An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01685

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target β-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target β-catenin actually function as mitochondrial pr

Full text of DOI:10.1021/acs.jmedchem.9b01685

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Article
An Underlying Mechanism of Dual Wnt Inhibition and AMPK
Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors
Wen Zhang, Vitaliy M Sviripa, Liliia Kril, Tianxin Yu, Yanqi Xie, William Brad
Hubbard, Pat Sullivan, Xi Chen, Chang-Guo Zhan, Yang Yang-Hartwich, B.
Mark Evers, Brett Spear, Roberto Gedaly, David S. Watt, and Chunming Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01685 • Publication Date (Web): 27 Nov 2019
Downloaded from pubs.acs.org on November 28, 2019
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An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation:
Mitochondrial Uncouplers Masquerading as Wnt Inhibitors
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Wen Zhang1,2, Vitaliy M. Sviripa3,4, Liliia M. Kril1,2,3, Tianxin Yu1,2, Yanqi Xie1,2,
W. Brad Hubbard5, Patrick G. Sullivan5, Xi Chen4, Chang-Guo Zhan4, Yang Yang-
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Hartwich6, B. Mark Evers2,7, Brett T. Spear2,8, Roberto Gedaly2,7,9
David S. Watt1,2,3* and Chunming Liu1,2*
,
1Department of Molecular and Cellular Biochemistry, College of Medicine, University of
Kentucky, Lexington, KY 40536
2Lucille Parker Markey Cancer Center, University of Kentucky, Lexington, KY 40536
3Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of
Kentucky, Lexington, KY 40536
4Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,
Lexington, KY 40536
5Department of Neuroscience, College of Medicine, University of Kentucky, Lexington,
KY 40536
6Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of
Medicine LSOG 209, 375 Congress Avenue, New Haven CT 06510
7Department of Surgery, College of Medicine, University of Kentucky, Lexington, KY
40536
8Department of Microbiology, Immunology and Molecular Genetics, University of
Kentucky, Lexington, KY 40536
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9Transplant Center, College of Medicine, University of Kentucky, Lexington, KY 40536
*Correspondence to: chunming.liu@uky.edu or dwatt@uky.edu
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Abstract
The importance of upregulated Wnt signaling in colorectal cancers led to efforts to
develop inhibitors that target -catenin in this pathway. We now report that several “Wnt
inhibitors” that allegedly target -catenin actually function as mitochondrial proton
uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling.
As expected for a process in which mitochondrial uncoupling diminishes ATP
production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor,
2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings,
a well-known “Wnt inhibitor”, FH535, functioned as a proton uncoupler, and in support
of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-
nitrophenyl)benzenesulfonamide (FH535-M) was inactive as an uncoupler and Wnt
inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and
AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition
alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the
electron transport chain.
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Introduction
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Aberrant activation of Wnt signaling is a hallmark of many human cancers, particularly
colorectal cancer (CRC), and the development of inhibitors that target this pathway and
the re-purposing of non-cancer related, FDA-approved drugs that target this pathway
represent promising venues for therapeutic advances in cancer treatment 1-5. The Wnt
inhibitors in current use include “off-label” drugs and new agents now under evaluation
in Phase 1/2 clinical trials 2, 4, 6. The mechanisms by which these agents affect the Wnt
signaling pathway are often unclear, and efforts to understand these events at a
biochemical level will facilitate the development of future Wnt inhibitors.
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In the absence of ligands that trigger Wnt signaling, -catenin undergoes sequential
phosphorylation by casein kinase-1 alpha (CK1) and glycogen synthase kinase-3
(GSK3) in the Axin/Adenomatous Polyposis Coli complex (Axin/APC) 7 to furnish
phosphorylated -catenin. In normal cells, phosphorylated -catenin undergoes
ubiquitination by -Transducin Repeat Containing E3 Ubiquitin Protein Ligase (-TrCP)
and subsequent proteasomal degradation 2-4, 7, 8. In colorectal cancer cells, mutations in
either -catenin or APC render this phosphorylation-ubiquitination-degradation sequence
dysfunctional, and either -catenin phosphorylation or -TrCP-mediated degradation fail.
These outcomes lead to aberrant -catenin accumulation and the translocation of
undesired levels of -catenin to the nucleus 3, 9. In the nucleus, these augmented levels of
-catenin bind to co-activators, including the T-cell factor/lymphoid enhancer factor
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(TCF/LEF), induce transcription of Wnt-target genes, such as c-Myc and cyclin D1, and
promote undesired growth 3.
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The complexity of the Wnt pathway lends itself to the development of inhibitors that
target various stages of these signaling events. High-throughput-screening methods using
stable cell lines containing Wnt reporter genes find frequent application for siRNA or in
small molecule screening 10, 11. Porcupine inhibitors 12, IWP-2 or LGK974, block the
secretion of Wnt ligands that initiate the signaling cascade and promote -catenin
degradation. Tankyrase inhibitors 13, XAV939 or IWR-1, stabilize Axin and also
promote -catenin degradation. Other inhibitors, such as ICG-001, bind and inhibit
CREB-binding Protein (CBP), a transcriptional co-activator of the -catenin/TCF
complex, and inhibit Wnt target gene expression 14. In contrast, several, alleged Wnt
inhibitors lack direct targets or clear mechanisms but find widespread use by many
investigators in research focused on understanding the Wnt pathway or cancer biology.
We studied alleged Wnt inhibitors in several chemical classes (e.g., N,N-diarylureas, N-
aryl benzenesulfonamides), but we failed, despite considerable effort, to elucidate precise
biological targets associated directly with the Wnt signaling pathway using biologically
active biotinylated analogs and pull-down assays 15, 16. For example, after screening a
library of more than 5,000 compounds using a stable HEK293T cell line containing a
modified TOPFlash reporter we identified 1-(1,1,1,4,4,4-hexafluoro-2-
(trifluoromethyl)butan-2-yl)-3-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)urea (FTU-11)
(Fig. 1A) as a potential Wnt inhibitor. We noted that FTU-11 resembled another
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fluorinated urea, 1-(4-(trifluoromethyl)phenyl)-3-(3,4,5-trifluorophenyl)urea (FDN-4E)
(Fig. 1A), reported to function as an AMPK activator 17, 18. Our chance observations that
FDN-4E also inhibited Wnt signaling and that FTU-11 also activated AMPK signaling
suggested a common mechanism for urea-mediated, Wnt inhibition and AMPK
activation. As an additional example, the N-aryl benezenesulfonamide, 2,5-dichloro-N-
(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), reported as an inhibitor of both β-
catenin in the Wnt signaling pathway and the Peroxisome Proliferator-activated Receptor
(PPAR) 19, also activated AMPK. A confluence of screening, and analog development
along several lines led a fortuitous intersection of experiments in which we recognized a
linkage among the Wnt pathway, AMPK activation, and oxidative phosphorylation. We
identified several soi-disant Wnt inhibitors that actually function as proton uncouplers or
electron-transport inhibitors.
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Results and Discussion
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To identify novel Wnt regulators by high throughput screening, a stable HEK293T cell
line containing the TOPFlash reporter was established 5. To identify Wnt inhibitors that
targeted molecular events downstream of -catenin, we treated these cells with lithium
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chloride to inhibit GSK3 and to stabilize -catenin. Screening a library previously
available from the Drug Discovery Center at University of Cincinnati (Cincinnati, OH,
USA) led to the identification of FTU-11 (Fig. 1A) that inhibited Wnt signaling at a 0.5
M concentration (96-well assay using stable cell line) (Fig. 1B). FTU-11 possessed
structural features that resembled another highly fluorinated urea, FDN-4E (Fig. 1A), that
functioned as a potent AMPK activator and repressed the growth of CRC cells 17, 18. A
comparison study of FTU-11 and FDN-4E using the luciferase assay revealed that FDN-
4E also inhibited Wnt signaling (24-well assay using stable cell line) (Fig. 1C). We also
validated the results by transient transfection of Super 8x TOPFlash or 8x FOPFlash into
HEK293T cells. Both FTU-11 and FDN-4E inhibited TOPFlash but not FOPFlash
activity (Fig. 1D). Since the results from the stable and transient transfection are
compatible, we used stable cell lines for all of the other experiments. In addition, the
reporter activity, FTU-11 and FND-4E also inhibited the proliferation of colon cancer
cells at sub-micromolar concentrations (Fig. 2A) and inhibited Wnt target genes in three
CRC cell lines (Fig. 2B).
Most importantly, we observed that FTU-11 functioned as an AMPK activator equal in
potency to FDN-4E, and as expected for an AMPK activator, FTU-11 inhibited acetyl-
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CoA carboxylase (ACC) that was subject to regulation by phosphorylated AMPK (Fig.
3). Taken together, these findings suggested a linkage between AMPK activation and the
inhibition of Wnt signaling. We probed this relationship using a series of AMPK
inhibitors and activators as well as inhibitors of the Wnt pathway in which we accepted at
face value the assertions in the literature that these Wnt inhibitors and AMPK activators
had exclusive selectivity for one of these targets. As a working hypothesis, we assumed a
direct relationship in which FTU-11 and FDN-4E disrupted some cellular process that
triggered AMPK activation. The phosphorylated AMPK in turn served as an inhibitor of
the ATP-dependent Wnt pathway.
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We treated HEK293T cells containing a modified TOPFlash reporter simultaneously with
either FTU-11 or FDN-4E and with a potent AMPK inhibitor, 4-(2-(4-(3-(pyridin-4-
yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine, known commonly as
“Compound C” 20. If the inhibition of Wnt signaling required activated, phosphorylated
AMPK, as proposed in our hypothesis, then Compound C should rescue the Wnt
signaling inhibited by either FTU-11 or FDN-4E. Compound C inhibited ACC
phosphorylation (i.e., conversion to its inactive form), as expected (Fig. 4A), but it had
no effect on either the FTU-11-mediated or FDN-4E-mediated Wnt inhibition (Fig. 4B).
Next, we treated HEK293T cells containing a modified TOPFlash reporter with an
AMPK activator, 4-hydroxy-3-(2'-hydroxy-[1,1'-biphenyl]-4-yl)-6-oxo-6,7-
dihydrothieno[2,3-b]pyridine-5-carbonitrile (A769662) (Fig. 4C), but unlike the results
using FTU-11 and FDN-4E, the direct AMPK activator, A769662 21-23, did not inhibit
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Wnt signaling (Fig. 4B). Taken together, these results suggested that the ureas, FTU-11
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and FDN-4E, inhibited Wnt signaling through an AMPK-independent mechanism.
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We hypothesized that either FTU-11 or FDN-4E affected a cellular process that altered
Wnt signaling and independently triggered the activation of the AMPK energy sensor.
We tested well-known Wnt inhibitors, such as (6S,9aS)-N-benzyl-6-(4-hydroxybenzyl)-8-
(naphthalen-1-ylmethyl)-4,7-dioxohexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-
carboxamide (ICG-001) and FH535 14, 19. These compounds had dramatically different
effects on AMPK activation. ICG-001 only inhibited Wnt signaling but had no effect on
AMPK (data not shown), and in contrast, FH535 not only inhibited Wnt signaling (Fig.
4D) but also induced AMPK phosphorylation and ACC phosphorylation (Fig. 4E). Since
ICG-001 was a well-characterized Wnt inhibitor with a specific target, namely the CREB
Binding Protein (CBP) 14, we concluded that FTU-11 and FH535 activated AMPK
through Wnt-independent mechanisms. In addition, we detected AMPK activation within
15 min of treatment with FH535. This rapid response was inconsistent with AMPK
regulation mediated by Wnt transcription. In conclusion, AMPK activation was an
unlikely a consequence of Wnt inhibition.
Since either FTU-11 or FH535, but not A769662, inhibited Wnt signaling, we concluded
that these N,N-diarylureas indirectly activated AMPK through a mechanism that altered
AMP/ATP ratio. To confirm this point, we analyzed ATP production in FTU-11-treated
and FH535-treated cells using a Seashorse XF Analyzer. As a control, we selected a well
known mitochondrial uncoupler, N-(4-(trifluoromethoxy)phenyl)carbonohydrazonoyl
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dicyanide (FCCP) 24, and we found that FTU-11, FH535 and FCCP decreased the rates of
ATP production in mitochondria (Fig. 5A). As expected, the decrease in mitochondrial
ATP production led to concomitant increase in glycolytic ATP production and AMPK
activation. These results suggested that these compounds induced AMPK activation by
inhibiting mitochondrial function.
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FCCP represented a potent, mitochondrial uncoupler that translocated protons across the
mitochondrial inner membrane, decreased oxidative phosphorylation that provided ATP
and increased oxygen consumption rate (OCR) as a means of compensating for the
increased pH in the intermembrane space. FCCP possessed both a hydrophobic
substructure and an ionizable nitrogen-hydrogen bond with a pKa sufficient to function as
a transmembrane proton transporter. Since uncouplers reduced membrane potential, we
analyzed the effect of FTU-11 on mitochondrial membrane potential using
tetramethylrhodamine methyl ester (TMRM) staining and found that FTC-11
significantly reduced the membrane potential of mitochondria (Fig. 5B). FDN-4E and
FH535 had similar effects (data not shown).
A previous study indicated that FH535 affected mitochondria respiration, but the exact
mechanism was unclear 25. The above data suggested that the ureas in this study and
other, soi-disant Wnt inhibitors functioned as mitochondrial proton uncouplers. We next
analyzed the effects of these compounds on OCR using Seashorse XF Analyzer, with
FCCP as a control (Figs. 5C and D). Oligomycin (Oligo) inhibited ATPase and thus
inhibited OCR 24. FCCP, FTC-11 and FH535 uncoupled mitochondrial
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oxidation/phosphorylation (OXPHOS) and increased OCR inhibited by oligomycin (Figs.
5E and F). Rotenone and antimycin A blocked the increase in OCR by inhibiting
electron-transport Complexes I and III, respectively 24.
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In the uncoupling assay, FH535 had the same function as the standard mitochondrial
uncoupler, FCCP. As was the case for FCCP, FH535 also possessed a hydrophobic
substructure and an ionizable, nitrogen-hydrogen bond that participated in proton
translocation. To confirm this point, we replaced this “active” hydrogen with a methyl
group (Fig. 6A), and as expected, we found that the N-methyl analog, 2,5-dichloro-N-
methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), no longer
functioned as a mitochondrial uncoupler (Fig. 6B) and did not activate AMPK or inhibit
Wnt (data not shown). Furthermore, we synthesized FH535 analogs and identified 2,5-
dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide analog, which we called Y3
(Fig. 6A), that strongly activated AMPK and inhibited Wnt signaling (Fig. 6C and D).
Just as in the case of FH535 and FH535-M, the methylated version of Y3, 2,5-dichloro-
N-methyl-N-(4-nitronaphthalen-1-yl)benzenesulfonamide (Y3-M) (Fig. 6A), failed as a
mitochondrial uncoupler (data not shown) and led to neither AMPK activation (Fig. 6C)
or Wnt inhibition (Fig. 6D).
The calculated pKa values for FH535 and Y3 using the ACD/pKa DB (version 11.1)
software (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada) were 5.83
and 5.39, respectively, and the calculated pKa values for FH535 and Y3 using the
ChemAxon (version 19.18) software (ChemAxon, Inc., Cambridge, MA) were 6.69 and
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6.46, respectively. In support of the validity of these pKa values, the calculated values for
another sulfonamide, trifluoromethanesulfonamide (CF3SO2NH2), using these same two
programs were 6.37 and 6.19, respectively, and these values compared well with the
experimentally determined value 26 of 6.3. Based on the reported pH values 27 for the
intermembrane space (pH 6.88±0.09) and the mitochondrial matrix (pH 7.78±0.17) and
assuming the release of these uncouplers into these two compartments and not simply the
retention in the inner membrane, the ChemAxon pKa values predicted that the
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percentages of the protonated forms of FH535 in the hydronium ion-rich intermembrane
space and the mitochondrial matrix were 39% and 8%, respectively (Fig. 7A). In the
same fashion, the percentages of the protonated forms of Y3 in the hydronium ion-rich
intermembrane space and the mitochondrial matrix were 28% and 5%, respectively.
A model for proton translocation entails the transport of the uncoupler and the conjugate
base of the uncoupler across the inner mitochondrial membrane. As suggested
diagrammatically (Fig. 7A), the inner membrane possesses several transporters for the
translocation of other anionic species (e.g., Pi, ADP), and these channels may participate
in translocating the conjugate base 28. In summary, these calculated pKa values and pH
values for the intermembrane space and matrix of mitochondria were consistent with both
FH535 and Y3 retaining reasonable, equilibrium concentrations of protonated and
unprotonated forms sufficient to translocate protons across the inner membrane. In
addition, FH535 and Y3 possessed calculated log P values (i.e., 5.83 and 5.39,
respectively), and these values were similar to other mitochondrial proton uncouplers,
again consistent with representing FH535 and Y3 as weak acids possessing the necessary
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lipophilicity to accomplish uncoupling. These results further supported that the
mitochondrial proton uncoupling activity of these compounds contributed to their
activities as AMPK activators and Wnt inhibitors.
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Since the above studies were performed in human cell lines, it was not clear whether the
test compounds targeted mitochondria directly, or indirectly through cell signaling
pathways, including the Wnt signaling pathway. To address this question, we performed
uncoupling assay using purified mitochondria from mouse livers. Similar to the results
in cell lines, the OCR was blocked by oligomycin rescued by control proton uncoupler,
FCCP. FH535 and Y3 also increased OCR in dose-dependent manner, but as expected,
the methylated analogs, FH535-M and Y3-M were inactive as mitochondrial proton
uncouplers (Figs. 7B, 7C). Although we cannot rule out the possibility that these
compounds may also have a mitochondrial-independent function, the experiments with
purified mitochondria suggested that these “Wnt inhibitors” directly target mitochondria
as proton uncouplers.
To test the hypothesis that the uncoupling effects inhibited Wnt signaling, we also
analyzed a classic mitochondrial proton uncoupler, FCCP, and found that FCCP strongly
activated AMPK (Fig. 8A) and inhibited Wnt signaling (Fig. 8B). We then tested
numerous inhibitors in the Seahorse assay, including oligomycin, rotenone and antimycin
and found that all of them inhibited oxidative phosphorylation, activated AMPK and
inhibited Wnt signaling (data not shown), an observation again consistent with the
inhibition of Wnt signaling by reduced ATP production. To test further this hypothesis,
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we treated cells with a metabolically inert, glucose analog, 2-deoxy-D-glucose (2-DG), to
reduce ATP levels by inhibiting glucose metabolism 29, and as expected 2-DG activated
AMPK (Fig. 8C), inhibited Wnt signaling (Fig. 8D) and reduced ATP levels in treated
cells (Fig. 8E). We analyzed the -catenin levels in LS174T cells. LiCl treatment
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increased both total and nucleus -catenin. FTU-11 and FH535 treatment reduced -
catenin induced by LiCl (Fig. 8F), suggesting that uncouplers may affect -catenin
protein synthesis or nuclear localization.
Conclusions
Many steps in the Wnt signaling pathway, including -catenin nuclear import and
chromatin remodeling, are ATP-dependent 3, 30, 31. We found the FTU-11 and FH535
reduced -catenin levels in LS174T cells (Fig. 8F), consisted with ATP requirements for
-catenin protein synthesis or nuclear localization. Other reports described ATP-
dependent steps in Wnt signaling. For example, the ATP-dependent chromatin
remodeling protein, Brg1, bound to -catenin and actived the transcription of Wnt-target
genes 30. Deletion of the ATPase domain of Brg1 inhibited Wnt signaling. Loss of Brg1
attenuated Wnt signaling and prevent Wnt-dependent tumorigenesis in the intestines 32.
A recent paper suggested that impaired mitochondrial ATP production inhibited Wnt
signaling via an induction of ER stress 33. Wnt signaling also regulates mitochondria
dynamics and membrane potential in a subset of cancers 34. Although ATP is not
specifically required for Wnt signaling, this study illuminated the linkage between this
ATP requirement and claims that some compounds, previously identified as direct Wnt
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inhibitors, in fact function as either inhibitors of mitochondrial oxidative phosphorylation
or the electron transport chain. This study not only suggested a need to re-examine some
of these prior claims but also suggested a new strategy for inhibiting Wnt signaling in the
development of new antineoplastic agents for cancer treatment. Many agents that
function as AMPK activators may also act as Wnt inhibitors and provide an impetus to
examine connections in which drugs for treating metabolic disorders find a potential
application in cancer therapeutics.
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Experimental Section
(1) Chemistry
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General Methods. FTU-11 was purchased from a chemical library once available
through the University of Cincinnati (Cincinnati, OH, USA) and purified by HPLC.
FDN-4E, FH535 and Y3 were synthesized as previously described 17, 18, 35. Solvents were
used from commercial vendors without further purification unless otherwise noted.
Nuclear magnetic resonance spectra were determined in DMSO-d6 using Varian
instruments (1H, 400; 13C, 100Mz; Varian, Inc., Palo Alto, CA, USA). High resolution
electrospray ionization (ESI) mass spectra were recorded on a LTQ-Orbitrap Velos mass
spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The FT resolution was set
at 100,000 (at 400 m/z). Samples were introduced through direct infusion using a syringe
pump with a flow rate of 5 µL/min. Melting points were determined in open capillarity
tubes with a Büchi B-535 melting point apparatus (Büchi Corp., New Castle, DE, USA)
and are uncorrected. Purity was established by combustion analyses performed by
Atlantic Microlabs, Inc. (Norcross, GA, USA). Further confirmation of purity of all
tested compounds was obtained by RP-HPLC that was performed on an Agilent
Technologies 1260 Infinity HPLC system by using the following general method: flow
rate = 0.5 mL/min; λ = 254 nm; column = Vydac 201SP C18, 250 mm × 4.6 mm, 90 Å, 5
μm. Eluent A: H2O + 0.1% TFA (v/v); Eluent B: acetonitrile; gradient profile, starting
from 5% B, increasing from 5% B to 100% B over 10 min, holding at 100% B from 10 to
20 min, and decreasing from 100% B to 5% B from 20 to 23 min. Prior to each injection,
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the HPLC column was equilibrated for 10 min with 1% B. All compounds tested were
determined to be ≥97% pure. Other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) with purify >95%.
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1-(1,1,1-Trifluoro-2-(trifluoromethyl)butan-2-yl)-3-(5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)urea (FTU-11). The purity of FTU-11 was confirmed by RP-HPLC: Rt
= 19.79 min (99% pure; Supporting Information, Figure S1).
1-(4-(Trifluoromethyl)phenyl)-3-(3,4,5-trifluorophenyl)urea (FDN-4E). To a stirred
solution of 77 mg (0.52 mmol) of 3,4,5-trifluoroaniline in 3 mL of benzene was added 97
mg (0.52 mmol, 1 eq) of 1-isocyanato-4-(trifluoromethyl)benzene. The mixture was
stirred at 50C for 3 h and was cooled to 25C. A precipitate was collected by filtration
to provide 90 mg (52%) of analytically pure FDN-4E as a white solid: mp 241-242oC.
1H NMR: δ 9.29 (s, 1H, NH), 9.14 (s, 1H, NH), 7.67 (d, 2H, J=8.8Hz), 7.62 (d, 2H,
J=8.8Hz), 7.42-7.38 (m, 2H). 13C NMR: δ 152.12 , 150.17 (ddd, J = 243.5, 9.9, 5.6 Hz),
142.95 (d, J = 1.2 Hz), 135.86 (td, J = 12.1, 3.5 Hz), 135.17 (t, J = 15.7 Hz), 132.75 (t, J
= 15.8 Hz), 128.52 , 126.07 (q, J = 3.8 Hz), 125.82 , 123.12 , 122.22 (q, J = 32.0 Hz),
120.42 , 118.22 , 102.71 (dd). HRMS (ESI) Calcd for C14H9F6N2O [MH+]: 335.0614.
Found: 335.0619. Anal. Calcd for C14H8F6N2O: C, 50.31; H, 2.41. Found: C, 50.09; H,
2.49. The purity of FDN-4E was further confirmed by RP-HPLC: Rt = 20.3 min (99%
pure; Supporting Information, Figure S2).
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2,5-Dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535). This
compound was synthesized as previously described 35. The purity of FH535 was
confirmed by RP-HPLC: Rt = 19.5 min (97% pure; Supporting Information, Figure S3).
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2,5-Dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M).
To a stirred solution of 360 mg (1 mmol, 1 eq) of FH535 and 830 mg (6 mmol, 6 eq) of
potassium carbonate in 2 mL of DMF was slowly added 0.12 mL (2 mmol, 2 eq) of
methyl iodide. After stirring at 25oC for 20 h, the mixture was poured into brine,
extracted with dichloromethane, dried over anhydrous MgSO4, and evaporated under
reduced pressure to afford a crude product. Purification by preparative layer silica gel
chromatography using 1:10 (v/v) ethyl acetate-hexane provided 0.2 g (54%) of FH-535-
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M: mp 142-143oC. H NMR: δ 8.23 (d, J = 2.7 Hz, 1H), 8.02 (dd, J = 8.7, 2.8 Hz, 1H),
7.87-7.77 (m, 3H), 7.26 (d, J = 8.7 Hz, 1H), 3.3 (s, 3H), 2.38 (s, 3H). 13C NMR: δ
146.87, 144.7, 140.33, 137.41, 134.79, 134.33, 132.38, 131.05, 130.2, 129.9, 126.05,
121.97, 39.39, 17.84. HRMS (ESI) Calcd for C14H13Cl2N2O4S [MH+]: 374.9968.
Found: 374.9968. Anal. Calcd for C14H12Cl2N2O4S: C, 44.81; H, 3.22; N, 7.47. Found:
C, 44.71; H, 3.17; N, 7.41. The purity of FH535-M was confirmed by RP-HPLC: Rt =
20.26 min (99% pure; Supporting Information, Figure S4).
2,5-Dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide (Y3). To a suspension
of 2.5 mmol (2.5 eq) of sodium hydride (60% dispersion in oil) in 5 mL of anhydrous
tetrahydrofuran (THF) was added a solution of 188 mg (1 mmol) of 4-nitro-1-
naphthylamine in 1 mL of THF. The mixture was stirred for 10 min at 0oC and 246 mg
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(1 mmol) of 2,5-dichlorobenzenesulfonyl chloride was added. The mixture was stirred
for 12 h at 25oC, quenched with saturated NaHCO3 and extracted with ethyl acetate. The
combined organic layers were washed with brine, dried over anhydrous MgSO4 and
concentrated in vacuum to provide crude product. Purification by recrystallization from
methanol provided 254 mg (64%) of Y3: mp 232-233oC. 1H NMR: δ 11.45 (br s, 1H),
8.41 (d, 2H, J=8.8Hz), 8.27 (d, 1H, J=8.4Hz), 7.94 (d, 1H, J=2.4Hz), 7.82-7.78 (m, 1H),
7.75-7.68 (m, 3H), 7.45 (dd, 1H, J=8.4 and 2.6Hz). 13C NMR: δ 143.38, 138.47, 138.11,
134.62, 133.88, 132.30, 130.35, 129.92, 129.67, 128.37, 127.62, 125.23, 124.73, 123.69,
122.74, 118.96. HRMS (ESI) Calcd for C16H9Cl2N2O4S [MH-]: 394.9666. Found:
394.9662. Anal. Calcd for C16H10Cl2N2O4S: C, 48.38; H, 2.54. Found: C, 48.63; H,
2.46. The purity of Y3 was confirmed by RP-HPLC: Rt = 19.82 min (99% pure;
Supporting Information, Figure S5).
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2,5-Dichloro-N-methyl-N-(4-nitronaphthalen-1-yl)benzenesulfonamide Y3-M. To a
solution of 100 mg (0.25 mmol, 1 eq.) of Y3 and 210 mg (6 mmol, 6 eq) of potassium
carbonate in 2 mL of DMF was slowly added 0.031 mL (2 mmol, 2 eq) of methyl iodide.
After stirring at 25oC for 20 h, the mixture was poured into brine and extracted with
dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and
evaporated under reduced pressure to afford a crude product. Purification by preparative
layer silica gel chromatography using 1:10 ethyl acetate-hexane (v/v) provided 70 mg
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(68%) of Y3-M: H NMR: δ 8.32 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 8.25 (d, J = 8 Hz, 1H),
8.21 (d, J = 8 Hz, 1H), 7.86-7.8 (m, 5H), 7.46 (d, J = 8 Hz, 1H), 3.48 (s, 3H). Anal.
Calcd for C17H12Cl2N2O4S: C, 49.65; H, 2.94; N, 6.81. Found: C, 49.78; H, 2.88; N,
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6.73. The purity of Y3-M was confirmed by RP-HPLC: Rt = 20.7 min (99% pure;
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Supporting Information, Figure S6).
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(2) Biology
Cell culture. LS174T colon cancer cells were cultured in EMEM (ATCC, 30-2003)
containing 10% (v/v) Fetal Bovine Serum (Sigma F0926, Sigma Aldrich Corp., St. Louis,
MO, USA). The DLD-1, SW480 and SW620 colon cancer cells were cultured in DMEM
(Sigma D6429) containing 10% (v/v) Fetal Bovine Serum (Sigma, F0926). For
proliferation assays, cells (3.5x104 cells per well) were split into 12-well plates. After 24
h, 1 L of each compound in DMSO solution were added to each well. DMSO was used
as a control. Each experiment was done in triplicate. Cell viability and number were
analyzed using the Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Indianapolis,
IN, USA).
Biochemistry. Western blotting: Cells were lysed in the appropriate volume of lysis
buffer: 50 mM HEPES, 100 mM NaCl, 2 mM EDTA, 1% (v/v) glycerol, 50 mM NaF, 1
mM Na3VO4, 1% (v/v) Triton X-100, with protease inhibitors. The following antibodies
were used: AMPK (Cell Signaling, 2532, Cell Signaling Technologies, Danver, MA,
USA), pAMPK (Cell Signaling, 2535), ACC (Cell Signaling, 3676), pACC (Cell
Signaling, 11818), Actin (Sigma, A1978). Antibodies for Axin 2, c-Myc and Cyclin D1
have been described previously 36. ATP analysis: Cells growing in 12-well plates were
treated with DMSO or inhibitors in DMSO solution and lysed by adding 1 mL boiling
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doubly distilled water. Supernatants were analyzed by luminescence using ATP
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Determination Kit (Invitrogen, A22066; Thermo Fisher Scientific, Waltham, MA, USA).
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Reporter assay. Wnt reporter assay and cell staining assay have been described
previously 5, 36. We subcloned Super 8xTOPFlash (provided by Professor Randall Moon,
University of Washington) into the pGL4.83 [hRlucP/Puro] Vector and transfected it into
HEK293T cells. A stable HEK293T cell line containing the TOPFlash reporter was
established using puromycin selection. To screen Wnt inhibitors that block the
downstream signaling transduction pathway of β-catenin, we treated the reporter cells
with 25 mM LiCl to stabilize β-catenin and activate Wnt signaling. The potential Wnt
inhibitors identified from screening were validated by transfecting TOPFlash or
FOPFlash plus control renilla reporters into HEK293T cells, and then treating the cells
with DMSO or testing compounds in DMSO solution. The Wnt signaling was activated
by LiCl or Wnt3A treatment.
Mitochondria Membrane potential assay: DLD-1 and LS174T cells were plated onto
24-well plates and maintained in DMEM (DLD-1) and RPMI1640 (LS174T) with 10%
(v/v) (DLD-1) and 5% (v/v) (LS174T) serum for one day. The cells were treated with
DMSO or a testing compound in DMSO solution for 2 h, followed by staining with 100
nM tetramethylrhodamine methyl ester perchlorate (TMRM) (Cayman Chemical
Company, MI, USA) for 15 min. Cells were rinsed once with PBS and then observed
under fluorescence microscopy at 568 nm.
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Seahorse assay. Approximately 3x104 cells were seeded in XF96 Cell Culture
microplate (80 L of 3.75x105 cells/mL) for all experiments. On the next day, cell
culture media were replaced with either Seahorse XF modified media with 25 mM
glucose and 1 mM pyruvate. After media exchange, cells were treated with 1 µM of
oligomycin A, 1.0 µM FCCP and mixture of 1.0 µM of rotenone and 1.0 µM of
antimycin A in standard mitochondrial stress test conditions. To determine the uncoupler
effects, FCCP was replaced with an equal volume of DMSO, or a compound to be tested
in DMSO solution. ATP production rate was analyzed using Agilent Seahorse XF Real-
Time ATP Rate Assay.
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Isolation of Mitochondria from Brain Tissue. A mitochondrial isolation protocol was
adapted from the previously described protocols with slight modifications 37, 38. All the
steps were carried out at 4 oC or on ice. After mice were euthanized with CO2 followed by
rapid decapitation, whole liver was quickly dissected out on a cold block and homogenized
using a Teflon-glass dounce homogenizer containing isolation buffer (215 mM mannitol,
75 mM sucrose, 0.1% BSA, 20 mM HEPES, 1 mM EGTA, adjusted to pH 7.2 with KOH).
The homogenate was transferred to a 2 mL-microcentrifuge tube and spun at 1,300 x g for
3 min. The supernatant was transferred to a fresh 2 mL-microcentrifuge tube and spun at
13,000 x g for 10 min. The supernatant was discarded, and the crude mitochondrial pellet
was resuspended in a 1.5 mL-microcentrifuge tubes and pelleted again at 10,000 x g for 10
min. The supernatant was discarded, and the mitochondrial pellets were resuspended in
isolation buffer to obtain an approximate concentration ≥ 10 mg/mL of mitochondria. The
absolute protein concentration was determined using a bicinchoninic acid (BCA) protein
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assay kit (Pierce, Cat # 23,227) by recording absorbance at 560 nm on a Biotek Synergy
HT plate reader (Winooski, VT, USA).
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Mitochondrial Bioenergetics Measurements. Mitochondrial bioenergetic measurements
were carried out using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent
Technologies, Santa Clara, CA, USA) to measure oxygen consumption rate (OCR) during
various states of respiration. The OCR were measured in the presence of different
substrates, inhibitors and uncouplers of the electron transport chain using previous methods
with slight modifications 37, 38. The stocks used for the assays were 500 mM pyruvate, 250
mM malate, and 30 mM adenosine diphosphate (ADP), and 1 M succinate (pH for all was
adjusted to 7.2). Stock assay solutions of 1 mg/mL (1.26 mM) oligomycin A, 1 mM N-(4-
(trifluoromethoxy)phenyl)carbonohydrazonoyl dicyanide (FCCP), and 1 mM rotenone
were prepared in ethanol. Stock solutions of FH535, Y3, FH535-M, and Y3-M were
prepared at 5mM in 100% DMSO. As per the instructions from XFe96 Extracellular Flux
kit, the sensor cartridge was hydrated with water and kept at 37oC overnight before the
experiment. One hour before the assay was conducted, water was removed from assay
plates and XF calibrant was added before re-incubation. The injection ports A to D of the
sensor cartridge were loaded with 25 µL of different combinations of the above
substrates/inhibitors/uncouplers as follows. Before loading, the stocks were diluted
appropriately in the respiration buffer (RB) (125 mM KCl, 0.1% BSA, 20 mM HEPES, 2
mM MgCl2, and 2.5 mM KH2PO4; pH 7.2) to get the final concentrations in the respiration
chamber of 5 µM pyruvate, 2.5 µM malate, 10 µM of succinate and 1 µM ADP (via Port
A), 1 µM oligomycin A (via Port B), 4 µM FCCP or 5 µM/1 µM FH535 or 5 µM/1 µM
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Y3 or 5 µM/1 µM FH535-M or 5 µM/1 µM Y3-M (via Port C) and 0.1 µM antimycin A
(via Port D) starting with the initial volume of 175 µL RB in the chamber and diluting it to
9X, 10X, 11X and 12X with every injection through ports A to D, respectively. Once
loaded, the sensor cartridge was placed into the Seahorse XFe96 Flux Analyzer for
automated calibration. Seahorse Standard XFe96 assay plates were used for loading
mitochondria. Initially total mitochondria were diluted to 6 µg/30 µL in RB and 30 µL
was loaded in each well resulting in 6 µg mitochondria/well. The assay plates were
centrifuged at 3,000 rpm for 4 min at 4oC to adhere liver mitochondria at the bottom of the
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wells. After centrifugation, 145 µL RB (pre-incubated to 37 C) was added without
disturbing the mitochondrial layer to obtain a final volume of 175 µL per well. After the
instrument calibration with the sensor cartridge was complete, the utility plate was replaced
by the plate loaded with mitochondria for bioenergetics analysis. The assays were carried
out under a previously optimized protocol. Briefly, it involved cyclic steps of mixing,
sequential injections of substrates/inhibitors via Ports A thru D, mixing, equilibration, and
measurement of the OCR through fluorimetric optical probes. The data output gives State
III respiration mediated through complex I and II in the presence of pyruvate, malate (PM),
succinate and ADP (Port A) followed by State IV rate in presence of oligomycin (Port B).
OCR was then measured in the presence of FCCP or compounds of interest in this study to
examine mitochondrial specific uncoupling activity State VCI+CII (Port C) and finally non-
mitochondrial respiration in presence of Antimycin A (Port D).
Statistics. All experiments were performed with at least three independent repeats.
Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, CA,
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USA). For all analyses, the significance of differences among groups was set at p < 0.05.
Error bars represent standard deviations. The Seahorse results were analyzed with
Seahorse Wave Desktop Software.
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Acknowledgments
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We are grateful to Professor Randall Moon for the Super 8x TOPFlash and 8x FOPFlash
plasmids. CL and DSW were supported by NIH R01 CA172379 from the National
Institutes of Health and by NIH UL1 TR000117 from the National Institutes of Health to
the University of Kentucky’s Center for Clinical and Translational Science. BME was
supported by NIH P30 CA177558 from the National Institutes of Health. DSW was also
supported in part by the Office of the Dean of the College of Medicine, the Center for
Pharmaceutical Research and Innovation in the College of Pharmacy, the Department of
Defense (DoD Prostate Cancer Research Program Award W81XWH-16-1-0635 [Grant
Log# PC150326P2]), and NIH P30 RR020171 from the National Institute of General
Medical Sciences to L. Hersh. VMS was supported by grant IRG 16-182-28 from the
American Cancer Society). The Redox Metabolism Shared Resource Facility (RM SRF)
that performed the Seahorse XF studies is supported by Markey Cancer Center (P30
CA177558).
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Conflict of Interest Statement
CL and DSW have partial ownership in a for-profit venture, Epionc, Inc., that seeks to
develop small-molecule inhibitors for cancer treatment. In accord with University of
Kentucky policies, CL and DSW have disclosed this work to the University of
Kentucky’s Intellectual Property Committee and to a Conflict of Interest Oversight
Committee.
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Ancillary Information
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a. Supporting Information Availability:
Supplemental Figures S1-S6 and Molecular Formula Strings Page.
b. Corresponding Author Information:
Correspondence to: chunming.liu@uky.edu or dwatt@uky.edu
c. List of Abbreviations Used:
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ACC: Acetyl-CoA carboxylase;
AMPK: Adenosine 5’ monophosphate-activated protein kinase;
BCA, Bicinchoninic acid;
CRC: colorectal cancer
2DG: 2-deoxy-D-glucose;
DMEM, Dulbecco’s Modified Eagle Medium
DMF, Dimethylformamide;
DMSO, Dimethyl sulfoxide;
EDTA, Ethylenediaminetetraacetic acid;
EMEM, Eagle’s minimum essential medium;
FDN-4E, 1-(4-(Trifluoromethyl)phenyl)-3-(3,4,5-trifluorophenyl)urea;
FH535, 2,5-Dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide
FH535-M, 2,5-Dichloro-N-methyl-N-(2-methyl-4-
nitrophenyl)benzenesulfonamide
FTU-11, 1-(1,1,1-Trifluoro-2-(trifluoromethyl)butan-2-yl)-3-(5-(trifluoromethyl)-
1,3,4-thiadiazol-2-yl)urea;
HEPES, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid;
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OCR: oxygen consumption rate;
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OXPHOS: oxidation/phosphorylation;
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RB, respiration buffer;
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RP-HPLC, reverse phase high pressure liquid chromatography;
Rt, retention time;
TMRM, Tetramethylrhodamine methyl ester;
Y3, 2,5-Dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide;
Y3-M, 2,5-Dichloro-N-methyl-N-(4-nitronaphthalen-1-yl)benzenesulfonamide.
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