In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase

Article abstract of DOI:10.1002/cbic.201402000

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in

Full text of DOI:10.1002/cbic.201402000

CHEMBIOCHEM
FULL PAPERS
DOI: 10.1002/cbic.201402000
In Vivo Structure–Activity Relationship Studies Support
Allosteric Targeting of a Dual Specificity Phosphatase
Vasiliy N. Korotchenko,[a, f] Manush Saydmohammed,[b] Laura L. Vollmer,[c] Ahmet Bakan,[d]
Kyle Sheetz,[b] Karl T. Debiec,[e] Kristina A. Greene,[e] Christine S. Agliori,[e] Ivet Bahar,[d]
Billy W. Day,[a, c, e] Andreas Vogt,*[c, d] and Michael Tsang*[b]
Dual specificity phosphatase 6 (DUSP6) functions as a feedback
attenuator of fibroblast growth factor signaling during devel-
opment. In vitro high throughput chemical screening attempts
to discover DUSP6 inhibitors have yielded limited success.
However, in vivo whole-organism screens of zebrafish identi-
fied compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here
we designed and synthesized a panel of analogues to define
the structure–activity relationship (SAR) of DUSP6 inhibition. In
vivo high-content analysis in transgenic zebrafish, coupled
with cell-based chemical complementation assays, identified
structural features of the pharmacophore of 1 that were essen-
tial for biological activity. In vitro assays of DUSP hyperactiva-
tion corroborated the results from in vivo and cellular SAR. The
results reinforce the notion that DUSPs are druggable through
allosteric mechanisms and illustrate the utility of zebrafish as
a model organism for in vivo SAR analyses.
Introduction
Dual specificity phosphatases (DUSPs) represent a family of en-
zymes that catalyze the dephosphorylation of proteins on
both phosphotyrosine and phosphoserine/phosphothreonine
residues within the same substrate. DUSP6, also known as mi-
togen-activated protein kinase phosphatase 3 (MKP3), belongs
to a subgroup of eleven dual specificity phosphatases that de-
phosphorylate and inactivate mitogen-activated protein kinas-
es (MAPKs).[1–3] During development, DUSP6 functions as a neg-
ative feedback regulator of fibroblast growth factor (FGF) sig-
naling.[4–6] The discovery of potent and selective inhibitors of
dual specificity phosphatases has been hindered by a high
degree of conservation between the DUSP active sites and
their shallow and feature-poor topology.[1] In addition, the
presence of a reactive, active site cysteine, which is critical for
enzymatic activity but displays high nucleophilicity and sensi-
tivity to oxidation, due to a low pKa sulfhydryl moiety, has
hampered drug discovery efforts.[7] Perhaps not too surprising-
ly, in vitro screens for DUSP inhibitors have yielded hit com-
pounds that were redox reactive,[8] lacked in vivo activity,[9] or
had activities not readily reconciled with DUSP inhibition.[10]
The advent of a whole organism live reporter for FGF activity
(Tg(dusp6:EGFP)pt6 [11,12]
enabled the discovery of a biologically
)
active inhibitor of zebrafish DUSP6, (E)-2-benzylidene-3-(cyclo-
hexylamino)-2,3-dihydro-1H-inden-1-one (BCI), designated here
as 1.[13] Chemical complementation assays revealed that 1 spe-
cifically inhibited DUSP6 and DUSP1, but not the related
DUSP5.[13] Interestingly, 1 lacked antiphosphatase activity in
a traditional biochemical assay with bacterially produced re-
combinant protein and therefore was missed in prior in vitro
screens for DUSP inhibitors.[14] Instead, 1 selectively inhibited
DUSP activity in the presence of ERK, which activates DUSP6
through a conformational change that brings a general acid
residue within close proximity to the active site cysteine, en-
hancing its nucleophilicity.[15] The zebrafish system therefore
captured the inhibitory activity of 1 against the biologically rel-
evant phosphatase activity of DUSPs and provided a useful
chemical probe to study the role of DUSP6 in embryonic devel-
opment and adult immunity.[13,16–21]
[a] V. N. Korotchenko,+ B. W. Day
Department of Pharmaceutical Sciences, University of Pittsburgh
3501 Fifth Avenue, Pittsburgh, PA 15213 (USA)
[b] M. Saydmohammed,+ K. Sheetz, M. Tsang
Department of Developmental Biology, University of Pittsburgh
3501 Fifth Avenue, Pittsburgh, PA 15213 (USA)
E-mail: tsang@pitt.edu
[c] L. L. Vollmer, B. W. Day, A. Vogt
The University of Pittsburgh Drug Discovery Institute
University of Pittsburgh
3501 Fifth Avenue, Pittsburgh, PA 15213 (USA)
E-mail: avogt@pitt.edu
[d] A. Bakan, I. Bahar, A. Vogt
Department of Computational & Systems Biology, University of Pittsburgh
3501 Fifth Avenue, Pittsburgh, PA 15213 (USA)
[e] K. T. Debiec, K. A. Greene, C. S. Agliori, B. W. Day
Department of Chemistry, University of Pittsburgh
3501 Terrace Street, Pittsburgh, PA 15213 (USA)
[f] V. N. Korotchenko+
Present address: Walter Reed Army Institute of Research
503 Forney Drive, Silver Spring, MD 20910 (USA)
[+] These authors contributed equally to this work.
To explore structure–activity relationship (SAR) of 1 and
DUSP6 inhibition, we synthesized a series of 29 analogues with
modifications in four functional groups of the 1 pharmaco-
phore. SAR was evaluated for FGF hyperactivation in vivo by
using transgenic zebrafish that report on FGF activity[11] and for
DUSP6 and DUSP1 inhibition in cell-based chemical comple-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402000.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1436

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
mentation assays.[13] These SAR studies revealed a strong corre-
lation between in vivo FGF hyperactivation and inhibition of
cellular DUSPs and a lack of correlation between biological ac-
tivity and whole organism toxicity. Biochemical assays for sub-
strate-induced DUSP6 hyperactivation corroborated the inhibi-
tory activities of the new analogues.
and hydrogen acceptor properties of the a,b-unsaturated
ketone through electron-donating or electron-withdrawing
substituents in rings II and III (Table S1, compounds 12–20)
and to investigate whether combinations of multiple structural
modifications would reach maximal potency (Table S1, com-
pounds 21–30).
The selective inhibition of substrate-induced DUSP6 activa-
tion by 1 was reconciled by molecular modeling studies of the
1–DUSP6 interaction. Unbiased docking simulations supported
that 1 would bind to the low-activity form of DUSP6, occupy-
ing a novel allosteric binding site adjacent to the phosphatase
active site.[13] Refined scoring of potential docking modes by
using Poisson–Boltzmann surface area (PBSA) binding free-
energy calculations indicated a preferred binding orientation
for 1, in which its cyclohexylamino side chain and a,b-unsatu-
rated ketone moiety form hydrogen bonds with DUSP6. Collec-
tively, these results support the hypothesis that DUSPs can be
targeted through allosteric mechanisms.
A convenient and flexible route to 1 and its analogues is
shown in Scheme 1B. This route allowed synthesis of the
parent compound and a series of analogues with a modified
fragment I in Scheme 1A. The condensation of 5-substituted 1-
indanones with appropriate benzaldehydes afforded the corre-
sponding 2-benzylidene-1-indanones in quantitative yield. The
bromination of 2-benzylidene-1-indanones with N-bromosuc-
cinimde (NBS) provided 3-bromo-2-benzylidene-1-indanones.[22]
The final synthetic step was the reaction of the 3-bromo-2-
benzylidene-1-indanones with amines, as developed by Crom-
well.[23] Reaction of unsubstituted 3-bromo-2-benzylidene-1
indanone (1b, X, Y=H) with amines proceeded in two steps,
initially giving a 2-[a-(alkylamino)benzyl]-1-indenone, which re-
arranged to the more thermodynamically stable 2-benzylidene-
3-(alkylamino)-2,3-dihydro-1H-inden-1-one.[23]
Results
The amine in position I was modified to examine steric ef-
fects, hydrogen-donating properties, effects of relative basicity
of the amino group, and effects of introduction of additional
hydrogen donors and acceptors. 3-Bromo-2-benzylidene-1-in-
danone (1b) was converted to analogues 1–11 by treatment
with two equivalents of the corresponding primary or secon-
dary amines in benzene at room temperature (Tables S2 and
S3). The reaction provided the desired compounds in generally
Chemical synthesis of BCI analogues
Compound 1 has four distinct potential sites of modification
(amine I, aromatic rings II and III, and the carbonyl group,
Scheme 1A). We created a small library of 29 analogues, de-
signed to probe spatial and hydrogen bonding requirements
of the aminoalkyl (ring I) system (compounds 2–11, Table S1 in
the Supporting Information) to modulate the electrophilicity
Scheme 1. Design and generation of 1 and analogues. A) Sites of modification of the scaffold. B) Synthesis of 1 and its analogues. a) KOH, RT, or, AcOH, H2SO4
(cat), RT; b) NBS, (PhCO)2O, CCl4, reflux; c) RR’NH (2 equiv), C6H6, RT. C) Synthesis of aminoalcohol 31.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1437

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
high yield; one notable exception was the 4-pipera-
zine analogue (9, BCI-187), which was afforded in
42% yield when using 1.9 equivalents of piperazine.
Using two equivalents of piperazine resulted in for-
mation of a mixture of the desired compound and
isomerization products with migration of the pipera-
zine group. Maury et al. reported that 1 and ana-
logues are labile compounds that can undergo allylic
rearrangements with migration of the amino group
or prototropic isomerization of the double bond into
an endocyclic alkene.[23] The rate of this isomerization
is dependent on the nature of the amine, but forma-
tion of the desired product was complete in our
hands within 24 h; the one exception was the reac-
tion of 1b with aniline, which required 120 h and
provided compound 4 (BCI-11) in 69% yield.
This method was found to be suitable for the syn-
thesis of analogues containing additional substitu-
ents in rings II or III, as well as analogues containing
combinations of different amines in position I. Acid-
or base-catalyzed condensation of substituted ben-
zaldehydes or indanones in the first step provided
corresponding 2-arylideneindan-1-ones 12a–30a in
good yield.[24] Following bromination with NBS, treat-
ment of the resulting 3-bromo-2-arylideneindan-1-
ones 12b–30b with cyclohexylamine in benzene pro-
vided final products bearing substituents in rings II
(12–17), III (18–20), and II and III (21, 22). Com-
pounds 23–30 were synthesized as described above
using morpholine.
To probe the importance of the carbonyl group,
we also synthesized 2-benzylidene-3-cyclohexylami-
noindan-1-ol (31, BCI-10), with a trans relationship
between the amine and hydroxy groups (as docu-
mented by the absence of a nuclear Overhauser
effect (NOE) between H-1 and H-3) through the re-
duction of 1 with LiAlH4 (Scheme 1C). The moderate
yield of alcohol 31 was attributed to over-reduction
of both the carbonyl group and the carbon–carbon
double bond.
Structure–activity studies in zebrafish embryos
We first analyzed all analogues for hyperactivation of
FGF signaling in the Tg(dusp6:EGFP)pt6 transgenic ze-
brafish model. In this assay, we previously established
transgenic zebrafish that express destabilized GFP
under the control of active FGF signaling.[11,12] These
transgenic embryos respond to FGF activation with
GFP expression in specific areas of the brain, which
can be quantified by automated image analysis using
cognition network technology (CNT).[25] To determine
optimal conditions for FGF reporter activation, and to
test whether the response was saturable, we per-
Figure 1. Quantitation of in vivo FGF hyperactivation by automated image analysis.
A) Upper panel. Representative fluorescence micrographs of 24 hpf Tg(dusp6:EGFP)pt6 em-
bryos treated for 5 h with vehicle (1% DMSO) or 20 mm 1. The major bright head struc-
tures are eye and retina, mid-hindbrain boundary, and trigeminal ganglia. Lower panel.
Archived scan images with CNT algorithm applied. Areas in red are regions of GFP ex-
pression in the head that exceeded a threshold relative to yolk sac fluorescence. B) Time
course of FGF activation. After 24 hpf, Tg(dusp6:EGFP)pt6 embryos were exposed to vehi-
cle (DMSO) or 20 mm 1 in 96-well plates, imaged every hour for 6 h, and analyzed by the
CNT rule set. Data show total GFP intensity in the head from eight embryos per condi-
tion ÆSEM. C) Dose–response of FGF activation by 1 at 5 h after treatment. D) Chemical
structures of important BCI analogues and numbering scheme used in this study.
formed time- and concentration-dependence experiments.
Twenty-four hours post-fertilization (hpf), embryos were treat-
ed with 20 mm of 1, and images were acquired as described
previously.[25,26] Figure 1A shows representative fluorescence
micrographs of vehicle- or 1-treated embryos before and after
CNT analysis.[26] Time course experiments in embryos treated
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1438

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
with 1 showed that after 5 h, GFP expression reached a maxi-
mum and remained stable for an additional hour (Figure 1B).
A concentration-dependence experiment at the 5 h time point
documented that FGF reporter activation was maximal at
20 mm and declined at higher concentrations (Figure 1C). We
tested all 29 analogues to determine if any of the new com-
pounds induced GFP expression similar to 1 (Table S4). Dose–
response curves were obtained, showing that nine compounds
were equipotent to 1, and one agent, 7 (BCI-9), had significant-
ly higher activity than 1 (EC50 =4.5 mm) (Table 1). In total, in
The pharmacophore of 1 contains an electrophilic a,b-unsatu-
rated ketone moiety, and although many marketed drugs are
electrophilic, the presence of such elements is often viewed as
a liability in drug development, due to possible non-selective
modification of cellular nucleophiles, leading to off-target ef-
fects and toxicity. Indeed, many of the active agents at the
highest doses started to show whole organism toxicity at the
time of imaging, as manifested by gross morphological
changes (data not shown). We therefore assessed toxicity
upon prolonged exposure to agents (24 h) by visual inspection
of larvae for morphological
changes, such as a bent tail phe-
Table 1. Activity of BCI analogues.
notype and the appearance of
opaque, necrotic cells (Figure 2;
Compound
Alias
FGF reporter activation
in zebrafish EC50 [mm]
p
Cellular inhibition of
Tables S4 and S5). Cellular toxici-
DUSP6
DUSP1
ty was confirmed by staining
1
2
3
4
5
BCI
10.6Æ0.8 (14)
13.3Æ3.3 (2)
12.3Æ0.8 (2)
@25
13.3Æ1.8 (12)
8.0Æ0.6 (11)
8.3Æ0.8 (3)
14.0Æ2.8 (3)
n.d.
n.d.
28.4Æ1.8 (6)
n.d.
25.5Æ6.1 (3)
n.d.
14.7Æ4.4 (3)
n.d.
with acridine orange (a vital dye
that labels cells with damaged
cell membranes), revealing the
presence of dead cells in the tail
(data not shown).
BCI-164
BCI-165
BCI-11
BCI-8
BCI-9
0.56
0.21
15.8Æ3.0 (3)
25.0Æ7.0 (3)
n.d.
n.d.
50.8Æ4.1 (7)
n.d.
64.7Æ15.7 (3)
n.d.
13.6 (1)
7
4.5Æ0.6 (7)
22.6Æ2.6 (3)
12.0Æ1.5 (5)
8.9Æ2.5 (2)
10.0Æ1.1 (4)
~10.0 (1)
10.4Æ0.6 (3)
12.0Æ3.0 (6)
@25
7.1Æ0.7 (2)
10.3 (2)
@25
@25
@25
!0.01
0.04
0.47
0.6
12
13
14
15
16
18
19
24
25
26
28
30
31
BCI-211
BCI-212
BCI-303
BCI-183
BCI-297
BCI-216
BCI-215
BCI-256
BCI-269
BCI-304
BCI-296
BCI-299
BCI-10
Several agents exhibited little
or no toxicity at concentrations
that hyperactivated FGF signal-
ing in vivo (Tables S4 and S5).
Conversely, we found some
agents that did not hyperacti-
vate FGF signaling but caused
developmental toxicity, and sev-
eral that were devoid of toxicity
and activity, including alcohol 31
(Tables S4 and S5 and Figure 2).
The latter observation suggests
that, although the a,b-unsaturat-
ed ketone moiety is important
for FGF hyperactivation, it also
0.66
37.8Æ11.8 (3)
n.d.
0.81
0.67
67.8Æ14.2 (3)
55.9Æ8.6 (9)
28.0Æ5.0 (3)
28.6Æ3.8 (11)
@100 (2)
@100 (2)
0.02
n.d.
n.d.
@100 (2)
n.d.
n.d.
n.d.
@100 (2)
n.d.
n.d.
n.d.
Average EC50 ÆS.E. of n experiments. p value: compared with BCI by Student’s t-test (two tailed, unequal var-
iances). n.d.: not determined.
vivo SAR identified 11 new compounds that showed concen-
tration-dependent hyperactivation of FGF signaling in Tg(dus-
p6:EGFP)pt6 embryos (Table 1, relevant structures shown in Fig-
ure 1D). Structural elements essential for activity included an
aliphatic amino-alkyl side chain at C-3 and the a,b-unsaturated
ketone moiety. Changes that were tolerated without loss of ac-
tivity were moderately electron-donating or -withdrawing sub-
stituents in rings II and III. A planar aromatic amine in ring I
and a strongly electron-withdrawing cyano substituent in rings
II or III were not tolerated (see Discussion for more details).
These data document that specific structural modifications af-
fected biological activity, suggesting that 1 is a bona fide phar-
macophore of in vivo FGF signaling.
could also be a contributing factor in embryo toxicity. To fur-
ther explore this hypothesis, we examined whether there was
a correlation between toxicity and electrophilicity by calculat-
ing Hammett s constants for 1 and nine analogues with or
without in vivo FGF enhancing activity. Hammett s constants
ranged from À0.54 (low electrophilicity) to 0.60 (high electro-
philicity), covering the entire spectrum of chemical reactivities
in the series (Figure 2).
At concentrations at or above their EC50 values (5–10 mm),
analogues with negative Hammett s values (lower electrophi-
licity) showed toxicity (Figure 2). In contrast, compounds with
positive Hammett s values appeared to be less toxic. Thus, sur-
prisingly, agents with predicted high electrophilicity were gen-
erally better tolerated. There seemed to be no correlation be-
tween toxicity and in vivo target activity, as two inactive ana-
logues (21 (BCI-266) and 24 (BCI-256)) were also toxic. Collec-
tively, the data demonstrate that electrophilicity does not sig-
nificantly contribute to toxicity. This suggests that, although
the unsaturated ketone is required for activity, it does not in-
discriminately modify essential cellular constituents, and that
the untoward effects of compounds are due to off-target ef-
Identification of a non-toxic analogue of 1 with cellular and
in vivo activity
Because the primary assay for biological activity involved the
use of a living vertebrate animal, we were able to observe
whole organism toxicities upon compound treatment and to
relate toxicity to in vivo target activity and chemical reactivity.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1439

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
icity experiment in EA.hy926 cells, a hybridoma cell line that
retains many properties of normal endothelial cells.[27–29] Al-
though both agents were relatively non-toxic under the condi-
tions of the assay, BCI showed signs of cell loss, nuclear con-
densation, and necrosis (PI staining) at concentrations above
25 mm (Figure S1). In contrast, BCI-215 was devoid of cellular
toxicity at concentrations up to 50 mm (Figure S1). Therefore,
the cellular assay recapitulated differences in developmental
toxicity.
Analogues of 1 inhibit DUSPs in a chemical
complementation assay
To test whether the FGF-hyperactivating activities of the new
analogues were due to DUSP inhibition, we tested seven
agents that had shown robust activity in zebrafish and that
were available in sufficient quantities for DUSP6 and DUSP1 in-
hibitory activity in our mammalian cell-based chemical comple-
mentation assay (Table 1).[30] In this assay, HeLa cells were
transfected with Myc-tagged DUSP1 or DUSP6 and stimulated
with phorbol ester (TPA) to activate the ERK pathway. The
expression of active phosphatases in the assay decreases TPA-
induced ERK phosphorylation. Thus, compounds that inhibit
DUSP activity restore pERK levels in DUSP-overexpressing cells.
Restoration of ERK phosphorylation can be quantified by com-
paring pERK distributions of treated and untreated cell popula-
tions by Kolmogorov–Smirnov (KS) statistics. Figure 3A shows
that 1, 7, and 19 increased pERK levels in DUSP-overexpressing
cells in a concentration-dependent manner, with IC50 values in
the micromolar range. Figure 3B shows representative images
for 19 that illustrate restoration of ERK phosphorylation in
DUSP-overexpressing cells. Table 1 shows that all agents with
activity in zebrafish also inhibited DUSP6 and DUSP1 in mam-
malian cells, whereas two inactive compounds (24 (BCI-256)
and 28 (BCI-296)) lacked antiphosphatase activity. Hence, these
data support the hypothesis that in vivo activity of the ana-
logues is due to DUSP inactivation and validate the core struc-
ture of 1 as a pharmacophore for DUSP inhibition.
Figure 2. Compound 19 lacks whole organism toxicity. Images show trans-
mitted light micrographs of embryos in the presence of vehicle (DMSO), or
the indicated concentrations of test agents after 24 h of treatment. Toxicity
manifests itself by the appearance of opaque cells, indicating necrosis (red
arrows demarcate examples of necrotic cells). Compound 19, 27, and 29, 30
exhibited minimal toxicity. Hammett s values indicate the degree of electro-
philicity of the a,b-unsaturated ketone moiety.
Analogues of 1 suppress ERK-stimulated activation of
DUSP6
DUSP6 activity is stimulated upon substrate binding. In the ab-
sence of ERK, DUSP6 has low basal catalytic activity, which is
significantly enhanced upon interaction with ERK.[15] In vitro
studies with 1 suggested that its mechanism of action involved
suppressing this ERK-stimulated activation of DUSP6.[13] We
confirmed that analogues of 1 also inhibited the activation of
DUSP6 by ERK binding (Figure 4A and B). Compounds 1, 7,
and 19 significantly suppressed activation of DUSP6 with simi-
lar magnitudes (Figure 4A and B). Consistent with previous
data,[13] none of the tested agents inhibited basal phosphatase
activity (Figure 4A). In contrast, suppression of DUSP6 activa-
tion by compound 31, which failed to hyperactivate FGF sig-
naling in the transgenic embryos, was insignificant (Figure 4A
and B). Thus, in vitro inhibition of DUSP6 by analogues of 1 cor-
related with in vivo activity.
fects unrelated to chemical reactivity. One agent in particular,
compound 19 (BCI-215), did not show any toxicity at concen-
trations two times higher than the EC50 value for FGF activa-
tion (Figure 2). To probe for possible developmental delays or
defects, we performed a hatching study and found that larvae
treated with 19 developed normally. After a 6 h exposure fol-
lowed by a washout, 100% of vehicle- or compound 19-treat-
ed embryos, at a concentration two times higher than the EC50
for FGF activation (20 mm), had hatched by 56 hpf (data not
shown). In contrast, no embryos hatched after exposure to
1 or 7 at their EC50 concentrations (10 or 5 mm, respectively).
Thus, 19 lacked whole organism toxicity at concentrations that
activated FGF signaling. To corroborate the results from the ze-
brafish developmental toxicity studies, we performed a cytotox-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1440

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
ed sum of van der Waals interactions and hydrogen bond for-
mation energies. The allosteric pocket of DUSP6, however, fea-
tures charged Asp262 and Arg299 side chains and hydropho-
bic patches, the interactions of which were not accounted for
in the pairwise atomic interactions. Hence, we rigorously evalu-
ated electrostatic and solvation effects to gain insights that
would complement the SAR studies. To this end, we refined
the structural model of the DUSP6–1 interaction by rescoring
docking modes with the help of PBSA calculations implement-
ed in the FRED application (OpenEye, Santa Fe, NM).[32] PB and
SA components of this scoring method describe electrostatic
interactions and solvation effects, respectively. The method is
computationally more expensive than force-field-based scor-
ing, and hence, is generally applied for refinement in post-
processing of docking poses.[33]
Application of this method permitted us to prioritize the
binding pose of 1 shown in Figure 4C and D. Consistent with
the existing model and the fact that deletion of the benzyli-
dene moiety abolishes biological activity,[13] the refined model
showed the indanone ring buried deep in the binding pocket.
Two previously undetected hydrogen bonds were observed by
using molecular dynamics simulations starting from this dock-
ing pose (Movie S2): the first between the ketone oxygen of
1 and the Arg299 side chain, and the second between the sec-
ondary amine of the cyclohexylamino side chain of 1 and the
Trp264 backbone oxygen. In addition, Trp264 makes hydro-
phobic contacts with the aminocyclohexane moiety. To confirm
these computational predictions, we analyzed docking of 31 to
DUSP6 by using the same parameters as DUSP6–1 (Figure 4E).
No hydrogen bond formation was observed between Arg299
and the a,b-unsaturated alcohol moiety in 31, in support of
a model where the a,b-unsaturated ketone moiety in 1 is re-
quired for DUSP6 inhibition through hydrogen bonding but
not covalent modification, consistent with experimental obser-
vations.
Figure 3. Inhibition of DUSP6 and DUSP1 by analogues of compound 1 in
mammalian cells. A) Concentration-dependent inhibition of DUSP1 (closed
symbols) and DUSP6 (open symbols) by 1 and analogues and a previously
described multi-targeted DUSP inhibitor (NSC95397). B) Representative fluo-
rescence micrographs of ERK phosphorylation (upper panel) and DUSP ex-
pression (lower panel) in the presence or absence of compound 19. Restora-
tion of ERK phosphorylation in DUSP-expressing cells is observed after treat-
ment with 19.
Discussion and Conclusions
Despite the evidence that DUSPs play important roles in a vari-
ety of maladies, including cancer,[34] inflammation,[35] and
immune dysfunction,[16] they have largely eluded attempts to
discover biologically active small molecule inhibitors. The rea-
sons for this are numerous and include overlapping substrate
specificity, shallow and feature-poor active sites, redox sensitiv-
ity, and the use of in vitro assays that do not represent the ac-
tivity of these enzymes within their biological context. We re-
cently identified a small molecule, compound 1, which inhibits
the activation of DUSP6 by ERK and presumably binds to a
novel allosteric site on DUSP6. In this report, we designed a
series of novel analogues of 1 to explore their SARs through in
vivo studies using transgenic zebrafish, with confirmation by
an in vitro phosphatase assay.
Refinement of the DUSP6-1 binding model by
computational modeling and simulation
We previously established the necessity of both the cyclohexyl-
amino substituent and the benzylidene substituent for the bio-
logical activity of compound 1.[13] These experimental findings
were consistent with a computational model of 1 binding to
an allosteric site on DUSP6 in an orientation in which both
rings make contact with the interior of the pocket through hy-
drophobic interactions. Occupation of this site by 1 is thought
to prevent a conformational change in DUSP6, which other-
wise would accommodate ERK binding upon positioning
Asp262 (in the general acid loop, GAL) close to Arg299 of the
phosphatase catalytic site (Figure 4C and Movie S1).[31] The en-
semble docking and clustering approach that we previously
utilized identified multiple potential orientations for 1 in the al-
losteric pocket with comparable interaction scores.[13] The scor-
ing incorporated only pairwise atomic interactions as a weight-
In vivo SAR supports the importance of:
Ring I: Changing the size of the aliphatic ring I in analogues 2
(BCI-164) and 3 (BCI-165) preserved activity, whereas replace-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1441

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
215)) substituents were tolerat-
ed, but a cyano group was not
(compound 20 (BCI-169)). Finally,
reduction of the a,b-unsaturat-
ed ketone to the a,b-unsatu-
rated alcohol (compound 31,
Scheme 1C) abolished activity.
These results are consistent with
the prediction by the model that
the carbonyl group of the inda-
none system engaged in a hydro-
gen bond with Arg299, with a
minor contribution from an addi-
tional hydrogen bond between
the cyclohexylamino moiety and
Trp264.
Double substitutions and com-
bination with a morpholino
substituent in ring I
Simultaneous substitutions in
both rings II and III with me-
thoxy groups or halogens ren-
dered the scaffold inactive (21,
22 (BCI-283)). Because of the
unique ability of the morpholino
substituent in ring I to increase
potency (analogue 7), we inves-
tigated whether inclusion of a
morpholino moiety in the ring I
position, along with substituents
in rings II and III would improve
the activity; this hypothesis was
not supported. Although the ac-
Figure 4. Compounds 1, 7, and 19 inhibit ERK2-stimulated activation of DUSP6 catalytic activity in vitro. A) DUSP6
phosphatase activity in the presence and absence of ERK2. Inclusion of 1, 7 and 19 partially suppressed DUSP6 ac-
tivation by ERK2 but not basal phosphatase activity. In contrast, compound 31 showed only a minor effect. Data
are from a single experiment that has been repeated twice with identical results. Vanadate was used as a positive
control. Note that in assays in the absence of ERK, results from treatment with 1 overlapped with 7 and 19 with
31. B) Graph showing average percent inhibition of DUSP6 hyperactivation ÆSD from three independent experi-
ments by 1, 7, 31 and 19. Percent inhibition was graphed from results at 10 min when the assay was within the
linear range. ***, p<0.001 by one-way ANOVA compared with vehicle control. C) Overview of putative allosteric
binding site of 1 and docking orientation (chloride ion highlights catalytic site). D) Close-up view of 1 s interac-
tions with allosteric binding site residues. Two hydrogen bonds are observed (black dotted lines), between the
ketone oxygen of 1 and Arg299 and the amine of 1 and the Trp264 backbone oxygen. E) Close-up view of the
interactions of 31 with allosteric binding site residues. Hydrogen bonding of the amine in 31 and the backbone
oxygen of Trp264 is present. In contrast, hydrogen bonding with Arg299, as noted for 1, is absent.
tivity of
a
fluoro-substituted
compound was preserved (14 vs.
26 (BCI-304)), the replacement of
cyclohexylamine with morpho-
line did not render previously in-
ment of the cyclohexyl ring with a phenyl ring in analogue 4
(BCI-11) abolished activity (Table 1). Shortening the side chain
by incorporating the nitrogen into the aliphatic ring preserved
activity (compound 5 (BCI-8)). Introduction of heteroatoms
into ring I abolished activity (analogues 8–11). A notable ex-
ception appeared to be the morpholine-substituted compound
7, which had the maximal in vivo activity among all tested BCI
analogues.
active compounds active (21 vs. 23 (BCI-267), 22 vs. 29 (BCI-
282)). Furthermore, the morpholino substituent abolished the
activity of three active agents (analogues 24, 27 (BCI-271), and
28 (BCI-296)). Thus, the ability of the morpholino group to pre-
serve or improve potency was limited to unsubstituted (7) and
para-fluoro-substituted (26) analogues.
In vivo activity and DUSP inhibition
Rings II and III: Substituents at the para position in ring II
with electron-donating (OMe: 12 (BCI-211), Me: 13 (BCI-212))
or electron-withdrawing (F: 14 (BCI-303), Cl: 15 (BCI-183), 3,4-
Cl2: 16 (BCI-297)) groups did not exhibit substantial changes in
activity compared to 1 (Table 1). The strongest electron-with-
drawing cyano group (compound 17 (BCI-7)), however, was
not tolerated. Similar results were obtained for compounds
with substitutions in ring III (compounds 18–20), where meth-
oxy (compound 18 (BCI-216)) and bromo (compound 19 (BCI-
There is considerable debate regarding whether potent and se-
lective inhibitors of DUSPs can be obtained. Multiple attempts
at discovering DUSP inhibitors have failed, and because ade-
quate probes are lacking, the question of what degree of spe-
cificity is required to elicit desired biological responses has
been intractable. The dearth of chemical probes for DUSP ac-
tivity has also prevented proof-of-principle studies in mam-
mals. Our studies demonstrate that DUSPs might be druggable
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1442

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
through exploitation of allosteric mechanisms. First, compound
1 and seven analogues that activated FGF signaling in vivo
also inhibited mammalian DUSP1 and DUSP6 in cultured cells,
whereas agents that lacked in vivo activity were devoid of anti-
phosphatase activity. This is a significant finding as, prior to
our current studies, only three agents had ever shown con-
firmed activity in the mammalian cell-based chemical comple-
mentation assay, namely the thiol poison, phenyl arsine
oxide;[36] the glutathione-depleting alkaloid, sanguinarine;[30]
and the para-quinone, NSC95397.[37] Second, experimental data
were consistent with computational modeling predictions
based on the DUSP6–1 interactions at the allosteric site, lend-
ing credence to the proposed mechanism of inhibition. Taken
together, the results validate the 1 scaffold as a bona fide
pharmacophore for allosteric DUSP6 inhibition.
at discovering redox-active, nonselective inhibitors with lack of
or promiscuous cellular activity. Our findings suggest that tar-
geting DUSPs by allosteric mechanisms can circumvent many
of the problems caused by the nature of DUSP’s catalytic
cavity. The zebrafish in particular has been indispensable in dis-
covering such inhibitors, and the present data create contin-
ued enthusiasm for the further identification of DUSP inhibitors
by phenotypic discovery in transgenic zebrafish.
Experimental Section
Chemical synthesis: The synthesis of and analytical data for all
compounds are described in the Supporting Information.
Zebrafish maintenance and compound treatment: All procedures
involving zebrafish were reviewed and approved by the University
of Pittsburgh Institutional Animal Care and Use Committee. Tg(dus-
p6:eGFP)pt6 embryos were obtained by natural mating and incubat-
ed at 28.58C.[26] One transgenic embryo was placed into every well
of a 96-well plate in E3 (200 mL; 5 mm NaCl, 0.17 mm KCl, 0.33 mm
CaCl2, 0.33 mm MgSO4). Compounds were dissolved as 100ꢁ stock
solutions in DMSO, and aliquots (2 mL) of each were added directly
to octuplicate wells. For the SAR studies, a negative control
(8 wells of DMSO (1%)) was included on every plate. A full dose–
response for 1 was run on each day of experiments.
All agents with in vivo activity were inhibitors of both
DUSP6 and DUSP1. Because there is no published X-ray crystal
structure of DUSP1, we created a homology model and
showed that the allosteric site also exists on DUSP1 (data not
shown). Therefore, the pharmacophore of 1 might not be ex-
pected to show selectivity for either DUSP.
Although we observed a correlation between in vivo and
cellular activity, there were differences in potency between
these assays. The most likely reasons for this are solubility,
uptake, and/or protein binding, as zebrafish assays are per-
formed in an unbuffered aqueous solution, whereas cellular
assays are conducted in complete growth medium with serum.
The most striking difference between assays was observed in
the in vitro DUSP hyperactivation assay, where compounds
showed only partial activity at high concentrations (100 mm).
Although we have observed this phenomenon before,[13] a de-
finitive explanation is lacking. One reason for the lackluster in
vitro activity could be that multiple binding processes and en-
zymatic reactions with different affinities and kinetics occur
concurrently, possibly affecting the enzyme–inhibitor interac-
tion. Alternatively, in vitro assays might not faithfully represent
biological conditions, due to lack of a proper microenviron-
ment and accessory/scaffolding proteins.
Automated imaging and analysis: At the end of compound treat-
ment, embryos were anesthetized with 40 mgmLÀ1 tricaine metha-
nesulfonate (MS222, Sigma) in E3. Plates were loaded into an
ImageXpress Ultra high-content reader (Molecular Devices) and
imaged by using a 4ꢁ objective at excitation/emission wave-
lengths of 488/525 nm (GFP).[26] Archived scan images were up-
loaded into Developer (Definiens AG) and analyzed for GFP expres-
sion in the head by using a simplified version of our previously de-
scribed CNT rule set.[26] A GFP threshold was set, based on well
background fluorescence, and regions within the zebrafish larva
were classified as positive for GFP expression if their fluorescence
intensity exceeded this threshold. GFP-expressing areas were
merged, and the four largest objects were selected for quantita-
tion. [Total head structure brightness=(mean GFP intensity)ꢁ(area
of the four head structures)]. EC50 values were determined from
dose–response curves by a four-parameter logistic equation where
the bottom and top were defined as the magnitude of FGF activa-
tion by 1% DMSO and by the maximum response elicited by the
positive plate control (usually seen with 20 mm of 1), respectively.
EC50 values in Table 1 are the averages ÆSEM of n independent
experiments.
Neither the dual inhibitory nature of 1 and analogues nor
the presence of an electrophilic a,b-unsaturated ketone ap-
peared to influence FGF hyperactivation. More importantly, the
fact that embryos treated with compound 19 developed nor-
mally suggests that neither a lack of selectivity nor the pres-
ence of a potentially electrophilic moiety were causes for toxic-
ity. These features make compound 19 not only an attractive
candidate for further evaluation in mammals but also provide
the research community with a much cleaner probe than 1 to
investigate the biological functions of DUSP1 and DUSP6. The
data demonstrate the rich potential of zebrafish in early drug
discovery and identify compound 19 as a candidate for proof-
of-principle studies to investigate the role of DUSP6 in embry-
onic development and disease models.
Developmental toxicity assessments: After drug treatment and
GFP quantitation, embryos in microplates were returned to the in-
cubator overnight in the continued presence of test agents. After
a total of 24 h treatment, wells were examined visually for signs of
toxicity, such as changes in gross morphology, necrosis, heart beat,
and circulation to tail. Selected larvae were photographed on
a transmitted light microscope to document toxicity.
Cell culture: HeLa cells were obtained from ATCC (Manassas, VA)
and were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS; HyClone, Logan,
UT), and 1% penicillin–streptomycin (Invitrogen) in a humidified at-
mosphere of 5% CO2 at 378C. EA.hy926 cells (ATCC CRL-2922), a
hybridoma cell line that retains many properties of normal endo-
thelial cells,[27–29] were maintained in DMEM supplemented with
DUSPs have long eluded drug discovery efforts using the
contemporary single-target, biochemical assay-based discovery
paradigm. Their active sites are shallow, and their catalytic ac-
tivity depends on a highly reactive, redox-sensitive cysteine.
Prior discovery efforts, therefore, have been exceedingly good
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1443

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
10% FBS, 100 mgmLÀ1 penicillin–streptomycin, and 2 mm gluta-
mine.
rescence was measured on an M5 multimode reader (Molecular
Devices) at excitation/emission wavelengths of 485/525 nm at ten
minute intervals for 1 h at room temperature.
Cytotoxicity of BCI analogues in cultured cells: EA.hy926 cells
(10000 cells per well) were plated in the wells of a 384-well micro-
plate, allowed to attach overnight, and treated with five-point, 1/3
dose reduction gradients of 1 and 19. The non-selective thiol
poison phenylarsine oxide (PAO), which induces apoptosis in
normal cells,[38] was included as a positive control. After 6 h of con-
tinuous exposure, cells were stained with 1 mgmLÀ1 propidium
iodide (PI) and 10 mgmLÀ1 Hoechst 33342 to visualize necrotic cells
and nuclei, respectively. Cells were imaged live on an ArrayScan II
high-content reader. Numbers of nuclei per imaging field, nuclear
condensation, and the percentage of PI-positive cells were deter-
mined by the target activation bioapplication as described.[38]
Statistical significance: In vivo and cell-based assay data were ana-
lyzed by using the two-tailed Student’s t-test, assuming unequal
variances. For in vitro phosphatase activity assays, one-way ANOVA
was performed between all groups, followed by post hoc compari-
son of means by using Tukeys multiple range test. P values of
<0.05 and <0.001 were considered statistically significant for in
vitro assays.
Molecular modeling: The scientific application FRED (OpenEye,
Santa Fe, NM) was used for rescoring docking poses of 1 at the pu-
tative binding site in DUSP6 (PDB: 1MKP). AM1-BCC partial charges
for 1 were calculated by using QUACPAC,[41] and conformers were
generated by using OMEGA[42] with default options. Conformational
changes in DUSP6 upon ERK binding (60 ns; Movie S1) and of the
1–DUSP6 complex (1.4 ns; Movie S2) were performed by using
NAMD[43] and CHARMM force field using water simulations.[44]
Antibodies and plasmids: Rabbit polyclonal phospho-ERK was
from Cell Signaling Technology (Beverly, MA). Mouse monoclonal
anti-c-myc (9E10) antibody was from Santa Cruz Biotechnology
(Santa Cruz, CA). Secondary antibodies were Alexa Fluor 488-conju-
gated goat anti-mouse and Alexa Fluor 594-conjugated goat anti-
rabbit (Invitrogen). c-Myc-DUSP6/MKP3 (PYST1) was in pSG5.[39,40] c-
Myc-DUSP1-pcDNA3.1 was subcloned from a pET15b vector encod-
ing DUSP1/MKP1/CL100 into pcDNA3.1 (both original plasmids
were gifts from Steve Keyse, CRUK, Dundee).
Movie S1: Dusp6 general acid loop motions: The movie shows
DUSP6 general acid loop (GAL) motions from an unbiased molecu-
lar dynamics simulation. Simulation was performed in the presence
of water (not shown) by using PDB structure 1MKP. The GAL con-
tains the catalytic residue Asp262, which is required for formation
of the “catalytic triad” (with Cys293 and Arg299). Upon formation
of the catalytic triad, DUSP6 performs its dephosphorylation func-
tion at a higher catalytic rate. This conformational shift is also re-
ferred to as the catalytic activation of DUSP6. The GAL in its active
form from DUSP10 structure (PDB id: 1ZZW) is shown in yellow for
comparison. Upon formation of the catalytic triad, Asp262 is
shown in sphere representation.
Chemical complementation assay for DUSP6 and DUSP1: Com-
pounds were analyzed for inhibition of DUSP1 and DUSP6 in intact
cells as described.[13] Briefly, HeLa cells were transfected in 384-well
plates with human c-Myc-DUSP6 or c-Myc-DUSP1 by using Fugene
HD (Roche). After 48 h in culture, cells were treated in quadrupli-
cate wells for 15 min with ten twofold concentration gradients of
1 and analogues or the nonselective DUSP inhibitor, NSC95397
(positive control), and stimulated for 15 min with phorbol ester
(TPA, 500 ngmLÀ1). Cells were immunostained with a mixture of
anti-pERK (1:200 dilution) and anti-c-Myc (1:100 dilution) antibod-
ies. Positive pERK and c-Myc-DUSP signals were visualized with
Alexa Fluor 594 (pERK) and Alexa 488 (c-Myc) conjugated secon-
dary antibodies, respectively. Plates were analyzed by three-chan-
nel multiparametric analysis for pERK and c-Myc-DUSP intensities
in an area defined by nuclear staining by using the target activa-
tion bioapplication on the ArrayScan II (Thermo Fisher Cellomics,
Pittsburgh, PA). DUSP transfected cells were classified as expressors
if their average c-Myc staining intensity exceeded a threshold
defined as the mean intensity+2 SD of untransfected cells. pERK
levels were quantified in the DUSP-expressing subpopulation by
Kolmogorov-Smirnov statistics, comparing the cumulative pERK
distribution of each test well to a reference distribution from 14
DUSP-transfected and vehicle-treated wells. High KS values denote
large differences in ERK phosphorylation levels compared with ve-
hicle control and indicate suppression of DUSP activity. KS values
were plotted against compound concentration, and IC50 values
were calculated by fitting curves to a four parameter logistic equa-
tion, with the top defined by the maximum KS value obtained in
the presence of the highest concentration of the positive control
(1 or NSC95397).
Movie S2: BCI and DUSP6 interactions: The movie shows interac-
tions of 1 in the allosteric pocket of DUSP6. Simulation was per-
formed in the presence of water (not shown) by using PDB struc-
ture 1MKP. The bound conformation of 1 was obtained by molecu-
lar docking. Compound 1 buries its hydrophobic surface in the
allosteric binding pocket and forms hydrogen bonds with Arg299
side-chain and Trp264 backbone oxygen. Hydrogen bond forma-
tion is indicated by blue dashed lines. Compound 1 binding in the
allosteric pocket prevents a GAL movement that brings Asp262
into proximity with the catalytic cysteine (Cys293), thereby inhibit-
ing catalytic activation of DUSP6.
Abbreviations
BCI: (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-
one; CNT: cognition network technology; DUSP: dual specificity
phosphatase; EC50: Half-maximal effective concentration; FGF: fi-
broblast growth factor; FITC: fluorescein isothiocyanate; GAL: gen-
eral acid loop; HBSS: Hank’s balanced salt solution; HCS: high-con-
tent screening; hpf: hours post fertilization; MAPK: mitogen-acti-
vated protein kinase or MAP kinase; MKP3: MAP kinase phospha-
tase 3; NBS: N-bromosuccinimide; PBSA: Poisson–Boltzmann sur-
face area; PDB: Protein Data Bank; PTP: protein tyrosine
phosphatase; SAR: structure–activity relationship
In vitro phosphatase assays: 3-O-Methylfluorescein phosphate
(OMFP)-based ERK2 induced activation of DUSP6 assays were per-
formed as described.[13] Recombinant His-tagged Dusp6 was ex-
pressed from a bacterial expression vector and 250 ng were incu-
bated with 100 mm 1, 7, 19, and 31. To assay activated DUSP6,
210 ng of recombinant ERK2 (Cell Signaling Technology, Danvers,
MA) was added to DUSP6/compound mixtures before the addition
of OMFP (100 mm). The final reaction volume was 15 mL. OMF fluo-
Acknowledgements
We thank Nina Senutovitch and Richard DeBiasio for technical
assistance with the EA.hy926 cell line and Donna Huryn for criti-
cally reading the manuscript. Grants from the National Institutes
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1444

CHEMBIOCHEM
FULL PAPERS
www.chembiochem.org
[19] Y. Niwa, H. Shimojo, A. Isomura, A. Gonzalez, H. Miyachi, R. Kageyama,
Genes Dev. 2011, 25, 1115–1120.
[20] F. C. Simoes, T. Peterkin, R. Patient, Development 2011, 138, 3235–3245.
[21] J. R. Meyers, J. Planamento, P. Ebrom, N. Krulewitz, E. Wade, M. E. Pow-
nall, Dev. Biol. 2013, 378, 107–121.
[22] B. Pearson, R. Ayer, N. Cromwell, J. Org. Chem. 1962, 27, 3038–3044.
[23] G. Maury, E. Wu, N. Cromwell, J. Org. Chem. 1968, 33, 1900–1907.
[24] R. Murray, N. Cromwell, J. Org. Chem. 1976, 41, 3540–3545.
[25] M. Saydmohammed, L. L. Vollmer, E. O. Onuoha, A. Vogt, M. Tsang, Birth
Defects Res. Part C 2011, 93, 281–287.
of Health supported this work [Grants R01 GM099738 and P41
GM103712 to I.B., CA78039 to A.V., HD053287 to M.T and A.V.,
and HL088016 to M.T.]. This project used the UPCI Chemical Biol-
ogy Facility, supported in part by award NIH P30A047904. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Heart,
Lung, and Blood Institute or the National Institutes of Health
(NIH).
[26] A. Vogt, H. Codore, B. W. Day, N. A. Hukriede, M. Tsang, J. Visualized. Exp.
2010, 40, e1900.
[27] C. J. Edgell, C. C. McDonald, J. B. Graham, Proc. Natl. Acad. Sci. USA
1983, 80, 3734–3737.
Keywords: BCI
signaling · high-content screening · zebrafish
·
cognition network technology
·
FGF
[28] C. J. Edgell, J. E. Haizlip, C. R. Bagnell, J. P. Packenham, P. Harrison, B. Wil-
bourn, V. J. Madden, In Vitro Cell. Dev. Biol. 1990, 26, 1167–1172.
[29] J. Bauer, M. Margolis, C. Schreiner, C. J. Edgell, J. Azizkhan, E. Lazarowski,
R. L. Juliano, J. Cell. Physiol. 1992, 153, 437–449.
[30] A. Vogt, J. S. Lazo, Pharmacol. Ther. 2005, 107, 212–221.
[31] J. D. Rigas, R. H. Hoff, A. E. Rice, A. C. Hengge, J. M. Denu, Biochemistry
2001, 40, 4398–4406.
[32] J. Grant, B. Pickup, A. Nicholls, J. Comput. Chem. 2001, 22, 608–640.
[33] B. Kuhn, P. Gerber, T. Schulz-Gasch, M. Stahl, J. Med. Chem. 2005, 48,
4040–4048.
[1] A. Farooq, M. M. Zhou, Cell. Signalling 2004, 16, 769–779.
[2] S. M. Keyse, Curr. Opin. Cell Biol. 2000, 12, 186–192.
[3] K. I. Patterson, T. Brummer, P. M. O’Brien, R. J. Daly, Biochem. J. 2009,
418, 475–489.
[4] Y. Kawakami, J. Rodriguez-Leon, C. M. Koth, D. Buscher, T. Itoh, A. Raya,
J. K. Ng, C. R. Esteban, S. Takahashi, D. Henrique, M. F. Schwarz, H. Asa-
hara, J. C. I. Belmonte, Nat. Cell Biol. 2003, 5, 513–519.
[5] C. Li, D. A. Scott, E. Hatch, X. Tian, S. L. Mansour, Development 2007,
134, 167–176.
[34] S. M. Keyse, Cancer Metastasis Rev. 2008, 27, 253–261.
[35] Y. Liu, E. G. Shepherd, L. D. Nelin, Nat. Rev. Immunol. 2007, 7, 202–212.
[36] A. Vogt, K. A. Cooley, M. Brisson, M. G. Tarpley, P. Wipf, J. S. Lazo, Chem.
Biol. 2003, 10, 733–742.
[6] M. Tsang, S. Maegawa, A. Kiang, R. Habas, E. Weinberg, I. B. Dawid, De-
velopment 2004, 131, 2769–2779.
[7] H. Kamata, S. Honda, S. Maeda, L. Chang, H. Hirata, M. Karin, Cell 2005,
120, 649–661.
[37] A. Vogt, P. R. McDonald, A. Tamewitz, R. P. Sikorski, P. Wipf, J. J. Skoko III,
J. S. Lazo, Mol. Cancer Ther. 2008, 7, 330–340.
[38] A. Vogt, P. A. McPherson, X. Shen, R. Balachandran, G. Zhu, B. S. Raccor,
S. G. Nelson, M. Tsang, B. W. Day, Chem. Biol. Drug Des. 2009, 74, 358–
368.
[8] M. Brisson, T. Nguyen, A. Vogt, J. Yalowich, A. Giorgianni, D. Tobi, I.
Bahar, C. R. Stephenson, P. Wipf, J. S. Lazo, Mol. Pharmacol. 2004, 66,
824–833.
[9] P. A. Johnston, C. A. Foster, T. Y. Shun, J. J. Skoko, S. Shinde, P. Wipf, J. S.
Lazo, Assay Drug Dev. Technol. 2007, 5, 319–332.
[39] S. Dowd, A. A. Sneddon, S. M. Keyse, J. Cell Sci. 1998, 111, 3389–3399.
[40] L. A. Groom, A. A. Sneddon, D. R. Alessi, S. Dowd, S. M. Keyse, EMBO J.
1996, 15, 3621–3632.
[41] A. Jakalian, D. B. Jack, C. I. Bayly, J. Comput. Chem. 2002, 23, 1623–1641.
[42] J. Bostrçm, J. R. Greenwood, J. Gottfries, J. Mol. Graphics Modell. 2003,
21, 449–462.
[10] K. Nemoto, A. Vogt, T. Oguri, J. S. Lazo, Prostate 2004, 58, 95–102.
[11] G. A. Molina, S. C. Watkins, M. Tsang, BMC Dev. Biol. 2007, 7, 62.
[12] M. Tsang, Birth Defects Res. Part C 2010, 90, 185–192.
[13] G. Molina, A. Vogt, A. Bakan, W. Dai, P. Q. de Oliveira, W. Znosko, T. E.
Smithgall, I. Bahar, J. S. Lazo, B. W. Day, M. Tsang, Nat. Chem. Biol. 2009,
5, 680–687.
[43] J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C.
Chipot, R. D. Skeel, L. Kale, K. Schulten, J. Comput. Chem. 2005, 26,
1781–1802.
[44] K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim,
E. Darian, O. Guvench, P. Lopes, I. Vorobyov, A. D. Mackerell, Jr., J.
Comput. Chem. 2010, 31, 671–690.
[14] J. S. Lazo, D. C. Aslan, E. C. Southwick, K. A. Cooley, A. P. Ducruet, B. Joo,
A. Vogt, P. Wipf, J. Med. Chem. 2001, 44, 4042–4049.
[15] M. Camps, A. Nichols, C. Gillieron, B. Antonsson, M. Muda, C. Chabert,
U. Boschert, S. Arkinstall, Science 1998, 280, 1262–1265.
[16] G. Li, M. Yu, W. W. Lee, M. Tsang, E. Krishnan, C. M. Weyand, J. J. Goron-
zy, Nat. Med. 2012, 18, 1518–1524.
[17] M. Maillet, N. H. Purcell, M. A. Sargent, A. J. York, O. F. Bueno, J. D. Mol-
kentin, J. Biol. Chem. 2008, 283, 31246–31255.
Received: February 1, 2014
[18] M. Matsuda, D. D. Nogare, K. Somers, K. Martin, C. Wang, A. B. Chitnis,
Development 2013, 140, 2387–2397.
Published online on June 6, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2014, 15, 1436 – 1445 1445