© 2009 Nature America, Inc. All rights reserved. nature CH nature Received 18 August 2008; accepted 30 April 2009; published online 5 July 2009; should Correspondence be addressed to M.T. ([email protected]). USA. of 4 modulators chemical 1 identifying for useful is zebra embryo the handling, fish of ease and screening development rapid size, small small-molecule its high-content for suited methods. genetic current with attainable been not has that goal useful tools for dissecting the roles of this pathway in development—a provide would FGF signaling modulate can that reversibly molecules signaling FGF limits during development and homeostasis these of presence the that revealed have mice in knockout and zebrafish in depletion Sprouty and (ERK) signal–regulated MAPK the family—extracellular of class one of dephosphorylation by only pathway the inhibits Dusp6 whereas pathway, the within points multiple at ing in levels optimal embryogenesis to adjusted is signaling FGF activities, concerted attenuators feedback pathway RAS/MAPK as tion to expression and (similar and Sef FGFs) proteins Spry4) Spry3 func Spry2, 3), (Spry1, Sproutys MAPK called also 6 (Dusp6; that suggest FGF must signaling ligands be tightly regulated. of phosphatase Dual-specificity of family multitude this the by and activated pathways FGFs ­signaling of roles biological wide-ranging The (MAPK) kinase rat (RAS)/mitogen-activated homolog the including pathways, signaling multiple class activate kinase to tyrosine receptor the of proteins transmembrane pass homeostasis and cellular proliferation of family secreted glycoproteins large that serve a important functions in development,of members are (FGFs) factors growth Fibroblast traditional high-throughput chemical screens to identify new compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded for Dusp6 in restricting cardiac progenitors and controlling heart organ size. This study highlights the power of catalytic activation by ERK2 substrate binding. We used BCI treatment at varying developmental stages to uncover a temporal role an allosteric binding site for BCI within the phosphatase domain. treatment blocked Dusp6 activity and enhanced FGF target in zebrafish embryos. Docking simulations predicted ( the activity of extracellular signal–regulated (ERKs) The phosphatase 6 dual-specificity (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit Wade Znosko MolinaGabriela lineages cell cardiac expands that Dusp6 of inhibitor an reveals screening chemical Zebrafish E Department Department of Biology,Computational Department of Microbiology and Molecular Genetics, )-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro- The zebrafish embryo is a vertebrate animal model that is well well is that model animal vertebrate a is embryo zebrafish The 7 Present Present address: SRI Center International, for Advanced Drug Research, Virginia, Harrisonburg, USA. E MIC 1,3 1 A . Sef and Spry proteins suppress RAS/MAPK signal . RAS/MAPK suppress proteins and Spry Sef , Thomas E Smithgall L BIOLOGY L 1 , 7 , 8 , Andreas Vogt

in in vitro advance online publication online advance 5 Department Department of Sciences Pharmaceutical and 1,4–6 1 screens. . These ligands bind .to bind single- ligands These . The of small identification 2 , 3 1 , , 8 3 2 , Ahmet Bakan Department Department of Pharmacology and Chemical Biology, , Ivet Bahar 1,3 . Through their their Through . 7,8 1 . Sef, Dusp6 Dusp6 Sef, . . Owing to to Owing . 1 H

cascade -inden- sarcoma sarcoma 3 , 4

, John S Lazo 1 4 and 2. We have identified a inhibitor small-molecule of Dusp6— , 8 2 , Weixiang Dai 1 - - ­ .

-one -one (BCI)—using a transgenic zebrafish chemical screen. BCI In In vitro d domain and suggested a plausible allosteric mechanism of action, action, of which was supported mechanism by allosteric plausible a suggested and phosphatase domain Dusp6 the on binding BCI for site favorable getically zebrafish in embryos activity and in cultured cells. Molecular Dusp6 modeling predicted an ener blocks BCI that revealed analyses Our NF- factor the ­transcription suppresses that compound a as described first was BCI naling. dihydro-1 molecule—( small a line and a an screen with transgenic FGF chemical reporter naling activity during early development ­signaling have been generated and allow for the live visualization of sig ­alternative of humans in deficiencies blood restore to order differentiation in HSCs the direct to molecules using to way have the studies opened animal and vertebrates, in conserved is pathway This E2 is a stem key cell hematopoietic (HSC) of regulator homeostasis prostaglandin that discovery the is screens zebrafish of relevance the the metabolism iron of regulating in pathway BMP importance the revealed have mice in dorsomorphin using treatment chemical upon defects patterning tor of bone morphogenetic protein (BMP), as embryos exhibited axial as an inhibi was identified screen, dorsomorphin In one phenotypic observations of phenotypes generated by exposure to small molecules. ­processes pathways signaling o 6 i Department Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, : 1 The generation of transgenic reporter lines in zebrafish offers offers zebrafish in lines reporter transgenic of generation The 0 . 1 0 studies supported a model in which BCI inhibits Dusp6 2 3 , 8 3 8,9 / , Billy W Day H n in vivo in c . Previous zebrafish chemical screens have relied on the the on relied have screens chemical zebrafish Previous . -inden-1-one (BCI, (BCI, -inden-1-one h 5 e , Pierre Queiroz de Oliveira m b i o tools for chemical screening. Reporters for FGF FGF for Reporters screening. chemical for tools . 3 in in vivo 1 8 Pittsburgh Pittsburgh Molecular Libraries Screening Center, These These authors contributed equally to this work. 9 0 E in in vitro )-2-benzylidene-3-(cyclohexylamino)-2,3-  B in cell-based luciferase reporter assays reporter luciferase B cell-based in and compounds that affect developmental developmental that affect and compounds 5 , 6 & Michael Tsang assays. Using BCI as a chemical probe, 1 —ht yeatvts G sig FGF hyperactivates )—that 1 2 . In this study, we performed 1 1 0 0 . Another example of of example . Another . Subsequent studies studies Subsequent . 2 in in vivo , 1 t r a

1 zebrafish 1 . L C I

identified identified

s e 1 1 3 1  - - - - . .

© 2009 Nature America, Inc. All rights reserved.  ­brachyury by whole mount signaling, we treated embryos before gastrulation (5 h.p.f.) and ( analyzed treatment post in a concentration-dependent manner and was detected as early as 2 h fluorescence d2EGFP enhanced BCI h. 6–8 after DMSO) (0.5% trol in treated embryos a was visually analyzed and of compared to vehicle well con each into arrayed 10 compoundsat containingtestplate 96-well were (h.p.f.) fertilization post h 24 at small-molecule for libraries chemical from assembled compounds diverse 5,000 over Tg(:EGFP) amino amino group, and 3-(cyclohexylamino)-2,3-dihydro-1 dene-2,3-dihydro-1 ( signaling FGF FGF target . the zebrafish embryo, resulting in the increased transcription of several gene) called FGF signaling, we measured an increase in the expression of ( studies implantation bead FGF from observations previous with tent and MHB, labels confirmed, as BCI increased expression of ( tailbud the and (r4) (MHB), rhombomere4 prospectiveboundary mid-hindbrain of sion in expression of another FGF target gene, the 1-somite stage to the 10-somite stage resulted in a marked increase stage in BCI-treated embryos ( greatly expanded within the notochord and the tailbud at the 6-somite cent zebrafish transgenic a line, of generation the described previously We modulator FGF an identifies screen chemical Zebrafish RESULTS size. organ heart regulate to field cardiac the in signaling studies These somitogenesis during activity expands Dusp6 of inhibition that found we t r a Fig. 1i We next determined the BCI structural features required to enhance DMSO g e d a

protein (d2EGFP) under the control of FGF signaling FGF of control the under (d2EGFP) protein Tg(dusp6:EGFP) 16–18 sef , j L C I )

) and cardiac progenitors at the expense of endothelial lineages. lineages. endothelial of expense the at progenitors cardiac 1 ), a known FGF target gene target FGF known a ), . These results confirmed that BCI enhanced FGF signaling in 5 . To further demonstrate that BCI treatment hyperactivated

spry4 suggest that Dusp6 functions as an attenuator of FGF FGF of attenuator an as functions Dusp6 that suggest s e DMSO DMSO

dusp6 Fig. modulators of this pathway. embryos this transgenic Fiveof modulators embryos as a biosensor for FGF signaling, we screened Fig. 1a r4 in situ ntla egr2b by semiquantitative RT-PCR ( H

1 -inden-1-one (BI, l b h Fig. 1g BCI pt6 ). We synthesized two analogs: ( analogs: two We). synthesized hybridization the expression of , which demarcates r3 and r5 identity,consis r5 and r3 demarcates which , – ta epess etblzd re fluores green destabilized expresses that , c ). To confirm that BCI hyperactivated FGF FGF hyperactivated BCI that To). confirm , h Fig. 1d ). The expanded brain structures were structures brain expanded The ). 10 10 µ µ M BCI M BCI dusp6 r4 ntla – f ). Similarly, BCI treatment from 1 4 2 c j i f BCI DMSO dusp6 . The expression of of expression The . ), which lacks the cyclohexyl engrailed 2b , as shown by the expan  M. intensity d2EGFP Fig. r3 r3 20 20 µ µ r5 r5 M BCI M BCI

1 ntla k H ( ntla ; E eng2b -inden-1-one -inden-1-one n )-2-benzyli = 3 for each il17rd

l k (zebrafish 1 ntla ), which 2 . Using . DMSO (also was was ------

10 O NH (ICD, MHB. Scale bar, 250 Scale MHB. ( concentrations higher fourfold at even effect, ( embryos ( BI h.p.f. 30 of 2 Figure analogs lacking either group were inactive ( inactive were group either lacking analogs as fluorescence, d2EGFP enhancing in required both were stituents Methods thereby resulting in a net increase in transcription of target genes genes target of ( transcription in FGF increase net the a of in resulting attenuator thereby feedback a block could BCI that ligand. We of absence the in signaling FGF enhance not did of embryos, MHB which are deficient in Fgf8 signaling ( the in expression d2EGFP are ( expressed FGFs where regions embryonic to restricted was expression d2EGFP increased the embryos, transgenic within BCI-treated acts pathway.In RAS/MAPK compound this a where identify probed to we and target, potential activity BCI’s for mechanism the determine To Dusp6 inhibits BCI µ

Fig. M BCI c ) and ICD ( ICD ) and advance online publication online advance 3

e 3

c a 20 ). To test this model, we whether BCI determined could ), which lacks the benzylidene group (see (see group benzylidene the lacks which ),

BCI structure-activity relationship studies. ( studies. relationship structure-activity BCI µ b

for synthesis). The cyclohexylamino and benzylidene sub benzylidene and cyclohexylamino The synthesis). for M BCI ), whereas related analogs, shown in inner panels, had no had panels, inner in shown analogs, related whereas ), histone spry4 sef Tg(dusp6:EGFFP) d Fig. ). d2EGFP fluorescence was enhanced in BCI-treated BCI-treated in enhanced was fluorescence d2EGFP ). H 4

1b  m. , c ). did not induce Furthermore, BCI treatment BCI. Scale bars, 250 RNA loading control. ( as measured by RT-PCR. Histone H4 served as expression of the FGF target genes DMSO treatment ( expression, respectively ( r3 and r5, as marked by somitogenesis stages expanded the MHB and demarcate the MHB. ( DMSO-treated embryos ( showed stronger embryos. Note that the MHB, r4 and somites ( compared to DMSO control treatment ( ntla with BCI during gastrulation had expanded treated with DMSO ( d2EGFP fluorescence as compared to embryos treated with BCI ( ( that hyperactivates FGF signaling in zebrafish. Figure 1 g a , – 20 O h c expression at the 6-somite stage ( DMSO ) pt6 ) µ

dusp6 Tg(dusp6:EGFP) M BI embryos treated with DMSO ( DMSO with treated embryos

Identification of a small molecule mRNA was increased in BCI-treated d b nature nature dusp6 Tg(dusp6:EGFP);ace b i ). ( , c a ) exhibited increased c,d ). (  k i l pt6 Fig. ) BCI treatment induced , ) The chemical structure of m; bar in staining ( j ) BCI treatment during d ). Red arrowheads mark mark arrowheads Red ). eng2b g embryos at 30 h.p.f. Fig. j ) when compared to – ). Red arrowheads ch

f 2 ) Embryos treated a–d ).

3a e and mic ) Lateral views views ) Lateral Supplementary – d h 20 d applies to ) than in 5 sef NH ) O a egr2b µ µ 1 M ICD M BCI 9 l a and e . Thus BCI

), BCI ( BCI ), d biology

, NH O reasoned reasoned f pathway, ). mutant mutant ) as

rescue rescue spry4

d – b

h ), ),

. -

© 2009 Nature America, Inc. All rights reserved. and substrate binding has little consequence for catalytic rate for catalytic consequence has little binding and substrate and Dusp4 (refs. 23–25). In contrast, Dusp5 is constitutively activated, below) shown as BCI, to sensitive is (which Dusp1 family,including Dusp the of members for several described been has activity catalytic catalytically stimulated by ERK interaction is to and binding subject can substrate be activity Dusp6 phosphatase nature CH nature binding. substrate with associated Dusp6 of activation overexpression Dusp5 not but Dusp6 rescue to BCI of ability the in noted we difference the different quite are activities catalytic and their conserved, highly pERK are dephosphorylate can Dusp5 and Dusp6 both Although ( by overexpression Dusp5 caused phenotype the in reversing no effect with observations to zebrafish of expression ectopic and (pERK), ERK activated dephosphorylate way same the signaling FGF suppress could it whether investigated and zebrafish characterized first we Dusps, other inhibit could BCI Dusp6 ( of higher ( to sion ( spry4 ( in XFD receptor negative dominant the of and Dusp6 and phenotypes generated by ectopic expression of the FGF inhibitors Spry4 Fig. Supplementary dusp6 spry4 e c b a dusp6

3n or Fig. S E T Fig. , or or Fig. P XFD o -injected embryos rescued rescued embryos -injected

P 3g ). These observations indicate that BCI is specific for Dusp6. suppresses FGF signaling FGF suppresses XFD

E 3h

– 3k Tg(dusp6:EGFP);ace MIC into 1-cell-stage zebrafish embryos decreased o Tg(dusp6:EGFP) , and DMSO DMSO , o o inhibited inhibited

mRNA, which suggests that BCI directly inactivated inactivated directly BCI that suggests mRNA, which

). In contrast, BCI treatment did not reverse the effects and A FGFR

FGF ERK Fig.

L BIOLOGY L

Supplementary FGFR d dusp6 Supplementary

1a P in vivo in P ) sef ME 16,18 sef XFD mRNA microinjections, BCI had little or or little had BCI microinjections, mRNA K Dusp6 transcription ( transcription Dusp5

. Injection of mRNA encoding encoding mRNA of Injection . suggests that BCI might suppress the the suppress might BCI that suggests Spr advance online publication online advance y4

Fig. BCI BCI sef 20,21

Fig. expression to control levels or or levels control to expression

1c . Dusp5 has been shown to shown been has Dusp5 . Uninj Uninj Uninj o l j i g f 22,23 ). The addition of 5 Scoring of sef expression (%) 100 1d 50 0 Fig. ). To whether determine Uninj. . . . . This substrate-induced – – n

3m = 200 dusp WT sef , 6 n

o inj. = 12 )

ex 2 0 – + 2

n p. . In contrast contrast In . = 15 n m dusp5 spr dusp6 sef

zebrafish zebrafish XFD 7 y4 2  expres n 2

M BCI inj. = 10 dusp6 . Thus inj. (--) dusp5 inj inj

22,23 – + . 5 .

n sef = 15 -

. , ex

dusp 4

n p. 5 = 186

suppress the phosphatase activity of several related . phosphatases. related several of activity phosphatase the suppress could BCI whether determined we selectivity, low exhibit inhibitors ( Dusp6 inhibited directly BCI that conclusion the ity, supports which ( orthovanadate tion as effectively as the generic tyrosine phosphatase inhibitor sodium dephosphoryla pERK2 Dusp6-mediated BCI prevented of Addition ( pERK-specific with immunoblotting by pERK2 dephosphorylated completely Dusp6 in an activity Dusp5. not but Dusp6, and Dusp1 inhibits specifically BCI that systems biological chemical the in ( assays activity ­complementation Dusp1 or Dusp6 block not did ICD with ( embryo zebrafish the in centrations FGF of activation 2.8 ± 4.0 ± 11.5 12.3 and were experiments independent six from inhibition (IC concentration like inhibitory activity, ( binding catalytic substrate by whose induced is Dusp1, Dusp6, human inhibited also ( BCI function Dusp6-Myc BCI suppresses that directly suggests which addition, TPA after levels pERK restored ( abolished ( Dusp6-Myc expressing cells in whereas cells, in untransfected led to this staining pERK strong was activated; ( to pERK inj. Fig. k h We next investigated whether BCI could directly inhibit Dusp6 Dusp6 inhibit directly could BCI whether investigated next We dusp6 dusp5 spr – + n = 167

(–) y4 4 f

spr inj. sef , lane 5). Because many known small-molecule phosphatase phosphatase small-molecule known many Because 5). lane , inj. inj. y4

n +BCI +BCI +BCI = 84 ex

Fig. inj. Fig. p. +

in in vitro n

= 133 4c

BCI 4 Fig.  , c d M, respectively, which is consistent with hyper with consistent is which respectively, M, ). BCI treatment of Dusp6-Myc–transfected cells cells Dusp6-Myc–transfected of treatment ). BCI

). Upon TPA treatment, the RAS/MAPK pathway pathway RAS/MAPK the ). TPAUpon treatment, signaling and d2EGFP expression at these con these at expression d2EGFP and signaling

4 pERK2 dephosphorylation assay. Recombinant dephosphorylation pERK2 f , lane 4 and 6). ICD did not activ block Dusp6 sef situ in showing embryos shield-stage ( signaling. block to act Dusp and Spry4 XFD, where showing pathway ( present not are ligands FGF where arrows), (red MHB the within d2EGFP of induction exhibit not did (10 BCI with treated Tg(dusp6:EGFP) Tg(dusp6:EGFP) ( Dusp6. of expression ectopic inhibits BCI 3 Figure bodies to c-Myc ( c-Myc to bodies (TPA, stimulated 12- with (Dusp6-Myc), Dusp6 human with Myc-tagged were transfected transiently assay plementation in com this a chemical hypothesis cell-based We tested Dusp6. overexpressing cells in els lev pERK restore should BCI pERK, rylates expression in green. Scale bars, 250 250 bars, Scale green. in expression (--) absent and yellow in expression (–) weaker blue, in expression (WT) normal denote that bars color by represented is embryos injected results. treatment ( with injected embryos in not in expression ( embryos control (uninj.) uninjected in that to compared spry4 a k – , ie ta Ds6 iety dephospho directly Dusp6 that Given Fig. n expression domain. Injection (inj.) of of (inj.) Injection domain. expression d ). ( ). )

or or Lateral views of 30 h.p.f. embryos. embryos. h.p.f. 30 of views Lateral hybridization. Red brackets demarcate demarcate brackets Red hybridization.

o 4 4 ) Graph depicting injection and BCI BCI and injection depicting ) Graph 50

e dusp5 d O ad muotie wt anti with immunostained and ) BCI activity require FGF ligand, and and ligand, FGF require activity BCI f ). Taken in we together, have shown ). ( ). ) values for DUSP6 and DUSP1 DUSP1 and DUSP6 for values ) , -tetradecanoylphorbol-13-acetate -tetradecanoylphorbol-13-acetate i , l e ). BCI treatment restored restored treatment BCI ). dusp6 ) Schematic of FGF/RAS/MAPK FGF/RAS/MAPK of ) Schematic Fig. reduced reduced pt6 pt6 Sef Fig. ;ace ( embryos

4 -injected embryos ( embryos -injected Fig. Fig. e  26,27 expression in mRNA- in expression ). In contrast, treatment treatment ). In contrast, M). The The M).

Fig. 4 4 embryos ( embryos f in vitro in – sef

a

4a n 4 ), ), pERK in which HeLa cells cells HeLa which in ) Lateral views of views ) Lateral e expression ( expression , ) b t r a spry4 2 a d ) 5 sef , ace and antibodies antibodies and ). In this assay, assay, this In ). . Half-maximal Half-maximal . b Fig. as determined determined as ) and ) and expression by expression c mutants mutants or or

, staining was was staining d

) were ) were 4 L C I  dusp5 f

m. sef , lane 3). lane , h g ), but but ), , dusp6 j

, m

s e  ), ), M  , ­ ­ - - - - - ­

© 2009 Nature America, Inc. All rights reserved.  (100 ICD analog, ( results. similar with times six repeated was that experiment a single from quadruplicates of ± s.d. averages the are Data cells. vehicle-treated and Dusp-transfected to normalized were levels pERK Methods. the in described as statistics Kolmogorov-Smirnov using phosphorylation ERK for analyzed and immunostaining c-Myc on based identified were panel) (bottom Dusp1 or panel) (top Dusp6 of levels high expressing Cells cells. expressing ( ( Dusp6-Myc of levels high expressing cells in in cells (red high remained levels pERK BCI, of presence the In yellow). in ( in cells (green Myc in cells (red phosphorylation ERK induced to TPA-stimulated were Dusp6-Myc with transfected transiently cells assay. HeLa complementation chemical ( assays. dephosphorylation pERK2 and complementation chemical both 4 Figure ( phos interactions Dusp5 favorable less the of energetically in resulted region domain phatase same the onto BCI of docking ( result, Dusp5 in accessible not is binding BCI dates accommo that Dusp6 in crevice particular the that showed Dusp6 and Dusp5 of superposition Structural Dusp5. not but Dusp6 block can BCI how understand to structures crystal phosphatase two the ( family phosphatase this of members certain of expression ( data activity with Dusp6 ( Dusp1 showed that BCI-Dusp1 interactions were comparable to those ( loop and the side chains of Trp264, Asn335 and Phe336 was predicted acid general the of backbone the with BCI of interaction close a site, or Asp262, Cys293 residues Arg299. At binding putative this catalytic helix and loop acid eral gen the between crevice a within fit preferentially to predicted was (ANM) model network ­analysis anisotropic by generated conformations them by was assessed further flexible docking ( forms high-activity and low- the both on sites binding potential of of analysis cluster From (Tripos). ORCHESTRAR using modeling logy 1MKP) identifier low- the Dusp6: activity form of determined by X-ray crystallography (Protein Data conformations Bank different two onto docked was ( sites binding BCI potential identify simulations docking unbiased perform to us enabled structures been six-stranded or five- have a domains catalytic Dusp determined several of structures Crystal Dusp6 to docking BCI predicts modeling Computational ( phosphatases MAPK of set a to limited is BCI of specificity indicates the that which activity, Dusp3/VHR or 1B) phosphatase tyrosine (protein PTP1B 25B), cycle division (cell Cdc25B block not did BCI t r a e d c Supplementary Fig. Fig. Supplementary

) Quantification of pERK levels in Dusp- in levels pERK of ) Quantification outlined shown a are in shown cells ; green ;

In the zebrafish microinjection assays, BCI inhibited ectopic ectopic inhibited BCI assays, microinjection zebrafish the In

the resulting BCI-bound conformations, we identified a number number a identified we conformations, BCI-bound resulting the green cells seen in b are outlined in yellow). yellow). in outlined b are in seen cells green

5 5 a b

and and L C I 3 ). Further docking simulations using a homology model of of model homology a using simulations docking Further ). BCI directly inhibits Dusp6 in Dusp6 inhibits directly BCI 4 and homology modeling ( modeling homology and c 28–31 , Supplementary Supplementary d Fig. ). In cells overexpressing Dusp6- overexpressing cells In ). s e f dusp6 ) pERK2 ) pERK2 . In each case, the phosphatase domain encompasses encompasses domain . In case, phosphatase the each 

Fig. M), was inactive (lane 5). Scale bar, 25 Scale 5). (lane inactive was M), 3

a 4 Fig. 1 ), pERK was abolished abolished was pERK ), e and the high-activity form obtained by homo by obtained form high-activity the and ). but not not but

2

). 4b  in vitro in  -sheet surrounded by five five by surrounded -sheet a 7, rather than interacting directly with the the with directly interacting than rather 7, ). The relative positions of Asp262 and and Asp262 of positions relative The ). – d ) Cell-based ) Cell-based

Fig. Fig. dephosphorylation assay shows that BCI (100 (100 BCI that shows assay dephosphorylation dusp5 d ), even even ),

4a 3 ). The most favorable site among among site favorable most The ). ), thus our rationalizing observed Supplementary Supplementary , exhibiting specificity toward toward specificity exhibiting , 3 3 using Dusp6 multiple Fig. Fig. e Dusp6-m a

pERK in Dusp-expressing 3  ). We compared ). Wecompared

cells (fold over control) 5 Methods Methods 10 -helices. These These -helices. 0 5 c  ) m. 0. 22,23,29 yc 0 0 Dusp6 log .5 ). BCI ). BCI ). BCI . As . µ 3 M compound 2 to DMSO 1.

0 a ­ - - - 1. that their basal activities are determined by the relative location of of location relative the by determined are activities basal their that suggests which the two of structures, superposition 5 Å optimal after the in Asp232 these catalytic residues ( residues catalytic these by substrate binding. substrate by that prevents the catalytic stimulation of phosphatase activity induced Fig. by suppressed ( BCI of the (30% addition inhibition) the assay activation resulted in a sevenfold enhancement that was also ( inhibition) enhancement was inhibited in substantially the presence this of BCI (57% and threefold, OMFP of dephosphorylation Dusp6 stimulated ( activity Dusp6 inhibited that concentration dephosphorylation ERK a at Indeed, OMFP. phosphatase toward Dusp6 basal activity block not would BCI that suggests This ( residues core the catalytic with interfacing OMFP with site, active phosphatase the within bind simultaneously could OMFP and BCI that predicted simulations Docking ERK2. of (OMFP, phosphate rescein dephospho 3- substrate, phosphatase the a of small-molecule rylation measured we predictions, modeling these test To Dusp6 of activation ERK2-mediated inhibits BCI binding. ERK by induced tion Such residues. activa Dusp6 catalytic to inhibit are likely motions on functional other constraints preventing the with thereby Asp262 loop, of this interaction of the flexibility the blocks form effectively low-activity the in crevice accessible the to binding BCI that ( loop to closer to the loop Asp262 acid phosphatase ent position its general that showed analysis to reori tendency suitably structure-induced has Our Dusp6 an intrinsic, loop. acid general conforma the at induce changes that tional modes ANM the on focusing particularly by dynamics Dusp6 explored we mechanistically, Dusp6 on action binding ERK upon activation catalytic high- activity, but a suppressed phosphatase basal did not abolish asparagine creating thereby loop, ­activity reorients phosphatase that the shift toward conformational Asp262 a induces Dusp6 to binding 2 5 Dusp6-m b  M) specifically suppressed Dusp6 activity (lane 4), whereas a related a related whereas 4), (lane activity Dusp6 suppressed specifically M) IC BCI

5c advance online publication online advance .0 D Supplementary ). These data suggest that BCI is an allosteric inhibitor of Dusp6 yc pERK in Dusp-expressing cells (fold over control) 10 15 Fig. 0 5

0. respective phosphatases Dusp6 and Dusp5 differ by by differ Dusp5 and Dusp6 phosphatases respective 3 0 Dusp1 Supplementary

5 5 . In support of this model, mutation of Asp262 to Asp262 of mutation model, this of .support In d BCI log ). Increasing the ratio of ).ERK2 to in of the (10:1) ratio Dusp6 Increasing 0. 1 5 µ M compound

c pERK Movies Fig. .0 in vitro in 5 ), by Dusp6 in the presence and absence absence and presence the in ), by Dusp6

1. 5 2 5 c

1 )

( Fig. 2

IC BCI and 9 Fig. . It was postulated that substrate substrate that postulated was It . D .0

5b pERK

f Dusp6

2 3 4 nature nature DMSO 1 ) f ). Addition of ERK2 protein protein ERK2 of ). Addition . To 3 ), BCI did not inhibit basal basal inhibit not did BCI ), 6 2 . Therefore, we proposed proposed we Therefore, . + – 6 5 4 3 2 1 Supplementary

further understand BCI understand further d pERK + – ch + + e mic Supplementary + + BCI O + + -methylfluo a ICD l

+ + biology Na

Fig. 3 VO pERK ERK2

4 BCI

5a 2 ). - - - - ­

© 2009 Nature America, Inc. All rights reserved. nature CH nature ( embryos BCI-treated in cardiomyocytes in increase an showed ( embryos untreated control to compared as hand2 ( populations progenitor cardiac processes. Dusp the of members developmental related block potentially could BCI later that Given in these biologi for the requirement the blocks probe to us allowed that Dusp1 and Dusp6 of molecule activity relevant small cally a of identification The size heart regulating in FGF and Dusp6 of The role Dusp6 from the gastrula stage to the somitogenesis stage resulted in cardiac expansion. Scale bars, 250 250 bars, Scale expansion. cardiac in resulted stage somitogenesis the to stage gastrula the from Dusp6 of expansion to correspond ( DMSO control with treated embryos in hearts show outlines Red hearts. enlarged had 8 h) for epiboly 40% (from ( respectively. domain, ( treatment expanded ( indicated. as BCI with treated embryos of 6 Figure c a acid loops General (red) expression in BCI-treated embryos. There is reduction of of reduction is There embryos. BCI-treated in expression (red) Asp262

f c a Dusp6 and FGFs regulate heart size. ( size. heart regulate FGFs and Dusp6 gata4 a ); blue box and brackets mark ALPM and caudal cardiac cardiac caudal and ALPM mark brackets and box blue ); E MIC expression in the caudal ALPM as compared to DMSO DMSO to compared as ALPM caudal the in expression A L BIOLOGY L loops Phosphatase c Asp232 , d ) BCI increased increased ) BCI vmhc d g b d

(compare (compare ) as compared to DMSO control treatment ( treatment control DMSO to compared ) as advance online publication online advance acid loop General d Asp262

RFU ×1,000 Cys293(Ser) b nkx2.5 10 20 30 0 a k e , 1 0 to to ). Red and green brackets show expression domains. ( domains. expression show brackets green and Red ). b ) BCI treatment ( treatment ) BCI α a l 4 2 0 Tr – ) and ) and expression and expanded expanded and expression Leu26 h p264 ) ) e h Phosphatase loop 3 0 In situ In Arg299 No Dusp + ERK&BC + ER No ER 8 Time (min cmlc2 Cl 0 K BC 40 K hybridization hybridization α Phe33 I 6 5 (compare (compare 50 ) I b h Asn33 ) 6 60 ), as compared to DMSO treated embryos ( embryos treated DMSO to compared as ), α 7 70 5

scl m 80 expression with a concomitant expansion of of expansion a concomitant with expression o l k to to i Percentage with -

c n expanded hearts 25 50 75 ). ( ). ) staining (red arrowheads). ( arrowheads). (red ) staining Methods dusp5 several of Expression fish. other of expression the examined we family, nm. 485/525 of wavelengths excitation/emission at measured were (RFU) units fluorescence Relative BCI. by blocked was OMFP,of induction this and dephosphorylation ( (Dusp6-Asp262). counterpart Dusp6 its than loop) (phosphatase pocket catalytic the to 5 Å closer approximately positioned is Dusp5-Asp232, acid, catalytic the that showing structures ( chains. side Phe336 and Trp262, Asn335 with interacts BCI backbone, loop acid general to addition In interactions. green) (lime BCI of view up ( (green). ion a chloride by distinguished is cavity catalytic The circled. are site active the of vicinity the in Clusters differently. colored is solutions close spatially of a cluster to corresponding pose docking Each domain. phosphatase Dusp6 of conformation low-activity ( mechanism. inhibition allosteric an 5 Figure ing; knockdown with antisense morpholino oligonucleotides results results oligonucleotides morpholino antisense with knockdown ing; FGF signal of regulator is an Dusp6 important embryogenesis, early In has a development. role in heart phosphatase this that ing the idea support thus mesoderm, plate lateral anterior the within expressed ­functions as a feedback regulator and of signaling FGF/MAPK/ERK is C. these, Of Thisse, to only direct submission Dusp6 http://zfin.org/). perturbation of their biological targets. biological their of perturbation transient and rapid to owing development of stages later at analysis development later in Dusp6 of role the of study the precludes which defects, polarity embryo in 0

e DMSO , f ) Fluorescent double double ) Fluorescent

Gastr n = 40 and

5 c Modeling of BCI-Dusp6 interactions and and interactions BCI-Dusp6 of Modeling ) Superposition of Dusp5 (yellow) and Dusp6 (white) crystal crystal (white) Dusp6 and (yellow) Dusp5 of ) Superposition

ula stage µ M and

n dusp22a

BCI = 41 10

Supplementary µ M n = 40 g DMSO (see in detail , described has been , h ) )

1-somite stage n cmlc2 = 19 in situ in  5 m. o dusp g µ ) Graph showing that temporal inhibition of inhibition temporal that showing ) Graph n m

). ( ). M n BCI expression in 18-somite-stage embryos embryos 18-somite-stage in expression = 20 1 j 8 hybridization showing showing hybridization

Fig. i 10 permit molecules small contrast, In . , genes, including including genes, j

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© 2009 Nature America, Inc. All rights reserved. The zebrafish embryo offers distinct advantages over traditional traditional over advantages vitro in distinct offers embryo zebrafish The DISCUSSION size. heart increases ultimately that progenitors myocardial of expansion an induce can inhibitor small-molecule a by Dusp6 of sensitive ( be signaling to Fgf8 to known is which tissue, ventricular for notable larly ( hearts (  ( stage later the at treated embryos in reduced was frequency ever, the how points; time both at hearts stages. We larger 8-somite observed Dusp6 activity limits heart organ size, we treated embryos at the 1- and in cardiac tissue ( 56 reached h.p.f. we larvae, In noted a expansion BCI-treated marked washout the next day. Embryos were then further incubated until compound they by followed DMSO, or BCI were with epiboly Embryos 40% at stage. treated larval at embryos treated analyzed we tissue, heart in increase an in resulted progenitors cardiac of expansion the diomyocytes (compare 2 chain stage 18-somite by an increase in cells positive for n expan concomitant cardiac of sion the with coupled was lineages hematopoietic and endothelial of loss The shown). not (data embryos BCI-treated n (compare progenitors endothelial or blood of that activation of FGF signaling expanded cardiac tissue at the expense Dusp6 resulted in a marked reduction in from embryos the of stage. ALPM in 1-somite Inhibition BCI-treated endothelial and blood lineages located within the rostral domain of the of expression analyzed size organ heart determines that progenitors on cardiomyocyte interaction occurs between the vascular and hematopoietic precursors other of expense lineages. Recent have studies shown that a repressive expansion of cardiac progenitors, it was not clear if this event was at the cursors (compare DMSO-treated embryos, which confirms a specific effect on heart pre to compared as pools progenitor cardiac expanded showed embryos the 10-somite stage. of Examination at situated are progenitors cardiac where to corresponds ALPM the pare embryos, a caudal expansion of size. In organ BCI-treated heart and progenitors cardiac restricts and signaling FGF limits Dusp6 whether test to BCI used tissue we Therefore, cardiac expanded stages somitogenesis during (Fgfr1) tor recep FGF activated a constitutively of expression size, ectopic heart agreement with the notion that FGF signaling plays a role in stipulating an an harboring embryos In development. heart zebrafish in Fgf8a for role by encoded factors transcription the express to begin and MHB) the behind just located (ALPM, mesoderm plate lateral anterior the as known tions genitor to cells undergo cellular migration form two popula bilateral embryo blastula-stage the of by layertor 5 can that cells be identified h.p.f.the mesodermal within progeni cardiac of group heart. small a from the develops heart of zebrafish The formation and patterning alter would activity Dusp6 t r a Fig. 6 vmhc = 15). Likewise, = 19). This surplus of cardiac progenitors was also noted at the the at noted also was progenitors cardiac of surplus This 19). = Using BCI as a chemical probe, we investigated how inhibiting inhibiting how investigated we probe, chemical a as BCI Using fgf8a Fig. 6 ) ) and o ( Fig. ). L C I and cell-based chemical screens. With the generation of of generation the With screens. chemical cell-based and mutation, both atria and ventricular cells are and mutation, reduced both ventricular atria myl7 In situ a

to to cmlc2 6l nkx2.5 , or or , , s e n hand2 Fig. analysis with probes for and and Fig. confirmed that treated embryos exhibited enlarged enlarged exhibited embryos that treated confirmed Fig. cmlc2 etv2 Fig. and and

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), a gene that is in expressed = 11). Though we noted an 7 expression, which suggests gata4 expression in BCI-treated ). Expansion was particu was ). Expansion n Fig. 6 = 16). To test whether caudal expansion of of expansion caudal cardiac cardiac myosin light e to Fig. 6 Fig.

6 42,43 f f ; 93%, ; 32%, 4 4 . We . In 4 2 ------.

context, no specific Dusp6 inhibitors with with inhibitors Dusp6 specific no context, assays do not faithfully recapitulate phosphatase activity in a bi used inhibitors mice in hearts enlarged with conserved likely was Dusp6 of recently found to is as cause other vertebrates, disruption size organ heart controlling in Dusp6 for this during role occur.to The morphogenesis regulated cardiac proper for allow to tightly period be must signaling FGF that suggests The in increase resulted cardiac progenitors in hearts, which enlarged lineages. at endothelial of field the the cardiac expense BCI expanded progenitors cardiac increased in signaling, resulted FGF which of activation global of studies with consistent were factors to begin transcription be ­specific expressed. The effects of BCI of Dusp6. of activation ERK-induced the suppresses BCI that Subsequent phosphatase assays signaling. and docking simulations provided FGF strong evidence enhances that molecule small a BCI, identified we compounds, 5,000 approximately of screen est that evoke nonspecific effects on embryo differentiation. From a mod molecules and toxic compounds of elimination the for allow screens pathway this modulate that pounds com new for screen to possible is it lines, reporter FGF ­transgenic AGCATAGATTGCCG-3 5 forward, Dusp5 primers: following the with zebrafish h.p.f. 24 from RT-PCR by amplified were frames reading described were as studies previously generated Zebrafish phenotype. same the exhibited embryos treated five the of four least at assay, and the of ducibility to times three show the repro was repeated ture the Each experiment images. cap to used were (Qimaging) camera Exi Retiga the and software Qimaging (Leica) microscope and fluorescentMZFLIII a illumination for using GFP using endow picture cube (Chroma).each for magnification and gain for exposure, settings same the under photographed were embryos Treated embryos. was as identified a compound that enhances fluorescence in treated NSC150117) as known study. (also this BCI in screened were ChemDiv from set a phosphatase-targeted and Systems Discovery MicroSource from library 10 at library each from compound with along added was solution DMSO (v/v) CaCl mM KCl,0.33 mM 0.17 NaCl, mM 200 (5 in plate 96-well a of well trans each Five into h.p.f. placed were 24 embryos reached genic they until °C 28.5 at incubated and crossings Care Animal Institutional Pittsburgh Committee. Use and of University the by approved and Zebrafish METHODS regeneration and repair are on such involving wound as FGF dependent signaling, conditions that diseases of for treatment agents new provide potentially it could and development, in Dusps of role the probe to compounds specific highly offer should This function. Dusp substrate-induced block to it should be to new design possible based on molecules rationally BCI ity activ biological lacked have or activity from promiscuous exhibited have identified Dusp1 targeting molecules inhibitors Small phosphatase small-molecule specific of identification pockets. catalytic features These structural have further hampered the all phosphatases, tyrosine and havestructures crystal revealed shallow identified

Previous Previous large-scale high-throughput screens for Dusp6 and Dusp1 role the probe directly to us allowed BCI of identification The 4 Dusp6 in heart formation during a critical period when cardiac- when period critical a during formation heart in Dusp6  7 M. The US National Cancer Institute diversity set, a natural products products natural a set, diversity Institute Cancer National US The M. . However, with the identification of a chemical Dusp inhibitor, inhibitor, Dusp chemical a of identification the .However, with advance online publication online advance

chemical mRNA 4 5 . The phosphatase catalytic site is highly conserved across across conserved site is highly catalytic . The phosphatase in in vitro

microinjection.

screens. Tg(dusp6:EGFP)  ; Dusp5 reverse, 5 reverse, Dusp5 ; assays with artificial substrates. Because these these Because substrates. artificial with assays 6 48,49 . All procedures involving were zebrafish reviewed .

dusp6  -AACTCGAGGCCATGAAGGTCTCC pt6 and and in vivo in embryos were obtained by natural natural by obtained were embryos

 nature nature 2 -AATCTAGATTAAGGCAGCGC , 0.33 mM MgSO mM 0.33 , XFD 1 8 . open and Both Dusp5 Spry4 . In addition, live embryo embryo live . addition, In in vivo in mRNA for microinjection microinjection mRNA for ch 4 activity have been been have activity 2 e . Treatment with with Treatment . mic n vitro in 4 a ), and a 0.5% 0.5% a and ), l biology

transgenic transgenic screens screens o n vitro in  logical logical l of E3 E3 of l 4 6 ------.

© 2009 Nature America, Inc. All rights reserved. nature CH nature UK) Research (Cancer Keyse S. by c-Myc–Dusp6 provided kindly Human was Inc.). (pSG5-PYST1) Technologies, (Life penicillin-streptomycin (v/v) 1% and (HyClone), serum bovine fetal (v/v) 10% with CO supplemented 5% of atmosphere humidified a in maintained and described as essentially out carried Chemical for stage shield at fixed and stage 1,000-cell the at were Embryos 500 with pg mRNA injected at previously. the or 1-cell stage, 2-cell 5 with treated described as synthesized were mRNAs and fied, GTTTTTCTGGCTGAC-3 GTTCCTCACCACATTC-3 AGTTATTGGACTC-3 M.T. wrote the paper help with from A.V., A.B., T.E.S., J.S.L., B.W.D. and I.B. A.B., T.E.S., J.S.L., I.B., B.W.D. and M.T. and experiments designed analyzed data. G.M., A.V., A.B., P.Q.O., W.D., W.Z. and M.T. experiments. performed G.M., A.V.,AUTHOR Fund. Discovery Drug Fiske by the and CA78039, and MH074411 CA52995, HD053287, grants by NIH supported also was work This (NIH). Health of Institutes National the or Institute Blood Lung, and Heart, National the of views official the represent necessarily not does and authors the of responsibility the solely is content The Institute. Blood to Lung, M.T.and Heart, National US the from R01HL088016 number by award part in supported was described project The compounds. individual of samples and set diversity Institute Cancer National the providing for Institute) Cancer National US Program, Therapeutics (Developmental R. Schultz We syntheses. the in thank Weassistance for M.S. Poslusney manuscript. thank the of reading critical for I. and Dawid We M.N. Rebagliati Hukriede, thank Ackn on the Note: (ref. 1MKP code accession under study previous a of part as deposited Accession in listed are BCI of and and assays, synthesis chemical modeling related analogs molecular Other vehicle. with treated and or Dusp1 with value Kolmogorov-Smirnov from Dusp6 four wells transfected Kolmogorov-Smirnov values for each condition were normalized to the average in ERK the phosphorylation Dusp-expressing cells after compound treatment, of restoration To activity. quantify Dusp of suppression indicate and control denote levels large compared differences in with vehicle ERK phosphorylation values Kolmogorov-Smirnov High cells. vehicle-treated and Dusp- Myc–expressing from function distribution cumulative reference a to compared and condition each for phosphorylation. established was function ERK distribution cumulative for pERK A analyzed and immunostaining c-Myc on based wells control vehicle-treated and DUSP-transfected using previously described as statistics Kolmogorov-Smirnov by quantified was by Dusp6 overexpressing phosphorylation cells in ERK BCI of Restoration (Cellomics). reader high-content IIArrayScan Compartmentalan Analysisbioapplicationthe on using staining nuclear by defined area an in intensities c-Myc–DUSP and pERK for analysis multiparametric three-channel by analyzed were Plates (c-Myc). Alexa-488 and (pERK) AlexaFluor-594 to conjugated antibodies secondary with alized Santa Cruz Biotechnology). Positive pERK and c-Myc–DUSP signals were visu dilution, to c-Myc (1:100 Technology) antibodies and Signaling Cell dilution, permeabilized and immunostained with a mixture of formaldehyde, antibodies (w/v) to4% in pERK 33342 (1:200 Hoechst with stained and fixed were Cells ml ng TPA(500 with min 15 for stimulated and ICD or BCI of gradients for wells 15 were ten min in with treated concentration twofold quadruplicate described as c-Myc–Dusp1 or c-Myc–Dusp6 and 384-well plate (Falcon Biocoat) in the presence of FuGene 6 (Roche Biosciences) in ­mammalianexpression cellsectopic for pcDNA3.1 into subcloned was Dusp1 Full-length Amplified PCR products were subcloned into pCS2+ and sequenced veri sequenced and pCS2+ into subcloned were products PCR Amplified Supplementary

owledgme

N methods. a t

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complementation codes. r O e Supplementary

2 E N C 7 MIC h TRIBUTIO . 1,000 individual cells were gated for Dusp–Myc expression expression Dusp–Myc for gated were cells individual 1,000 . e 5 Detailed materials and methods for for methods and materials Detailed m 0 : The low-activity form of Dusp6 was was Dusp6 of form low-activity The Bank: Data Protein . HeLa cells (2,000) were plated in the wells of a collagen-coated i informatio A c n a L BIOLOGY L ts l

B  ; Spry4 forward, 5 forward, ; Spry4 i o l N o  g . S  y

; Spry4 reverse, 5 reverse, Spry4 ; Methods

n

w assays and e b s

i t e advance online publication online advance chemica 2 .

7 in . HeLa cells were obtained from ATCC from obtained were cells HeLa . .

HeLa  -ACTCGAGCCATGGAGTCAAGG l l compound informati 

cells. -AATCTAGATCATGAGGCTT 2 7 in situ in . cells culture, h in 20 After These experiments were were experiments These in vitro in hybridization. 2 at 37 °C, DMEM DMEM °C, 37 at o n phosphatase phosphatase is available  M M BCI 31).

–1

5 ). 0 - - .

31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturechemicalbiology/. 16. 15. 14. 13.

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