Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2

Article abstract of DOI:10.1002/cmdc.201700028

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca²⁺-dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition.

Full text of DOI:10.1002/cmdc.201700028

Accepted Article
Title: Target deconvolution efforts on Wnt pathway screen reveal dual
modulation of oxidative phosphorylation and SERCA2
Authors: Matias Casas-Selves, Andrew Zhang, James E. Dowling,
Stefan Hallen, Aarti Kawatkar, Nicholas J Pace, Christopher
Denz, Timothy Pontz, Farzin Garahdaghi, Qing Cao, Alan
Sabirsh, Kumar Thakur, Nichole O'Connell, Jun Hu, Ivan
Cornella-Taracido, Eranthie Weerapana, Michael Zinda, and
Robert A. Goodnow
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.201700028
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700028
A Journal of
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10.1002/cmdc.201700028
ChemMedChem
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Target deconvolution efforts on Wnt pathway screen reveal dual modulation of oxidative
phosphorylation and SERCA2
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Dr. Matias Casás-Selves,1,7,+ Dr. Andrew X. Zhang,2,+,* Dr. James E. Dowling,1 Dr. Stefan Hallén,3 Aarti Kawatkar,2
Dr. Nicholas J. Pace,4 Christopher R. Denz,1 Timothy Pontz,1 Farzin Garahdaghi,1,8 Qing Cao,5 Dr. Alan Sabirsh,3
Kumar Thakur,1 Dr. Nichole O’Connell,6,9 Dr. Jun Hu,6,10 Dr. Iván Cornella-Taracido,2,11 Prof. Dr. Eranthie
Weerapana,4 Dr. Michael Zinda,1 Dr. Robert A. Goodnow Jr.,2,12 Dr. M. Paola Castaldi2
1Oncology, Innovative Medicines and Early Discovery Unit, AstraZeneca, 35 Gatehouse Drive, Waltham,
Massachusetts 02451, US
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2Discovery Sciences – Chemical Biology, Innovative Medicines and Early Discovery Unit, AstraZeneca, 35
Gatehouse Drive, Waltham, Massachusetts 02451, US
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3Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Discovery Unit, AstraZeneca,
Pepparedsleden 1, Molndal, 431 83, SE
4Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, US
5Discovery Sciences – Computational Chemistry, Innovative Medicines and Early Discovery Unit, AstraZeneca, 35
Gatehouse Drive, Waltham, Massachusetts 02451, US
6Discovery Sciences – Structure and Biophysics, Innovative Medicines and Early Discovery Unit, AstraZeneca, 35
Gatehouse Drive, Waltham, Massachusetts 02451, US
7Present address: Drug Discovery Program, Ontario Institute for Cancer Research, 661 University Avenue, Suite 510,
Toronto, Ontario M5G 0A3, Canada
8Present address: Synageva BioPharma Corp., 33 Hayden Avenue, Lexington, Massachusetts 02421, US
9Present address: Nurix, Inc. 1700 Owens St. Suite 290, San Francisco, California 94158, US
10Present address: Shire, 300 Shire Way, Lexington, Massachusetts 02421, US
11Present address: Discovery Chemistry, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston,
Massachusetts 02115, US
12Present Address: Pharmaron, 303 Wyman Street, Room 322, Waltham, Massachusetts 02451, US
+Co-first authors
*Correspondence: Andrew.Zhang@astrazeneca.com
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Abstract
Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting
in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter
identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-M potency. We report
here a comprehensive mechanism of action deconvolution study towards identifying the efficacy target(s) and
biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and
biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory
cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis through uncoupling the
mitochondrial potential. Affinity chemoproteomics experiments identified SERCA2 as a putative binding partner of
the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2
towards Wnt pathway inhibition.
Introduction
The Wnt signaling pathway is an evolutionarily conserved pathway among metazoans[1] critical for development,
regeneration, and adult tissue maintenance. Mutations in this pathway have been implicated in the development of
various types of cancers, most notably colon cancer[2]. The presence or absence of active Wnt signaling is dependent
on the state and cellular localization of -catenin: in the absence of Wnt ligand-promoted signaling, -catenin is
captured by the “destruction complex,” resulting in its phosphorylation by GSK3β, subsequent ubiquitination, and
proteasomal degradation. Activation of Wnt signaling begins with Wnt 3a forming a ternary complex with LRP6 and
Frizzled, which recruits Axin and prevents it from facilitating the formation of the destruction complex, thus
preventing -catenin phosphorylation and degradation. Stabilized -catenin then accumulates in the cytoplasm and is
shuttled to the nucleus to function as a transcription factor[3] for genes affecting cell proliferation and differentiation.
The Wnt pathway has become a focal point for drug discovery efforts in both academia and industry and while drug
discovery efforts have yielded new small molecules and targets at various points in this pathway[4], the clinical
applicability of inhibiting this pathway still remains unknown[5].
High throughput screens (HTS) using cell-based reporter gene assays (RGA-HTS) provide a unique
opportunity to discover new small molecule hits for a given signaling pathway[6]. One of the challenges of this
approach is that even though the structure of the lead series is known, usually its molecular target is not, and thus
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target deconvolution can be a challenging endeavor[7, 8, 9]. In the context of the Wnt pathway, the identification of
XAV939 as the inhibitor of Tankyrase acts as a seminal example of a chemical genetics-based target deconvolution
effort from a HTS that provided novel mechanistic insights into a disease-relevant signaling pathway[10]
.
Herein, we describe the identification of the 1,2,3-thiadiazole-5-carboxamides (TDZ) chemical series (shown
in Figure 1) using a high-throughput Wnt-driven reporter gene assay transfected into L-cells. Hits from this series
were used to design “clickable” photoaffinity probes for chemoproteomics-based experiments. The results of these
studies, coupled with metabolic studies, suggest that rather than acting directly on the Wnt pathway, this class of
compounds impairs the mitochondrial function and also acts as ionophores through modulation of SERCA2.
Results and Discussions
Design and Wnt inhibitory properties of chemical probes around hit chemical series
We carried out a cell-based high throughput screen of the AstraZeneca compound collection to identify selective
inhibitors of Wnt-3A-mediated induction of a luciferase reporter gene in mouse L S/L cells[11]. The use of unstimulated
L S/L cells, then, provided the ability to quickly identify false positives at the initial stage of screening (non-specific
inhibitors of the pathway, luciferase inhibitors). Subsequent testing using colorectal cancer lines with constitutive
activation of the pathway (e.g. DLD-1, HCT-116) allowed for prioritizing compounds for near neighbor testing and
SAR studies. The primary screen was carried out at 10 µM test concentration which yielded ~20,000 hits (2.6%) that
exhibited greater than 40% inhibition of signal. A portion of these primary screen hits, ~13,000 compounds, were
selected for reconfirmation at the same concentration in triplicate. Compounds with greater than 40% inhibition of
signal in two out of the three replicates were subsequently evaluated in concentration-response experiments in the
reporter assay and counter screened for effects on general cell health using the Alamar Blue assay[12] on L-cells.
Figure 1. a) Summary of SAR around 1,2,3-thiadiazole-5-carboxamide (TDZ) series and b) design of photoaffinity
chemical probes. See Tables S1 and S2, and Figures S1 and S2 for more detailed SAR data.
From this screening cascade, a small cluster of N-[1-(4-cyanophenyl)-1H-pyrazol-5-yl]-4-methyl-1,2,3-
thiadiazole-5-carboxamides (subsequently abbreviated as TDZ) (1a) was identified. Using compound 1a as a starting
point, we initiated a series of chemistry campaigns to establish SAR in the series (summarized in Figure 1). From
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these efforts we observed that electron-withdrawing substituents on the N1 aryl ring (2) contributed to greater
inhibition in the Wnt DLD-1 reporter gene assay activity while replacement of the 1,2,3-thiadiazole ring with other
heterocycles (1b) dramatically reduced potency. In order to address the moderate to high lipophilicity (clog P > 3) and
poor aqueous solubility exhibited by compounds in this series, we conducted all subsequent SAR exploration using
an iso-structural series of 4-aryl-thiazol-5-amino carboxamides (3). We determined, using matched molecular pair
analysis, that members of the thiazole series possess lower lipophilicity (clog P = 0.5) than those derived from the
pyrazole scaffold (see supporting information). In addition, we were able to more readily explore substitution at the
C2 position (equivalent to C4 in the pyrazole series) using late-stage transformations and began to introduce
functionality that led to analogs with further reduction in lipophilicity (4) and which informed the design of affinity-
based probes for chemical proteomics (5). However, we were unable to conduct affinity-based pulldown experiments
due to the apparent loss of Wnt reporter activity in the initial construct (6). Despite the high degree of structural
variability tolerated at this site we had previously observed that introduction of polar functionality, such as amino and
amido groups (see SI), led to reduced Wnt reporter activity even in compounds with good in vitro cellular permeability
and low to moderate efflux (6; Caco2 AB Papp = 2.75, ER = 7). We subsequently shifted our chemistry efforts towards
identifying a suitable photoreactive probe for in-cell photoaffinity experiments[13]. We established that the C4 aryl
SAR supported introduction of a photolabile p-CF3-diazirin-yl group (7a) which could be combined with a clickable
homopropargyl substituent at C2 (8). Consistent with our earlier SAR, the CF3-diazirin-yl containing 1,2,3-
thiadiazole-5-carboxamides inhibit Wnt DLD-1 reporter gene activity whereas the corresponding thiophenes (7b)
exhibit no activity in the concentration range tested (Wnt DLD-1 reporter IC50 >38 M) and represented a suitable
negative control.
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Figure 2. Biochemical analysis of TDZ series on known proteins nodes of the Wnt signaling pathway. a) Effect of
probe 8 on levels of biomarkers relevant to Wnt signaling at 24 hours post-treatment and on levels of active β-catenin
at 48 hours post-treatment in SW480 cells. b) Treatment with probe 8 does not affect levels of β-catenin mRNA and
LRP6 mRNA 24 hours post-treatment in SW480 cells. c) Effect of probe 8 on levels of active β-catenin is consistent
across multiple cell lines (HCT116 and DLD-1) with Wnt pathway mutations 24 hours post-treatment.
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Probe 8 and active competitor 4 showed nanomolar potency in the Wnt-driven luciferase reporter with modest
(2-5 fold) selectivity for a generic Renilla reporter and greater than 100-fold selectivity in Alamar Blue assays for cell
viability in L-cells. We also confirmed that this chemical series was able to directly modulate active β-catenin levels
and affect other Wnt signaling nodes (Figure 2A). SW480 cells treated with probe 8 showed a dose-dependent decrease
in protein levels of active β-catenin at 24 hours, and further treatment for 48 hours reduces active β-catenin levels to
undetectable amounts (even at lower doses). Further, a critical membrane receptor for Wnt signaling, LRP6[14], and
GSK3β, another key player for Wnt signaling[15] were inactivated in a dose-dependent manner (as evidenced by Ser9
phosphorylation for GSK3β), further indicating overall dysfunction of the Wnt pathway. Importantly, mRNA levels
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of both active β-catenin and LRP6 were not affected, suggesting that β-catenin modulation is occurring at the
posttranslational level (Figure 2B). Lastly, we found that this effect is also seen in other cell lines with Wnt pathway
mutations (Figure 2C), namely HCT116 and DLD-1.
Metabolic studies reveal perturbations to mitochondria and oxidative phosphorylation
Figure 3. Metabolic effects of TDZ series. a) Accumulation of extracellular lactic acid after 24 hours in the presence
of increasing concentrations of probe 8. b) Time course for change in levels of metabolic markers in SW480 cells at
250 nM probe 8; β-catenin levels are notably unchanged. c) Measurement of TMRE fluorescence as an indication of
mitochondrial membrane potential indicates that our TDZ chemical series has an inhibitory effect on mitochondrial
activity. Data points for TMRE fluorescence measurements represent the average of duplicates ± standard deviation.
d) Effect of carbon source on viability of cells treated with TDZ series. DLD-1-TF-EF1α cell viability in glucose and
galactose-based medium is presented upon treatment with probe 8. Data show the average of duplicates experiments
± standard deviation. e)-f) Time course for Wnt reporter assay inhibition at 4 hours post-treatment and 24 hours post-
treatment. Inhibition of Wnt reporter signal by probe 8 and XAV939 e) at 4 hours post treatment, and f) at 24 hours
post treatment. Data points represent the average of duplicates ± standard deviation. See also Figure S3.
Intriguingly, over the course of our target validation efforts, we observed that treating SW480 cells with probe 8
resulted in marked acidification of the medium and a dose-dependent increase in lactic acid excretion by the cell,
which suggested an increase in glycolytic output (Figure 3A). In light of this observation, we carried out an expanded
signaling analysis and cell biology experiments in parallel to our chemoproteomic efforts to deepen our understanding
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of the biological effects of the TDZ probes. Given that cells shift from oxidative to glycolytic metabolism quickly[16]
,
we investigated the immediate signaling events following the addition of a probe 8. SW480 cells treated with probe 8
(at 250 nM) showed increased Akt signaling and elevated levels of glycolysis marker pAS160[17] as early as 15 minutes
post-treatment, and an increase in pAMPK 4 hours post-treatment (Figure 3B), both of which suggest an energetic
imbalance[18], [19]. Importantly, non-phosphorylated β-catenin levels did not change during this early timeframe but
only decreased at 24 hours. Using a fluorescence based assay to measure mitochondrial function, we confirmed that
treatment with the TDZ series (probe 8 and compound 4) did indeed suppress the mitochondrial membrane potential
and induces mitochondrial dysfunction (Figure 3C).
Given that mitochondrial electron transport chain dysfunction can trigger a shift from oxidative to glycolytic
metabolism, we chose to alter the growth condition of DLD-1 cells by replacing glucose with galactose, thus blocking
the ability of the cell to become glycolytic and increase the dependence on oxidative catabolism of glutamine and the
mitochondrial electron transport chain for energy production[20], [21]. Treatment of cells with probe 8 grown in media
containing galactose instead of glucose resulted in complete loss of cell viability at high concentrations, a phenotype
that was not observed in glucose-based medium (Figure 3D). Interestingly, we found that probe 8 caused a decrease
in the Wnt reporter assay 4 hours post treatment (Figure 3E), but did not modulate the levels of β-catenin within the
same timeframe. In contrast, XAV939 (Figure 3F), a bona-fide inhibitor of the Wnt signaling pathway through
inhibition of Tankyrase did not show antagonism in the same window of measurement, but showed inhibition at 24
hours. Known inhibitors of the electron transport chain showed a Wnt inhibition phenotype similar to that of probe 8
at 24 hours (Figure S3), linking mitochondrial dysfunction to inhibitory readouts in the Wnt reporter assay.
Oxygen consumption measurements suggest mitochondrial uncoupling mechanism based on proton leak
With this new experimental insight, we set out to identify the mechanism by which the TDZ series perturbed oxidative
phosphorylation by utilizing the XF Extracellular Flux Analyzer (Seahorse Bioscience) to measure oxygen
consumption rate (OCR) and extracellular acidification rate (ECAR) as orthogonal readouts of metabolism and
mitochondrial function. After determining the basal bioenergetic characteristics of the DLD-1 cells (Figure S4), we
next evaluated the effects of our TDZ series on ECAR and OCR. Effects due to injection of compounds 4 and 7b
(inactive analogue) are shown in Figure 4A. Read-outs of ECAR demonstrated that compound 4 but not compound
7b increases medium acidification at a concentration of 0.1 M, close to its IC50 in the Wnt reporter), in agreement
with the observed increase in lactic acid.
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Figure 4. XF96 experiments looking at the effect of the TDZ series on extracellular acidification and oxygen
consumption in DLD-1 cells. a) Effect of 0.1 μM compound 4 and inactive analogue 7b on extracellular acidification
rate (ECAR). b) Effect of compound 4 on oxygen consumption rate in XF96 basal medium in XF96 basal medium +
5 mM galactose, 5 mM pyruvate, and 100 μM palmitate/BSA. c) Effect of inactive analogue 7b on oxygen
consumption rate in XF96 basal medium in XF96 basal medium + 5 mM galactose, 5 mM pyruvate, and 100 μM
palmitate/BSA. Data points for all experiments represent the average of quadruplicates ± standard deviation. See also
Figures S3 and S4.
In order to further investigate this observation, we monitored the effect of compounds 4 and 7b on oxygen
consumption rate (OCR) in the presence of galactose, pyruvate and palmitate (focusing on the electron transport
chain). Interestingly, as seen in panel B, OCR by DLD-1 cells did not change in the presence of compound 4, and
were largely unaffected by the subsequent injection of 1 M oligomycin A. This observation suggests that the TDZ
series does not directly inhibit any of the complexes of the electron transport chain, unlike the phenotypes associated
with rotenone, antimycin A, and oligomycin A. Injections of 200 M DNP to fully uncouple mitochondria through
inducing a massive proton leak across the mitochondrial membrane stimulated respiration only in the control cells and
at 0.1 M compound 4, indicating that a proton leak in the cells had already occurred in the presence of higher levels
of compound 4, rendering the effect of DNP redundant. This observation suggests that a mechanism of action of
compound 4, and of the TDZ series, likely mirrors that of DNP, which is the dissipation of the potential across the
mitochondrial membrane[22]. Such a mode of action would not affect the actual oxidation-reduction function of the
electron transport chain, thus maintaining sensitivity to rotenone and antimycin, but would effectively abrogate the
function of ATP synthase, thus masking the effect of oligomycin A. Inactive analogue 7b (Figure 4C) did not affect
cellular respiration under any of these conditions. Taken together, we have shown that the the TDZ series inhibits
ATP synthesis by inducing a proton leak in the mitochondria, and that this phenotype correlates with its Wnt inhibitory
properties. This effect may, at least in part, also explain the phenomenon of compounds containing TDZs showing
fungicidal or insecticidal activities, with the TDZ moiety being identified as a key pharmacophore[23], [24], [25]. Notably,
to the best of our knowledge, this study is the first to directly link the 1,2,3-thiadiazole-5-carboximide (TDZ)
functionality to mitochondrial uncoupling effects.
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In-cell chemoproteomics experiments identify potential binding partners
Our metabolic studies showed that the inhibitory effects of the TDZ series on the Wnt reporter assay occurred along
the same time scale as its effects on mitochondrial function. However, we did not see a corresponding decrease in the
levels of active -catenin at 4 hours, as we would have expected; instead, active -catenin levels decreased at a much
later time point. Furthermore, we found that treatment with 2,4-dinitrophenol did not cause a decrease in the levels of
active -catenin, in contrast to other mitochondrial inhibitors (Figure S3). We questioned whether there could be
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another mechanism contributing to the depletion of active -catenin along with mitochondrial perturbation. To further
elucidate this mechanism, we set out to identify potential protein targets of the series by in-cell chemoproteomics
experiments[26] using the photoaffinity probes previously described. We cultured cells in SILAC[27] heavy and light
media and treated SW480 cells respectively with probe 8 alone (500 nM) or in the presence of excess competition
compound (4, 10 μM), photocrosslinked by UV irradiation at 365 nm and lysed the cells. Following, light and heavy
lysates were combined and subjected to click chemistry conditions to conjugate a biotin group for subsequent
streptavidin affinity purification. Affinity purified proteins were subjected to an on-bead trypsin digestion and
subsequent analysis using a multidimensional protein identification technology (MudPIT) platform[28]. Putative
protein targets were expected to show enrichment in the heavy-labeled sample (incubation with probe 8 alone) and
decreased enrichment in the light-labeled sample (probe 8 with competition compound 4) due to competition by the
non-photoactivatable parent compound, affording heavy:light ratios greater than 1 (illustrated in Figure 5 and Table
S1). Four proteins showed greater than 1.5-fold decrease in enrichment upon competition: AUP1 (ancient ubiquitous
protein), HO-2 (heme oxygenase 2), ALDH1A3 (aldehyde dehydrogenase 1A3), and ATP2A2 (sarco(endo)plasmic
reticulum Ca2+ dependent ATPase, or SERCA2).
Figure 5. Depiction of chemoproteomics data for two replicates. Unique peptide intensity ratios between the absence
of competition (DMSO treated) and presence of competition (compound 4) were calculated and replicates were plotted
against one another. Highly competed targets are identified and labeled according to their Gene-ID. See also Table S3
and Figures S5.
Target validation experiments suggests mechanism Wnt inhibition related to SERCA modulation
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Figure 6. Target validation of SERCA2. a) Fluorescence microscopy was used to determine subcellular localization
of probe 8 to the endoplasmic reticulum. b) siRNA knockdown experiments of SERCA2 and in combination with
other SERCA isoforms showed modest decrease in levels of active -catenin when multiple isoforms are knocked
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down. c) Treatment of cells with pan-SERCA inhibitor thapsigargin showed knockdown of -catenin signal at 24
hours, but not at 4 hours. d) Measurement of intracellular Ca2+ levels after treatment with thapsigargin, compound 4,
and inactive analogue 7b. Compound 4 treatment resulted in an immediate increase in Ca2+ levels consistent with
treatment with thapsigargin and SERCA modulation. Data show the average of duplicates experiments ± standard
deviation.
Through in-cell fluorescence imaging experiments where probe 8 was conjugated to a TMRE-azide, we observed that
probe 8 localizes to the endoplasmic reticulum (Figure 6A), consistent with the identification of ER proteins AUP1,
HO-2, and SERCA2 as highly competed proteins in our chemoproteomics experiments. From our list of potential
targets, we were particularly intrigued by the identification of SERCA2, a regulator of intracellular calcium[29]
.
SERCA is a class of sarco(endo)plasmic reticulum Ca2+ ATPases responsible for modulating the intracellular levels
of calcium. Inhibition of SERCA activity by its known inhibitor, thapsigargin[30], diminishes Ca2+ uptake by the ER,
resulting in excess Ca2+ levels in the cytosol and the mitochondria, ultimately causing mitochondrial dysfunction and
apoptosis[31]. Additionally, intracellular calcium is an important component of non-canonical Wnt signaling pathways
and an increase in intracellular Ca2+ is known to downregulate the canonical, -catenin based pathway[32]. To validate
the relevance of SERCA2 as a putative target for Wnt signaling, we performed siRNA knockdown experiments on
SERCA2. While knockdown of SERCA2 alone did not give an appreciable effect on the levels of active -catenin
(Figure 6B), simultaneous knockdown of SERCA2 along with SERCA1 and 3 did show a modest decrease in the
levels of active -catenin (Figure 6B). Notably, siRNA knockdowns of the other three proteins (Figure S5) did not
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show any changes in the levels of active -catenin. To corroborate this observation, we treated SW480 cells with the
ionophore thapsigargin, a known pan-SERCA inhibitor and monitored the levels of active -catenin at 4 hours and 24
hours. Thapsigargin treatment did not reduce the levels of active -catenin after 4 hours; rather, we see a complete
reduction of active -catenin after 24 hours (Figure 6C), in agreement with published observations of thapsigargin
modulation of the Wnt signaling pathways[33]. These observations suggest that SERCA2 inhibition results in a Wnt
inhibition phenotype, and more importantly, this phenotype replicates the pattern of decrease in active -catenin
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observed following treatment with the TDZ series. We postulate that while the TDZ series is very likely to be pan-
SERCA modulators, our observed effect is likely dominated by SERCA2, as the levels of SERCA1 and SERCA3 are
minimal in SW480 cells and not detectable by Western Blot (data not shown).
Inhibition of SERCA by thapsigargin is known to cause an immediate but transient increase in levels of
intracellular calcium[34]. Given the relevance of SERCA2 to the observed effects on the Wnt pathway, we asked
whether our TDZ series behaves in a similar manner to thapsigargin in modulating intracellular Ca2+. To this end, we
monitored changes in the levels of intracellular Ca2+ upon treatment with TDZ series using a Ca2+ dependent
fluorophore and compared this effect to treatment with thapsigargin (Figure 6D). As expected, treatment with
thapsigargin increased the fluorescence intensity by 50% at its height of activity. We found that treatment with
compound 4 at concentrations close to the Wnt IC50, indeed resulted in an immediate, 20% increase in the levels of
intracellular calcium. While the effect of compound 4 is not as potent and not as efficacious as treatment with
thapsigargin, it is nonetheless consistent with the expected increase in intracellular Ca2+ upon SERCA modulation.
Furthermore, this observation is dependent on the presence of the TDZ functionality, as treatment with the inactive
analogue 7b did not result in the same increase in intracellular Ca2+.
Conclusion
As presented, a series of 1,2,3-thiadiazole-5-carboxamides (TDZ) were identified as putative Wnt pathway inhibitors
from a RGA-HTS in L-cells. We set out to discover and validate the molecular target(s) of the series through
chemoproteomics, cellular and biochemical techniques. Intriguingly, we found two possible mechanisms to explain
the observed phenotypes: rapid onset of mitochondrial dysfunction through depolarization of the membrane potential
(in line with the decrease in the Wnt luciferase reporter assay at 4 hours), and modulation of SERCA2 and intracellular
Ca2+ (that results in the subsequent, more gradual decrease in the levels of active -catenin).
In conclusion, we must further emphasize the importance of utilizing an integrated approach in target
deconvolution, as the use of a single technique did not provide sufficient information to comprehensively elucidate
the biological impact of the TDZ series. The mechanism of action of mitochondrial uncouplers may not necessarily
be driven by binding to a single protein, but rather by properties of the small molecule that can induce chemical
changes in the mitochondria (such as the acidity of 2,4-dinitrophenol). Similarly, performing photoaffinity
chemoproteomics experiments may indicate additional targets responsible for parallel mechanisms. Given that there
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are no previous reports directly implicating the TDZ pharmacophore to mitochondrial depolarization or intracellular
calcium modulation, further studies to elucidate these mechanisms are warranted.
Methods
Wnt reporter assay
DLD-1-TopFlash-EF1α Renilla cells expressing a Wnt-responsive firefly luciferase and EF1α-driven Renilla
luciferase were trypsinized and resuspended with reporter assay medium (RPMI1640 + 2% FBS +
penicillin/streptomycin) to 250,000 cells mL-1. 60 μL of this suspension (15,000 cells) were seeded into white 96 well
plates (Corning Life Sciences 3917). The cells were allowed to incubate at 37oC humidified air supplemented with
5% CO2 for 24 hours, after which test compounds were added at twice the desired concentration using 60 μL of
reporter assay medium for a total volume of 120 μL. The cells were allowed to incubate at 37oC humidified air
supplemented with 5% CO2 for 24 hours. DualGlo Luciferase substrate solution (Promega, 60 μL) prepared according
to manufacturing protocols was added to the plate with thorough mixing. The plate was allowed to incubate at room
temperature in the dark for 20 minutes, after which wells containing cells were read for luminescence. Stop and Glo
Renilla substrate solution (Promega, 60 μL) prepared according to manufacturing protocols was added to the plate
with thorough mixing. The plate was allowed to incubate at room temperature in the dark for 20 minutes, after which
wells containing cells were read for luminescence. Percent signal was determined by setting the cells without
compound treatment as maximum signal.
XF96 Extracellular Flux Analyzer Assay
Effects on oxygen consumption and extracellular acidification in DLD-1 cells (ATCC CCL-221) were detected at 37
°C using the XF96 Extracellular flux analyzer from Seahorse Bioscience. DLD-1 cells were seeded at a density of
35000 cells per well in 100 µl RPM1 1640 medium containing 10% FCS (Gibco 10270) 20-24 hours prior to
experiment. Medium was switched to 150 µl per well of Seahorse Bioscience XF Base medium (102353-100) using
the XF prep station (Seahorse Bioscience) 30 min prior to read-outs. The following additions were made to the assay
medium: 5 mM galactose, 5 mM sodium pyruvate, 100 µM palmitate and 25 µM fatty acid free BSA; before adjusting
the pH to 7.4. Injections to yield desired final concentrations were made by loading ports A-D with 25 µl of stock
solutions containing compounds/reagents with no or less than 0.2% DMSO as solvent. OCR and ECAR data was
captured by 3 min measures with 1 min mixing intervals and data analyzed using the XF software. Data was
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normalized to cell number by fixation in 3.7% formaldehyde for 20 min at room temperature and stained for nuclei
using 0.5 μg mL-1 Hoescht (Molecular Probes, 33342) for 30 min. Images were captured with a ImageXpress Micro
from Molecular Devices and data analyzed using the MetaExpress software.
Cell Culture for SILAC Experiments
SW480 cells were cultured in medium containing heavy or light amino acids for SILAC proteomics experiments. To
label with heavy amino acids, cells were grown in heavy amino acid medium (SILACTM RPMI1640 with L-Arginine-
HCl, 13C6, 15N4, and L-Lysine-2HCl, 13C6, 15N2, Pierce Biotechnology) with 10% FBS + penicillin/streptomycin
for 7 passages. To label with light amino acids, cells were grown in light amino acid medium (SILACTM RPMI + L-
Arginine-HCl and L-Lysine-2HCl, Pierce Biotechnology) with 10% FBS + penicillin/streptomycin for 7 passages.
After 7 passages, cells were treated with appropriate compounds and harvested as described above.
In-cell proteomics and target enrichment
SW480 cells were treated with probe 8 (500 nM) or probe 8 (500 nM) + compound 4 (10 μM) for one hour in
OptiMEM at 37°C in humidified air supplemented with 5% CO2. Following incubation, cells were washed with 20
mL of OptiMEM and irradiated with a hand-held UV lamp (365 nm) for 15 min at 4oC. Post-irradiation, cells were
washed with cold PBS and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1% NP40 and 5 mM
EDTA at pH 7.4) supplemented with 1X Halt Protease and Phosphatase Inhibitors (Pierce). The lysates were briefly
sonicated, and protein concentration was determined using DC Protein Assay (BioRad) and adjusted to 2 mg mL-1
aliquots with lysis buffer. To 1 mg of proteins (500 μL lysate) was added 20 μL of a reaction mixture containing
Biotin-azide (Life Technologies, final concentration 100 μM), THPTA (Sigma, final concentration 100 μM), CuSO4
(final concentration 1 mM), and sodium ascorbate (final concentration 1 mM) in PBS. The reaction was allowed to
incubate in the dark for 1 hour, after which lysates were precipitated in 50%/50% methanol/acetone mixture at -20oC
for one hour. Precipitated proteins were pelleted by centrifugation at maximum speed and washed three times with
cold methanol. After washing, methanol was fully removed and the protein pellet was resuspended in 1% SDS (in
PBS) until all protein was in solution. Samples were diluted 1 to 10 in PBS, mixed with 100 μL of streptavidin coated
agarose beads (Thermo Scientific) and incubated overnight at 4oC. After overnight incubation, the mixture was
centrifuged at 1200 rpm for 2 minutes at room temperature, after which supernatant was removed slowly and the beads
were washed with 1% SDS in PBS (400 μL). The mixture was centrifuged at 1200 rpm for 2 minutes and most of the
supernatant was removed, leaving behind 100 μL. The beads were transferred using the remaining supernatant to a
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1.5 mL Eppendorf tube and centrifuged at 1200 rpm for 2 minutes. The remaining supernatant was removed and the
beads were washed three times with PBS, then three times with deionized water, and then frozen down and stored at
-80oC until analysis by Western Blot or by mass spectrometry.
Supplemental Information
Supplemental information includes Synthetic and Experimental Procedures, list of reagents, 3 tables, 5 figures, and
1H NMR spectra of compounds 1-8.
Acknowledgements
The authors thank the AstraZeneca Postdoctoral Program for its generous financial support of this work. We thank
Emma Louise Jenkins, Daniel Russell, and Nicholas Keen for their contributions to the HTS screening strategy. We
thank Dr. Sylvie Guichard and Dr. Susan Critchlow for fruitful discussions and for their valuable insights. We are
grateful to Prof. Dr. Herbert Waldmann for discussions, as well as profound advice and feedback on the preparation
of this manuscript.
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Figure 1. a) Summary of SAR around 1,2,3-thiadiazole-5-carboxamide (TDZ) series and b) design of photoaffinity
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chemical probes. See Tables S1 and S2, and Figures S1 and S2 for more detailed SAR data.
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Figure 2. Biochemical analysis of TDZ series on known proteins nodes of the Wnt signaling pathway. a) Effect of
probe 8 on levels of biomarkers relevant to Wnt signaling at 24 hours post-treatment and on levels of active β-catenin
at 48 hours post-treatment in SW480 cells. b) Treatment with probe 8 does not affect levels of β-catenin mRNA and
LRP6 mRNA 24 hours post-treatment in SW480 cells. c) Effect of probe 8 on levels of active β-catenin is consistent
across multiple cell lines (HCT116 and DLD-1) with Wnt pathway mutations 24 hours post-treatment.
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Figure 3. Metabolic effects of TDZ series. a) Accumulation of extracellular lactic acid after 24 hours in the presence
of increasing concentrations of probe 8. b) Time course for change in levels of metabolic markers in SW480 cells at
250 nM probe 8; β-catenin levels are notably unchanged. c) Measurement of TMRE fluorescence as an indication of
mitochondrial membrane potential indicates that our TDZ chemical series has an inhibitory effect on mitochondrial
activity. Data points for TMRE fluorescence measurements represent the average of duplicates ± standard deviation.
d) Effect of carbon source on viability of cells treated with TDZ series. DLD-1-TF-EF1α cell viability in glucose and
galactose-based medium is presented upon treatment with probe 8. Data show the average of duplicates experiments
± standard deviation. e)-f) Time course for Wnt reporter assay inhibition at 4 hours post-treatment and 24 hours post-
treatment. Inhibition of Wnt reporter signal by probe 8 and XAV939 e) at 4 hours post treatment, and f) at 24 hours
post treatment. Data points represent the average of duplicates ± standard deviation. See also Figure S3.
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Figure 4. XF96 experiments looking at the effect of the TDZ series on extracellular acidification and oxygen
consumption in DLD-1 cells. a) Effect of 0.1 μM compound 4 and inactive analogue 7b on extracellular acidification
rate (ECAR). b) Effect of compound 4 on oxygen consumption rate in XF96 basal medium in XF96 basal medium +
5 mM galactose, 5 mM pyruvate, and 100 μM palmitate/BSA. c) Effect of inactive analogue 7b on oxygen
consumption rate in XF96 basal medium in XF96 basal medium + 5 mM galactose, 5 mM pyruvate, and 100 μM
palmitate/BSA. Data points for all experiments represent the average of quadruplicates ± standard deviation. See also
Figures S3 and S4.
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Figure 5. Depiction of chemoproteomics data for two replicates. Unique peptide intensity ratios between the absence
of competition (DMSO treated) and presence of competition (compound 4) were calculated and replicates were plotted
against one another. Highly competed targets are identified and labeled according to their Gene-ID. See also Table S3
and Figures S5.
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Figure 6. Target validation of SERCA2. a) Fluorescence microscopy was used to determine subcellular localization
of probe 8 to the endoplasmic reticulum. b) siRNA knockdown experiments of SERCA2 and in combination with
other SERCA isoforms showed modest decrease in levels of active -catenin when multiple isoforms are knocked
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down. c) Treatment of cells with pan-SERCA inhibitor thapsigargin showed knockdown of -catenin signal at 24
hours, but not at 4 hours. d) Measurement of intracellular Ca2+ levels after treatment with thapsigargin, compound 4,
and inactive analogue 7b. Compound 4 treatment resulted in an immediate increase in Ca2+ levels consistent with
treatment with thapsigargin and SERCA modulation. Data show the average of duplicates experiments ± standard
deviation.
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