Chemical Genetics Reveals a Role of Squalene Synthase in TGFβ Signaling and Cardiomyogenesis

Article abstract of DOI:10.1002/anie.202100523

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFβ signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFβ signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFβ/SMAD signaling.

Full text of DOI:10.1002/anie.202100523

Angewandte
Research Articles
Chemie
How to cite:
International Edition: doi.org/10.1002/anie.202100523
German Edition: doi.org/10.1002/ange.202100523
Cell Signaling
Chemical Genetics Reveals a Role of Squalene Synthase in TGFb
Signaling and Cardiomyogenesis
Yasushi Takemoto, Shin Kadota, Itsunari Minami, Shinya Otsuka, Satoshi Okuda,
Masahiro Abo, Louvy Lynn Punzalan, Yan Shen, Yuji Shiba,* and Motonari Uesugi*
Abstract: KY02111 is a widely used small molecule that boosts
cardiomyogenesis of the mesoderm cells derived from pluri-
potent stem cells, yet its molecular mechanism of action
remains elusive. The present study resolves the initially
perplexing effects of KY02111 on Wnt signaling and subse-
quently identifies squalene synthase (SQS) as a molecular
target of KY02111 and its optimized version, KY-I. By
disrupting the interaction of SQS with cardiac ER-membrane
protein TMEM43, KY02111 impairs TGFb signaling, but not
Wnt signaling, and thereby recapitulates the clinical mutation
of TMEM43 that causes arrhythmogenic right ventricular
cardiomyopathy (ARVC), an inherited heart disease that
involves a substitution of myocardium with fatty tissue. These
findings reveal a heretofore undescribed role of SQS in TGFb
signaling and cardiomyogenesis. KY02111 may find its use in
ARVC modeling as well as serve as a chemical tool for
studying TGFb/SMAD signaling.
ity has greatly been enhanced by in vitro modeling using
pluripotent stem cells (PSCs); however, challenges remain
regarding efficiency of differentiation and maturation for
therapeutic applications and disease modeling.
Our previous screening of a chemical library and structure
optimization led to the discovery of KY02111 (Figure 1A),
a small-molecule probe that boosts cardiomyogenesis of
a range of PSCs including humans.[3] Brief 3-day exposure of
PSC-derived mesoderm cells to KY02111 elicits the differ-
entiation to cardiomyocytes. Despite the utility of KY02111 in
Introduction
The heart is the first organ to become functional in the
vertebrate embryo.[1] Although the heart develops early,
cardiogenesis is a highly regulated process involving differ-
entiation and cellular specialization. Cardiomyocytes com-
prise approximately 40% of the total heart cells and originate
from mesoderm cells through an organized interplay of
extracellular factors including members of the bone morpho-
genetic proteins (BMPs), including Activin and NODAL,
fibroblast growth factor (FGF), and Wingless (Wnt) fami-
lies.[1,2] Our understanding of the cardiomyogenesis complex-
[*] Dr. Y. Takemoto, S. Otsuka, S. Okuda, Dr. M. Abo, Dr. L. L. Punzalan,
Dr. Y. Shen, Prof. M. Uesugi
Institute for Chemical Research (ICR), Kyoto University
Uji, Kyoto 611-0011 (Japan)
E-mail: uesugi@scl.kyoto-u.ac.jp
Dr. S. Kadota, Prof. Y. Shiba
Figure 1. Analysis of KY02111’s effects on Wnt signaling. A) Structure
of KY02111. B) Effects of KY02111 on GSK3b inhibitor-induced TCF
promoter activation. HEK293 cells were transfected with TOPflash and
b-galactosidase plasmids and cultured for 24 h. The cells were then
treated with various concentrations of KY02111 in a medium contain-
ing BIO (3 mM), CHIR99021 (10 mM), or LiCl (40 mM). After 24 h,
luciferase activity was measured. Values are mean ÆSD. (n=3)
C) Effects of KY02111 on the Wnt signal proteins. HEK293 cells were
treated with various concentrations of KY02111 in a medium contain-
ing BIO (3 mM), CHIR99021 (10 mM), or LiCl (40 mM) for 24 h. The
lysates were immunoblotted with the indicated antibodies. The sam-
ples were derived from the same experiment, and they were analyzed
in parallel.
Institute for Biomedical Sciences, Shinshu University
Matsumoto, Nagano 390-8621 (Japan)
E-mail: yshiba@shinshu-u.ac.jp
Dr. I. Minami, Prof. M. Uesugi
Institute for Integrated Cell-Material Sciences (WPI-iCeMS)
Kyoto University, Kyoto 606-8501 (Japan)
Prof. M. Uesugi
School of Pharmacy, Fudan University
Shanghai 201203 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202100523.
&&&&
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
These are not the final page numbers!

Angewandte
Research Articles
Chemie
the xeno-free preparation of cardiomyocytes, the mechanistic
KY02111 potently inhibited the ability of BIO to drive
underpinning for its effect on the cardiomyogenesis remains
incompletely understood. Our previous study showed that
KY02111 potently blocked the Wnt signaling that is pharma-
cologically induced by BIO, a small-molecule GSK3b inhib-
itor. The Wnt signaling inhibition of KY02111 appears
consistent with the earlier notion that, although Wnt signaling
is essential during the initial stage of PSC differentiation to
mesoderm cells, its repression increases the yield of cardio-
myocytes at later stages.[4] However, since our earlier tran-
scriptome analysis showed a marginal effect of KY02111 in
the downregulation of the Wnt-dependent genes, its effects on
the Wnt target genes could not be clearly established.[3]
Here we resolve the initially perplexing effects of
KY02111 on Wnt signaling and then identify squalene
synthase (SQS) as a bona fide molecular target of KY02111.
The present study demonstrates that KY02111 primarily
impairs TGFb signaling, but not Wnt signaling, by disrupting
the interaction of SQS with cardiac ER-membrane protein
TMEM43. Our study of KY02111 and their analogs highlights
a heretofore unknown role for SQS in TGFb signaling and
cardiomyogenesis.
a reporter gene in which the expression of luciferase is
controlled by TCF, a far downstream transcription factor of
the Wnt signaling.[3]
We examined the effects of KY02111 on the ability of two
other GSK3b inhibitors (CHIR99021 and lithium chloride) to
stimulate the TCF reporter gene.[12] Perplexingly, while
KY02111 itself failed to nullify the ability of the two inhibitors
to stimulate the reporter gene in HEK293 cells, it abrogated
the reporter activation by BIO (Figure 1B). We also checked
the status of b-catenin by western blot analysis. Treatment of
HEK293 cells with BIO decreased the phosphorylation of b-
catenin at Ser33, Ser37, and Thr41 through the inhibition of
GSK3b and thereby increased the amounts of b-catenin
(Figure 1C). Note that the accumulation of b-catenin is
accompanied by its Ser45 phosphorylation as the CK1-
mediated Ser45 phosphorylation precedes the GSK3b-medi-
ated phosphorylation (Figure 1C). However, when the cells
were co-treated with BIO and KY02111, the phosphorylation
of b-catenin was maintained with a concomitant degradation
of cellular b-catenin (Figure 1C). In sharp contrast, KY02111
caused no similar effects for the cells treated with CHIR99021
or lithium chloride (Figure 1C). This finding led us to
conclude that KY02111 impairs the ability of BIO, but not
others, to inhibit GSK3b.
Results
With these puzzling results in hand, we next examined
whether KY02111 influences the interaction of BIO with
GSK3b, by taking an advantage of desthiobiotin ATP probe,
which permits monitoring of ATP occupancy in kinases
through its reaction with Lys residues in the ATP-binding
sites[13] (Figure 2A). After treatment of cell lysate of HEK293
cells with the ATP probe, the probe-labeled proteins were
purified by avidin beads and western blotted with a GSK3b
antibody for estimating the ATP occupancy of GSK3b
(Figure 2B). BIO, which is known to bind to the ATP-binding
pocket of GSK3b, competitively displaced the ATP probe,
and the competition was prevented by co-treatment with
KY02111. These results and those described previously
suggest that KY02111 blocks the interaction of BIO with
GSK3b.
One possible explanation of the above results is that
KY02111 exerts its effect by interacting directly with BIO. To
test this possibility, varied concentrations of KY02111 were
mixed with BIO (30 mM) in PBS containing 33% of DMSO in
vitro. BIO has visible light absorbance at 507 nm exhibiting
a pink color in day light, while KY02111 solutions are
transparent without no apparent visible light absorption
(Figure 2C–E). Addition of KY02111 displayed a decrease
of the pink color and 507-nm absorption of BIO in a dose-
dependent manner. Centrifugation of the samples showed
precipitation of pink-colored materials at the bottom of the
tubes, suggesting that KY02111 co-aggregates with BIO to
form insoluble assemblies. On the other hand, SO140, a close
analog of KY02111 with the amide nitrogen methylated
(Figure S1), failed to co-aggregate with BIO and thereby to
affect the BIOꢀs ability to bind to GSK3b, stabilize b-catenin
and stimulate the TCF reporter gene (Figures S2 and S3).
When HEK293 cells were treated both with BIO and
KY02111, lysed in lysis buffer, and centrifuged, similar
KY02111 Does Not Inhibit Wnt Signaling
Canonical Wnt signaling is tightly regulated by the
amount of the transcriptional co-activator b-catenin that
dictates key developmental gene expression patterns. In the
absence of Wnt, cytoplasmic b-catenin protein is constantly
degraded by the action of the Axin complex, which contains
casein kinase 1 (CK1) and glycogen synthase kinase 3b
(GSK3b). CK1 and GSK3 sequentially phosphorylate the
amino terminal region of b-catenin at Ser45 and Ser33/Ser37/
Thr41, respectively, resulting in b-catenin recognition by b-
TrCP, an E3 ubiquitin ligase subunit, and subsequent b-
catenin degradation.[5,6] This continual elimination of b-
catenin leads to the repression of Wnt target genes. When
a Wnt ligand binds to a transmembrane Frizzled (Fz) receptor
and its co-receptor, low-density lipoprotein receptor related
proteins (LRPs), the Wnt-Fz-LRP membrane complex
squelches the Axin complex to the membrane, leading to
the stabilization of b-catenin and the subsequent activation of
Wnt target genes.[7]
The Wnt signaling is readily impaired by two well-known
chemical inhibitors: XAV939 (tankyrase inhibitor)[8] and
IWP-2 (porcupine inhibitor).[9] These two potent inhibitors
have been shown to promote the differentiation to cardio-
myocytes from PSC-derived mesoderm cells.[10] Since their
target proteins are located upstream of b-catenin in the Wnt
signaling pathway, their cardiomyogenesis activity can be
canceled by adding (2’Z,3’E)-6-bromoindirubin-3’-oxime
(BIO), a Wnt signal activator that inhibits GSK3b.[11] In
contrast, the addition of BIO has no impacts on the
cardiomyogenesis activity of KY02111. This observation
previously led us to believe that KY02111 acts on the
downstream of the Wnt signaling pathway.[3] In fact,
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
KY02111 potentiates the generation of cardiomyocytes even
in the absence of BIO, suggesting the existence of a BIO-
independent mode of activation by KY02111.[3]
To obtain structure–activity profiles, we chemically syn-
thesized eight analogs of KY02111 and examined their ability
to potentiate cardiomyogeneis and to block the BIO-induced
Wnt signaling (Figure S1). Removal of the Cl group or
methylation of the amide group abolished the cardiomyo-
genesis activity, while substitution of Cl with Br or I (SO087
or KY-I, respectively) maintained or enhanced the activity.
The potent activity of KY-I was lost or diminished with the
modification of its dimethoxyphenyl group (SO2123, SO3027,
and SO2127) or methylene linker (SO3030). On the other
hand, TCF reporter assays showed that SO2068 and SO2123
readily inhibited the BIO-induced Wnt signaling although
they had no detectable cardiomyogenesis activity. The
structure–activity profiles were not necessarily paralleled
with the BIO inhibitory activities (Figure S1). We thus
hypothesized that KY02111 has a distinct endogenous target
unrelated to BIO or GSK3b.
To identify a bona fide target of KY02111, we designed
a photo-affinity probe of KY02111. Among the KY02111
analogs we synthesized, SO2093 (Figure 3A) exhibited car-
diomyogenesis activity at a comparable level to that of
KY02111 (data not shown), suggesting a conjugation site
suited for a probe design. Conjugation of a benzophenone
photoreactive group and an alkyne tag at that site yielded
a photo-affinity probe, SOB1102 (Figure 3A). The benzo-
phenone group would permit covalent photoaffinity labeling
of the cellular target while the alkyne tag would facilitate
identification of the photo-crosslinked proteins. HEK293 cells
were treated with the probe, followed by UV irradiation and
lysis of the cells. The lysates were separated into a PBS-
soluble cytosolic fraction (fraction S) and an insoluble
1%NP-40-containing membrane fraction (fraction I). After
Cu-catalyzed azide-alkyne cycloaddition with a rhodamine-
azide molecule, probe-crosslinked proteins were separated by
SDS-PAGE and visualized by in-gel fluorescence scanning
(Figure 3B). We found a ꢀ 45 KDa band in the insoluble
membrane fraction that was competed out with an excess
amount of KY02111 (indicated by an arrow). The ꢀ 45 KDa
protein was purified by avidin agarose resins after click
reaction with a biotin-azide. Mass spectrometric sequencing
identified the 45 KDa protein as squalene synthase (SQS), an
ER-membrane enzyme that catalyzes the first step committed
to the biosynthesis of sterols within the isoprenoid pathway.
Western blot analysis confirmed the binding of probe to SQS,
and the interaction was competed with an excess amount of
KY02111 (Figure 3C). Importantly, the interaction between
SQS and the probe was also prevented by the addition of KY-
I, a KY02111 analog that exerts cardiomyogenesis more
potently than KY02111, but no effects was observed with
SO3030, which is devoid of cardiomyogenesis activity (Fig-
ure 3D).
Figure 2. KY02111 forms co-aggregates with BIO. A) Schematic illus-
tration of the desthiobiotin-ATP probe pulldown assay. B) Results of
the ATP probe pulldown assay. KY02111, BIO, and ATP-probe were
added to HEK293 cell lysates. After purification of destiobiotin-bound
GSK3b with avidin beads, GSK3b was detected by western blotting. C–
E) Co-aggregation of KY02111 with BIO. Various concentrations of
KY02111 were added to a PBS:DMSO=2:1 solution in the absence or
presence of 30 mM of BIO. After mixing, photographs of the samples
were captured (C), and spectra of the solutions in the absence (D) or
presence (E) of BIO were measured.
insoluble pink-colored materials were observed in the pellets,
supporting the notion that the two compounds co-aggregate
in a cellular environment (Figure S4). These results collec-
tively solidify the conclusion that KY02111 impairs the BIO-
mediated GSK3b inhibition through co-aggregation with
BIO. KY02111 does not directly inhibit endogenous Wnt
signaling.
Squalene Synthase Is a KY02111-Binding Protein
Although Wnt signaling is essential during the initial stage
of PSC differentiation to mesoderm cells, it needs to be
repressed for increasing the yield of cardiomyocytes from
mesoderm cells.[4] In the xeno-free production of cardiomyo-
cytes from PSCs where BIO is often used as a substitute for
costly Wnt ligands, initial pharmacological activation of Wnt
signaling by BIO must be removed for the late stage
differentiation from mesoderm cells to cardiomyocytes. Such
a control over BIO can be achieved by addition of KY02111
at the late stage of differentiation. Thus, the co-aggregation of
KY02111 with BIO may provide a mechanistic rationale for
the observed KY02111-induced enhancement of cardiomyo-
genesis in a BIO-containing culture medium. However,
Cellular engagement of KY02111 with SQS was validated
by cellular thermal-shift assays, in which thermal stabilization
of proteins upon ligand binding is monitored in a cellular
context. SQS protein was denatured upon heating the cells at
558C and thereby precipitated out from the soluble fraction.
&&&&
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
These are not the final page numbers!

Angewandte
Research Articles
Chemie
KY02111 Binds to SQS without Affecting Its Enzymatic Activity
SQS is a mevalonate pathway enzyme that catalyzes the
conversion of farnesyl diphosphate to squalene for the
biosynthesis of sterols. We first examined whether KY02111
has affinity to SQS(31–370), a truncation mutant of SQS
encompassing the enzyme domain (Figure S6A). The photo-
affinity probe of KY02111 (SOB1102) exhibited photo-
reaction with FLAG-tagged SQS(31–370) as much as it did
with FLAG-tagged full-length SQS in HEK293 cells (Fig-
ure S6B), and the reaction was competed with an excess
amount of KY02111 (Figure S6C). To confirm the direct
interaction between SQS(31–370) and KY02111, we prepared
SOB1046, a fluorescein-conjugated fluorescent probe of
KY02111, and examined its association with bacterially
expressed SQS(31–370) in vitro. Addition of SQS(31–370)
dose-dependently increased the degree of fluorescent polar-
ization of SOB1046, indicating the reduced rotation of
SOB1046 due to its physical association with the SQS protein.
The KD value was estimated to be 167.2 nM (Figure S7).
The direct interaction of KY02111 with SQS(31–370)
prompted us to examine the effects of KY02111 on the
enzymatic activity of SQS in vitro. The SQS enzymatic
activity was monitored by measuring the production of
NADP+, which is accompanied with the conversion of
farnesyl diphosphate to squalene (Figure S8). Unexpectedly,
KY02111 failed to display detectable inhibitory effects on the
production of NADP+ up to 30 mM, whereas zaragozic acid
(ZA-A), a natural product that is known to inhibit the
enzymatic activity of SQS,[14] readily impaired the production
of NADP+ in a dose-dependent manner (Figure S8B). The
effects of KY02111 on the SQS activity was further evaluated
in cells by monitoring the transcriptional activation capability
of sterol regulatory element-binding protein (SREBP) (Fig-
ure S8C). Lipid-depletion from the medium or addition of
compactin (an HMG-CoA reductase inhibitor) or ZA-A each
increased the signals from an SREBP reporter gene by
limiting the intracellular levels of sterols, endogenous inhib-
itors of SREBP. In contrast, KY02111 had no detectable
effects on the SREBP activity up to 30 mM (Figure S8C).
These results indicate that KY02111 directly binds to SQS
without affecting the SQS enzymatic activity in vitro and in
cells.
Figure 3. Identification of squalene synthase as a binding protein of
KY02111. A) Structures of KY02111, KY-I, SO2093, and SOB1102.
B) Photoaffinity labeling. HEK293 cells were pretreated with 0 or
30 mM of KY02111 for 20 min and treated with 1 mM of SOB1102 for
40 min. After UV irradiation for 30 min, the cells were collected and
fractionated into PBS-soluble (S) and PBS-insoluble (I) fractions. To
each fraction, rhodamine-azide was added for click chemistry. The
rhodamine-conjugated proteins were separated by SDS-PAGE and
visualized by in-gel fluorescence scanning (left). Whole proteins were
detected by silver stain (right). C,D) Confirmation of photo-reacted
SQS. HEK293 cells were pretreated with DMSO, KY02111 (30 mM) (C),
KY-I (30 mM) (D), or SO3030 (30 mM) (D) for 20 min and treated with
1 mM of SOB1102 for 40 min. After UV irradiation for 30 min, the cells
were collected. To the PBS-insoluble fraction, biotin-azide was added
for click chemistry. After purification of biotin-conjugated proteins with
avidin beads, SQS was detected by western blotting. E) Cellular
thermal-shift assay. HEK293 cells were treated with various concen-
trations of KY02111 for 1 h. The cells were collected and treated with
heat at 558C. The cell extracts were immunoblotted with an a-SQS
antibody.
When the cells were treated with increasing amounts of
KY02111, more SQS protein remained in the soluble fraction
(Figure 3E), corroborating that KY02111 binds to SQS in
cells. The eight analogs of KY02111 were also subjected to
cellular thermal-shift assays. Analogs SO087 and KY-I, which
exerted cardiomyogenesis as much as or greater than
KY02111, respectively, stabilized SQS at similar or higher
levels than that of KY02111 (Figures S1 and S5). On the other
hand, the analogs that had little cardiomyogenesis activity
failed to thermally stabilize SQS in cells. The ability of the
bioactive analogs to potentiate cardiomyogenesis mirrored
their levels of thermal stabilization of SQS in cells (Fig-
ure S5B). The excellent correlation encouraged us to further
pursue SQS as the most likely target protein of KY02111.
KY02111 Impairs TGFb Signaling by Disrupting the SQS-
TMEM43 Interaction
The inability of KY02111 to modulate the enzymatic
activity of SQS prompted us to examine its effects on the
protein-protein interactions of SQS. The protein-protein
interaction database of BioGRID indicates four potential
SQS-interacting proteins that are localized in ER: SRPRB,[15]
ARL6IP1,[16] SYVN1,[17] and TMEM43.[18] The Myc-tagged
versions of these proteins were expressed in HEK293 cells,
and their potential interactions with FLAG-tagged SQS were
evaluated by co-immunoprecipitation (Figures 4A and S9).
The results showed that SQS associates with TMEM43 but
not with SRPRB, ARL6IP1, and SYVN1. The SQS-TMEM43
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
TMEM43 or SQS on the TGFb-induced expression of N-
cadherin (Figure 4E). The knockdown of either TMEM43 or
SQS suppressed the TGFb-induced expression of N-cadherin,
although the suppression was weaker in the SQS knockdown.
Interestingly, the SQS knockdown lowered the protein level
of TMEM43. It is likely that the physical association of
TMEM43 with SQS stabilizes TMEM43 to upregulate the
TGFb signaling.
TGFb cytokines regulate gene expression by receptor-
mediated activation of SMAD transcription factors. Receptor
binding of a TGFb cytokine stimulates the phosphorylation of
SMAD2 and SMAD3 to form a hetero-trimeric complex with
SMAD4 that binds to DNAwith partner transcription factors.
Three independent siRNA knockdown of TMEM43 in A549
cells all reduced the protein level of SMAD4 while a control
siRNA of EGFP had no significant effects (Figure 4F). The
protein levels of SMAD2 and SMAD3 were unchanged,
indicating that the TMEM43 knockdown selectively elimi-
nates SMAD4. Treatment of the cells with KY02111 or KY-I
similarly led to the decreased protein level of SMAD4 but not
SMAD2 and SMAD3, while SO3030, an inactive analog of
KY02111, showed no detectable effects on the protein levels
of SMADs (Figure 4D). Overall, our data led us to propose
a model in which KY02111 decreases the protein level of
SMAD4 and thereby represses TGFb signaling by destabiliz-
ing TMEM43 through the disruption of the SQS-TMEM43
interaction. Mounting evidence indicates that TGFb signaling
plays a key role in cardiomyogenesis, and inhibitors of TGFb
receptors have been reported to promote cardiomyogene-
sis.[19] KY02111 potentiates cardiomyogenesis most likely by
inhibiting TGFb signaling.
Figure 4. KY02111 affects TGFb signaling by disruption of SQS-
TMEM43 interaction. A,B) Effects of KY02111 and its derivatives on
the interaction between SQS and TMEM43. HEK293 cells were trans-
fected with pCMV-3Tag-1a or pCMV-3Tag-1a-SQS, and pCMV-3Tag-9-
TMEM43. After 6 h, various concentrations of KY02111 (A), DMSO,
KY-I (30 mM), or SO3030 (30 mM) (B) were added. Immunoprecipitated
proteins were analyzed by immunoblotting. C,D) Effects of KY02111
and its derivatives on the TGFb-induced expression of N-cadherin.
A549 cells were treated with TGFb (0 or 10 ngmLÀ1) for 2 days and
then with various concentrations of KY02111, DMSO, KY02111
(10 mM), KY-I (10 mM), or SO3030 (10 mM) for another 2 days. The
lysates were analyzed by immunoblotting. E) Effects of SQS/TMEM43
knockdown on the TGFb-induced expression of N-cadherin. A549 cells
were transfected with each siRNA (siEGFP, siSQS, or siTMEM43).
After 2 days, the cells were then treated with various concentrations of
TGFb for another 2 days. The lysates were analyzed by immunoblot-
ting. F) Effects of TMEM43 knockdown on the SMADs expression.
A549 cells were transfected with each siRNA (siEGFP or siTMEM43).
After 2 days, the cells were treated with 0 or 10 ngmLÀ1 of TGFb for
another 2 days. The lysates were analyzed by immunoblotting.
Prolonged KY02111 Exposure Induces ARVC-like Phenotype in
Cells
TMEM43 is a nuclear and ER membrane protein that is
conserved from bacteria to human.[18] Although the biological
roles of TMEM43 remain unclear, its mutations lead to
arrhythmogenic right ventricular cardiomyopathy (ARVC),
an inherited heart disease that involves a substitution of
myocardium with fatty tissue. The most aggressive arrhyth-
mogenic cardiomyopathy/ARVC subtype is ARVC type 5
(ARVC5), caused by a S358L mutation in TMEM43.[20] Mice
expressing TMEM43-S358L recapitulate the human disease,
displaying cardiomyocyte death with severe fibrofatty re-
placement.[21] These clinical and experimental observations of
TMEM43 properties led us to investigate the effects of the
S358L mutation on the interaction with SQS (Figure S10).
Co-immunoprecipitation experiments showed that the mu-
tated TMEM43 failed to associate with SQS, suggesting that
the disrupted interaction of TMEM43 with SQS might be
related to the ARVC phenotype.
interaction was abolished by addition of KY02111 or KY-I but
not SO3030, a KY02111 analog that lacks in cardiomyo-
genesis activity, suggesting that the pharmacological disrup-
tion of the SQS-TMEM43 interaction is related to the
cardiomyogenesis activity (Figures 4A and 4B).
During the course of our analysis, the biological activity of
KY02111 was also profiled by a series of cellular assays in the
MEXT molecular profiling platform program, a nation-wide
consortium for profiling biological activities of small mole-
cules. KY02111 was found to dose-dependently diminish the
TGFb-induced expression of N-cadherin in human LoVo
colon adenocarcinoma cells. Similar results were reproduced
in human A549 lung cancer cells (Figure 4C), and KY-I, but
not SO3030, also decreased the TGFb-induced expression of
N-cadherin (Figure 4D).
We speculated that the continued disruption of the SQS-
TMEM43 interaction by KY02111 during the entire process
of cardiomyogenesis may also lead to the ARVC phenotype.
To test the hypothesis, we added KY02111 to the culture
medium throughout an 18-day cardiomyogenesis protocol of
human iPS cells (Figure S11A). As previously reported, this
The functional link between TMEM43 and TGFb has not
been well-documented in the literature. To probe this
possibility, we examined the effects of siRNA knockdown of
&&&&
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
These are not the final page numbers!

Angewandte
Research Articles
Chemie
established protocol generated 80% cTNT positive cells with
However, even when pregnant mice carrying SQS knockout
high TNNT2 expression levels. The expression levels of these
two cardiomyocyte markers were significantly reduced by the
consecutive treatment with KY02111 (Figure S11BC). The
KY02111-treated cells displayed increased levels of preadi-
pocyte markers (DLK1 and PPARG) (Figure S11DE).
SO3030, a KY02111 analog that does not affect the SQS-
TMEM43 interaction, had limited effects. Taken together,
these data suggest that the disruption of the SQS-TMEM43
interaction contributes to the ARVC phenotype and that this
KY02111-induced activity serves as a tool to mimic the
ensuing disease.
mice are given excessive amounts of squalene and cholesterol
in their diet, SQS knockout mice remain embryonic lethal,[29]
suggesting that SQS has other important developmental
functions besides the synthesis of squalene. The regulation
of TGFb signaling through its interaction with TMEM43 may
represent one of those additional functions.
Another unexpected lesson arising from our study is due
to the surprising finding that KY02111 exerts its effect by
inhibiting the activity of BIO through molecular co-assembly.
BIO has a structure with two linked indole rings. Such
a chemical feature can be found in indigo, which in fact
similarly co-assembled with KY02111 in vitro (data not
shown). In contrast, tryptophan, which has only one indole
ring, failed to aggregate with KY02111 (data not shown).
Presumably, KY02111 aggregates and squelches small mole-
cules bearing two indole rings. Small-molecule targeting by
a bioactive small-molecule has been barely documented,
except by those of theonellamide[30] and adhesamine,[31] which
exert their biological activity by binding to ergosterol and
heparansulfate, respectively. The KY02111-BIO interaction
presents an example in which two exogenous small molecules
selectively interact and condensate each other. Such finding
would complicate the understanding of pharmacological and
chemical biological investigations, as exemplified in our study.
Our limited experience serves as cautionary warnings of the
risks in using multiple small molecules simultaneously in
cellular and molecular investigations.
Our work has generated several important questions that
will be the basis for further deep investigations. Foremost, we
do not understand how TMEM43 maintains the SMAD4
protein levels. In canonical TGFb signaling, SMAD4 serves as
a central mediator in transmitting the signal to the nucleus. Its
expression level is tightly regulated and frequently decreased
in human cancer. Ubiquitin (E3) ligase Skp2[32] and b-
TrCP1[33] interacts with SMAD4, which leads to the increased
ubiquitination and accelerated proteolysis. It has also been
reported that BRK (BRK-Y447F) phosphorylates
SMAD4,[34] resulting in its degradation through the recog-
nition by the ubiquitin-proteasome system. We found that
knockdown of TMEM43 decreases the protein levels of
SMAD4 (Figure 4D), suggesting that TMEM43 activates
TGFb signaling by maintaining the steady-state SMAD4
level. Future work will be critical for a better understanding
of the underlying molecular basis for the finding.
Discussion
Our chemical genetic study demonstrates that KY02111
and its optimized analog KY-I bind to SQS and thereby
impair TGFb signaling through the disruption of the SQS-
TMEM43 interaction. This mechanism is fully consistent with
the ability of KY02111 to boost mesodermal-to-cardiomyo-
cyte differentiation. The timing of TGFb signaling blockade
has been shown to be important for inducing cardiomyoge-
nesis.[19a] Adding an inhibitor of TGFb signaling during the
differentiation of mesodermal cells into cardiomyocytes
promotes myocardial differentiation. In contrast, the addition
of a TGFb signaling inhibitor during iPS cell differentiation
into mesodermal cells does not display such pro-differentia-
tion activity. Importantly, our study showed that KY02111
exposure throughout cardiomyogenesis induces ARVC-like
phenotype in cells, analogous to the phenotype of clinically
observed TMEM43 mutation. The findings suggest the
interesting possibility that the prolonged disruption of the
SQS-TMEM43 interaction contributes to the ARVC pheno-
type and that KY02111 serves as a unique tool to mimic the
disease.
In addition to the mutations of TMEM43, those of several
other genes have clinically been identified as responsible for
the development of ARVC. Among them are TGFb3,[22]
a member of the TGFb superfamily, and desmosome con-
stituents such as desmoplakin,[23] plakophilin-2,[24] desmo-
glein-2,[25] desmocollin-2[26] and plakoglobin,[27] which are
important for cell-cell adhesion and are abundant in the
epidermis and myocardium. The expression of plakoglobin,
desmoplakin, and plakophilin-2 is regulated by TGFb signal-
ing.[28] Thus, myocardial degeneration and fibrosis in patients
with ARVC may be the result of dysfunction and disruption
of intercellular adhesion structures due to abnormal TGFb
signaling. The present study revealed that TMEM43 also
plays a key role in TGFb signaling through its interaction with
SQS. Significantly, the SQS-TMEM43 interaction is disrupted
by the clinically observed mutation of TMEM43. The
TMEM43 mutation contributes to the development of ARVC
most likely through dysregulation of TGFb signaling.
Another question is how SQS stabilizes TMEM43 through
protein-protein interaction. Protein-protein interactions of
ER-membrane bound proteins often increase protein stabil-
ity. A representative example is the physical association of
SREBPs with SREBP cleavage-activating protein (SCAP), an
escort protein of SREBPs, which stabilizes SREBPs by
masking their degradation signals.[35] Another interesting case
is HMG-CoA reductase (HMGCR), an ER-membrane-
bound rate-limiting enzyme in mevalonate pathway. Under
low geranylgeranyl pyrophosphate (GGPP) concentrations,
ER-resident UbiA prenyltransferase domain-containing pro-
tein 1 (UBIAD1) binds and stabilizes HMGCR. Under
GGPP excess, UBIAD1 translocates to the Golgi, releasing
HMGCR for proteasomal degradation.[36] Our future studies
Our results underscore an unexpected role of SQS in
TGFb signaling. SQS is the traditional metabolic enzyme that
has long been known to convert FPP to squalene for
cholesterol biosynthesis. SQS knockout mice are embryonic
lethal, indicating that SQS is essential for development.[29]
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
will focus on elucidating the molecular underpinning of the
TMEM43 stabilization by SQS.
Conflict of Interest
Furthermore, we need more fully to understand how
KY02111 displayed weak downregulation of Wnt-dependent
genes in our earlier transcriptome analysis,[3] which was the
reason for focusing our initial studies on Wnt signaling as
a potential target pathway of KY02111. Efficient differentia-
tion of mesoderm cells into cardiomyocytes requires the
inactivation of BMP, activin, and TGFb signaling as well as
Wnt signaling.[19b] Ample studies have described the signaling
crosstalk among these signal transduction pathways. BMP,
Activin, TGFb signalings share a number of SMAD members
including SMAD4 in transmitting the signals.[37] The Wnt-
signaling scaffolding protein Axin and its associated kinase,
GSK3b, interact with TGFb-regulated SMAD3 for protea-
some-dependent degradation. One of the downstream tran-
scription factors in Wnt signaling, lymphoid enhancer factor
1 (LEF1), is activated by phosphorylated SMAD2 and
SMAD4 to control the E-cadherin gene expression,[38] while
the protein stability of Snail, a downstream factor of TGFb
signaling, is regulated by GSK3b through phosphorylation.[39]
These previously reported crosstalk between Wnt and TGFb
signaling pathways and the findings of the present study
indicate that KY02111 indirectly affects Wnt signaling
through the decrease of SMAD4 levels.
The authors declare no conflict of interest.
Keywords: cardiomyogenesis · chemical genetics · SQS ·
TGFb signaling
[1] T. Brand, Dev. Biol. 2003, 258, 1 – 19.
[2] a) M. Wagner, M. A. Siddiqui, Exp. Biol. Med. 2007, 232, 852 –
865; b) W. Liu, A. C. Foley, Wiley Interdiscip. Rev. Syst. Biol.
Med. 2011, 3, 191 – 205; c) T. Brade, L. S. Pane, A. Moretti, K. R.
Chien, K. L. Laugwitz, Cold Spring Harbor Perspect. Med. 2013,
3, a013847; d) C. Sun, M. I. Kontaridis, Curr. Opin. Physiol. 2018,
1, 123 – 139.
[3] I. Minami, K. Yamada, T. G. Otsuji, T. Yamamoto, Y. Shen, S.
Otsuka, S. Kadota, N. Morone, M. Barve, Y. Asai, T. Tenkova-
Heuser, J. E. Heuser, M. Uesugi, K. Aiba, N. Nakatsuji, Cell Rep.
2012, 2, 1448 – 1460.
[4] X. Lian, C. Hsiao, G. Wilson, K. Zhu, L. B. Hazeltine, S. M.
Azarin, K. K. Raval, J. Zhang, T. J. Kamp, S. P. Palecek, Proc.
Natl. Acad. Sci. USA 2012, 109, E1848 – E1857.
[5] a) M. J. Hart, R. de los Santos, I. N. Albert, B. Rubinfeld, P.
Polakis, Curr. Biol. 1998, 8, 573 – 581; b) A. Salic, E. Lee, L.
Mayer, M. W. Kirschner, Mol. Cell 2000, 5, 523 – 532; c) C. Liu,
Y. Li, M. Semenov, C. Han, G. H. Baeg, Y. Tan, Z. Zhang, X. Lin,
X. He, Cell 2002, 108, 837 – 847.
[6] G. Wu, G. Xu, B. A. Schulman, P. D. Jeffrey, J. W. Harper, N. P.
Pavletich, Mol. Cell 2003, 11, 1445 – 1456.
[7] a) M. Molenaar, M. van de Wetering, M. Oosterwegel, J. Peter-
son-Maduro, S. Godsave, V. Korinek, J. Roose, O. Destrꢁe, H.
Clevers, Cell 1996, 86, 391 – 399; b) J. Behrens, J. P. von Kries, M.
Kꢂhl, L. Bruhn, D. Wedlich, R. Grosschedl, W. Birchmeier,
Nature 1996, 382, 638 – 642.
[8] S. M. Huang, Y. M. Mishina, S. Liu, A. Cheung, F. Stegmeier,
G. A. Michaud, O. Charlat, E. Wiellette, Y. Zhang, S. Wiessner,
M. Hild, X. Shi, C. J. Wilson, C. Mickanin, V. Myer, A. Fazal, R.
Tomlinson, F. Serluca, W. Shao, H. Cheng, M. Shultz, C. Rau, M.
Schirle, J. Schlegl, S. Ghidelli, S. Fawell, C. Lu, D. Curtis, M. W.
Kirschner, C. Lengauer, P. M. Finan, J. A. Tallarico, T. Bouw-
meester, J. A. Porter, A. Bauer, F. Cong, Nature 2009, 461, 614 –
620.
Conclusion
Our mechanistic study of KY02111 and its analogs
revealed a heretofore undescribed critical role of SQS in
TGFb signaling. It shows that KY02111 disrupts the SQS-
TMEM43 by binding to SQS and thereby recapitulates an
ARVC familial mutation of TMEM43. KY02111 and its
analogs could be used in ARVC modeling, as well as serve as
a research tool for studying TGFb/SMAD signaling. The
findings of the present study provide a fresh perspective for
the study of the TGFb signaling pathways and serve as
a starting point for further investigations in more physiolog-
ical settings.
[9] B. Chen, M. E. Dodge, W. Tang, J. Lu, Z. Ma, C. W. Fan, S. Wei,
W. Hao, J. Kilgore, N. S. Williams, M. G. Roth, J. F. Amatruda, C.
Chen, L. Lum, Nat. Chem. Biol. 2009, 5, 100 – 107.
[10] a) H. Wang, J. Hao, C. C. Hong, ACS Chem. Biol. 2011, 6, 192 –
197; b) X. Lian, J. Zhang, S. M. Azarin, K. Zhu, L. B. Hazeltine,
X. Bao, C. Hsiao, T. J. Kamp, S. P. Palecek, Nat. Protoc. 2013, 8,
162 – 175.
Acknowledgements
We thank Norio Nakatsuji for his guidance. This work was
supported by JSPS (26220206 and 19H00922 to M.U.;
17K13267 and 19K05733 to Y.T.; JP16H06276 (AdAMS)),
Suzuken Memorial Foundation, and the International Col-
laborative Research Program of Institute for Chemical
Research (2020-94). This work was inspired by the interna-
tional and interdisciplinary environments of WPI-iCeMS and
JSPS CORE-to-CORE Program, “Asian Chemical Biology
Initiative.” This study used 600-MHz and 800-MHz NMR
spectrometers in the Joint Usage/Research Center (JURC) at
the Institute for Chemical Research, Kyoto University.
[11] L. Meijer, A. L. Skaltsounis, P. Magiatis, P. Polychronopoulos, M.
Knockaert, M. Leost, X. P. Ryan, C. A. Vonica, A. Brivanlou, R.
Dajani, C. Crovace, C. Tarricone, A. Musacchio, S. M. Roe, L.
Pearl, P. Greengard, Chem. Biol. 2003, 10, 1255 – 1266.
[12] a) C. N. Bennett, S. E. Ross, K. A. Longo, L. Bajnok, N. Hemati,
K. W. Johnson, S. D. Harrison, O. A. MacDougald, J. Biol.
Chem. 2002, 277, 30998 – 31004; b) W. J. Ryves, A. J. Harwood,
Biochem. Biophys. Res. Commun. 2001, 280, 720 – 725.
[13] M. P. Patricelli, A. K. Szardenings, M. Liyanage, T. K. Nomanb-
hoy, M. Wu, H. Weissig, A. Aban, D. Chun, S. Tanner, J. W.
Kozarich, Biochemistry 2007, 46, 350 – 358.
[14] J. D. Bergstrom, M. M. Kurtz, D. J. Rew, A. M. Amend, J. D.
Karkas, R. G. Bostedor, V. S. Bansal, C. Dufresne, F. L. Van-
Middlesworth, O. D. Hensens, et al., Proc. Natl. Acad. Sci. USA
1993, 90, 80 – 84.
[15] S. Tajima, L. Lauffer, V. L. Rath, P. Walter, J. Cell Biol. 1986, 103,
1167 – 1178.
&&&&
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
These are not the final page numbers!

Angewandte
Research Articles
Chemie
[16] H. M. Lui, J. Chen, L. Wang, L. Naumovski, Mol. Cancer Res.
2003, 1, 508 – 518.
[17] M. Kikkert, R. Doolman, M. Dai, R. Avner, G. Hassink, S.
van Voorden, S. Thanedar, J. Roitelman, V. Chau, E. Wiertz, J.
Biol. Chem. 2004, 279, 3525 – 3534.
[18] L. Bengtsson, H. Otto, J. Cell Sci. 2008, 121, 536 – 548.
[19] a) E. Willems, J. Cabral-Teixeira, D. Schade, W. Cai, P. Reeves,
P. J. Bushway, M. Lanier, C. Walsh, T. Kirchhausen, J. C.
Izpisua Belmonte, J. Cashman, M. Mercola, Cell Stem Cell
2012, 11, 242 – 252; b) D. Schade, A. T. Plowright, J. Med. Chem.
2015, 58, 9451 – 9479.
[20] N. D. Merner, K. A. Hodgkinson, A. F. Haywood, S. Connors,
V. M. French, J. D. Drenckhahn, C. Kupprion, K. Ramadanova,
L. Thierfelder, W. McKenna, B. Gallagher, L. Morris-Larkin,
A. S. Bassett, P. S. Parfrey, T. L. Young, Am. J. Hum. Genet.
2008, 82, 809 – 821.
[21] a) G. Zheng, C. Jiang, Y. Li, D. Yang, Y. Ma, B. Zhang, X. Li, P.
Zhang, X. Hu, X. Zhao, J. Du, X. Lin, Protein Cell 2019, 10, 104 –
119; b) L. Padron-Barthe, M. Villalba-Orero, J. M. Gomez-
Salinero, F. Dominguez, M. Roman, J. Larrasa-Alonso, P. Ortiz-
Sanchez, F. Martinez, M. Lopez-Olaneta, E. Bonzon-Kulichen-
ko, J. Vazquez, C. Marti-Gomez, D. J. Santiago, B. Prados, G.
Giovinazzo, M. V. Gomez-Gaviro, S. Priori, P. Garcia-Pavia, E.
Lara-Pezzi, Circulation 2019, 140, 1188 – 1204.
[27] G. McKoy, N. Protonotarios, A. Crosby, A. Tsatsopoulou, A.
Anastasakis, A. Coonar, M. Norman, C. Baboonian, S. Jeffery,
W. J. McKenna, Lancet 2000, 355, 2119 – 2124.
[28] J. Xu, S. Lamouille, R. Derynck, Cell Res. 2008, 19, 156 – 172.
[29] R. Tozawa, S. Ishibashi, J. Osuga, H. Yagyu, T. Oka, Z. Chen, K.
Ohashi, S. Perrey, F. Shionoiri, N. Yahagi, K. Harada, T. Gotoda,
Y. Yazaki, N. Yamada, J. Biol. Chem. 1999, 274, 30843 – 30848.
[30] S. Nishimura, Y. Arita, M. Honda, K. Iwamoto, A. Matsuyama,
A. Shirai, H. Kawasaki, H. Kakeya, T. Kobayashi, S. Matsunaga,
M. Yoshida, Nat. Chem. Biol. 2010, 6, 519 – 526.
[31] a) S. Yamazoe, H. Shimogawa, S. Sato, J. D. Esko, M. Uesugi,
Chem. Biol. 2009, 16, 773 – 782; b) N. Takemoto, T. Suehara,
H. L. Frisco, S. Sato, T. Sezaki, K. Kusamori, Y. Kawazoe, S. M.
Park, S. Yamazoe, Y. Mizuhata, R. Inoue, G. J. Miller, S. U.
Hansen, G. C. Jayson, J. M. Gardiner, T. Kanaya, N. Tokitoh, K.
Ueda, Y. Takakura, N. Kioka, M. Nishikawa, M. Uesugi, J. Am.
Chem. Soc. 2013, 135, 11032 – 11039.
[32] M. Liang, Y. Y. Liang, K. Wrighton, D. Ungermannova, X. P.
Wang, F. C. Brunicardi, X. Liu, X. H. Feng, X. Lin, Mol. Cell.
Biol. 2004, 24, 7524 – 7537.
[33] M. Wan, Y. Tang, E. M. Tytler, C. Lu, B. Jin, S. M. Vickers, L.
Yang, X. Shi, X. Cao, J. Biol. Chem. 2004, 279, 14484 – 14487.
[34] S. Miah, C. A. S. Banks, Y. Ogunbolude, E. T. Bagu, J. M. Berg,
A. Saraf, T. T. Tettey, G. Hattem, G. Dayebgadoh, C. G. Kempf,
M. Sardiu, S. Napper, L. Florens, K. E. Lukong, M. P. Washburn,
Sci. Adv. 2019, 5, eaaw3113.
[35] D. L. Kober, S. Xu, S. Li, B. Bajaj, G. Liang, D. M. Rosenbaum,
A. Radhakrishnan, Proc. Natl. Acad. Sci. USA 2020, 117, 28080 –
28091.
[36] a) M. M. Schumacher, R. Elsabrouty, J. Seemann, Y. Jo, R. A.
DeBose-Boyd, eLife 2015, 4, e05560; b) M. M. Schumacher, D. J.
Jun, Y. Jo, J. Seemann, R. A. DeBose-Boyd, J. Lipid Res. 2016,
57, 1286 – 1299.
[37] F. Dituri, C. Cossu, S. Mancarella, G. Giannelli, Cells 2019, 8,
1130.
[38] A. Nawshad, D. Medici, C. C. Liu, E. D. Hay, J. Cell Sci. 2007,
120, 1646 – 1653.
[22] G. Beffagna, G. Occhi, A. Nava, L. Vitiello, A. Ditadi, C. Basso,
B. Bauce, G. Carraro, G. Thiene, J. A. Towbin, G. A. Danieli, A.
Rampazzo, Cardiovasc. Res. 2005, 65, 366 – 373.
[23] A. Rampazzo, A. Nava, S. Malacrida, G. Beffagna, B. Bauce, V.
Rossi, R. Zimbello, B. Simionati, C. Basso, G. Thiene, J. A.
Towbin, G. A. Danieli, Am. J. Hum. Genet. 2002, 71, 1200 – 1206.
[24] B. Gerull, A. Heuser, T. Wichter, M. Paul, C. T. Basson, D. A.
McDermott, B. B. Lerman, S. M. Markowitz, P. T. Ellinor, C. A.
MacRae, S. Peters, K. S. Grossmann, J. Drenckhahn, B. Michely,
S. Sasse-Klaassen, W. Birchmeier, R. Dietz, G. Breithardt, E.
Schulze-Bahr, L. Thierfelder, Nat. Genet. 2004, 36, 1162 – 1164.
[25] K. Pilichou, A. Nava, C. Basso, G. Beffagna, B. Bauce, A.
Lorenzon, G. Frigo, A. Vettori, M. Valente, J. Towbin, G. Thiene,
G. A. Danieli, A. Rampazzo, Circulation 2006, 113, 1171 – 1179.
[26] A. Heuser, E. R. Plovie, P. T. Ellinor, K. S. Grossmann, J. T.
Shin, T. Wichter, C. T. Basson, B. B. Lerman, S. Sasse-Klaassen,
L. Thierfelder, C. A. MacRae, B. Gerull, Am. J. Hum. Genet.
2006, 79, 1081 – 1088.
[39] B. P. Zhou, J. Deng, W. Xia, J. Xu, Y. M. Li, M. Gunduz, M. C.
Hung, Nat. Cell Biol. 2004, 6, 931 – 940.
Manuscript received: January 12, 2021
Accepted manuscript online: August 10, 2021
Version of record online: && &&
,
&&&&
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
Cell Signaling
KY02111 is a widely used small molecule
that boosts cardiomyogenesis. Chemical
genetics of KY02111 identified squalene
synthase (SQS) as a molecular target of
KY02111. By disrupting the interaction of
SQS with cardiac ER-membrane protein
TMEM43, KY02111 impairs TGFb/SMAD
signaling and recapitulates the clinical
mutation of TMEM43 that causes an
inherited heart disease.
Y. Takemoto, S. Kadota, I. Minami,
S. Otsuka, S. Okuda, M. Abo,
L. L. Punzalan, Y. Shen, Y. Shiba,*
M. Uesugi*
&&&& — &&&&
Chemical Genetics Reveals a Role of
Squalene Synthase in TGFb Signaling and
Cardiomyogenesis
&&&&
www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
These are not the final page numbers!