide group in the terminal of PEG chain in
2cA−2eA, 3dB, and 4cC also shows a favorable hydrogen
bonding or salt-bridge with neighboring amino acid residues,
G324(B), S188(B), A190(B), N212(B), S189(B), or E375(B).
In contrast, we did not observe any complementary binding
between the azide groups of 2aA−2bA, 3cB, and 4dC and
ST6GALI. Overall, the increased binding affinity of ST
inhibitors, 2aA−2eA, 3cB−3dB, and 4cC−4dC, is primarily
due to hydrogen bond formation, electrostatic attraction, or
salt−bridge interaction within the binding region of protein.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals and reagents were commercially
available and used without further purification unless indicated
otherwise. All solvents were of anhydrous grade unless indicated
otherwise. Reactions were monitored by thin-layer chromatography
on silica gel. Flash chromatography was performed on silica gel of
60−200 μm particle size. NMR spectra were recorded on Bruker
AV400, AMX400, or AV500 MHz instruments. Chemical shifts (δ)
were reported in parts per million (ppm) and referenced to the
chemical shift of residual solvent. High-resolution mass spectra were
recorded on a Bruker Daltonics spectrometer. Chromatography was
performed at room temperature using a high-performance liquid
chromatography (HPLC) system consisting of a Waters 1525 Binary
System with a Waters 2487 or 2489 dual λ absorbance detector, using
a Kromasil 300-5C4 column (250 × 10 mm) equilibrated with 0.1%
TFA/H2O and 0.05% TFA/CH3CN as eluents, at a flow rate of 2
mL/min, and the eluted materials were monitored using a UV
absorbance detector that measured the absorbance at 254 nm, unless
indicated otherwise. The HPLC fractions containing products were
lyophilized in a vacuum concentrator (FreeZone plus 4.5,
LABCONCO). The purity of lithocholic acid derivatives was
established as >95% by employing HPLC and 1H NMR spectroscopy.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-Azidoethoxy)-5-
oxopentan-2-yl)-10,13-di-methylhexadecahydro-1H-cyclopenta-
[a]phenanthren-3-yl)oxy)-3-((7-nitrobenzo[c][1,2,5] oxadiazol-4-
yl)amino)-4-oxobutanoic Acid (2aA). Yield: 17% (powder); mp:
78−79 °C; 1H NMR (400 MHz, CDCl3): δ 8.46 (d, J = 8.4 Hz, 1H),
6.97 (d, J = 8.4 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H), 4.87−4.80 (m,
2H), 4.23 (t, J = 4.8 Hz, 2H), 3.46 (t, J = 4.8 Hz, 2H), 3.21−3.06 (m,
2H), 2.42−2.34 (m, 1H), 2.29−2.21 (m, 1H), 1.95−1.89 (m, 1H),
1.86−1.75 (m, 5H), 1.67−1.64 (m, 1H), 1.55−1.52 (m, 2H), 1.50−
1.29 (m, 8H), 1.28−1.20 (m, 3H), 1.16−0.97 (m, 6H), 0.91−0.88
(sd, J = 6.4 Hz, 6H),0.62 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
174.2, 173.3, 168.6, 144.3, 143.8, 142.3, 135.7, 125.5, 100.0, 77.8,
62.9, 56.4, 55.9, 52.6, 49.8, 42.7, 41.9, 40.5, 40.1, 35.7, 35.3, 34.8,
34.6, 32.0, 31.1, 30.9, 28.1, 26.9, 26.4, 26.3, 24.2, 23.3, 20.8, 18.2,
12.0. HRMS calcd for C36H48N7O9 (M − H)−, 722.3514; found,
722.3510.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-Azidoethoxy)-
ethoxy)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-3-yl)oxy)-3-((7-nitrobenzo[c][1,2,5]-
oxadiazol-4-yl)amino)-4-oxobutanoic Acid (2bA). Yield: 11%
1
(powder); mp: 75−76 °C; H NMR (400 MHz, CDCl3): δ 8.45
(d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.27 (d, J = 8.4 Hz, 1H),
44.86−4.81 (m, 2H), 4.22 (t, J = 4.8 Hz, 2H), 3.70−3.65 (m, 4H),
3.37 (t, J = 4.8 Hz, 2H), 3.20−3.05 (m, 2H), 2.40−2.33 (m, 1H),
2.27−2.19 (m, 1H), 1.94−1.91 (m, 1H), 1.86−1.73 (m, 5H), 1.67−
1.64 (m, 1H), 1.54−1.52 (m, 2H), 1.44−1.29 (m, 8H), 1.27−1.19
(m, 3H), 1.15−0.97 (m, 6H), 0.90−0.86 (sd, J = 6.4 Hz, 6H), 0.61 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 174.5, 173.5, 168.7, 144.3,
143.8, 142.3, 135.8, 125.3, 100.0, 77.7, 70.0, 69.2, 63.3, 56.4, 56.0,
52.6, 50.6, 42.7, 41.9, 40.5, 40.0, 35.7, 35.3, 34.8, 34.5, 32.0, 31.1,
30.9, 28.1, 26.9, 26.4, 26.3, 24.1, 23.2, 20.8, 18.2, 12.0. HRMS calcd
for C38H52N7O10 (M − H)−, 766.3776; found, 766.3767.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-(2-
Azidoethoxy)ethoxy)ethoxy)-5-oxopentan-2-yl)-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-4-oxobutanoic Acid
(2cA; FCW34). Yield: 12% (powder); mp: 72−73 °C; 1H NMR
(400 MHz, CDCl3): δ 8.46 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 6.0 Hz,
1H), 6.27 (d, J = 8.8 Hz, 1H), 4.87−4.81 (m, 2H), 4.21 (t, J = 5.2 Hz,
2H), 3.71−3.66 (m, 8H), 3.38 (t, J = 5.2 Hz, 2H), 3.19−3.05 (m,
CONCLUSIONS
■
In summary, we have designed and synthesized a series of cell-
permeable and potent ST inhibitors and have demonstrated
that one of these inhibitors, FCW34, markedly reduces tumor
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2H), 2.40−2.32 (m, 1H), 2.26−2.18 (m, 1H), 1.95−1.92 (m, 1H),
1.86−1.72 (m, 5H), 1.67−1.63 (m, 1H), 1.55−1.52 (m, 2H), 1.45−
1.29 (m, 8H), 1.27−1.19 (m, 3H), 1.15−0.97 (m, 6H), 0.91−0.87
(sd, J = 6.4 Hz, 6H), 0.61 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
174.5, 172.9, 168.6, 144.3, 143.8, 142.3, 135.7, 125.4, 100.0, 77.7,
70.7, 70.6, 70.1, 69.3, 63.4, 56.4, 55.9, 52.6, 50.7, 42.7, 41.9, 40.5,
40.1, 35.7, 35.3, 34.8, 34.6, 32.0, 31.9, 31.1, 30.9, 28.2, 26.9, 26.5,
26.4, 26.3, 24.2, 23.3, 20.8, 18.3, 12.0. HRMS calcd for
C40H57N7O11Na (M + Na)+, 834.4014; found, 834.4014.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-13-oxo-
3,6,9,12-tetraoxaheptadecan-16-yl)-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-((7-nitro benzo[c]-
[1,2,5]oxadiazol-4-yl)amino)-4-oxobutanoic Acid (2dA; FCW66).
Yield: 10% (oil); 1H NMR (500 MHz, CDCl3): δ 8.44 (d, J = 8.5 Hz,
1H), 7.16 (br s, 1H), 6.26 (d, J = 8.0 Hz, 1H), 4.81 (br s, 2H), 4.19
(t, J = 4.5 Hz, 2H), 3.68−3.64 (m, 12H) 3.70 (t, J = 5.0 Hz, 2H),
3.14−3.07 (m, 2H), 2.37−2.31 (m, 1H), 2.24−2.18 (m, 1H), 1.92−
1.90 (m, 1H), 1.85−1.72 (m, 5H), 1.64 (br s, 1H), 1.53−1.52 (m,
2H), 1.43−1.27 (m, 8H), 1.25−1.19 (m, 3H), 1.13−0.96 (m, 6H),
0.89−0.85 (sd, J = 6.0 Hz, 6H), 0.60 (s, 3H). 13C NMR (125 MHz,
CDCl3): δ 174.4, 172.8, 168.8, 144.3, 143.8, 142.6, 135.8, 125.2,
100.1, 77.6, 70.7, 70.6, 70.5, 70.0, 69.2, 63.4, 56.4, 55.9, 52.9, 50.7,
42.7, 41.9, 40.5, 40.1, 35.7, 35.3, 34.8, 34.6, 32.0, 31.1, 30.9, 28.1,
26.9, 26.4, 26.3, 24.1, 23.3, 20.8, 18.3, 12.0. HRMS calcd for
C42H60N7O12 (M − H)−, 854.4300; found, 854.4293.
20.8, 18.3, 12.0. HRMS calcd for C39H55N5O10Na (M + Na)+,
776.3847; found, 776.3840.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-(2-
Azidoethoxy)ethoxy)ethoxy)-5-oxo pentan-2-yl)-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(4-nitro
benzamido)-4-oxobutanoic Acid (3cB). Yield: 24% (powder); mp:
52−53 °C; 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.8 Hz, 2H),
7.95 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.2 Hz, 1H), 4.98−4.95 (m,
1H), 4.83−4.77 (m, 1H), 4.20 (t, J = 4.8 Hz, 2H), 3.70−3.65 (m,
8H), 3.37 (t, J = 4.8 Hz, 2H), 3.16−3.00 (m, 2H), 2.39−2.32 (m,
1H), 2.26−2.18 (m, 1H), 1.95−1.92 (m, 1H), 1.89−1.73 (m, 5H),
1.66−1.64 (m, 1H), 1.55−1.52 (m, 2H), 1.40−1.26 (m, 8H), 1.23−
1.16 (m, 3H), 1.14−0.96 (m, 6H), 0.90−0.87 (sd, J = 6.4 Hz, 6H),
0.62 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 174.4, 169.8, 165.2,
149.8, 139.1, 128.4, 123.8, 70.7, 70.6, 70.1, 69.3, 63.4, 56.4, 56.0, 50.7,
49.3, 42.7, 41.9, 40.4, 40.1, 35.7, 35.3, 34.9, 34.6, 32.0, 31.1, 30.9,
28.1, 27.0, 26.4, 26.3, 24.1, 23.3, 20.8, 18.3, 12.0. HRMS calcd for
C41H59N5O11Na (M + Na)+, 820.4109; found, 820.4103.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-13-oxo-
3,6,9,12-tetraoxaheptadecan-16-yl)-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(4-nitro benzami-
do)-4-oxobutanoic Acid (3dB). Yield: 21% (oil); 1H NMR (400
MHz, CDCl3): δ 8.27 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H),
7.36 (d, J = 7.6 Hz, 1H), 4.7−4.96 (m, 1H), 4.80−4.79 (m, 1H),
3.72−3.58 (m, 14H), 3.38 (t, J = 4.8 Hz, 2H), 3.18−2.98 (m, 2H),
2.36−2.33 (m, 1H), 2.27−2.20 (m, 1H), 1.95−1.92 (m, 1H), 1.89−
1.78 (m, 5H), 1.67−1.66 (m, 1H), 1.55−1.52 (m, 2H), 1.40−1.34
(m, 8H), 1.23−1.17 (m, 3H), 1.15−0.97 (m, 6H), 0.91−0.87 (sd, J =
4.0 Hz, 6H), 0.62 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 175.3,
174.4, 169.9, 165.3, 149.8, 139.1, 128.5, 123.8, 72.4, 70.6, 70.5, 70.2,
70.0, 69.2, 63.4, 61.6, 56.4, 55.9, 50.6, 49.3, 42.7, 41.9, 40.4, 40.1,
36.1, 35.7, 35.4, 34.9, 34.6, 32.0, 30.9, 30.7, 28.2, 27.0, 26.3, 24.1,
23.3, 20.9, 18.3, 12.0. HRMS calcd for C43H63N5O12Na (M + Na)+,
864.4371; found, 864.4362.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-16-oxo-
3,6,9,12,15-pentaoxaicosan-19-yl)-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(4-nitrobenzamido)-4-
oxobutanoic Acid (3eB). Yield: 22% (oil); 1H NMR (400 MHz,
CDCl3): δ 8.25 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.44 (d,
J = 6.4 Hz, 1H), 4.95 (br s, 1H), 4.78 (br s, 1H), 4.18 (t, J = 4.0 Hz,
2H), 3.63−3.62 (m, 16H), 3.35 (t, J = 4.4 Hz, 2H), 3.14−2.97 (m,
2H), 2.37−2.30 (m, 1H), 2.24−2.16 (m, 1H), 1.93−1.90 (m, 1H),
1.84−1.76 (m, 5H), 1.64−1.62 (m, 1H), 1.53−1.51 (m, 2H), 1.39−
1.33 (m, 8H), 1.25−1.19 (m, 3H), 1.15−0.94 (m, 6H), 0.89−0.86
(sd, J = 6.4 Hz, 6H), 0.60 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
174.4, 169.9, 165.2, 149.8, 139.1, 128.5, 123.8, 70.6, 70.5, 70.4, 69.9,
69.1, 63.4, 56.4, 55.9, 50.6, 49.4, 42.7, 41.9, 40.4, 40.0, 35.7, 35.3,
34.8, 34.5, 32.0, 31.1, 30.8, 28.1, 26.9, 26.4, 26.2, 24.1, 23.2, 20.8,
18.2, 12.0. HRMS calcd for C45H67N5O13Na (M + Na)+, 908.4633;
found, 908.4630.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-16-oxo-
3,6,9,12,15-pentaoxaicosan-19-yl)-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-((7-nitrobenzo[c][1,2,5]-
oxadiazol-4-yl)amino)-4-oxobutanoic Acid (2eA). Yield: 10% (oil),
1H NMR (500 MHz, CDCl3): δ 8.44 (d, J = 8.5 Hz, 1H), 6.26 (d, J =
7.5 Hz, 1H), 4.81 (br s, 2H), 4.20 (t, J = 4.5 Hz, 2H), 3.68−3.63 (m,
16H) 3.37 (t, J = 4.5 Hz, 2H), 3.14−3.06 (m, 2H), 2.37−2.31 (m,
1H), 2.24−2.18 (m, 1H), 1.92−1.90 (m, 1H), 1.85−1.75 (m, 5H),
1.65 (br s, 1H), 1.52−1.51 (m, 2H), 1.40−1.31 (m, 8H), 1.22−1.19
(m, 3H), 1.13−0.96 (m, 6H), 0.89−0.86 (sd, J = 6.0 Hz, 6H), 0.60 (s,
3H). 13C NMR (125 MHz, CDCl3): δ 174.4, 168.8, 144.3, 143.8,
142.6, 135.8, 125.1, 100.1, 77.5, 70.6, 70.5, 70.0, 69.2, 63.4, 56.5, 55.9,
53.0, 50.7, 42.7, 41.9, 40.5, 40.1, 35.7, 35.3, 34.8, 34.6, 32.0, 31.0,
30.9, 28.1, 26.9, 26.4, 26.3, 24.1, 23.2, 20.8, 18.3, 12.0. HRMS calcd
for C44H64N7O13 (M − H)−, 898.4562; found, 898.4556.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-Azidoethoxy)-5-
oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]-
phenanthren-3-yl)oxy)-3-(4-nitrobenzamido)-4-oxobutanoic Acid
1
(3aB). Yield: 32% (powder); mp: 61−62 °C; H NMR (400 MHz,
CDCl3): δ 8.29 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.37 (d,
J = 7.6 Hz, 1H), 5.00−4.97 (m, 1H), 4.86−4.80 (m, 1H), 4.24 (t, J =
5.2 Hz, 2H), 3.47 (t, J = 5.2 Hz, 2H), 3.17 (dd, J = 17.4 Hz, 4.0 Hz,
1H), 3.06 (dd, J = 17.2 Hz, 4.0 Hz, 1H), 2.43−2.36 (m, 1H), 2.31−
2.23 (m, 1H), 1.98−1.95 (m, 1H), 1.91−1.80 (m, 5H), 1.68−1.66
(m, 1H), 1.57−1.54 (m, 2H), 1.43−1.37 (m, 8H), 1.30−1.23 (m,
3H), 1.19−0.98 (m, 6H), 0.93−0.91 (sd, J = 7.6 Hz, 6H), 0.65 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 174.0, 169.8, 165.2, 149.9,
139.1, 128.4, 123.8, 62.8, 56.4, 55.9, 49.8, 49.3, 42.7, 41.9, 40.4, 40.0,
35.7, 35.3, 34.8, 34.5, 32.0, 31.0, 30.8, 28.1, 26.9, 26.4, 26.2, 24.1,
23.2, 20.8, 18.2, 12.0. HRMS calcd for C37H51N5O9Na (M + Na)+,
732.3584; found, 732.3593.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-Azidoethoxy)-5-
oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]-
phenanthren-3-yl)oxy)-3-(2-(7-methoxy-2-oxo-2H-chromen-4-yl)-
acetamido)-4-oxobutanoic Acid (4aC). Yield: 35% (powder); mp:
1
151−152 °C; H NMR (400 MHz, CDCl3): δ 7.58 (d, J = 8.8 Hz,
1H), 7.31 (d, J = 7.6 Hz, 1H), 6.83 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.73
(d, J = 2.4 Hz, 1H), 6.28 (s, 1H), 4.78−4.70 (m, 2H), 4.21 (t, J = 5.0
Hz, 2H), 3.82 (s, 3H), 3.74 (s, 2H), 3.45 (t, J = 5.0 Hz, 2H), 3.03−
2.87 (m, 2H), 2.41−2.33 (m, 1H), 2.28−2.20 (m, 1H), 1.93−1.91
(m, 1H), 1.84−1.74 (m, 5H), 1.61−1.53 (m, 2H), 1.49−1.46 (m,
1H), 1.39−1.27 (m, 8H), 1.23−1.14 (m, 4H), 1.10−0.93 (m, 5H),
0.89−0.88 (sd, J = 4.4 Hz, 6H), 0.61 (s, 3H); 13C NMR (100 MHz,
CDCl3): δ 174.3, 174.0, 169.8, 167.9, 163.0, 161.5, 155.4, 149.9,
126.0, 112.9, 112.8, 112.5, 101.0, 62.8, 56.3, 55.9, 55.8, 49.8, 49.2,
42.7, 41.9, 40.4, 40.0, 39.7, 35.8, 35.3, 34.9, 34.5, 32.0, 31.1, 30.9,
28.1, 27.0, 26.4, 26.3, 24.1, 23.3, 20.8, 18.2, 12.0. HRMS calcd for
C42H56N4O10Na (M + Na)+, 799.3894; found, 799.3890.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-Azidoethoxy)-
ethoxy)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-3-yl)oxy)-3-(4-nitrobenzamido)-4-oxo-
butanoic Acid (3bB). Yield: 30% (powder); mp: 57−58 °C; 1H NMR
(400 MHz, CDCl3): δ 8.26 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.4 Hz,
2H), 7.34 (d, J = 6.8 Hz, 1H), 4.97−4.95 (m, 1H), 4.83−4.77 (m,
1H), 4.21 (t, J = 4.6 Hz, 2H), 3.70−3.64 (m, 4H), 3.37 (t, J = 5.2 Hz,
2H), 3.16−3.02 (m, 2H), 2.40−2.33 (m, 1H), 2.27−2.19 (m, 1H),
1.95−1.92 (m, 1H), 1.89−1.74 (m, 5H), 1.65−1.63 (m, 1H), 1.55−
1.52 (m, 2H), 1.40−1.27 (m, 8H), 1.24−1.20 (m, 3H), 1.16−1.00
(m, 6H), 0.90−0.87 (sd, J = 6.4 Hz, 6H), 0.62 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 174.4, 169.8, 165.3, 149.9, 139.1, 128.4, 123.8,
70.0, 69.2, 63.2, 56.4, 56.0, 50.6, 49.3, 42.7, 41.9, 40.4, 40.1, 35.7,
35.3, 34.9, 34.6, 32.0, 31.1, 30.9, 28.1, 26.9, 26.4, 26.3, 24.1, 23.3,
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-Azidoethoxy)-
ethoxy)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-3-yl)oxy)-3-(2-(7-methoxy-2-oxo-2H-
chromen-4-yl)acetamido)-4-oxobutanoic Acid (4bC). Yield: 29%
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1
(powder); mp: 142−143 °C; H NMR (400 MHz, CDCl3): δ 7.58
rat ST3GALI (CalBiochem #566227) were obtained from CalBio-
chem at specific activities of 2.4 and 7.0 U/mg, respectively, stored at
−80 °C, and consumed within 1 week. The donor substrate, CMP-
Neu5Ac (Sigma #C8271·), and acceptors, p-nitrophenyl T-antigen
(Calbiochem #575303) and Galβ1-4GlcNAc (N-acetyllactosamine,
Calbiochem, Millipore 345250), were purchased from commercially
available vendors.
(d, J = 9.2 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.83 (dd, J = 8.8 Hz, 2.4
Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.28 (s, 1H), 4.77−4.70 (m, 2H),
4.21 (t, J = 4.8 Hz, 2H), 3.82 (s, 3H), 3.74 (s, 2H), 3.69−3.64 (m,
4H), 3.36 (t, J = 4.8 Hz, 2H), 3.02−2.87 (m, 2H), 2.40−2.32 (m,
1H), 2.26−2.18 (m, 1H), 1.93−1.91 (m, 1H), 1.83−1.74 (m, 5H),
1.61−1.46 (m, 3H), 1.39−1.26 (m, 8H), 1.23−1.10 (m, 4H), 1.08−
0.94 (m, 5H), 0.88−0.86 (sd, J = 6.0 Hz, 6H), 0.60 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ 174.3, 169.8, 167.9, 162.9, 161.5, 155.4,
149.9, 126.0, 112.9, 112.7, 112.5, 101.0, 70.0, 69.2, 63.2, 56.3, 56.0,
55.8, 50.6, 49.1, 42.7, 41.9, 40.4, 40.0, 39.7, 35.7, 35.3, 34.8, 34.5,
32.0, 31.1, 30.9, 28.1, 26.9, 26.4, 26.2, 24.1, 23.3, 20.8, 18.2, 12.0.
HRMS calcd for C44H60N4O11Na (M + Na)+, 843.4156; found,
843.4147.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-5-(2-(2-(2-
Azidoethoxy)ethoxy)ethoxy)-5-oxopentan-2-yl)-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(2-(7-
methoxy-2-oxo-2H-chromen-4-yl)acetamido)-4-oxobutanoic Acid
(4cC). Yield: 25% (powder); mp: 134−135 °C; 1H NMR (400 MHz,
CDCl3): δ 7.57 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H), 6.82
(dd, J = 8.8 Hz, 2.0 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 6.27 (s, 1H),
4.77−4.69 (m, 2H), 4.20 (t, J = 4.8 Hz, 2H), 3.81 (s, 3H), 3.74 (s,
2H), 3.70−3.64 (m, 8H), 3.36 (t, J = 5.2 Hz, 2H), 3.02−2.85 (m,
2H), 2.38−2.31 (m, 1H), 2.25−2.17 (m, 1H), 1.92−1.90 (m, 1H),
1.82−1.73 (m, 5H), 1.57−1.44 (m, 3H), 1.38−1.25 (m, 8H), 1.22−
1.13 (m, 4H), 1.08−0.93 (m, 5H), 0.87−0.86 (sd, J = 5.2 Hz, 6H),
0.60 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 174.4, 174.3, 169.8,
168.0, 162.9, 161.5, 155.3, 149.9, 126.0, 113.0, 112.7, 112.5, 100.9,
70.6, 70.5, 70.0, 69.2, 63.3, 56.3, 56.0, 55.7, 50.6, 49.1, 42.7, 41.9,
40.3, 40.0, 39.6, 35.7, 35.3, 34.8, 34.5, 32.0, 31.2, 30.9, 28.1, 26.9,
26.3, 26.2, 24.1, 23.2, 20.8, 18.2, 12.0. HRMS calcd for
C46H64N4O12Na (M + Na)+, 887.4418; found, 887.4417.
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-13-oxo-
3,6,9,12-tetraoxaheptadecan-16-yl)-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(2-(7-methoxy-2-
oxo-2H-chromen-4-yl)acetamido)-4-oxobutanoic Acid (4dC).
Yield: 21% (powder); mp: 126−127 °C; 1H NMR (500 MHz,
CDCl3): δ 7.57 (d, J = 9.0 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 6.83
(dd, J = 9.0 Hz, 2.0 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 6.27 (s, 1H),
4.77−4.69 (m, 2H), 4.19 (t, J = 4.5 Hz, 2H), 3.82 (s, 3H), 3.74 (s,
2H), 3.68−3.64 (m, 12H), 3.36 (t, J = 4.5 Hz, 2H), 3.02−2.85 (m,
2H), 2.37−2.31 (m, 1H), 2.24−2.17 (m, 1H), 1.92−1.90 (m, 1H),
1.82−1.74 (m, 5H), 1.60−1.45 (m, 3H), 1.37−1.26 (m, 8H), 1.25−
1.10 (m, 4H), 1.07−0.93 (m, 5H), 0.87−0.86 (sd, J = 6.5 Hz, 6H),
0.60 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 174.3, 174.0, 169.8,
167.9, 163.0, 161.4, 155.4, 149.8, 126.0, 113.0, 112.7, 112.5, 101.0,
70.7, 70.6, 70.5, 70.0, 69.2, 63.4, 56.4, 56.0, 55.8, 50.7, 49.2, 42.7,
41.9, 40.4, 40.0, 39.7, 35.8, 35.3, 34.9, 34.6, 32.0, 31.2, 30.9, 28.1,
27.0, 26.4, 26.3, 24.1, 23.3, 20.8, 18.3, 12.0. HRMS calcd for
C48H68N4O13Na (M + Na)+, 931.4681; found, 931.4681.
α2,3-O-Sialyltransferase (α2,3-O-ST) Inhibition Assay.26,28 For
detection of sialyltransferase activity, reverse-phase HPLC was applied
to detect the end product of sialylated saccharides. The assay mixtures
contained the following components: 200 mM MES buffer; 100 mM
NaCl; 0.5 mM EDTA; 0.01% Triton X-100; 1.4 mU ST3GALI; 2.5
mM p-nitrophenyl T-antigen; 1 mM CMP-Neu5Ac; and different
concentrations of sialyltransferase inhibitors to a total volume of 50
μL. The assay mixture was incubated at 37 °C for 15 min and then
quenched by heating up to 101 °C for 10 min. The sialylated product
was resolved by reversed-phase HPLC. For the IC50 value, we chose
six to eight concentrations of compounds within the range of 0 to
100% inhibition in response to enzyme activity. The internal standard
(acceptor or sialylated product) was used to improve the precision of
quantitative analysis in HPLC. The residual activities (%) versus
compound concentrations were plotted to fit the sigmoidal curve (or
the dose−response curve) equations, and the IC50 values were
calculated using GraphPad Prism software. Kinetic data were obtained
by fitting the initial rate data to the nonlinear regression equation.
α2,3-N-Sialyltransferase (α2,3-N-ST) Inhibition Assay.26 For the
detection of sialyltransferase activity, the reverse-phase HPLC was
applied to detect the end product of sialylated saccharides. The assay
mixtures contained the following components: 200 mM MES buffer;
100 mM NaCl; 0.5 mM EDTA; 0.01% Triton X-100; 1.4 mU
ST3GALIII; 25 μM Galβ1-4Glc; 1mM CMP-Neu5Ac; and different
concentrations of test compounds to a total volume of 50 μL. The
assay mixture was incubated at 37 °C for 1.5 h and then quenched by
heating up to 101 °C for 10 min. The sialylated product was identified
and analyzed by reversed-phase HPLC. For the IC50 value, we chose
six to eight concentrations of compounds within the range of 0 to
100% inhibition in response to enzyme activity. The internal standard
(acceptor or sialylated product) was used to improve the precision of
quantitative analysis in HPLC. The residual activities (%) versus
compound concentrations were plotted to fit the sigmoidal curve (or
the dose−response curve) equations, and the IC50 values were
calculated using GraphPad Prism software. Kinetic data were obtained
by fitting the initial rate data to the nonlinear regression equation.
α2,6-N-Sialyltransferase (α2,6-N-ST) Inhibition Assay.26 For
detection of sialyltransferase activity, the reverse-phase HPLC was
applied to detect the end product of sialylated saccharides. The assay
mixtures contained the following components: 25 mM Tris buffer;
150 mM NaCl; 5 mM CaCl2; 10 mM MnCl2; 0.5 μg ST6GALI; 25
μM Galβ1-4GlcNAc; 1 mM CMP-Neu5Ac; and different concen-
trations of sialyltransferase inhibitors to a total volume of 50 μL. The
assay mixture was incubated at 37 °C for 15 min and then quenched
by heating up to 101 °C for 10 min. The sialylated product was
resolved by reversed-phase HPLC. For the IC50 value, we chose six to
eight concentrations of compounds within the range of 0 to 100%
inhibition in response to enzyme activity. The internal standard
(acceptor or sialylated product) was used to improve the precision of
quantitative analysis in HPLC. The residual activities (%) versus
compound concentrations were plotted to fit the sigmoidal curve (or
the dose−response curve) equations, and the IC50 values were
calculated using GraphPad Prism software. Kinetic data were obtained
by fitting the initial rate data to the nonlinear regression equation.
Cell Culture. Human breast carcinoma MDA-MB-231, MCF-7,
and Hs 578T cells were purchased from Bioresource Collection and
Research Center (BCRC 60425, 60436 and 60120, respectively) and
maintained in Dulbecco’s modified Eagle’s medium (DMEM;
HyClone) with 10% fetal bovine serum (FBS; Biological Industries),
2 mM L-glutamine, and antibiotics (containing 100 mg/L
Streptomycin and 100 U/mL Penicillin G). Hs 578T was
supplemented with 10 μg/mL bovine insulin (Sigma) additionally.
All the cell lines were maintained in a humidified incubator with 5%
(3S)-4-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-1-Azido-16-oxo-
3,6,9,12,15-pentaoxaicosan-19-yl)-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(2-(7-methoxy-2-oxo-
2H-chromen-4-yl)acetamido)-4-oxobutanoic Acid (4eC). Yield:
1
21% (powder); mp: 113−114 °C; H NMR (500 MHz, CDCl3): δ
7.57 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 7.0 Hz, 1H), 6.82 (dd, J = 9.0
Hz, 2.0 Hz, 1H), 6.73 (d, J = 2.0 Hz, 1H), 6.27 (s, 1H), 4.75−4.71
(m, 2H), 4.18 (t, J = 5.0 Hz, 2H), 3.81 (s, 3H), 3.73 (s, 2H), 3.67−
3.62 (m, 16H), 3.35 (t, J = 5.0 Hz, 2H), 3.01−2.85 (m, 2H), 2.37−
2.31 (m, 1H), 2.23−2.17 (m, 1H), 1.92−1.90 (m, 1H), 1.79−1.73
(m, 5H), 1.59−1.44 (m, 3H), 1.35−1.26 (m, 8H), 1.25−1.10 (m,
4H), 1.07−0.92 (m, 5H), 0.87−0.85 (sd, J = 6.5 Hz, 6H), 0.59 (s,
3H); 13C NMR (125 MHz, CDCl3): δ 174.2, 169.8, 167.9, 162.9,
161.3, 155.4, 149.7, 126.0, 113.1, 112.7, 112.5, 101.0, 76.6, 70.7, 70.6,
70.6, 70.5, 70.0, 69.2, 63.4, 56.3, 56.0, 55.7, 50.7, 49.2, 42.7, 41.9,
40.4, 40.0, 39.7, 35.8, 35.3, 34.9, 34.5, 32.0, 31.2, 30.9, 28.1, 26.9,
26.4, 26.3, 24.1, 23.2, 20.8, 18.3, 12.0. HRMS calcd for
C50H72N4O14Na (M + Na)+, 975.4943; found, 975.4945.
Biology. Human ST6GAL1 was purchased from R&D systems
(#7620-GT) at specific activity >150 pmol/min/μg, stored at −80 °C,
and used within 1 week. Rat ST3GAL3 (CalBiochem #566218) and
537
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CO2 at 37 °C. The mammary epithelial M10 cell line was purchased
from Bioresource Collection and Research Center (BCRC 60197)
and maintained in the Minimum Essential Medium (MEM; Gibco
BRL) with 10% FBS (Biological Industries), 2 mM L-glutamine, and
antibiotics (containing 100 mg/L Streptomycin and 100 U/mL
Penicillin G). Cell lines were maintained in a humidified incubator
with 5% CO2 at 37 °C. The human breast carcinoma MDA-MB-468
cell line was purchased from American Type Culture Collection
(ATCC HTB-132) and maintained in Leibovitz’s L-15 Medium
(Gibco; 11415-064) with 10% FBS (Biological Industries), 2 mM L-
glutamine, and antibiotics (containing 100 mg/L Streptomycin and
100 U/mL Penicillin G). Cell lines were maintained in a humidified
incubator at 37 °C. The human breast carcinoma BT-549 cell line was
purchased from American Type Culture Collection (ATCC HTB-
122) and maintained in the RPMI-1640 medium (Hyclone;
SH30027.02) with 10% FBS (Biological Industries), 0.023 U/mL
insulin (Sigma), 2 mM L-glutamine, and antibiotics (containing 100
mg/L Streptomycin, 100 U/mL Penicillin G). Cell lines were
maintained in a humidified incubator with 5% CO2 at 37 °C. Human
cervical adenocarcinoma HeLa cells were purchased from American
Type Culture Collection (ATCC CCL-2) and maintained in DMEM
(HyClone) with 10% FBS (Biological Industries), 2 mM L-glutamine,
and antibiotics (containing 100 mg/L Streptomycin and 100 U/mL
Penicillin G). Cell lines were maintained in a humidified incubator
with 5% CO2 at 37 °C.
Western Blot. Samples containing equal amounts of protein (30
μg) were separated on a 6 to 10% SDS-PAGE and electroblotted onto
Immobilon-P membranes (Millipore Corp.) in a transfer buffer.
Immunoblotting was performed using antibodies against talin-1, FAK,
paxillin, IKKα, IKKβ, NFκB, IκBα, β-tubulin (Cell signaling),
phospho-talin, phospho-FAK (Tyr 397) (Tyr 576/577) (Tyr925),
phospho-IKKα/β, phospho-NFκB, phospho-IκBα, phospho-paxillin
(Cell Signaling), ST3GALIII (Santa Cruz), β-actin (BD Biosciences),
and secondary antibodies antimouse IgG and antirabbit IgG (Perkin
Elmer). The antibody reaction was visualized using SuperSignal West
Pico reagent (Thermo Scientific).
Detection of Endogenous Sialyltransferase Expression by
Western Blot Analysis. To understand the endogenous ST
expression levels in M10, MCF7, Hs578T, BT549, MDA-MB-468,
MDA-MB-231, and HeLa cell lines. Cells were harvested and lysed
with cell lysis buffer (Cell Signaling Technology). Protein
concentrations were determined and equalized before loading.
Samples containing equal amounts of protein (20 μg) were separated
on 10% SDS-PAGE gel and electroblotted onto a polyvinylidene
fluoride-transfer membrane (PVDF, Pall Corporation) in a transfer
buffer. Immunoblotting was performed using specific antibodies
including ST6GALI (Thermo Fisher Scientific; #MA5-11900),
ST6GALNACI (Thermo Fisher Scientific; #PA5-31200), ST3GALIII
(Santa Cruz Biotechnology; #sc-134040), ST3GALI (GeneTex;
#GTX106121), and β-actin (EMD Millipore; #MAB1501) as internal
control. The antibody reaction was visualized by the western blot
chemiluminescence reagent (PerkinElmer) using an FL1000 imaging
system.
random views were photographed and the mean number of invasive
cells was counted under a microscope.
Transient Transfection of siRNA Against ST3GALIII.
STEALTH siRNA against human ST3GALIII was purchased from
Thermo Fisher; the sequences are shown in Table 2.
Table 2
siRNA ID
name
sequence
HSS143938 siST3GalIII
(A)
GGA CGC ACA AUA UCC AGC GAG
AGA A
UUC UCU CGC UGG AUA UUG UGC
GUC C
HSS143939 siST3GalIII
(B)
GGA AGC UGG UGA AAG CUC GCG
UCA U
AUG ACG CGA GCU UUC ACC AGC
UUC C
HSS143940 siST3GalIII
(C)
GGG ACU CUU GGU AUU UGU GCG
CAA U
AUU GCG CAC AAA UAC CAA GAG
UCC C
Human breast cancer cells MDA-MB-231 were seeded onto 6-well
plates to 40−60% confluency. The siRNA against human ST3GALIII
(Invitrogen) was dissolved in DEPC water to form 20 μM solutions.
The lipofectamine RNAiMAX transfection reagent was diluted in
Opti-MEM medium. siRNA was also diluted in Opti-MEM medium,
mixed with the lipofectamine RNAiMAX reagent in a 1:1 ratio, and
then added to cells. After 72 h, cells were harvested for further
analysis.
Real-Time PCR. RNA was isolated from MDA-MB-231 cells using
the PureLink RNA Mini Kit (Ambion Inc.) according to
manufacturer’s instructions. For reverse transcription, 5 μg of total
RNA was used for reverse transcription with Superscript III
(Invitrogen Co.) using olio-dT and random primers. One-twentieth
of reverse-transcription products were used as a template for real-time
PCR in iQ5 (BioRad) using a SYBR Green I assay. PCR was
performed in 20 μL of SYBR Green PCR Master Mix (Thermo)
containing 0.5 μM forward primers and reverse primers, and
approximately 30 ng of cDNA. Amplification and detection were
performed as follows: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C
for 5 s, 62 °C for 5 s, and 72 °C for 10 s. The primer sequences for
Human ST3GALIII were the following: the forward primer 5′-
TAGCCCTCTGCCTCTTTCT-3′ and the reverse primer 5′-
TTGCGTACTTGGTGGCTAAT-3′, which amplified a 204-bp
ST3GALIII cDNA fragment. The β-actin mRNA level was
determined using the following: the forward primer 5′-TCACCCA-
CACTGTGCCTATCTACGA-3′ and the reverse primer 5′-CAGCG-
GA ACCGCTCATTGCCAATGG-3′, which amplified a 295-bp β-
actin cDNA fragment.
Lectin Affinity Assay and IP-Western Blotting. The lectin
affinity assay was modified according to the previous study reported.1
The cell surface α-2,3-sialylated antigen-expressing proteins were
specifically captured using biotinylated Maackia amurensis Lectin II
(MALII) (Vector Laboratories, Inc.) About 500 μg of total cell lysate
proteins were incubated with MALII for 16 h at 4 °C with rotation.
The MALII−protein complexes were captured using Streptavidin-
conjugated agarose beads, followed by washing with RIPA buffer three
times. Antigen-expressed proteins were released into the SDS-PAGE
sample buffer and immunoblotted with target protein integrin-β1, -β3,
-β4, and -β5 (Cell Signaling, Inc.).
MTT Cytotoxicity Assay. About 104 of MDA-MB-231 cells were
seeded on 96-well culture dishes in DMEM (Gibco) containing 10%
FBS (Gibco). Different concentrations of compounds were added to
each plate and incubated for 48 h. Cell viabilities at different
concentrations of compounds-−treatments were determined by the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,
Sigma) assay. In the untreated control, 0.1% DMSO-containing
medium was used.
Transwell Migration Assay. The cell migration assay was
performed using transwell chambers with a filter insert (8 μm pore;
Falcon, BD Biosciences). Cells were seeded onto the transwell insert
with a serum-free medium at a density of 8 × 104 cells/mL and then
treated with different doses of sialyltransferase inhibitors or DMSO
for 16 h. Conditioned medium obtained from 5% fetal bovine serum
in tissue culture medium was added to the lower wells as a
chemoattractant. Thereafter, invading cells were fixed and stained
after 16 h. These experiments were carried out in triplicate. Three
Immunofluorescence Assay. Cells were cultured on cover slides
and treated without or with Fcw34 and Fcw66 (10 and 20 μM) for 48
h. Cells were fixed with 4% paraformaldehyde for 5 min at 4 °C. After
fixation, cells were permeabilized by 0.3% Triton X-100 at room
temperature for 30 min, followed by blocking with 2% BSA for 30
min. Cells were probed with the rabbit-antiphospho-talin antibody (1
mg/mL) and mouse-anti-integrin β1 or integrin β3 at 4 °C O/N,
followed by Alexa405-labeled goat-antimouse and Alexa546-labeled
goat-antirabbit secondary antibodies. DRAQ7 (Abcam) was used for
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counter staining. Finally, slides were mounted using the Fluoromount-
G reagent and observed under a fluorescence microscope (Zeiss LSM
780 plus ELYRA S.1, Germany).
sides. The acceptor solution, equilibrium standard (200 μM
permeability control and the test compound), and blank control
solutions were then added to wells of UV plates (Thermo Fisher
Scientific, #8404). UV absorption was measured from 230 to 500 nm
in 10 nm intervals to determine the peak absorbance of the control
and test compounds (Enspire Multimode Plate Reader, PerkinElmer).
The apparent permeability (Papp) and permeability rate (Pe) values
were calculated using the determined peak absorbance for each
respective test compound and permeability control.
Zebrafish Angiogenesis Model. Zebrafish (Daniorerio) trans-
genic lines Tg(kdrl:mCherry)ci5 × Tg(fli1a:negfp)y72,3 were from
Taiwan Zebrafish Core Facility (Academia Sinica, Taipei, Taiwan)
and maintained at 28.5 °C in an incubator with approval from the
Animal Care Committee at National Sun Yat-sen University
(Kaohsiung, Taiwan). Embryos were treated with 0.003% 1-phenyl-
2-thiourea (PTU; Sigma, St. Louis, MO, USA) at 6 h postfertilization
(hpf) to prevent pigment formation and used to monitor the effects of
Fcw34 on embryonic angiogenesis.4 Zebrafish embryos were
generated by natural pair-wise mating and raised at 28 °C in embryo
water (0.2 g/L of Instant Ocean Salt in distilled water).
Approximately, 20 healthy embryos were placed in 6 cm dishes and
various concentrations of Fcw34 were separately added to the embryo
water at 6 h postfertilization (hpf). The embryo water containing
Fcw34 was replaced daily. At 30 hpf, the embryos were anesthetized
using 0.05% 2-phenoxyethanol in embryo water and examined for
vessel development, especially in the ISV and the CVP; at 48 hpf, the
migration and proliferation of endothelial cells were observed in the
ISV, using a fluorescence microscope with a digital imaging system
(Olympus; Tokyo, Japan). The length of the ISV was measured with
the NIH Image program.
Immunoprecipitation Assay. Cells were lysed in RIPA buffer
(20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,
1% NP-40, 1% sodium deoxyphosphate, 2.5 mM pyrophosphate, 1
mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL Leupeptin, and 1
mM PMSF). After clarification, 800 μg of total proteins was
immunoprecipitated with the antibody to p-talin-1 (Cell Signaling,
Inc.) and protein A Dynabeads (Life Technology, Inc.). Immuno-
precipitates were subjected to Western blotting using the indicated
antibodies.
Xenograft Study. Athymic nude mice (BALB/cAnN.Cg-Foxn1nu/
CrlNarl) of 4−5 weeks of age were obtained from the National
Laboratory Animal Center (Taipei, Taiwan) and were raised in a
specific pathogen-free environment. The animal studies were
approved by the Animal Core Facility at Academia Sinica.
The breast cancer MDA-MB-231/Luc cells were kindly provided
by Dr. Pei-wen Hsiao (ABRC Laboratory Animal Core Facility,
Agricultural Biotechnology Research Center, Academia Sinica,
Taipei); first, they were re-suspended in normal saline and then
subcutaneously injected into the abdominal mammary gland area of
nude mice. Tumor volumes and body weight were measured every
week and calculated using the formula, V = 1/2 × length × (width)2.
While the tumor grew to about 70−100 mm3 (Day 20), we divided
the animals into two groups randomly and all mice received 10 mg/kg
FCW34 every other day. Vehicle (40% PEG400 + 60% normal saline)
treatment was used as control. The growth and spontaneous
metastasis of tumors were observed under an IVIS50 in vivo imaging
system (Xenogen) with Firefly D-Luciferin substrate (NanoLight)
injection. The metastasized tumor tissues were dissected on day 56.
Ethics Statement. The procedures of animal experiments were
performed according to the guidelines provided by the Council of
Agriculture Executive Yuan, Taiwan (ROC). The protocol used for
the mouse study was approved by the Academia Sinica Institutional
Animal Care and Use Committee (Protocol ID 11-12-297) and
conducted in accordance with the guidelines. The mice were raised in
a specific pathogen-free environment and efforts were made to
minimize animal suffering.
Statistical Analysis. Differences between the groups were
statistically evaluated using the unpaired Student’s t-test. The results
are presented as mean SEM. All P values were two-tailed, and a P
value of less than 0.05 was considered to be statistically significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01477.
■
sı
Histological Analysis. Tissues were fixed in either 4%
paraformaldehyde or Bouin’s solution for 24 h following standard
sectioning methods and staining in hematoxylin/eosin.
Effect of 2aA−2eA on migration in MDA-MB-231 cells;
effect of FCW34 on migration in MDA-MB-231 cells;
effect of FCW66 on migration in MDA-MB-231 cells;
effects of ST3GALIII siRNA knockdown efficiency in
MDA-MB-231 cells; inhibition of integrin sialylation by
sialyltransferase inhibitors in MDA-MB-231 cells; effects
of sialyltransferase inhibitor FCW34 on tumor meta-
stasis in MDA-MB-231 breast cancer cells in vivo; effects
of sialyltransferase inhibitor FCW34 on toxicity assess-
ment in MDA-MB-231 breast cancer cells in vivo; effects
of sialyltransferase inhibitors on the integrin−FAK−
paxillin pathway in MDA-MB-231 cells; effects of
sialyltransferase inhibitors on the NFκB pathway in
MDA-MB-231 cells; apparent permeability (Papp) values
obtained for the reference compounds, FCW34, and
FCW66 using the PAMPA model; endogenous
expression of ST6GALI, ST3GALIII, ST3GALI, and
ST6GALNACI proteins; comparison of binding modes
of 2aA−2eA, 3cB−3dB, 4cC−4dC, AL10, LCAG, and
Lith-O-ASP in the binding sites of two asymmetric
polypeptide chains (A/B) of ST6GALI (PDB 6QVT);
computed physicochemical properties of the selected
compounds used in this study; computational binding
values of complex structure of ST6GALI-inhibitor;
sensitization of tumors to ST inhibitors by regulation
of the talin/integrin/FAK/paxillin and integrin/NFκB-
signaling pathways; and experimental procedures, details
Immunohistochemistry. The paraffin-embedded tissue blocks
were sectioned into 3 μm slices and mounted on the poly-L-lysine-
coated slides. After deparaffinization, the slides were blocked with 3%
hydrogen peroxide for 10 min and subjected to antigen retrieval with
microwaving in 10 mM citrate buffer for 15 min. The CD31 (1:100
dilution; BD) and Ki67 (1:200 dilution; Novocastra) antibodies were
applied onto the sections and incubated at 4 °C overnight, followed
by repeated wash with phosphate-buffered saline (PBS). The
horseradish peroxidase/Fab polymer conjugate (Polymer Detection
System, Zymed, USA) was then applied to the sections and the
sections were incubated for 30 min. After rinsing with PBS, the
sections were incubated with the peroxidase substrate diaminobenzi-
dine (1:20 dilution, Zymed) for 5 min. Thereafter, the sections were
counterstained with Gill’s hematoxylin for 2 s, dehydrated with serial
dilutions of ethyl alcohol, cleared with xylene, and finally mounted.
Parallel Artificial Membrane Permeability Assay. The assay
was performed as described in the protocol of the parallel artificial
permeability assay kit (Bioassay Systems, PAMPA-096). Working
solutions of each compound were prepared from 10 mM stock
solution in DMSO, which was then diluted into a final concentration
of 200 μM PBS (pH 7.4, 2% DMSO). Solution containing 5%
lecithin/dodecane was added to the donor side of the well membrane.
Reference compounds, chloramphenicol, diclofenac, and theophylline,
with 200 μM concentrations were chosen as the permeability control
and the test compounds (200 μM), FCW34 and FCW66, were then
added to duplicate the donor wells. The acceptor side of the well was
filled with PBS. The plates were then kept at room temperature for 18
h. On the following day, samples were collected from the acceptor
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of HPLC-determined purity data, HRMS results, and 1H
and 13C NMR spectra of all final compounds and
intermediates (PDF)
020, MOST 108-3111-Y-001-056, and MOST-108-3114-Y-
001-002).
ABBREVIATIONS
■
Molecular formula strings and some data (CSV)
ASP, aspartic acid; CD31, cluster of differentiation 31; Co-IP,
coimmunoprecipitation; CVP, caudal vein plexus; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DCC, dicyclohexylcarbodii-
mide; DIPEA, N,N-diisopropylethylamine; DMAP, 4-
(dimethylamino)pyridine; EC, ethylene glycol; ECM, extrac-
ellular matrix; FAK, focal adhesion kinase; FMOC, fluo-
renylmethoxycarbonyl; HBTU, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HPF, hours
postfertilization; H&E, hematoxylin and eosin; IKK, inhibitory
kappa B kinase; ISV, intersegmental vessels; LCA, lithocholic
acid; MC, methoxycoumarin; NBD, N-nitrobenzoxadiazole;
NF-κB, nuclear factor kappa-light-chain-enhancer of activated
B cells; PECAM-1, platelet-endothelial cell adhesion molecule-
1; PEG, polyethylene glycol; ST, sialyltransferase; t-BU, tert-
butyl; TFA, trifluoroacetic acid
AUTHOR INFORMATION
■
Corresponding Author
Wen-Shan Li − Institute of Chemistry, Academia Sinica,
Taipei 115, Taiwan; Doctoral Degree Program in Marine
Biotechnology, National Sun Yat-Sen University, Kaohsiung
804, Taiwan; Ph.D Program in Biotechnology Research and
Development, Taipei Medical University, Taipei 110,
Taiwan; Department of Medicinal and Applied Chemistry,
Kaohsiung Medical University, Kaohsiung 807, Taiwan;
Department of Chemistry, College of Science, Tamkang
University, New Taipei City 251, Taiwan; orcid.org/
0000-0002-8359-4582; Email: wenshan@
gate.sinica.edu.tw
Authors
REFERENCES
■
Chih-Wei Fu − Institute of Chemistry, Academia Sinica,
Taipei 115, Taiwan; Department of Chemistry, National
Central University, Taoyuan City 320, Taiwan
Han-En Tsai − Institute of Chemistry, Academia Sinica, Taipei
115, Taiwan
Wei-Sheng Chen − Institute of Chemistry, Academia Sinica,
Taipei 115, Taiwan; Department of Chemistry, National
Central University, Taoyuan City 320, Taiwan
Tzu-Ting Chang − Institute of Chemistry, Academia Sinica,
Taipei 115, Taiwan
Chia-Ling Chen − Institute of Chemistry, Academia Sinica,
Taipei 115, Taiwan
Pei-Wen Hsiao − Agricultural Biotechnology Research Center,
Academia Sinica, Taipei 115, Taiwan
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ACKNOWLEDGMENTS
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We acknowledge the use of NMR and Mass Spectrometry
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