Heterotaxin: A TGF-β signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos

Article abstract of DOI:10.1016/j.chembiol.2010.12.008

Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin,

Full text of DOI:10.1016/j.chembiol.2010.12.008

Chemistry & Biology
Article
Heterotaxin: A TGF-b Signaling Inhibitor
Identified in a Multi-Phenotype Profiling Screen
in Xenopus Embryos
Michael K. Dush,1 Andrew L. McIver,2 Meredith A. Parr,1 Douglas D. Young,2 Julie Fisher,3 Donna R. Newman,1
1,
Philip L. Sannes,1 Marlene L. Hauck,3 Alexander Deiters,2, and Nanette Nascone-Yoder
*
1Department of Molecular Biomedical Sciences, College of Veterinary Medicine
2Department of Chemistry
*
3Department of Clinical Sciences, College of Veterinary Medicine
North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA
*Correspondence: alex_deiters@ncsu.edu (A.D.), nanette_nascone-yoder@ncsu.edu (N.N.-Y.)
DOI 10.1016/j.chembiol.2010.12.008
SUMMARY
(Zhu et al., 2006). Also associated with heterotaxia is intestinal
malrotation, which occurs in as many as one in 500 births and
Disruptions of anatomical left-right asymmetry result predisposes affected individuals to life-threatening conditions
(Kamal, 2000).
The initial establishment of the left-right axis eventually results
in the expression of genes exclusively on the left side of the
in life-threatening heterotaxic birth defects in vital
organs. We performed a small molecule screen
for left-right asymmetry phenotypes in Xenopus
embryos and discovered a pyridine analog, hetero-
taxin, which disrupts both cardiovascular and diges-
tive organ laterality and inhibits TGF-b-dependent
left-right asymmetric gene expression. Heterotaxin
analogs also perturb vascular development, melano-
embryo, including the TGF-b family members nodal and lefty,
and the transcription factor Pitx2 (Levin, 2005). Although it has
been demonstrated that situs inversus or heterotaxia can result
if these genes are misexpressed, how such left-right cues are
translated into the asymmetric morphology of developing organs
is poorly understood. Such knowledge is essential for under-
standing the etiology of congenital deformities.
genesis, cell migration, and adhesion, and indirectly
inhibit the phosphorylation of an intracellular medi-
In recent years, whole organism chemical-genetic strategies,
ator of TGF-b signaling. This combined phenotypic in which pharmacologically well-characterized small molecules
are screened in living embryos for their ability to induce a devel-
profile identifies these compounds as a class of
opmental phenotype of interest, have been successfully used to
illuminate the mechanisms that establish the initial left-right axis
TGF-b signaling inhibitors. Notably, heterotaxin
analogs also possess highly desirable antitumor
properties, inhibiting epithelial-mesenchymal transi-
tion, angiogenesis, and tumor cell proliferation in
mammalian systems. Our results suggest that
assessing multiple organ, tissue, cellular, and molec-
of the early embryo (e.g., Adams et al., 2006). However, the effi-
cacy of such screening strategies is limited by the availability of
known bioactives capable of exerting specific effects on devel-
oping model organisms. To our knowledge, no discovery-based
screens have been employed to uncover novel compounds that
ular parameters in a whole organism context is a valu-
perturb left-right asymmetric organ morphogenesis. Identifying
able strategy for identifying the mechanism of action
of bioactive compounds.
novel heterotaxia-inducing small molecules may not only
offer an increased understanding of the molecular etiology of
common birth defects but may also reveal new classes of small
molecules capable of modulating pathways that play important
roles in development and disease.
INTRODUCTION
Unfortunately, uncovering the mechanism of action of a novel
Vertebrate embryos develop with left-right asymmetry, evident in molecule identified in a whole organism or phenotype-based
the asymmetric anatomical positioning of the heart and other screen remains a major challenge. Increasingly, multiparameter
vital organs. Correct asymmetries are essential for the function phenotypic profiling is being used to categorize small molecules
of the cardiovascular and digestive systems, and severe malfor- discovered in high-throughput biochemical assays or cell-based
mations are linked to disruptions of organ laterality. Complete screens, providing insight into mechanism of action by similari-
reversal of normal left-right asymmetries (situs inversus) occurs ties to reference compounds with known cellular targets (Feng
in one in 8,500 births (Brueckner, 2007), whereas ‘‘heterotaxia,’’ et al., 2009). However, even compounds identified in multiplex
in which one or more organs deviate from normal by appearing strategies may still be ineffectual or have unpredictable or toxic
independently and randomly oriented with respect to left effects in vivo.
and right, occurs in one in 10,000 births (Brueckner, 2007).
Here, we describe an approach to small molecule discovery
Heterotaxia is often accompanied by intracardiac defects and that combines the advantages of whole organism screening
is associated with at least 3% of all congenital heart disease and multiplex profiling by generating a multiparameter profile
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
Figure 1. A Mixture of Pyridine Regioisomers
Causes Heterotaxia in Xenopus
Embryos were treated with (A) DMSO or (B) 200 mM active
pool. (C) Ventral view of organs in intact DMSO control
tadpole. Ventral (D) and left-ventral (E) views of tadpole
in (C) with skin removed, showing the normal asymmetry
(arrowhead, D) of the foregut loop, and the normal coun-
terclockwise rotation of the intestine (curved arrow, E).
(F) Tadpole exposed to active pool. Ventral (G) and right-
ventral (H) view of tadpole in (F) with skin removed,
showing the reversed position (arrowhead, G) of the fore-
gut loop. The intestine is coiling in the normal direction
(curved arrow, H) but is located on the opposite side of
the animal. Ventral views of tadpole exposed to active
pool (I–K) show normal foregut looping (arrowhead, J)
but the intestine coiling in the reversed direction (i.e.,
clockwise; curved arrow, K). (L) Ventral view of heart of
DMSO control, with outflow tract (conus, c) and ventricle
(v) indicated, with the normal direction of cardiac looping
(curved arrow). (M and N) Two examples of heterotaxin-
induced reversed heart looping (curved arrows).
RESULTS
The Discovery of Heterotaxin (1)
A solid-supported multicomponent cyclotrimeri-
zation reaction (Young et al., 2007) was utilized
to generate a pilot library of ꢀ130 novel com-
pounds as 44 pools of regioisomers. We arrayed
X. laevis embryos in individual wells of a 24-well
plate in growth media supplemented with pools
of regioisomers (200 mM). After the embryos
had completed asymmetric morphogenesis, the
morphology of the heart and digestive tract was
assessed in anesthetized tadpoles. All controls,
i.e., untreated or exposed to DMSO alone, had
normal organ asymmetries (Figures 1A, 1C–1E,
and 1L), as did the embryos in 33/44 (75%) pools.
In our initial screen, 2/44 pools (4.5%) were lethal
at 200 mM, and 8/44 pools (18%) elicited multiple
defects (in organ, eye, craniofacial and tail devel-
opment) in exposed embryos. However, one
of embryonic phenotypes. Recent studies have illustrated the pyridine pool induced clear heterotaxia (heart and/or gut looping
promise of embryos of the aquatic frog, Xenopus laevis, for small defects) in 100% of the exposed individuals (Figures 1B, 1F–1K,
molecule discovery (e.g., Tomlinson et al., 2009). A unique 1M, and 1N). In three repeat trials with embryos derived from
feature of Xenopus embryos, not found in other models (e.g., oocytes obtained from different mothers, this pool was effective
zebrafish), is the morphogenesis of their left-right asymmetric at inducing heterotaxia in at least 50% of embryos exposed at
organs, which is highly analogous to humans and easily visible a concentration of 100–200 mM. Overall, 89% (n = 28) of the
in situ (Muller et al., 2003). Therefore, we employed Xenopus embryos in all trials that were exposed to the active regioisomer
embryos in a screen for heterotaxia-inducing compounds and pool at 100–200 mM exhibited heterotaxia phenotypes, strongly
identified a novel pyridine-based molecule, which we named implicating one or more regioisomers in this pool as an inhibitor
‘‘heterotaxin.’’ Further analyses of the in vivo phenotypic profile of a cellular target(s) required for normal left-right asymmetric
of this compound revealed that it elicits multiple TGF-b-depen- organ development.
dent phenotypes throughout development and inhibits TGF-b-
dependent intracellular signaling events, identifying it as a new Identification of the Active Heterotaxia-Inducing
TGF-b signaling inhibitor. Importantly, heterotaxin analogs also Regioisomer
disrupt invasive phenotypes, angiogenesis, and tumor cell To facilitate the isolation of the regioisomer responsible for the
proliferation in mammalian systems, revealing a new class of observed heterotaxia phenotypes, the solid-phase synthesis
compounds with TGF-b-inhibitory therapeutic potential.
used to generate the original pool was conducted on a 5-fold
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
Figure 2. Purification, Identification, and Synthesis of Heterotaxin (1)
(A) GC trace of the regioisomers in the active pool.
(B) Active regioisomer (1) after separation (see Supplemental Experimental Procedures), phenotypic assay, and structural assignment.
(C) Regioselective synthetic route toward gram quantities of heterotaxin (1) and analogs (27–31).
larger scale. A GC trace of the heterotaxia-inducing pool prior to Despite the differences between the X. laevis and X. tropicalis
purification indicated the presence of several different regio- species in culture temperature (15ꢁC versus 24ꢁC, respec-
isomers (Figure 2A). After separating these components by tively), size (1.2 versus 0.7 mm, respectively), and growth
flash-column chromatography on silica gel, we found that only rate (7–8 versus 3–4 days, respectively), the regioisomerically
the 2,4,6-regioisomer 1 (Figure 2B; GC retention time of 17.17 pure compound induced identical heterotaxic organ
min) displayed the ability to induce the desired phenotype in deformities in both the heart and gut as observed in
Xenopus embryos (data not shown). X. laevis (data not shown). Due to its ‘‘heterotaxia-inducing’’
As further confirmation, we exposed embryos of a related propensity, we named the purified, active regioisomer
species, Xenopus tropicalis, to this purified component. ‘‘heterotaxin.’’
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
A Regioselective Synthetic Route to Heterotaxin
controls (Figure 4A), the melanocytes in heterotaxin-treated
Our original synthesis route toward heterotaxin necessitated tadpoles exhibit a striking increase in pigmentation and den-
laborious purification of the 2,4,6-regioisomer 1 from a mixed dricity (Figures 4B and 4C), and the number of melanocytes
pool and was, therefore, inefficient for generating the large is reduced by as much as 50% (Figure 4D). Exposure to
(milligram) quantities of heterotaxin required for further charac- heterotaxin also results in dilated vitelline vessels and ventral
terization. Consequently, we developed a robust, scalable hemorrhaging (Figures 4E–4G), suggesting that the compound
synthetic approach to heterotaxin, with a sequence of chemical perturbs the normal processes of vasculogenesis and/or
transformations that provided
synthesis (Figure 2C).
a
completely regioselective angiogenesis.
Finally, in addition to being malrotated, the digestive tracts of
The synthesis commenced with the condensation of the lithi- embryos exposed to heterotaxin are wider and shorter than
ated trityl-protected propargyl alcohol 2 and commercially avail- controls (e.g., see Figures 4E and 4G). The elongation of the
able chlorodiisopropylsilane (3) to afford the silane 4 in 76% yield digestive tract is driven by the rearrangements of endoderm cells
(Petit et al., 2003). The silane 4 was then subjected to in situ within the embryonic gut tube, a process that also facilitates the
bromination by NBS, followed by a subsequent reaction with formation of the gut lumen and epithelial lining (Reed et al., 2009).
propargyl alcohol in the presence of TEA and DMAP in DCM to The endoderm cells are initially round but adopt an elongated
afford the silyl ether 5 in 85% over two steps (Petit et al., shape and remodel their intercellular adhesive junctions as they
2003). The key step of the synthesis was a microwave-mediated, move toward the circumference of the gut, intercalating between
cobalt-catalyzed [2+2+2] cyclotrimerization reaction between each other to form a single layer (Reed et al., 2009). In controls the
the diyne 5 and propionitrile under open-vessel conditions. resultant simple columnar epithelium can be observed lining the
This delivered the pyridine 6 in 98% yield. The silicon tether lumen of the lengthened coils of gut (arrows, Figure 4H). In
allowed for a completely regioselective pyridine formation contrast, in heterotaxin-treated embryos, the digestive epithe-
(Bols and Skrydstrup, 1995), with the C-N bond being formed lium is still stratified and contains clumps of rounded cells that
with the substituted sp-carbon center bearing the CH2OTrt have failed to rearrange into a single layer (arrows, Figures 4I
group. The silicon tether was then removed using TBAF in reflux- and 4J). These dysmorphic cells block the gut lumen and exhibit
ing THF to afford the regioisomerically pure 2,4,6-substituted increased intercellular adhesion, as indicated by their intensified
pyridine 9 in 99% yield. The alcohol 9 was converted into the E-cadherin staining (arrows and inset, Figures 4I and 4J). This
aldehyde 12 in 90% yield using a Swern oxidation. Following unusual morphology suggests that the normal acquisition or
the oxidation, a Wittig reaction was employed to install the C4 maintenance of migratory cell properties in the embryonic endo-
chain at the 4-position affording the alkene 15 in a moderate yield derm is inhibited in these embryos, preventing the cell rearrange-
of 78%. Reduction of the double bond in 15 with Pd/C under 1 ments required for normal gut elongation.
atm of H2 furnished the pyridine 21 in almost quantitative yield.
The acid-catalyzed deprotection of the trityl group proceeded Identifying the Phenocritical Period
smoothly and delivered heterotaxin (1) in 100% yield. This sche- for the Effects of Heterotaxin
matic route allowed us to synthesize reasonable (37 mg) quanti- To determine the phenocritical period in which heterotaxin elicits
ties of heterotaxin, and analogs thereof (see below), which we each of the above phenotypes, we exposed embryos to hetero-
used to further investigate the mechanism by which it induces taxin for limited times (Figure 4K). The expression of left-right
heterotaxia, and to elucidate its mechanism of action.
genes in the Xenopus embryo peaks between stages 19 and
26 (Ohi and Wright, 2007). To determine whether heterotaxin
directly affects left-right gene expression or function, we
Multiple Phenotypes Are Induced by Heterotaxin
Heterotaxin perturbs organ laterality, suggesting that it interferes exposed embryos to heterotaxin at successively later stages of
with global embryonic left-right patterning. Indeed, heterotaxin development beginning at stage 12 (gastrula), 18 (neurula), 26
perturbs left-right asymmetric gene expression patterns in the (late neurula), or 32 (tailbud). The compound can elicit robust
lateral plate mesoderm (LPM). For example, in heterotaxin- heterotaxic organ phenotypes when applied as late as stage 18,
treated embryos, the pattern of the Xenopus nodal homolog, but will induce heterotaxia only at low frequency at stage 26, and
Xnr-1, is randomized with respect to left and right (Figures 3A– has no effect on organ asymmetry when applied at stage 32 (Fig-
3D). Although some embryos exhibit normal left-side nodal ure 4K). We then exposed embryos to heterotaxin through stage
expression (36%, n = 28), some show bilateral nodal expression 26, washed away the compound with multiple rinses in fresh
on both the left and right sides (18%, n = 28), some exhibit media, and cultured in the absence of heterotaxin through
reversed (i.e., right-sided) nodal expression (11%, n = 28), and organogenesis. We found that even this limited exposure can
some (36%, n = 28) completely lack nodal expression on either disrupt organ asymmetries (Figure 4K), suggesting that hetero-
side (Figure 3I). As expected, the expression patterns of the taxin affects left-right asymmetry between stages 18 and 26,
Xenopus homolog of Pitx2, a transcription factor whose expres- coinciding with the peak of left-right gene expression.
sion is dependent on nodal activity, is similarly randomized by
We used similar exposures to determine the period in which
exposure to heterotaxin (Figures 3E–3H and 3J). These results heterotaxin induces other phenotypes. The melanogenesis
confirm that the organ anomalies induced by heterotaxin repre- phenotype can be elicited even when heterotaxin is applied as
sent bona fide left-right asymmetry defects.
late as stage 32 (Figure 4K), suggesting that the compound
To gain further information about heterotaxin’s mechanism of acts directly on developing melanocyte precursors, which
action, we carefully assessed heterotaxin-treated embryos migrate and differentiate between stage 30 and 40 (Collazo
throughout development (Figure 4). Compared to DMSO-treated et al., 1993). Likewise, the effect on gut elongation can also be
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
A
B
I
C
D
E
F
J
G
H
Figure 3. Heterotaxin Perturbs Left-Right Asymmetric Gene Expression Patterns
(A–H) In situ hybridization for nodal (Xnr-1; A–D), and Pitx2 (XPitx2; E–H) genes was performed on DMSO- (A, B, E, and F) and heterotaxin-treated (C, D, G, and H)
embryos. The expression pattern (arrows) is shown for both the left (A, C, E, and G) and right (B, D, F, and H) sides of the embryo (A–D, stage 23; E–H, stage 26).
The frequency of embryos exhibiting left only (Left, black), right only (Right, red), bilateral (gray), or absent (None, white) expression of nodal (I) or Pitx2 (J) is
quantified in the bar graphs.
elicited by exposure to the compound as late as stage 32 Structure-Activity Relationship Studies of Heterotaxin
(Figure 4K), just prior to when migratory properties are acquired To further explore heterotaxin’s multifunctionality, we conducted
by endoderm cells in the embryonic gut (Reed et al., 2009), SAR studies with heterotaxin analogs. Our regioselective route
indicating that heterotaxin exerts a direct effect on these cells. to heterotaxin (Figure 2C) was purposely designed in a flexible
In contrast the frequency of heterotaxin-induced vasculo- fashion to enable the introduction of other substituents on the
genesis/angiogenesis defects declines at stage 32 (Figure 4K). pyridine ring through the selective replacement of the butyl and
Because the genes that regulate the formation of the vitelline the ethyl side chain with additional functional groups. This
veins are expressed between stage 18 and 28 (Walmsley et al., enabled the assembly of a small set of analogs from a common
2002), and the vascular vitelline network is already forming at intermediate. The R1 and R2 groups were selected as methyl,
stage 30 (Inui and Asashima, 2006), the observed window of ethyl, propyl, phenyl, and hydroxymethylene, based on the orig-
susceptibility to heterotaxin is consistent with the timing of inal side chains found in heterotaxin and in order to probe the
neovascularization. Overall, these results suggest that multiple size of the putative cellular protein-binding pocket. Thus, we
independent phenotypes in heterologous tissues result from het- synthesized substantial quantities of the diyne 5 and used that
erotaxin acting directly and specifically on discrete populations to branch out to the synthesis of analogs. The key step was again
of embryonic cells at different stages of development.
a cobalt-catalyzed [2+2+2] cyclotrimerization reaction between
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
Figure 4. Heterotaxin Perturbs Melanogen-
esis, Angiogenesis, and Cell Rearrange-
ments
Compared to the round melanocytes seen in
DMSO controls (small arrows, A and C), hetero-
taxin-treated embryos (two different embryos are
shown in B and C) exhibit highly dendritic pigment
cells (large arrows, B and C).
(D) Number of abdominal melanocytes in embryos
exposed to either DMSO, the active pool identified
in the original pilot screen, or different concentra-
tions of purified heterotaxin.
(E–G) Heterotaxin induces mild (arrows, E and F) to
severe (arrow, G) defects in vasculogenesis
(compare the three different examples in E–G to
DMSO control in A).
(H–J) Immunohistochemical staining for E-cad-
herin (green), laminin (red), and nuclei (blue;
DAPI) on frontal sections (1003) through the gut
tube of embryos treated with DMSO (H, control),
100 mM heterotaxin (I), or 200 mM heterotaxin (J).
A single-layer columnar epithelium lines the diges-
tive tract of the DMSO tadpole (arrows, H; inset at
4003), whereas the cells lining the heterotaxin-
treated gut are round, exhibit high levels of
E-cadherin (green), and have failed to rearrange
into a single layer (arrows, I [inset at 4003], J).
(K) Time course studies reveal the sensitivity of
each phenotype to heterotaxin exposure. Left-
right asymmetry, reversal of heart looping, foregut
looping, and/or intestinal rotation; Vasculo-
genesis/Angiogenesis, hemorrhaging or enlarged
blood vessels; Melanogenesis, decreased number
and increased dendricity of melanocytes; Migra-
tion, perturbed cell rearrangement, indicated by
shortening of the primitive gut tube. ‘‘+’’ indicates
that at least 75% of embryos exhibited the pheno-
type in two or more independent trials; ‘‘+/À‘‘ indi-
cates that at least 50% of embryos exhibited the
phenotype in two or more independent trials.
See Figure S1 for a similar phenotypic profile
induced by a known TGF-b inhibitor, SB-505124.
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In Vivo Profiling Identifies TGFb Inhibitors
Figure 5. Synthesis of Additional Hetero-
taxin Analogs from Common Precursors
See Table S1 for SAR analyses of heterotaxin ana-
logs and Supplemental Experimental Procedures.
did not have a major effect on activity,
benzylation (35) produced a very active
compound (EC50 value = 10 mM) (Table
S1). Interestingly, for each analog, the
EC50 for inducing defects in asymmetric
organogenesis was identical to the EC50
for perturbing melanogenesis, vasculo-
genesis/angiogenesis, and/or gut elon-
the diyne 5 and a variety of different nitriles (propionitrile, valero- gation, although the severity of each phenotype varied between
nitrile, and benzonitrile), delivering the fused, regioisomerically analogs (Table S1). These results show that replacing individual
pure pyridines 6–8 (R1 = ethyl, butyl, phenyl) in 82%–98% yield. substituents does not isolate the various activities of the
The silicon tether was then removed using TBAF to afford compound, suggesting that the individual phenotypes induced
the 2,4,6-substituted pyridines 9–11 in 86%–99% yield. The by heterotaxin are not chemically separable and may result
alcohols 9–11 were converted into the aldehydes 12–14 in from perturbation of the same biological target.
84%–90% yield using the previously employed Swern oxidation,
followed by a Wittig reaction with several different alkylphospho- Identifying the Cellular Target of Heterotaxin Analogs
nium bromides to install different chain lengths at the four posi- Our phenocritical timing studies suggest that heterotaxin
tion in 15–20 (R2 = methyl, propyl, butyl, pentyl). Reduction of the perturbs left-right asymmetry during the stages when asymmet-
double bond in 15–20 with Pd/C under 1 atm of H2 furnished the rically expressed TGF-b ligands, such as nodal, establish organ
pyridines 21–26 in almost quantitative yields. The acid-catalyzed laterality. Therefore, we hypothesized that TGF-b signaling is
deprotection of the trityl group proceeded smoothly and deliv- inhibited by heterotaxin. Because the above SAR studies indi-
ered the heterotaxin analogs 27–31 in 71%–100% yield.
cate that the various phenotypes induced by heterotaxin are
Two additional analogs were synthesized by deprotection of not chemically separable, it is possible that the complete pheno-
the trityl group at different stages of the synthesis (Figure 5). typic profile of this compound is attributable to inhibited TGF-b
One deprotection was conducted after removal of the silicon signaling. This hypothesis is strongly supported by the fact that
tether from 9 to afford the diol 32 in 66% yield, and the second exposure to a known small molecule TGF-b signaling inhibitor,
was performed on the alkene 16 to obtain the compound 33 in SB505124 (DaCosta Byfield et al., 2004), induced the same
63% yield. Installation of different hydrocarbon substituents on phenotypic profile as our compounds, including heterotaxia,
the hydroxyl group of heterotaxin (1) was accomplished by sub- vasculogenesis and melanogenesis defects, and aberrant
jecting 1 to deprotonation with NaH, followed by the addition of migratory cell properties, within the same phenocritical periods
the appropriate alkyl halide (methyl, benzyl, and hexyl) to afford (Figure S1). Importantly, other signaling pathways that also influ-
the ethers 34–36 in 42%–86% yield. Finally, the hydroxyl group ence all four of these developmental processes are unaffected
of heterotaxin (1) was oxidized to the carboxylic acid 38 by by heterotaxin (Figure S1).
a two-step oxidation process. First, the aldehyde 37 was formed
To test the hypothesis that heterotaxin interferes with TGF-b
in moderate yield by oxidation of the alcohol 1 with MnO2, fol- signaling, we evaluated the expression of Xantivin, the Xenopus
lowed by a Lindgren oxidation (Stangeland and Sammakia, homolog of lefty, which is normally expressed in the left LPM as
2004; Lindgren and Nilsson, 1973) to form the carboxylic acid a direct consequence of nodal-type TGF-b signaling (Ho et al.,
38 in 89% yield (Figure 5).
2006). Although DMSO-treated control embryos exhibit normal
The length of the alkyl chain at the CH2R2 substituent expression of this target gene in the left LPM, Xantivin could
was found to be critical for the specific activity of this class of not be detected in the left or right LPM of heterotaxin-treated
molecules, with the highest activity being observed for butyl embryos (Figure 6A; Figure S2), strongly suggesting that
(heterotaxin, 1) and pentyl (28), whereas ethyl (27), ethylene nodal-type TGF-b signaling is inhibited by heterotaxin. These
(33), hydroxymethylene (32), and hexyl (29) were inactive (or data are consistent with previous reports in which embryos
toxic) (see Table S1 available online). Although the size of the exposed to a known TGF-b signaling inhibitor failed to express
R1 group does not appear to be critical, as ethyl (heterotaxin, 1), Xantivin (Ho et al., 2006).
butyl (31), and phenyl (30) are tolerated, both the butyl (31) and
TGF-b receptor activation is conveyed by the phosphorylation
phenyl (30) substitutions did yield more potent analogs, which of intracellular mediators, known as Smads, which ultimately
exhibited activity at lower concentrations than the original effect transcription (ten Dijke and Hill, 2004). Nodal signaling
heterotaxin molecule (EC50 values = 10 and 50 mM, respectively) occurs primarily via phosphorylation of Smad2; thus, the
(Table S1). Furthermore, although modifications of the CH2OH level of phosphorylated Smad2 in Xenopus extracts may be
group through methylation (34), oxidation (38), or alkylation (36) used as an indicator of embryonic nodal-type TGF-b signaling
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Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
Figure 6. Heterotaxin Inhibits TGF-b
Signaling
(A) The frequency of embryos exhibiting left only
(Left, black), right only (Right, red), bilateral
(gray), or absent (None, white) expression of
Xantivin is quantified in the bar graphs. (See Fig-
ure S2 for images of in situ hybridization).
(B) Western blotting of embryos exposed from 10
hpf for 24 hr to DMSO, 200 mM heterotaxin (1),
200 mM phenotypically inactive analog (32),
80 mM active analog (35), and a known nodal
signaling inhibitor (SB-505124; 50 mM). Although
unmodified Smad2 is detected under all condi-
tions, phosphorylated Smad2 (pSmad2) is only
detected in the presence of DMSO or the inactive
heterotaxin analog 32 (albeit at reduced levels).
ERK is total protein control.
(C) Western blotting of embryos exposed to
DMSO, 1, 32, 35 and the BMP signaling inhibitor,
dorsomorphin (DM; 50 mM). BMP-specific
Smad1/4/5 levels are unaffected by heterotaxin
analogs.
(D) Activin-induced elongation in Xenopus animal
cap explants is inhibited by active heterotaxin
analogs 1 and 35, but not DMSO or analog 32.
The box plot shows that activin-induced length-
ening (results pooled from two identical trials) is
inhibited significantly only by compounds 1 and
35, as calculated by one-way ANOVA (*p < 0.05).
Error bars indicate the first through third interquar-
tile range of the measured lengths.
(E) Western blotting of A549 cells exposed to
10 ng/ml TGF-b1 and DMSO, 100–200 mM 1, 32,
35, or 10 mM SB-431542 for 48 hr. TGF-b1-
induced upregulation of Vimentin and Snail is
inhibited by analogs 1 and 35, but not DMSO or
analog 32. GAPDH is total protein control.
(F) Western blotting of A549 cells exposed to
10 ng/ml TGF-b1 and DMSO, 100–200 mM 1, 32,
35, or 10 mM SB-431542 for 1 hr. TGF-b1-induced
phosphorylation of Smad2 is unaffected by
heterotaxin analogs. GAPDH controls for total
protein.
See Figure S2 for effect of 1 on non-Smad-depen-
dent TGF-b signaling via PI3K/Akt.
(Ho et al., 2006). As expected, the level of phosphorylated otherwise remain naive to TGF-b signals and fail to elongate at
Smad2 is unaffected by exposure to DMSO (Figure 6B, lane 1). all (Green, 1999). We employed this assay to quantify the degree
However, Smad2 phosphorylation is abolished in embryos to which heterotaxin analogs interfere with TGF-b-ligand-depen-
exposed to heterotaxin (1) (Figure 6B, lane 2), or to the more dent signaling. In contrast to DMSO or the inactive analog 32,
potent heterotaxin analog 35 (Figure 6B, lane 4), but is only mildly heterotaxin (1) and the potent analog 35 significantly inhibit acti-
downregulated by exposure to the phenotypically inactive vin-induced animal cap elongation (Figure 6D); thus, heterotaxin
heterotaxin analog 32 (Figure 6B, lane 3). The inhibition of analogs inhibit activin-dependent activity in Xenopus.
Smad2 phosphorylation by heterotaxin is comparable to that
To determine if heterotaxin analogs inhibit the activity of other
induced by SB-505124 (Figure 6B, lane 5). Importantly, the level TGF-b ligands, we assessed their activity in human cell culture.
of phosphorylated Smad1/5/8, which indicates signaling via A549 cells undergo an epithelial-mesenchymal transition in
other TGF-b superfamily ligands such as BMP, remains unaf- response to TGF-b1 (Kim et al., 2007), as indicated by the upre-
fected by heterotaxin analogs (Figure 6C); thus, the effect of gulation of mesenchymal markers such as Snail and Vimentin
heterotaxin is specific for Smad2-dependent TGF-b signaling.
(Figure 6E, compare lanes 1–2). Heterotaxin (1) and the potent
A unique advantage of amphibian embryos is the ability to analog 35 inhibit the upregulation of these markers (Figure 6E,
culture specific embryonic tissues ex vivo in order to isolate lanes 4–7), whereas DMSO or the inactive analog 32 has no
the effects of exogenous growth factors on cell behavior. It is effect (Figure 6E, lanes 3 and 8–9); thus, heterotaxin analogs
well established that the addition of the Smad2-mediated inhibit TGF-b1-dependent activity in human cells.
TGF-b ligand activin to Xenopus ‘‘animal cap’’ explants can elicit To determine if heterotaxin compounds directly affect ligand-
concentration-dependent elongation in a tissue that would dependent Smad2 phosphorylation, we assessed levels of
Chemistry & Biology 18, 252–263, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 259

Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
Figure 7. Heterotaxin Inhibits Angiogenesis
in Human Cells
HUVECs form tubes when cultured in the pres-
ence of media alone (A), DMSO (B), unsubstituted
pyridine (C), or the inactive heterotaxin analog 32
(J). In contrast, HUVECs cultured in the presence
of a known anti-angiogenic agent, sulforaphane
(D) (Bertl et al., 2006), are unable to form tubes.
Although the original heterotaxin molecule 1 (F)
exhibits only very mild anti-angiogenic effects in
this assay (at 6 hr), the other active heterotaxin
analogs 36 (G), 35 (H), and 30 (I) have obvious
concentration-dependent anti-angiogenic proper-
ties comparable to
a known TGF-b signaling
inhibitor (E, SB-505124). Cells were stained with
Calcein AM to confirm viability.
See Figure S3 for effect of analog 30 on tumor cell
growth.
pressed by our compound (data not
shown), TGF-b1-induced activation of
PI3K (1 hr induction), as indicated by the
levels of phosphorylated Akt (Ser-473)
(Figure S2E, lanes 1–2), was inhibited by
heterotaxin (1) (Figure S2E, lanes 4–5),
whereas DMSO and the inactive analog
32 had no effect (Figure S2E, lanes 3
and 6). Taken together, these results
indicate that 2,4,6-substituted pyridines
function as both indirect (Smad2 depen-
dent) and direct (non-Smad dependent)
TGF-b signaling inhibitors.
Heterotaxin Analogs Exhibit
Anti-Angiogenic Properties
in Mammalian Cells
In addition to development, the TGF-b
pathway also plays a multiphasic role in
tumor progression. Although early in
tumorigenesis TGF-b is tumor suppres-
sive (Jakowlew, 2006), later tumor
cells are resistant to TGF-b-mediated
growth inhibition, and upregulation of
TGF-b facilitates metastatic invasion,
phosphorylated Smad2 in these cells with a 1-hr TGF-b1 promoting cell migration and epithelial-to-mesenchymal transi-
induction (Figure 6F). Compared to the effect of SB431542, tion (Yingling et al., 2004; Tian et al., 2003), as well as new blood
a known TGF-b type I receptor inhibitor (Inman et al., 2002), vessel growth and angiogenesis (ten Dijke and Arthur, 2007),
TGF-b1-induced Smad2 phosphorylation remained relatively crucial requirements for tumor growth and metastasis.
unaffected by our compounds in this time frame, suggesting
Therefore, TGF-b signaling inhibitors, like heterotaxin and its
that the effect of heterotaxin on Smad2 phosphorylation in vivo analogs, may be useful for blocking the tumor-promoting effects
may not involve direct inhibition of TGF-b receptors or may of TGF-b. Because our compounds inhibit vascular development
inhibit a non-Smad-dependent TGF-b signaling pathway (Zhang, in vivo, we assessed their anti-angiogenic potential in a mamma-
2009).
lian system (Figure 7). The human umbilical vein endothelial cell
We tested the latter possibility by assessing the effect of (HUVEC) assay provides a visual readout of the ability of exoge-
heterotaxin on TGF-b1-induced activation of phosphatidylinosi- nous factors to inhibit the formation of microcapillary tubes (e.g.,
tol 3-kinase (PI3K), as well as mitogen-activated protein kinases Figure 7D). Compared to solvent or pyridine controls (Figures 7B
(MAPKs), including p38, c-Jun amino-terminal kinase (JNK), and 7C), the heterotaxin analogs that inhibited vascular develop-
and extracellular signal-regulated kinase (ERK). Although the ment in Xenopus (e.g., 1, 30, 35, 36) (see Table S1) were also able
activation of most of these non-Smad pathways was not sup- to inhibit tube formation (Figures 7F–7I) in HUVEC cultures.
260 Chemistry & Biology 18, 252–263, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
The effects were comparable to those elicited by a known TGF-b tory cell morphology and behavior, and concomitantly increases
receptor inhibitor (Figure 7E). However, whereas TGF-b receptor E-cadherin levels, as might be predicted for an inhibitor of TGF-b
inhibitors can also block the growth-inhibitory effects of TGF-b signaling. The effect of heterotaxin on gut morphogenesis
(e.g., SB-431542; Halder et al. [2005]), promoting tumorigenesis, provides a novel inroad for investigating the role of TGF-b
heterotaxin analogs appear not to have this limitation. In fact, signaling in the poorly understood processes of gut elongation
compound 30 not only inhibits angiogenesis (Figure 7I) but also and rotation.
significantly inhibits growth in several mammalian tumor cell
lines (Figure S3). Thus, 2,4,6-substituted pyridine analogs may The Cellular Target of Heterotaxin
be broadly applicable in the development of anti-angiogenic Heterotaxin compounds disrupt Smad2 phosphorylation in vivo,
antitumor compounds in mammalian systems.
although this is not a direct effect. Possible mechanisms of action
of heterotaxin and its analogs include inhibiting the synthesis,
secretion, or processing of TGF-b receptors or ligands. Alterna-
tively, these compounds could be influencing non-Smad-depen-
DISCUSSION
A multi-phenotype-based whole organism screen of small mole- dent pathways downstream of TGF-b receptors. Indeed, we
cules in X. laevis embryos identified a novel class of pyridines found that heterotaxin directly inhibits TGF-b-induced PI3K
with TGF-b inhibitory activity. Our data have implications for activity. Although activation of PI3K by TGF-b requires the activity
understanding the role of the TGF-b pathway in the development of TGF-b receptors, the molecular interactions underlying the
of left-right asymmetry, gut morphogenesis, melanogenesis, and activation of non-Smad-dependent TGF-b signaling events are
vascular development, and for the employment of heterotaxin extremely complex and context dependent (Zhang, 2009). There-
analogs in the development of TGF-b inhibitory lead compounds fore, further investigations of the role of PI3K-mediated TGF-b
with therapeutic potential.
signaling during Xenopus development will be required before
the cellular target(s) of 2,4,6-substituted pyridines can be fully
resolved. Nonetheless, because non-Smad-dependent TGF-b
pathways are frequently involved in activating the pro-oncogenic
effects of TGF-b signaling during tumor progression, e.g., PI3K-
Heterotaxin and the Role of TGF-b in Left-Right
Asymmetry, Melanogenesis, Vasculogenesis,
and Gut Development
Our results validate our phenotypic screen for heterotaxia Akt signaling is required for Smad-dependent transcriptional
because TGF-b ligands are well known to play an evolutionarily responses as well as tumor cell migration (Bakin et al., 2000),
conserved role in the development of left-right asymmetry (Whit- our results raise the exciting possibility that heterotaxin
man and Mercola, 2001). Moreover, in addition to left-right compounds might be able to selectively target TGF-b-dependent
patterning, TGF-b signaling has also been implicated in the other tumor-promoting outcomes without also blocking the tumor-
biological processes disrupted by heterotaxin. For example, suppressive effects of TGF-b. This property could be important
TGF-b signaling is required for the assembly of the embryonic for selectively controlling distinct TGF-b responses in different
vasculature (ten Dijke and Arthur, 2007), the establishment of therapeutic contexts.
vessel wall integrity (Pepper, 1997), and the regulation of
vascular homeostasis (Bertolino et al., 2005). As might be pre- Heterotaxin Analogs as Therapeutic Agents
dicted to occur in the presence of a TGF-b inhibitor, heterotaxin Because of their important roles in tumorigenesis, TGF-b
dramatically impairs angiogenesis both in vivo and in vitro. pathway components are excellent chemotherapeutic targets,
Because in vitro tube formation may also be influenced by other although compounds that can appropriately modulate this
factors, confirmation of a direct effect of heterotaxin on human multifunctional pathway in vivo are still in development. We
angiogenesis must await further studies in mammalian models. discovered compounds that specifically inhibit nodal-dependent
Nonetheless, the similarity of the anti-angiogenic activity profiles gene expression and multiple TGF-b-dependent biological
of heterotaxin analogs in both frog embryos and human cells processes in a whole vertebrate embryo, including neovascula-
suggests that these compounds could have broader rization and migratory behavior. In addition, heterotaxin analogs
applicability.
inhibit TGF-b-induced epithelial-mesenchymal transition and
In addition, TGF-b signaling normally increases melanocyte angiogenesis in human cells, while inhibiting the growth of
precursor proliferation (Kawakami et al., 2002) but inhibits multiple mammalian tumor cell lines. Thus, heterotaxin analogs
melanogenic differentiation (Kim et al., 2004). Consistent with exhibit highly desirable biological activity and may be valuable
the expected outcome of inhibiting TGF-b signaling, heterotaxin in the development of TGF-b-inhibitory chemotherapeutics for
exposure during melanocyte precursor migration and differenti- blocking tumor proliferation and/or metastasis.
ation results in decreased melanocyte number but increased
dendricity. Because nodal is expressed in aggressive mela- SIGNIFICANCE
nomas, which reacquire melanocyte precursor-like properties,
heterotaxin analogs may be promising in the development of In the developing embryo, a myriad of cellular processes
differentiation-based anti-melanoma therapies (Hendrix et al., form organs in a dynamic and complex three-dimensional
2007).
milieu. In disease states these same processes occur
Finally, in multiple contexts, TGF-b signaling induces cell inappropriately in equally complicated adult environments.
motility and decreased E-cadherin-mediated intercellular adhe- Identifying small molecules that can predictably modulate
sion in cells undergoing epithelial-to-mesenchymal transitions cellular processes in their multifarious biological contexts
(Xu et al., 2009). In the developing gut, heterotaxin inhibits migra- is imperative for the discovery of effective drugs and stem
Chemistry & Biology 18, 252–263, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 261

Chemistry & Biology
In Vivo Profiling Identifies TGFb Inhibitors
used according to manufacturer’s protocol. Subconfluent HUVECs at passage
5 (ꢀ80% confluent) were incubated with 2 mM Calcein AM for 30 min at 37ꢁC to
allow for fluorescent monitoring of cell viability and tube formation. Cells were
treated with DMSO, sulforaphane, or heterotaxin analogs/SB-505124 at the
time of seeding. Tube formation was assessed 6 hr after treatment.
cell therapies. However, many lead compounds are initially
identified in target-based biochemical or simplified cell-
based assays because such assays are amenable to high-
throughput screening; consequently, the in vivo effects of
such compounds are often unpredictable. Although multi-
plexed profiling can provide important information about
potential toxicity and mechanism of action, such knowledge
is not necessarily predictive of efficacy in vivo. Moreover,
even when a compound has been identified in a whole
organism phenotype-based screen, there is as yet, to our
knowledge, no reliable or systematic way to determine its
cellular target(s).
Western Blotting
St 10 embryos were exposed to DMSO, heterotaxin analogs, SB-505124,
or dorsomorphin for 24 hr. Ten embryos from each treatment were pelleted,
resuspended in 100 ml lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
b-glycerophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin), and lysed
by mechanical disruption, followed by freeze-thaw cycles. Approximately
20 mg of each cleared lysate was run on a 10% NuPAGE Bis-Tris gel and
then transferred to a PVDF membrane. Membranes were blocked for 1 hr in
TBST (20 mM Tris [pH 7.6], 136 mM NaCl, 0.1% Tween 20) + 5% nonfat dry
milk, and incubated overnight at 4ꢁC with primary antibodies (see below).
Membranes were washed four times in TBST, incubated for 2 hr in TBST+
5% nonfat dry milk and HRP-conjugated donkey anti-rabbit IgG (Upstate),
washed three to four times in TBST, then visualized using chemiluminescence
with SuperSignal West Pico (Pierce).
We have shown that a whole organism multi-phenotype
profiling strategy can be used to both identify compounds
capable of modulating important biological processes
in vivo and to infer mechanism of action. Using a combina-
tion of independent tissue-level developmental phenotypes,
immunohistochemical analyses, gene expression patterns,
tissue culture, and biochemical assays, we discovered
a class of TGF-b signaling inhibitors. Remarkably, these
compounds also elicit TGF-b-dependent phenotypes in
human cells that mirror their activity profiles in vivo, sug-
gesting that they may be valuable in the development of
therapeutic agents to inhibit pathologic conditions medi-
ated by excess TGF-b signaling (e.g., metastasis, fibrosis).
Our discovery suggests that multi-phenotype profiling in
whole organisms is a powerful strategy for identifying the
pathway-level mechanism of action of small molecules.
A549 cells (ATCC) were maintained in DMEM with antibiotics and 10% FBS.
Cells were starved overnight in DMEM with 1% FBS and incubated with
DMSO, heterotaxin analogs, or SB-431542 in DMEM with 10% FBS for
30 min before treatment with rhTGFb1 (R&D Systems) for 1–48 hr. Whole-
cell lysates were prepared in RIPA lysis buffer (Pierce) with complete Mini
EDTA Protease Inhibitor and Phos-STOP (Roche) and sonication. Approxi-
mately 40 mg of each cleared lysate was run on a 4%–12% NuPAGE Bis-Tris
gel and then transferred to a nitrocellulose membrane before blocking and
antibody staining as above. Autoradiography bands were scanned and quan-
titated with ImageJ freeware. The integrated optical density of each band was
normalized to GAPDH or b-actin and the fold change determined by dividing
each normalized value by the lowest normalized sample value.
Primary antibodies used were Smad2, phospho-Smad2, phospho-
Smad1/5/8, phospho-p38, Erk, phospho-Erk, phospho-SAPK/JNK, phos-
pho-Akt (Ser473), and Snail from Cell Signaling Technology, and Vimentin,
b-actin, and GAPDH from Santa Cruz Biotechnology.
EXPERIMENTAL PROCEDURES
Embryo Screening and Animal Caps
Experiments involving live animals were performed in accordance with
national regulations, and approved by the NC State University Institutional
Animal Care and Use Committee. X. laevis embryos were obtained by
in vitro fertilization, de-jellied with 2% cysteine-HCl (pH 7.8–8.1), sorted to
eliminate anomalous individuals, and cultured in 0.13 MMR at 15ꢁC–22ꢁC
(Sive et al., 1998). Staging was according to Nieuwkoop and Faber (1994).
Stock solutions were prepared in DMSO (20 mM). For the screen, approxi-
mately 130 compounds were diluted to 200 mM in 2 ml 0.13 MMR in a 24-well
plate; 1% DMSO was used as a solvent control. Four embryos were exposed
in each well starting at ꢀ10 hr after fertilization. Organs were evaluated in anes-
thetized tadpoles (0.05% MS-222) when controls reached stage 44–46.
Animal caps were dissected (Green, 1999) and cultured in 5 ng/ml human
activin A (R&D Systems), or activin plus DMSO, 200 mM 1, 32, or 35 for 2 hr.
Caps were then cultured 8 hr in 0.753 MMR + gentamycin (Sive et al.,
1998). Final explant lengths were calculated using Photoshop CS2 (Measure
tool). The significance of decreased elongation was determined by one-way
ANOVA between groups.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and one table and can be found with this article online at
doi:10.1016/j.chembiol.2010.12.008.
ACKNOWLEDGMENTS
We thank Dr. K. Symes for experimental advice and Brooke Griff for technical
assistance. This work was supported by NSF (IOB 0642012; NNY), NIH
(RO1 DK085300-01A1; NNY), and NCSU CCMTR (NNY, AD, MLH). A.D. is
a recipient of an NSF CAREER Award (CHE 0846756).
Received: January 20, 2010
Revised: December 3, 2010
Accepted: December 5, 2010
Published: February 24, 2011
In Situ Hybridization and Immunohistochemistry
Embryoswerefixedatst23/26inMEMFA(Siveetal., 1998). Digoxigenin-labeled
riboprobes for Xnr-1, Xantivin (gift of C. Wright), and XPitx2c were synthesized
from linearized plasmids (Muller et al., 2003). In situ hybridization was as
described (Lipscomb et al., 2006). St 44 embryos were fixed for immunohisto-
chemistry and processed for cryosectioning (Fagotto and Gumbiner, 1994).
Staining was performed using anti-E-cadherin (DSHB; 5D3 at 1:200) and anti-
laminin (Sigma; L9393 at 1:200) primary antibodies and Alexa-conjugated
secondary antibodies (Invitrogen; 1:2000), as described (Reed et al., 2009).
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