Small molecule-mediated disruption of Wnt-dependent ... · either Shh or an activated Notch (NICD)...
Transcript of Small molecule-mediated disruption of Wnt-dependent ... · either Shh or an activated Notch (NICD)...
FL < -3xSDRL > -3xSD
FL > 4x SD; 0.8 uM-2x SD < RL > 2xSD; 0.8 uM-2x SD < RL > 2xSD; 7.5 uM
-1x SD < FL > +1xSD; 247 removed-1x SD < GL > +1xSD; 17 removed
FL/RL > 80% control
FL/RL > 80% control
Wnt-GL < 60% control
FL/RL < -5xSD FL/RL > -1xSD
198,080 compounds
Primary screen (2.5uM) Dose-dependent test(0.8, 2.5, 7.5 uM)
2,319 inhibitors
385 inhibitors
FL inhibitor and protein exocytosis test
(2.5 uM)
121 inhibitors
62 inhibitors of Wnt response (IWRs)
5 IWRs
59 inhibitors of Wnt production (IWPs)
4 IWPs
Wnt secretion test(2.5 uM)
Exogenous Wnt test (2.5 uM)
Hh pathway test(2.5 uM)
Notch pathway test(2.5 uM)
SHH
RLRL
RL
FLFL
RL
Shh pathway activity = FL activityGeneral cell health = RL activity
Culture medium
Cytoplasm
CMV
SHHpRK
SHH
SV40
RL
8xGLI
FLSV40 -RL
8xGliBS -FL
SHH
SHH
CMV
Wn
t3A
pRKWnt3A
SV40
RLSV40-RL
8xTCF
FLSTF
Wnt3AWnt3A
Wnt3A
RLRL
RL
FLFL
RL
Wnt pathway activity = FL activityGeneral cell health = RL activity
Culture medium
Cytoplasm
L-Wnt-STF cell line
CMV
GLCMV
-GL
CMV
FLCMV- FL
GLGL
GL
Exocystosis = GL activity
FLFL
FL
FL
Culture medium
FL inhibitor/exocytosis test
Hh pathway response assay
RLRL
RL
FLFL
RL
General cell health = RL activityNotch pathway activity = FL activity
NICD
NICD
SV40
RL
CMV 4xCBF
NIC
D
FLCBF -FL
SV40 -RL
NICD
Notch pathway response assay 55 IWPs
55 IWPs
16 IWRs
Consistutive FL activity used to identify FL inhibitors
Supplemental Figure 1. Identification of small molecule antagonists of the Wnt/β-catenin signal transduction pathway. The ~200K com-pound UTSouthwestern chemical library was screened using a cell line with constitutive Wnt/β-catenin pathway activity maintained by cell-autonomous Wnt3A protein production (L-Wnt-STF cells; Primary screen). Potential Wnt/β-catenin pathway antagonists were identified using a stably transfected Wnt-responsive firefly luciferase (FL) and control Renilla luciferase (RL) reporters. Approximately 1% of the compounds in the library that scored as hits was tested again in a dose-dependent manner to identify the most potent compounds with minimal cellular toxicity (Dose-dependent test). Compounds that abrogated FL activity by inhibition of FL activity, or that generally blocked cellular secretion of proteins were removed (FL inhibitor and exocytosis test). To separate compounds that either inhibit Wnt/β-catenin pathway response or Wnt3A protein production, compounds were tested in HEK293 cells using the same assay as described in the Primary screen with the exception that exogenous Wnt3A protein (provided in conditioned medium) is used to stimulate pathway response (Exogenous Wnt test). Compounds that retained their anti-pathway activity in this test were considered Inhibitors of Wnt response (IWRs), whereas those that did not were considered Inhibitors of Wnt production (IWPs). Compounds from both categories were tested for effects on two other stem cell-associated signal transduction pathways (the Hh and Notch pathways) using cultured cell-based assays similar to those used to identify Wnt/β-catenin pathway antagonists (Hh and Notch pathway tests). Hh and Notch pathways were stimulated using either Shh or an activated Notch (NICD) cDNA construct, respectively. Those compounds that minimally impacted these two pathways were considered to have specific activity for the Wnt/β-catenin pathway. Lastly, IWPs were directly tested for their ability to inhibit Wnt3A protein secretion (Wnt secretion test; see Supp. Fig. 2b). Criteria for selecting hits are provided. In the end, five IWRs and four IWPs with high specificity for attacking the Wnt/β-catenin pathway were selected for further analysis (see Fig. 1). Concentration of compounds used in each test is noted. Insets show schematics of assays used in the screen and secondary tests with the utility of each luciferase signal. Criteria used to identify compounds of interest are noted for each test.
Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer
Supplementary Material
Baozhi Chen, Michael E. Dodge, Wei Tang, Jiangming Lu, Zhiqiang Ma, Chih-Wei Fan, Shuguang Wei, Wayne Hao, Jessica Kilgore, Noelle S. Williams, Michael G. Roth, James F. Amatruda, Chuo Chen, and Lawrence Lum
Nature Chemical Biology: doi: 10.1038/nchembio.137
.2
.4
.6
.8
1.0
1.2
1.4
DMSO IWP-2 IWR-1 DMSO IWP-2 IWR-1 DMSO IWP-2 IWR-1
Wnt 1 Wnt 2 Wnt 3a
0.01 ug 0.025 ug0.05 ug
DNA:
No
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ized
Wn
tp
ath
way
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A
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leve
ls o
f W
nt-
GL
in m
ediu
m
(% o
f co
ntr
ol)
IWP-1
IWP-2
IWP-3
IWP-4
DMSO
Wnt3A GLN C
Compound:
Culture medium
GL
Wnt-GL
C
1 2 3 4 5
Wnt pathway (HEK293; Wnt CM)
0.2
0.4
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0.8
1
1.2
Compound:
no
rmal
ized
FL
acti
vity
SHH pathway (NIH-3T3)
Notch pathway (HEK293)
Wnt pathway (L-Wnt-STF)
1 2 3 4IWR compounds IWP compounds
B
Supplemental Figure 2. IWR and IWP compounds specifically inhibit the Wnt/β-catenin pathway. (a) Summary of results relating to IWR and IWP compounds from the screening process. Wnt pathway tests were performed in either cells responding to autonomously produced Wnt protein (L-Wnt-STF cells) or exogenously provided Wnt in conditioned medium (HEK293 cells). (b) IWP compounds inhibit Wnt3A secretion. Left: schematic of Wnt-Gaussia luciferase (Wnt-GL) fusion protein used to monitor levels of secreted Wnt protein in the cell medium. Right: levels of Wnt-GL but not GL secreted from cells treated with IWP compounds are decreased as compared to cells treated with carrier. The Wnt-GL protein elicits levels of Wnt/β-catenin pathway response similar to that of Wnt3A protein (data not shown). (c) IWR and IWP compounds generally inhibit Wnt/β-catenin pathway response induced by Wnt proteins. Pathway activity induced by Wnt1, Wnt2, or Wnt3A, and monitored using the STF reporter, is decreased in cells treated with either IWR-1 or IWP-2. FL activity was normalized to control RL activity as before.
Nature Chemical Biology: doi: 10.1038/nchembio.137
IWR-5
IWR-4
IWR-3
IWR-3,4,5
Supplemental Figure 3. IWR-3, -4, and -5 share structural similarity. Three-dimensional representation of IWR-3, -4, and -5 in equilibrium geometry using AM1 semi-empirical methods reveals similarities in structure. All three structures are superimposed on the right.
Nature Chemical Biology: doi: 10.1038/nchembio.137
IWP-2
Supplemental Figure 4. Synthetic scheme for IWP-2. A similar synthetic route wastaken to generate IWP-1.
Nature Chemical Biology: doi: 10.1038/nchembio.137
DMSOIWP-1
(2.5µM)F H F H− −no
trea
tmen
t
LRP6
Actin
Deglycosidase:
C
pBSK ShhN ShhN ShhN-C25S ShhN ShhN ShhN-
C25S ShhN ShhN ShhN-C25S
Total lysate Aqueous DetergentPhase:
DNA:
IWP-2(2.5µM): ++ +− − − − − − −
fatty acyl ShhNfatty acyl and non-acyl ShhN
B
A
Wnt5b:IWP-2 (2µM):
+−
++
−−
Tota
lTo
tal
Tota
l
Aq.
Aq.
Det.
Det.
Supplemental Figure 5. Speci�city tests for IWP compounds. (a) IWP compounds inhibit palmitoylation of Wnt5A, a Wnt protein that does not presumably activate β-catenin (so “non-canonical” Wnts). (b) IWP compounds do not a�ect maturation of LRP6, a process dependent upon palmitoyl modi�cation of its cytotail domain. Lysate derived from DLD-1 cells treated with DMSO or IWP-1 was incubated with either Endo H (H) or PNGaseF (F), and then probed for LRP6 protein. IWP-1 does not alter LRP6 acquisition of EndoH resistance, suggesting it does not inhibit intracellular palmitoylation of LRP6. Sensitivity of LRP6 to PNGaseF con�rms that LRP6 is glycosylated. (c) IWP on phase separation of the N-terminal signaling domain of the Shh protein (ShhN), a protein palmitoylated by Hhat/Rasp, another MBOAT family member.
Nature Chemical Biology: doi: 10.1038/nchembio.137
A
250
150
10075
50
37
2520
15
10
control Porcn-Myc
IWP-PB: IWP-2:
--
--
+-
++
Transfected DNA:
B
(IWP-PB)
C
37
50
Column treatment: DMSO
IWP-PB
IWP-1: +−
IWP-PB
−
Porcn-myc DNA: +− + + +
N/A N/A
N/A N/A
Total lysate Pull-down
Supplemental Figure 6. Synthesis of IWP-PB and further characterization of its interaction with Porcn. (a) Synthetic route for a biotinylated IWP compound (IWP-PB). (b) Silver stain analysis of IWP-PB-bound material using identical conditions to those used inFig. 3g. (c) IWP-1 can compete for Porcn binding to the structurally distinct IWP-2. A similar experiment as described in Fig. 3g except IWP-1 is used for competition.
Nature Chemical Biology: doi: 10.1038/nchembio.137
DMSO IWP-2A B
N
SNH
O
S
N
N
O
S
0.2
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IWP-2
-v1
No
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Wn
t p
ath
way
re
spo
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IWP-2
-v2
IWP-2
-v3
N
SNH
O
S
N
N
O
S
IWP-2-v1
N
SNH S
N
N
O
S
O
IWP-2-v2
IWP-2-v3
N
SNH
O
S
N
N
O
S
IWP-2
IWP-2
DMSO IWP2 DMSO IWP2 DMSO IWP2 DMSO IWP20 µg 0.1 µg 0.05 µg 0.025 µgPorcn DNA:
C
Tubulin
Porcn
Supplemental Figure 7. Characterization of IWP action and speci�city. (a) IWP-2 does not induce destruction of Porcn. Levels of overexpressed Porcn increase in the presence of IWP-2. (b) IWP-2 does not appear to alter localization of Porcn. (c) Chemical structure and activity of several compounds related to IWP-2. IWP-2-v2 retains activity against the Wnt/β-catenin pathway as measured using the STF reporter (right), whereas IWP-2-v1 and –v3 do not, suggesting that the benzothiazole group is essential to IWP activity against Porcn.
Nature Chemical Biology: doi: 10.1038/nchembio.137
F Axin2ΔDAX Axin2 Axin2
Helacells
Cos-1cells
A
DMSO IWR1 DMSODNA:
Chemical:
Axin1-GFP: - - + + + + + + + +- -IWR-1 (10µM): - - - - - -+ + + + + +
Total Lysate IP Axin1 IP IgG
250
100
5050
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Axin1
Dvl2
CK1ε
GSK3β
PP2A
Axin1-GFP
LRP6-myc DNA:Axin2-myc DNA:
-++
++ + +
+ +
LRP6
Axin2
---
IWR-1(10µM): - - - -+ +- -
+ +
- -
- - - -+ + + ++ + + +- - - -
- + + + +- - -Total lysate IP LRP6
0 2 4 6.5
CHX
IWR-1CHX
Axin2
Tubulin
Axin2
Hrs:
Tubulin
DLD-1 cells
0 1 2 3Hrs:Axin1
β−catenin
Actin
MG132 (2.5µM)
IWR1 (10µM): - + - + - +Total lysate IP IgG IP β-cat
C
Axin2
β−catenin
B
1:31:3
D E
Supplemental Figure 8. Characterization of IWR/Axin interactions. (a) IWR inhibits Axin2 protein destruction. Lysate from DLD-1 cells treated for with IWR-1 for increasing time durations in the presence or absence of cycloheximide (100µM) was subjected to Western blot analysis. In the absence of protein synthesis, IWR is able to block degradation of Axin2 protein. (b) Chemical inhibition of the proteasome does not phenocopy the effects of IWR-1 on Axin1 protein levels. Lysate from L-Wnt cells treated with either IWR-1 or the proteasome inhibitor MG132 for increasing time durations were subjected to Western blot analysis. Consistent with the inability of IWR-1 to inhibit proteasome function, we do not observe accumulation of β-catenin in cells treated with IWR compounds (see Fig. 2). (c) IWR does not alter the affinity between Axin2 and β-catenin. A similar ratio of Axin2 protein from cells treated with either DMSO or IWR-1 is observed from both β-catenin immunoprecipitation and total lysate, suggesting that IWR-1 induces little or no change in the affinity of β-catenin for Axin2. (d) IWR does not disrupt interaction of Axin1 with known binding partners. Lysate from HEK293 cells transfected with control or Axin-GFP and treated with or without IWR-1 was subjected with immunoprecipitation using an Axin1 antibody. Bound material was Western blotted for proteins previously shown to interact with Axin protein. We were unable to observe interaction between Axin and PP2A. (e) IWR does not disrupt LRP6-Axin interaction. Lysate from HEK293 cells transfected with LRP6-myc and/or Axin2-myc cDNAs and treated with or without IWR-1 was subjected to immunoprecipitation using an anti-LRP6 antibody. Precipitated material was subjected to Western blot analysis. (f) IWR compounds do not induce subcellular re-distribution of Axin2 protein. HeLa or Cos1 cells transfected with Axin2 cDNA and treated either with DMSO or IWR-1, were immunostained with an anti-Axin2 antibody. Subcellular localization of Axin2 protein does not appear to be altered in the presence of IWR-1. Axin2 lacking the DAX domain, which has been previously shown to be important to normal Axin2 subcellular localization, is diffusely localized in the cytoplasm and is used here as a positive control.
Nature Chemical Biology: doi: 10.1038/nchembio.137
A
C
IWR-1 (Endo or Exo)
D
endoor exo
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β-catenintubulin
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DMSO IWR-1 linker-biotin
IWR-PB
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linker
DMSO
F
IWR-PEG-Biotin (IWR-PB)
Axin2 OE hAxin2-DAXpcDNAM
arke
r IWR-PB: - - + + - + + IWR-1: - - - + - - +
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OEX Axin2: - - + + ++IWR-PB: - - - -
-- - -+
+-
-IWR-1:
Total lysate Pull-down
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DLD-1 cells
Axin1-GFP DNA (µg): .01 .01 .05 .05--
IWR-IS-myc
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IWR1 IWR-Frag
Perc
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espo
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RL)B
IWR-Frag
DMSO2.5 µM
N
O
H
H
O
O
OH
Supplemental Figure 9. Synthetic scheme for IWR-1 and IWR-PB compounds. (a) Synthetic route for IWR-1. Endo and exo diastereomers result depending on the starting material. (b) A fragment of IWR-1 (IWR-Frag) is incapable of inhibiting Wnt pathway response in L-Wnt cells. (c) Synthetic scheme for a biotinylated IWR-1 (IWR-PB). (d) IWR-PB is active in cultured cells. (e) IWR-PB blocks accumulation of β-catenin in response in L-Wnt-STF cells. (f) Silver stain analysis of eluted proteins associated with IWR-PB/streptavidin agarose resin derived from HEK293 cells transfected with control or Axin2 expression constructs. The identical conditions were used in Western blot analysis of full-length Axin2 protein shown in Fig. 4f. (g) Axin2 interacts with IWR independently of other known Wnt pathway components. Material associated with IWR-PB under identical conditions used in Fig. 4f was probed by Western blot for various Wnt pathway components known to interact with Axin2. (h) IWR-IS abrogates IWR-induced Axin protein stabilization. Cells expressing IWR-IS exhibit mitigated Axin protein stabilization in response to IWR-1 as measured using Western blot analysis of a co-expressed Axin1-GFP protein. The IWR-1-dependent changes in endogenous Axin1 protein levels confirms the effectiveness of IWR-1 and reveals the low efficiency of DNA transfection in these experiments.
Nature Chemical Biology: doi: 10.1038/nchembio.137
100
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10 20 30 10 20 300 0
IWR-1 (10µM)DMSOTrypsinization time (sec):
Chemical:
∗∗
∗
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Sufu
Axin2-Myc
lysate from HEK293 cells
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IWR-1 (30µM)
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DMSO
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IWR-3 (30µM)
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∗100
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Supplemental Figure 10. Altered proteolytic sensitivity of Axin2 protein in cells treated with IWR. (a) Lysates derived from HEK293 cells expressing Axin2-myc protein treated with or without IWR-1 for 24 hrs were incubated with trypsin for indicated time periods in the presence or absence of additional IWR-1. Partial trypsinization of Axin2 (top) reveals IWR-1-sensitive cleavage sites and overall increased susceptibility to proteolysis. Re-probing of the same samples with an antibody recognizing an irrelevant protein [Suppressor of fused (Sufu)] reveals no IWR-dependent di�erences in the partial trypsinization pro�le. (b) Lysates derived from HEK293 cells expressing Axin1-GFP protein treated with IWR-1 or IWR-3 for 1hr on ice prior to addition of trypsin for indicated time periods at room temperature. IWR-1 and IWR-3 are both able to alter the partial trypsinization pro�le of Axin1 (top), suggesting that their mechanism of action on Axin1 is similar to that in the case of Axin2. Sufu Western blot is a loading and trypsinization control (bottom). “*” denote Axin trypsinfragments with increased stability in the presence of IWR compounds.
Nature Chemical Biology: doi: 10.1038/nchembio.137
Supplementary Methods
Reagents.
L-Wnt-STF cells were generated by transfecting L-Wnt cells (ATCC) with SuperTopFlash (STF; R.
Moond) and SV-40 Renilla luciferase plasmids and selecting for clones resistant to G418 and Zeocin.
The following expression constructs were generously provided by: P. Beachy (Shh and Wnt3A), R.
Kopan (NICD), P.-T. Chuang (ShhN-C25S), and M. Brown and J. Goldstein (MBOAT family
members). Wnt1, Wnt2, Wnt3, and Wnt5B expression constructs were purchased from
OpenBiosystems. Notch reporter construct was provided by J. LaBorda. The Wnt-GL expression
construct was generated by ligating Wnt3A coding sequence to GL (AA15-185) via an XbaI site. CMV-
GL was generated by inserting the CMV promoter into the pGluc-Basic vector (New England Biolabs).
Expression constructs for mPorcn-myc and hAxin2-myc constructs were engineered using PCR-
based cloning and mutagenesis strategies. Axin-GFP construct was from M. Bienz. The source of
primary antibodies used in this study are as follows: β-catenin, Kif3A, Actin, and β-tubulin (Sigma); p-
β-catenin (Ser33/37/Thr41), GSK3β, LRP6, P-Lrp6 (Ser1490), Dvl2, Shh, Axin1, and Axin2 (from Cell
Signaling Technology); E-Cadherin and PP2A (BD Transduction Laboratories); Myc, Apc, and
CK1ε (Santa Cruz Biotechnology); and Gsk3β (Stressgen). Pools of four pre-designed siRNA
reagents used in RNAi experiments were purchased from either Qiagen (APC) or from Dharmacon (β-
catenin). Axin2 primers used in RT-PCR experiments: 5'-AGCTCTGAGCCTTCAGCATC (reverse)
and 5'-TCAGCAGAGGGACAGGAATC (forward).
Radiolabeling experiments.
Wnt3A-myc and murine Porc expression constructs were transfected into Cos7 cells (6 well format,
150K cells/well) using Fugene6 transfection reagent (1:3 DNA:Fugene, Roche). After 48 hrs, the
medium was replaced with DMEM (HyClone), 2.5% FBS and supplemented with 2.5 µM IWP-2 (0.2%
DMSO final) as appropriate. 37.5 µCi/mL H3-Palmitate (47.7 Ci/mmol, Perkin Elmer) was added to all
wells, and media, compound, and radiation replaced after 2 hours. After 4 hrs total incubation time,
cells were lysed (PBS/ 1% NP-40/ protein inhibitors) and Wnt3A-myc pulled down with anti-Myc
monoclonal crosslinked to Protein G agarose. Precipitated material was run out on a 4-20% gradient
gel (Biorad), transferred to nitrocellulose, and exposed through a LE Transcreen to film at -80oC for 14
days.
Nature Chemical Biology: doi: 10.1038/nchembio.137
Chemical synthesis, continued.
IWP-1: 1H NMR (400 MHz, DMSO-d6) δ 12.42 (br, 1H, NH), 8.98 (t, J = 5.9 Hz, 1H, NH), 8.73 (d, J = 7.8 Hz, 1H), 8.38 (d, J = 7.8 Hz, 1H), 7.99 (dd, J = 7.8, 7.6 Hz, 1H), 7.92 (dd, J = 7.8, 7.6 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 2.3 Hz, 1H), 7.08 (d, J = 8.2 Hz, 2H), 7.02 (dd, J = 8.5, 2.3 Hz, 1H), 4.28 (d, J = 5.9 Hz, 2H), 3.83 (s, 3H), 3.80 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 179.7, 168.3, 163.7, 158.7, 158.4, 156.2, 155.7, 137.9, 134.3, 133.9, 132.8, 132.3, 128.0, 127.5, 127.3, 126.8, 126.7, 121.2, 114.9, 113.7, 104.7, 55.6, 55.5, 42.4; MS(ES+) calcd for C26H22N5O5S (M+H)+ 516.1, found 516.1.
2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511 .011.512.012.513.013.5
7.16
2.15
1.12
2.22
1.10
3.33
2.14
1.01
1.00
1.00
0.95
DMSO
H2O
30405060708090100110120130140150160170180190200
DMSO
Nature Chemical Biology: doi: 10.1038/nchembio.137
IWR-3: 1H NMR (400 MHz, DMSO-d6) δ 8.24 (t, J = 6.0 Hz, 1H, NH), 8.09 (dd, J = 7.8, 0.4 Hz, 1H), 7.70–7.64 (m, 2H), 7.32–7.19 (m, 9H), 6.92 (d, J = 6.9 Hz, 1H), 6.05 (s, 1H), 5.25 (s, 2H), 4.23 (d, J = 6.0 Hz, 2H), 3.88 (d, J = 6.9 Hz, 2H), 2.90 (s, 3H), 2.16–2.11 (m, 1H), 1.77–1.71 (m, 5H), 1.37–1.28 (m, 2H), 1.09–1.00 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 175.1, 161.5, 161.2, 160.6, 153.4, 151.0, 143.6, 139.9, 136.9, 135.4, 128.3, 128.2, 127.1, 126.7, 124.5, 123.0, 118.9, 115.1, 114.8, 101.9, 47.2, 46.9, 44.0, 41.8, 35.7,
29.7, 28.9, 24.1; MS(ES+) calcd for C33H34N5O4 (M+H)+ 564.3, found 564.1.
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5
2.14
2.22
5.88
1.20
3.49
2.15
2.32
2.11
1.04
1.07
9.03
2.17
1.00
1.11
DMSOH2O
2030405060708090100110120130140150160170180
DMSO
Nature Chemical Biology: doi: 10.1038/nchembio.137
IWR-1: 1H NMR (400 MHz, CDCl3) δ 10.72 (s, 1H, NH), 8.89 (dd, J = 7.3, 1.1 Hz, 1H), 8.81 (dd, J = 4.1, 1.2 Hz, 1H), 8.17 (dd, J = 8.2, 1.1 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 7.56 (dd, J = 8.2, 7.3 Hz, 1H), 7.53 (dd, J = 8.2, 1.2 Hz, 1H), 7.46 (dd, J = 8.2, 4.1 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 6.28 (s, 2H), 3.53 (s, 2H), 3.47 (s, 2H), 1.80 (d, J = 8.8, 1H), 1.62 (d, J = 8.8, 1H); 13C
NMR (100 MHz, CDCl3) δ 176.5, 164.6, 148.4, 138.8, 136.5, 135.2, 134.9, 134.8, 134.5, 128.2, 128.0, 127.5, 126.9, 122.0, 121.8, 116.6, 52.4, 46.0, 45.7; MS(ES+) calcd for C25H20N3O3 (M+H)+ 410.2, found 410.1.
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0
1.11
1.12
4.45
2.06
2.21
1.17
2.27
3.38
1.09
1.06
1.00
404550556065707580859095100105110115120125130135140145150155160165170175180
CHCl3
Nature Chemical Biology: doi: 10.1038/nchembio.137