Chemical Genetics Reveals a Role of dCTP Pyrophosphatase 1 in Wnt Signaling

Article abstract of DOI:10.1002/anie.201905977

Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/β-catenin signaling most likely through a direct interaction with USP7.

Full text of DOI:10.1002/anie.201905977

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Chemical genetics reveals a role of dCTP pyrophosphatase 1 in
Wnt signaling
Authors: Herbert Waldmann, Alexandra Friese, Shobhna Kapoor,
Tabea Schneidewind, Srinivasa Rao Vidadala, Juhi Sardana,
Alexandra Brause, Tim Förster, Matthias Bischoff, Jessica
Wagner, Petra Janning, and Slava Ziegler
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201905977
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Link to VoR: http://dx.doi.org/10.1002/anie.201905977
http://dx.doi.org/10.1002/ange.201905977

10.1002/anie.201905977
Angewandte Chemie International Edition
Chemical genetics reveals a role of dCTP pyrophosphatase
1 in Wnt signaling
Alexandra Friese1,#, Shobhna Kapoor1,2,#, Tabea Schneidewind1, Srinivasa Rao Vidadala1,3,
Juhi Sardana1, Alexandra Brause1, Tim Förster1, Matthias Bischoff1, Jessica Wagner1,4, Petra
Janning1, Slava Ziegler1, and Herbert Waldmann1,4,
*
Affiliations:
1Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11,
44227 Dortmund, Germany
2Present address: Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai -
400076, Maharashtra, India
3Present address: Syngene International Limited, Bengaluru, Karnataka 560099, India
4Faculty of Chemistry and Chemical Biology, Technische Universität Dortmund, Otto-Hahn Straße 6,
44227 Dortmund, Germany
#These authors contributed equally to this work.
*Correspondence to: herbert.waldmann@mpi-dortmund.mpg.de
1
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10.1002/anie.201905977
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Abstract
Cell-based screening is a powerful approach to identify novel chemical modulators and
biological components of relevant biological processes. The canonical Wnt pathway is
essential for normal embryonic development and tissue homeostasis, and its deregulation
plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members
and regulators is of significant interest. By means of a cell-based assay monitoring Wnt
signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling.
Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP
pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with
ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors.
These findings indicate that dCTPP1 plays a role in regulation of Wnt/β-catenin signaling
most likely through a direct interaction with USP7.
2
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10.1002/anie.201905977
Angewandte Chemie International Edition
Canonical Wnt/β-catenin signaling is a fundamental growth control pathway essential for e.g.
embryonic development, stem cell maintenance, cell proliferation and tissue homeostasis[1].
Upon activation the central Wnt pathway component β-catenin is no longer phosphorylated
and degraded by the so-called destruction complex[2], but rather stabilized and translocated
to the nucleus, where it activates the expression of Wnt target genes[3]. Hyperactivated Wnt
signaling has been associated with carcinogenesis, in particular colorectal cancer[4]. Thus,
the discovery of new canonical Wnt pathway members and new small-molecule modulators
of Wnt signaling is of major interest[5].
We report the identification of the pyrrolocoumarin Pyrcoumin, which modulates Wnt
signaling by competitive inhibition of dCTP pyrophosphatase 1 (dCTPP1). dCTPP1 is a
positive regulator of canonical Wnt signaling, interacts with Ubiquitin carboxyl-terminal
hydrolase (USP7) in Wnt signal-dependent manner, and this interaction is weakened by
dCTPP1 inhibitors. These findings reveal a yet unknown interaction between dCTPP1 and
USP7 and a role for dCTPP1 in the Wnt/β-catenin pathway.
Screening of 16,574 in-house compounds at 10 µM concentration in a Wnt-responsive
reporter gene assay in HEK293 cells stably transfected with the human Frizzled-1 receptor
and a TOPFlash reporter plasmid[6] identified pyrrolocoumarins as promising inhibitor class
(Figure S1a, Table S1). Activity was confirmed in a manual reporter gene assay using
HEK293T cells transiently transfected with the SuperTOPflash reporter (Figure S1b).
Compound 1 (termed Pyrcoumin, Figure 1a) inhibited Wnt signaling with a half-maximal
inhibitory concentration (IC50) of 8.4 µM in the SuperTOPFlash reporter gene assay (Figure
1b) and was selected for further characterization. Pyrcoumin dose-dependently decreased
the expression of Wnt target genes AXIN2, CCND1 and LEF1 in Wnt-activated HEK293 cells
(Figure 1c) and inhibited Wnt pathway activity also in HCT116 cells (Figure 1b), harboring a
Ser45del mutation in β-catenin that constitutively activates the Wnt pathway[7]. Pyrcoumin did
not affect the abundance of β-catenin (Figure 1d, Figure S1c), or other pathway components
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10.1002/anie.201905977
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such as Axin2 and phosphorylated (Ser33/Ser37/Thr41) β-catenin (Figure 1e). These results
suggest a mode of action downstream of β-catenin.
Figure 1: Pyrcoumin antagonizes Wnt signaling downstream of β-catenin. (a)
Structure of Pyrcoumin (1). (b) Wnt-responsive reporter gene assays in HEK293T and
HCT116 cells transiently transfected with SuperTOPFlash (STF) plasmid upon
treatment with Pyrcoumin or DMSO in Wnt-3a conditioned medium (WCM) for 22 h
(mean ± S.D., n = 6 for HEK293T, n = 3 for HCT116). (c) mRNA expression levels of
the Wnt target genes AXIN2, LEF1 and CCND1 in HEK293 cells treated with L-cell
conditioned medium (LCM) and compound or DMSO in WCM for 24 h as determined
using RT-qPCR (mean ± S.D., n = 3). (d) In-Cell-Western in HEK293 and HCT116 cells
treated with Pyrcoumin in WCM (for HEK293) or DMEM (for HCT116) to quantify β-
catenin levels (mean ± S.D., N = 4, n = 3). (e) Representative immunoblots for β-
catenin, Axin2, phosphorylated β-catenin and β-actin, used as loading control, using
HEK293 cells treated with LCM, WCM or 10 µM Pyrcoumin in WCM for 24 h.
Uncropped blots in Figure S5a.
4
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For target identification by affinity-based chemical proteomics we synthesized active probe 2
(Figure 2a) (IC50 = 14.8 µM for the corresponding azide analog (Figure S1d-e)) and a control
probe (Figure S1f; the corresponding azide analog was inactive (Figure S1d-e)). As bulky
substituents at the nitrogen of the pyrrole moiety impaired inhibitory activity (Figure S1a), the
linker was attached to the carboxylic acid. To allow quantification of enriched proteins, we
employed stable isotope labeling of amino acids in cell culture (SILAC)[8]. To this end, probes
were immobilized on solid support and the active probe 2 was incubated with normal (“light”)
HCT116 cell lysate, whereas the control probe was exposed to 13C- and 15N-labeled
(“heavy”) HCT116 cell lysate (and vice versa). Beads enriched with proteins by 2 and the
control probe were mixed prior to identification of bound proteins by means of mass
spectrometry. Probe 2, but not the control probe significantly enriched deoxycytidine-
triphosphate (dCTP) pyrophosphatase 1 (dCTPP1) and AIFM2 (Data Sheets S1-2) with
SILAC ratios of >13:1. dCTPP1 is an all-α-nucleoside triphosphate (NTP) pyrophosphatase
that eliminates non-canonical NTPs, thus acting as a house-keeping nucleotide hydrolase,
which preserves genomic integrity[9]. It catalyzes the hydrolysis of dNTPs to their
corresponding monophosphates and displays higher affinity towards 5-modified non-
canonical dNTPs (e.g., 5-I-dCTP>5-Br-dCTP>5-Me-dCTP>dCTP), thereby decreasing their
intracellular levels and hence subsequently their insertion into nascent DNA during
replication[9-10]. dCTPP1 is considered an attractive anti-cancer target, because it is
overexpressed in many carcinomas (e.g., breast and gastric)[11]
.
Reversible binding of dCTPP1 to probe 2 was confirmed by means of concentration-
dependent competitive pull-down using immunoblotting for dCTPP1 (Figure 2b). Physical
engagement of dCTPP1 by Pyrcoumin in a complex cellular matrix was proven by means of
the cellular thermal shift assay (CETSA) in HCT116 and HEK239T cell lysates[12]. Pyrcoumin
induced thermal stabilization of dCTPP1 with a shift in melting temperature of 6 °C (Figure
2c-d, and Figure S2). In isothermal mode at 52 °C, Pyrcoumin dose-dependently stabilized
dCTPP1 with a half-maximal effective concentration (EC50) of 5.9 µM (Figure 2e-f). These
results demonstrate engagement of dCTPP1 by the compound in cellular lysate.
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10.1002/anie.201905977
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In contrast, AIFM2 enrichment by 2 could not be confirmed using immunoblotting after affinity
pull-down, and binding of Pyrcoumin to AIFM2 could not be detected by differential scanning
fluorimetry (DSF) and CETSA in HCT116 lysates (Figure S3). Thus, AIMF2 was not further
investigated as potential target.
dCTPP1 has not been linked to Wnt signaling. To analyze the role of dCTPP1, we depleted
dCTPP1 by means of short interfering RNA (siRNA) in HEK293T cells by approx. 98%
(Figure 2g) which led to a 50% reduction in the Wnt-responsive reporter gene activity (Figure
2h). Thus, depletion of dCTPP1 phenocopies the effect of Pyrcoumin and further confirms
dCTPP1 as novel positive regulator of the Wnt/β-catenin pathway.
Figure 2: dCTPP1 is the target of Pyrcoumin. (a) Structure of the active probe 2
used in the affinity pull-down (PD) in HCT116 cells. (b) Immunodetection of dCTPP1
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10.1002/anie.201905977
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binding to immobilized probe 2 using HCT116 cell lysates pre-incubated with
Pyrcoumin. Uncropped blot in Figure S5b. (c) Representative immunoblots of CETSA
for dCTPP1 in HCT116 cell lysates treated with 30 µM of Pyrcoumin or DMSO (n = 3).
Uncropped blots in Figure S5c. (d) Quantification of band intensities from (c), which
were normalized to the intensity of the band at 44 °C. (e) Representative immunoblots
of CETSA-based isothermal dose-response for dCTPP1 in HCT116 cell lysates treated
with Pyrcoumin or DMSO and incubated at 52 °C (n = 3). Uncropped blot in Figure
S5d. (f) Quantification of band intensities from (e), which were normalized to the band
intensity of the control cells. (g) Quantification of dCTPP1 knockdown. HEK293T cells
were transiently transfected with siRNA targeting dCTPP1 or control siRNA for 48 h,
and 22 h later harvested for immunoblotting (n = 3). Uncropped blots in Figure S5e. (h)
Wnt-responsive reporter gene assay in HEK293T cells transiently transfected with
SuperTOPFlash (STF) plasmid and siRNA targeting dCTPP1 or control siRNA (mean ±
S.D., n = 3).
Investigation of dCTPP1 inhibition by Pyrcoumin revealed that the compound partially inhibits
(43% residual activity) the enzymatic activity with an IC50 value of 3.3 µM (Figure 3a, Table
S1, Figure S4a). Inactive derivative 3 (Figure 3b) does not inhibit dCTPP1. dCTPP1 inhibition
by the pyrrolocoumarin hits correlated with inhibition of Wnt signaling (Figure S4b, Table S1).
Pyrcoumin is a competitive inhibitor of dCTPP1, as it increased the Michaelis-Menten
constant Km of dCTPP1 for its substrate 5-Me-dCTP by four fold without altering the
maximum reaction velocity Vmax (Figure 3c-d).
Different dCTPP1 inhibitors have been developed recently, e.g. benzimidazole 4[11a] (Figure
3b) (IC50 = 44 nM in this study), and triazolothiadiazoles[13] like 5 (Figure 3b) (IC50 = 1.1 µM in
this study) (Figure 3a). Both compounds inhibited the Wnt-responsive reporter gene activity
in HEK293T cells with IC50 values of 10.1 µM and 4.1 µM, respectively (Figure 3e, Figure
S4c). These findings demonstrate that dCTPP1 inhibitors with different chemotypes inhibit
Wnt signaling and indicate that dCTPP1 may play a role in Wnt pathway regulation.
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Figure 3: Pyrcoumin is a competitive inhibitor of dCTPP1. (a) dCTPP1 enzymatic
assay in presence of Pyrcoumin, the inactive derivative 3, benzimidazole 4 and
triazolothiadiazole 5 (mean ± S.D., n = 3). IC50 values are 3.3 ± 0.3 µM for Pyrcoumin,
44 ± 3 nM for 4 and 1.07 ± 0.03 µM for 5. (b) Structures of pyrrolocoumarin 3 and
dCTPP1 inhibitors 4 and 5. (c) Michaelis-Menten plot of dCTPP1 in presence of
Pyrcoumin or DMSO determined with dCTPP1 enzymatic assay (mean ± S.D., n = 3).
(d) Michaelis-Menten constant Km and maximum reaction velocity Vmax of dCTPP1 in
presence of Pyrcoumin or DMSO calculated from (c). (e) Wnt-responsive reporter gene
assay in HEK293T cells transiently transfected with Wnt-3a and SuperTOPFlash (STF)
plasmids, treated with Pyrcoumin, 4 or 5 for 24 h (mean ± S.D., n = 3).
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10.1002/anie.201905977
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dCTPP1 has not been linked to Wnt signaling before and a STRING analysis for known
interaction partners[14] did not reveal a link either. To establish a connection between
dCTPP1 and Wnt signaling, we performed co-immunoprecipitation coupled to mass
spectrometry using HCT116 cells that expressed FLAG-tagged dCTPP1. Ubiquitin carboxyl-
terminal hydrolase 7 (USP7) and 3-hydroxyacyl CoA dehydrogenase type-2 (HSD17B10) co-
purified with dCTPP1 (Data Sheets S3-4). HSD17B10 has not been linked to Wnt signaling
either. However, the deubiquitinating enzyme (DUB) family member USP7 mediates
deubiquitination and prevents proteasomal degradation of tumor suppressors and
oncogenes[15], and is involved directly or indirectly in Wnt signaling. Thus, USP7 acts as
positive feedback regulator of Wnt by enhancing the stability of β-catenin, and its knockdown
impairs Wnt signaling[16]
.
Co-immunoprecipitation in HCT116 cell lysates using anti-dCTPP1 antibody-coated beads
confirmed the interaction between dCTPP1 and USP7 (Figure 4a). In addition, pull-down and
gel filtration shift assays using recombinant proteins confirmed a direct dCTPP1-USP7
interaction (Figure 4b-d). Fluorescence polarization measurements revealed a Kd of 1.2 µM
for binding of fluorescein-labeled dCTPP1 to USP7 (Figure 4e), and Pyrcoumin concentration
dependently weakened this interaction (Kd = 10.5 µM for 20 µM Pyrcoumin and 5.5 µM for 3
µM Pyrcoumin). By analogy, dCTPP1 inhibitors with different chemotypes impaired the
dCTPP1-USP7 interaction (Figure 4f, Kd = 19.1 µM for 20 µM 4 and 9.5 µM for 20 µM 5),
whereas the inactive pyrrolocoumarin 3 had no effect on dCTPP1-USP7 complex formation
(Figure S4d). Notably, the dCTPP1 substrate 5-Me-dCTP also compromised dCTPP1
binding to USP7 (Figure 4f, Kd = 5.9 µM for 100 µM 5-Me-dCTP), hence, the binding event as
such and its consequences, and not primarily the inhibition itself may be at the heart of the
inhibition of the protein-protein interaction, e.g. by modulation of the protein conformation.
These findings suggest that the dCTPP1-USP7 interaction may be functionally relevant to
Wnt signaling and that it is amenable to small-molecule perturbation.
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10.1002/anie.201905977
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Figure 4: Pyrcoumin impairs the direct interaction of dCTPP1 and USP7. (a) Co-
immunoprecipitation of dCTPP1 and USP7 in HCT116 cell lysate using anti-dCTPP1
antibody-coated beads (n = 3). L: HCT116 lysate, E: eluate with dCTPP1-bound
proteins, FT: flow-through. Uncropped blots in Figure S5f. (b) dCTPP1 co-elutes with
USP7 in gel filtration shift assays with recombinant USP7 and dCTPP1 in presence of
20 µM Pyrcoumin or DMSO. Representative gel filtration chromatograms. (c)
Representative gel images of respective gel filtration runs from (b). (d) Pull-down
assays with recombinant His6-USP7 and dCTPP1 in presence of 20 µM Pyrcoumin or
DMSO (n = 3). (e) Fluorescence polarization assays using 20 nM fluorescein-labeled
dCTPP1 and USP7 in presence of Pyrcoumin or DMSO (mean ± S.D., n = 3). Kd
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values are 1.2 ± 0.1 µM for DMSO control, 10.5 ± 2.8 µM for 20 µM Pyrcoumin, 5.5 ±
0.6 µM for 3 µM Pyrcoumin. (f) Fluorescence polarization assays using 20 nM
fluorescein-labeled dCTPP1 and USP7 in presence of Pyrcoumin, 4, 5, 5-Me-dCTP or
DMSO (mean ± S.D., n = 3). Kd values are 1.2 ± 0.1 µM for DMSO control, 10.5 ± 2.8
µM for 20 µM Pyrcoumin, 19.1 ± 2.7 µM for 20 µM 4, 9.5 ± 0.5 µM for 20 µM 5, 5.9 ±
0.9 µM for 100 µM 5-Me-dCTP.
Through a cell-based assay and subsequent target identification and validation we have
discovered the new Wnt pathway inhibitor Pyrcoumin and shown that it targets the nucleotide
pyrophosphatase dCTPP1. dCTPP1 is a novel positive regulator of Wnt signaling. Pyrcoumin
partially inhibits the enzymatic activity of dCTPP1 in a substrate competitive manner and
both Pyrcoumin, and structurally unrelated dCTPP1 inhibitors, as well as 5-Me-dCTP
modulate the interaction of dCTPP1 with its newly identified binding partner USP7. These
findings suggest that impaired complex formation with USP7 may result from a
conformational change of dCTPP1 upon inhibitor binding and that this indirect interference
with the dCTPP1-USP7 interaction might be at the heart of Wnt pathway inhibition by
dCTPP1 inhibitors.
Acknowledgments
This research was supported by the Max Planck Gesellschaft and the European Research
Council under the European Union's Seventh Framework Programme (FP7/2007-2013) /
ERC Grant agreement n°268309. We thank Dr. Sonja Sievers and the Compound
Management and Screening Center (COMAS), Dortmund, Germany, for screening; the
Dortmund protein facility for cloning of nFLAG-tagged dCTPP1; Andreas Brockmeyer and
Malte Metz for mass spectrometry; Jana Flegel and Michele Saretzki for WST assays; Hacer
Karatas for help with chemical synthesis; Dennis Schade for the protocol for the STF reporter
gene assay and Beate Schölermann and Christine Nowak for technical assistance.
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COMMUNICATION
A cell-based screen for inhibitors of
canonical Wnt signaling identified the
pyrrolocoumarin Pyrcoumin, a new
Alexandra Friese, Shobhna Kapoor,
Tabea Schneidewind, Srinivasa Rao
Vidadala, Juhi Sardana, Alexandra
Brause, Tim Förster, Matthias Bischoff,
Jessica Wagner, Petra Janning, Slava
Ziegler, and Herbert Waldmann*
competitive
inhibitor
of
dCTP
pyrophosphatase
1
(dCTPP1).
Pyrcoumin counteracts the yet
unknown interaction of dCTPP1 with
ubiquitin carboxyl-terminal hydrolase
(USP7).
Chemical genetics reveals a role of
dCTP pyrophosphatase 1 in Wnt
signaling
Keywords: natural products, protein-protein interactions, small-molecule inhibitors, target
identification, Wnt pathway
13
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