Design and characterization of a potent and selective dual ATP- and substrate-competitive subnanomolar bidentate c-Jun N-terminal kinase (JNK) inhibitor

Article abstract of DOI:10.1021/jm200479c

c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC₅₀ = 18 nM; K _i = 1.5 nM) and JNK/substrate association in a displacement assay (IC₅₀ = 46 nM; K_i = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.

Full text of DOI:10.1021/jm200479c

ARTICLE
pubs.acs.org/jmc
Design and Characterization of a Potent and Selective Dual
ATP- and Substrate-Competitive Subnanomolar Bidentate c-Jun
N-Terminal Kinase (JNK) Inhibitor
||
||
John L. Stebbins,†, Surya K. De,†, Petra Pavlickova,† Vida Chen,† Thomas Machleidt,‡ Li-Hsing Chen,†
Christian Kuntzen,§ Shinichi Kitada,† Michael Karin,§ and Maurizio Pellecchia*,†
†Infectious and Inflammatory Disease Center, Cancer Center, Sanford-Burnham Medical Research Institute,
10901 North Torrey Pines Road, La Jolla, California 92037, United States
‡Life Technologies, Discovery and ADMET Services, 501 Charmany Drive, Madison, Wisconsin 53719, United States
§Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California at San Diego, 9500 Gilman Drive,
MC0723, La Jolla, California 92093-0723, United States
S Supporting Information
b
ABSTRACT: c-Jun N-terminal kinases (JNKs) represent valuable targets in the
development of new therapies. Present on the surface of JNK is a binding pocket for
substrates and the scaffolding protein JIP1 in close proximity to the ATP binding
pocket. We propose that bidentate compounds linking the binding energies of
weakly interacting ATP and substrate mimetics could result in potent and selective
JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK.
19 inhibits JNK kinase activity (IC50 = 18 nM; Ki = 1.5 nM) and JNK/substrate
association in a displacement assay (IC50 = 46 nM; Ki = 2 nM). Our data demon-
strate that 19 targets for the ATP and substrate-binding sites on JNK concurrently.
Finally, compound 19 successfully inhibits JNK in a variety of cell-based experi-
ments, as well as in vivo where it is shown to protect against Jo-2 induced liver
damage and improve glucose tolerance in diabetic mice.
’ INTRODUCTION
related MAP kinases p38 and Erk.11ꢀ13 The mechanism of this
inhibition is thought to be due to competition of 20 with the
D-domains of JNK substrates or upstream kinases.12,14 To
increase stability and increase cell permeability of 20, an all-D
retro-inverso amino acid of compound 20 fused to the cell
permeable HIV-TAT peptide, 11 (D-JNKI), was devised
(sequence Ac-tdqsrpvqpflnlttprrprpprrrqrrkkrg-CONH2; MW
= 3395).15 11 significantly decreases c-Jun phosphorylation by
JNK when tested in cell, however, albeit very selective, inhibi-
tion studies suggest that 11 is only a modest JNK inhibitor.16 In
comparison, the small molecule ATP mimetic, 21 (SP600125),
is very potent in vitro but not very selective for JNK.17ꢀ19
Hence, most of the current efforts focus on optimization of 21
and other ATP mimetics for the design of JNK inhibitors.1,9,20
Recently, using a combination of structure-based design guided
by the X-ray structure of JNK1 in complex with 20 and 21, as well
as NMR fragment-based drug discovery approaches,21 we pro-
posed thatbylinking molecules thatspan these two sites weshould
be able to generate selective, high affinity bidentate JNK mod-
ulators. Indeed, we describe here a bidentate molecule with the
The c-Jun N-terminal kinases (JNKs) are a series of serine/
threonine protein kinases belonging to the mitogen activated
protein kinase (MAPK) family. In mammalian cells, three distinct
genes encoding JNKs have been identified, JNK1, JNK2, and JNK3,
and at least 10 different isoforms exist.1ꢀ3 JNK1, JNK2, and JNK3
share more than 90% amino acid sequence identity, and the ATP
pocket is >98% homologous. JNK1 and JNK2 are ubiquitously
expressed, whereas JNK3 is most commonly found in the brain,
cardiac muscle, and testis.2,4 JNK activation in response to stimuli
such as stress or cytokines results in activation of several transcrip-
tion factors and cellular substrates implicated in inflammation,
insulin signaling, mRNA stabilization, and cell proliferation and
survival.3,5ꢀ7 Because of the link between these pathways and
the pathogenesis of diseases such as Parkinson’s and Alzheimer’s
and inflammatory diseases, cancer, diabetes, atherosclerosis, and
stroke, JNK inhibitors are expected to be useful therapeutic
agents.1,3,8,9
JNK binds to substrates and scaffold proteins, such as JIP-1,
that contain a D-domain, as defined by the consensus sequence
R/K(2ꢀ3)X(1ꢀ6)L/I-X-L/I.10 A JIP1 D-domain peptide corresponding
to amino acids 153ꢀ164, 20 (pepJIP1; sequence RPKRPTTLNLF;
MW 1343), inhibits JNK activity in vitro and in cell while displaying
extraordinary selectivity with negligible inhibition of the closely
Received: April 20, 2011
Published: August 04, 2011
r
2011 American Chemical Society
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Figure 1. Fragment-based design and synthesis of bidentate JNK inhibitors. (A) Schematic representation of the proposed approach overlaid on the
surface representation of JNK1 in complex with 20 (RPKRPTTLNLF) and the ATP mimic 21 (PDB-ID 1UKI). The surface generated with
MOLCAD50 and color coded according to cavity depth (blue, shallow; yellow, deep). (B) Docked structure of 20 and 21 on the surface of JNK1. (C)
Docked structure of the bidentate compound 9 on the surface of JNK1. (D) Scheme for the synthesis of 8 and the bidentate 9 (see methods for
experimental details). (E) In vitro JNK kinase activity inhibition by 9. (F) Displacement of 20 from GST-JNK1 by 9 in absence (circles) and presence
(squares) of a saturating amount of staurosporine (0.5 μM).
aforementioned characteristics that functions as a JNK inhibitor
in vitro, in cell, and in a variety of in vivo models.
suggesting the possibility of obtaining high affinity and selective
compounds by designing appropriate bidentate molecules. In-
deed, this approach has been successfully executed for a variety of
other proteins and kinases.25ꢀ30 Hence, our hypothesis is that by
tailoring a weak docking site binder to a weakly interacting ATP-
mimic, it should be likewise possible to develop potent and
selective inhibitors of JNK.
’ RESULTS AND DISCUSSION
In the realm of drug discovery, fragment-based drug design
approaches are becoming increasingly successful in tackling
challenging targets, such as those involving proteinꢀprotein
interactions.22 A common fragment-based drug design approach
consists of designing bidentate compounds chemically linking
two weakly interacting scaffolds that occupy adjacent pockets on
the target’s surface (Figure 1AꢀC). In this case, the free energy
of binding of the resulting bidentate compound with respect to
those of the individual fragments can be expressed as:
To define an optimal interacting docking peptide sequence for
JNK we tested 20 peptide sequences derived from its putative
substrates and scaffolding proteins, all presenting a D-domain
consensus motif10 (Supporting Information Table S1). Each pep-
tide was tested for its ability to displace 20 (residues 153ꢀ164) from
JNK1 by using a dissociation enhanced lanthanide fluoro-immuno
assay (DELFIA) platform. DELFIA is a heterogeneous assay
whereby a biotin-linked 20 is adsorbed onto a streptavidin-coated
plate followed by incubation with GST-JNK1. Detection of the 20/
GST-JNK1 complex is facilitated by a highly fluorescent anti-GST
Eu-antibody conjugate (PerkinꢀElmer). On the basis of these data
(Supporting Information Table S1), a minimal peptide sequence,
10 (RPTTLNL), was identified as necessary and sufficient to
displace full-length 20 in this assay. We are confident that inhibition
is not at the level of either GST or streptavidin as 10 did not
significantly displace a previously described and unrelated binding
pair (SIAH1/phyllopod peptide)31 in a similar DELFIA assay plat-
form when tested up to 45 μM.
ΔGAB ¼ ΔHA þ ΔHB ꢀ TΔSAB
¼ ꢀ RT lnðKAD ꢁ KDB ꢁ EÞ
Where, R represents the Boltzmann constant, T is the tempera-
ture of the system, ΔHA and ΔHB are the enthalpy of binding of
fragments A and B respectively, ΔSAB represents the entropy loss
upon binding of the bidentate compound, and KDA and KDB are
the dissociation constants of the individual initial binders and E is
the linking coefficient.23 The recently determined X-ray structure
of JNK1 in complex with 20 and the ATP-mimic 21,24 reveals a
close proximity between the ATP and the docking binding sites,
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Table 1. In Vitro Activity Data for Reported JNK Inhibitors
The design of a weak ATP mimetic JNK inhibitor was based
on the structure of 21 (Figure 1B).24 Given that 21 was found to
potently inhibit several protein kinases,17,18,20 in order to increase
JNK selectivity and also facilitate subsequent synthetic efforts, we
removed the keto group from 21. Furthermore, on the basis of its
docking mode, a convenient position to elongate the ATP-mimetic
toward the docking site was identified in our previous work by using
a combination of structure-based design guided by NMR-relaxation
measurements using paramagnetic spin-labeling approaches.21
These efforts resulted in the design of 8 (Figure 1D). The synthesis
of 8 starts from the iodination of 1-azaindole with iodide and its
protection with trimethylsilylethoxy methyl chloride, followed by a
Suzuki coupling between 3 and 4-methoxycarbonylphenylboronic
acid and attachment of the designed 3-carbon linker (Figure 1D).
8 is a relatively weak ATP mimetic, inhibiting JNK1 phos-
phorylation of the substrate ATF2 in the LanthaScreen time-
resolved fluorescence resonance energy transfer (TR-FRET)
based kinase activity assay (Invitrogen, Carlsbad, CA) with 14
μM IC50 value. Similarly, 10 was able to displace 20 in the
DELFIA assay (IC50 25 μM) but was unable to inhibit JNK1
phosphorylation of the substrate ATF2 in the same TR-FRET
based kinase activity assay at concentrations up to 25 μM, hence
representing a fairly weakly interacting substrate binding scaffold.
However, when linked to the indazole moiety of 8, the resulting
compound 9 (Figure1D) was able to compete with 20 for JNK1
binding and inhibit JNK1 kinase activity with remarkable sub-
nanomolar affinities (Table 1). To properly link compounds 8
and 10, two Gly residues were inserted as part of the linker based
on molecular modeling and NMR-based considerations and the
X-ray structure of the ternary complex, as described in our
previous studies.21
Consistent with the bidentate binding mode of 9 to JNK1, the
preincubation of JNK1 with an excess of ATP-mimetic stau-
rosporine,32 thus eliminating one binding site, reduces the
ability of 9 to compete with 20 binding to JNK1 by 600-fold
(Figure 1F). As expected, under these circumstances, the IC50 for
9 was similar to that of the peptide alone (Table 1). Direct
comparison of kinase inhibition properties of 9 (MW 1204) with
20 (MW 1343) clearly demonstrated an improvement of over
100-fold (Figure 1E). Hence, by linking the binding energy of a
minimal 20 sequence with a modest 21 derived ATP mimetic, we
successfully produced a very potent bidentate molecule repre-
senting a new class of JNK inhibitors.
To confer further favorable pharmacological properties to this
compound for in vivo studies, analogous to the clinical candidate
11,16 we produced an all-D retro-inverso version of 9 fused to the cell
penetrating HIV-TAT sequence. As expected, the resulting com-
pound, 19 (Table 1), efficiently competes with 20 for binding to
JNK1 as well as strongly inhibiting JNK1 kinase activity (Figure 2A,
B), with IC50 values of 18 and 46 nM, respectively. Using the same in
vitro kinase activity assay and the same ATF2 substrate, 19 was found
to be inactive versus p38R at concentrations up to 100 μM, a mem-
ber of the MAPK family with highest structural similarity to JNK, thus
demonstrating selectivity. LineweaverꢀBurk analysis indicates that
19 is competitive with both ATP and ATF2 for binding to JNK1
as the data with an apparent K(i) of 2 and 1.5 nM, respectively
(Figure 2C,D). Consistent with the proposed bimodal binding
of 19 to JNK1, the data fit very well with both the mixed and the
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Figure 2. In vitro characterization of 19. (A) Dose dependent displacement of biotinylated 20 from GST-JNK1 and (B) JNK kinase activity inhibition
by 19. (C and D) LineweaverꢀBurk analysis with compound 19.
competitive inhibition modes and very poorly with either the non-
competitive or the uncompetitive inhibition models (Supporting
Information Table S2).
NONcNZO10/LtJ mice reflects most human obesities. For this
analysis, glucose intolerant NONcNZO10/LtJ mice were in-
jected intraperitoneally daily for four days with 25 mg/kg 19. The
ability of mice to process glucose injected intraperitoneally was
then measured. 19 was remarkably effective in restoring normo-
glycemia without inducing hypoglycemia compared to both the
vehicle control and 11 (Figure 4A). The ability of 19 to improve
glucose tolerance is consistent with its proposed function as an
effective JNK inhibitor, while the observed shortcoming of
compound 11 under our current experimental conditions can
simply be a reflection of its limited potency against the target, as
very recently corroborated by a comparative study.38
The link between hapototoxicity and JNK function has been
established using JNK1ꢀ/ꢀ and JNK2ꢀ/ꢀ mice.39ꢀ41 Thus, to
extend our in vivo analysis of 19, we tested its ability to block Jo2-
induced liver damage as measured by the release of liver enzymes
alanine-aminotransferase (ALT) and aspartate aminotransferase
(AST) into the blood. 19 effectively blunted the Jo2-induced
elevation of AST and ALT levels relative to control animals
(Figure 4B). Consistent with its proposed ability to effectively
inhibit JNK function in vivo, 19 treatment resulted in signifi-
cantly reduced levels of phosphorylated c-Jun in the liver of
treated mice as compared to the control (Figure 4C).
In an attempt to further profile the biological properties of 19,
we compared its ability to function in the context of a complex
cellular milieu with that of 11. For this analysis, we employed a cell-
based TR-FRET assay.33 In this assay, 19 was significantly more
effective at inhibiting tumor necrosis factor-R (TNF-R) stimulated
phosphorylation of c-Jun in B16ꢀF10 melanoma cells (EC50
=
14 μM), while both 11 and 21 were significantly less effective under
the same experimental conditions (Figure 3A). The cell-based
system employed makes use of a GFP-c-Jun stable expression system.
As a result, the levels of GFP-c-Jun in these cells are higher than endo-
genous levels. This could have an inflationary effect on the EC50
values obtained with this assay when testing substrate competitive
compounds. Thus, in an orthogonal assay, we measured the ability of
19 to inhibit anisomycin stimulated endogenous c-Jun phosphoryla-
tion in HEK293T cells. Indeed, we found 19 to be significantly more
effective in this system (EC50 = 2 μM; Figure 3B). To further profile
19 efficacy in cell as well as demonstrate its selectivity, we tested the
ability of 19 to inhibit the release of cytokines from RAW 264.7
mouse macrophage cells in response to lipopolysaccharide (LPS).
LPS stimulated secretion of TNF-R from macrophages is dependent
on JNK activation34 whereas IL-1βsecretion from RAW 264.7 cells is
known to be p38 dependent.35 We found that 19 was able to inhibit
JNK dependent TNF-R release, while p38R dependent IL-1β secre-
tion was unaffected (Figure 3C). Taken together, these findings
conclusively establish that 19 as a potent and selective dual substrate
and ATP competitive JNK bidentate inhibitor able to function
efficiently and selectively in a cellular context.
’ CONCLUSION
In conclusion, by applying the principles of fragment-based
drug discovery to the design of dual ATP- and substrate-
competitive kinase inhibitors, we identified bidentate molecules
with superior JNK inhibitory properties. We anticipate that this
approach will find wide applications in the design and synthesis
of other potent and selective bidentate kinase inhibitors. Regard-
ing the reported bidentate compounds, given that 11 is currently
a clinical candidate (under XG102 by Xigen Corp., Lausanne,
Switzerland), we can speculate that 19, having markedly improved
biochemical and pharmacological properties and even reduced
JNK activation has been linked to the impaired glucose
tolerance associated with type 2 diabetes.8,36 Therefore we tested
the ability of 19 to restore glucose tolerance in the type 2 diabetes
mouse model NONcNZO10/LtJ37 (Jackson Laboratories, Bar
Harbor, Maine 04609, United States ), chosen because obesity in
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Figure 4. In vivo characterization of 19. (A) Effect of 19 and 11
(25 mg/kg) on glucose tolerance in 26-week-old NONcNZO10/LtJ
mice from Harlan (Jackson Laboratories, Bar Harbor, Maine 04609,
United States ). Solid diamonds, vehicle control; solid squares, 25 mg/kg
19; solid triangle, 25 mg/kg 11.). p < 0.0001 for the comparison of
compound 19 with control, by two-way ANOVA using all the measure-
ments. (B) Effect of 25 mg/kg 19 (open bars) on AST and ALT levels after
Jo2 treatment as compared to vehicle control (filled bars). Data are mean (
SE for four mice. AST levels significantly different as compared to control
mice p< 0.05. ALT levels significantly different as compared to control mice
p< 0.02. (C) Effect of 25 mg/kg 19 on phospho-c-Jun levels in C57/B6 mouse
liver. Phospho-c-Jun levels measured by sandwich immunoassay (Meso Scale
Discovery). Results shown as percent of vehicle control ( SD (n = 4). p <
0.0001 for the comparison of compound 19 with control, by unpaired t test.
Figure 3. Cell-based characterization of 19. (A) TR-FRET analysis of
c-Jun phosphorylation upon TNF-R stimulation of B16ꢀF10 cells in the
presence of increasing 19 (closed triangles), 21 (inverted triangles) and 11
(closed squares). (B) Inhibitory effect of 19 on phospho-c-Jun upon
anisomycin stimulation of HEK293T cell. (C) Compounds 19 and 11
effect on TNF-R and IL-1β levels after 5 h of exposure to LPS as compared
to vehicle control. Results shown as percent of vehicle control (SD (n=3).
Cytokine production was measured directly from cell culture medium by a
sandwich immunoassay (Meso Scale Discovery).
molecular weight over 11, could equally well enter further clinical
investigations. Furthermore, our current efforts focused on the iden-
tification of small molecules 20 mimetics42ꢀ45 could likewise lead,
based on the reported results, to the design of additional bidentate
compounds voided of the peptidyl nature.
Given the tremendous efforts of the past decade dedicated to
the design of potent and selective kinase inhibitors for both
therapeutics and basic cell biology studies, we are confident that
the bidentate approach proposed will find wide applications in
both the pharmaceutical and basic chemical biology arenas.
concentration of 10 nM was added. After 1 h incubation at 0 °C, each well
was washed five times to eliminate unbound protein and the Eu-antibody if
displaced by a test compound. Subsequently, 200 μL of enhancement
solution (Perkin-Elmer) was added to each well and fluorescence measured
after 10 min incubation (excitation wavelength, 340 nm; emission wave-
length, 615 nm). Controls include unlabeled peptide and blanks receiving
no compounds. Protein and peptide solutions were prepared in DELFIA
buffer (Perkin-Elmer). Staurosporine (Calbiochem; San Diego, CA; cata-
logue no. 569397) was included at 20-times molar equivalent GST-JNK1
and preincubated for 15 min on ice.
In Vitro Kinase Assay. Assay platform from Invitrogen was
utilized. The time-resolved fluorescence resonance energy transfer assay
(TR-FRET) was performed in 384-well plates. Each well received JNK1
(0.8 nM), ATF2 (200 nM), and ATP (1 μM) in 50 mM HEPES, 10 mM
MgCl 2, 1 mM EGTA and 0.01% Brij-35, pH 7.5 and test compounds.
The kinase reaction was performed at room temperature for 1 h.
After this time, the terbium labeled antibody and EDTA were added
into each well. After an additional hour incubation, the signal was
measured at 520/495 nm emission ratio on a BMG Pherastar fluo-
rescence plate reader.
’ MATERIALS AND METHODS
DELFIA Assay (Dissociation Enhanced Lanthanide Fluoro-
Immuno Assay). To each well of 96-well streptavidin-coated plates
(Perkin-Elmer), 100 μL of a 100 ng/mL solution of biotin-labeled 20
(Biotin-lc-KRPKRPTTLNLF, where lc indicates a hydrocarbon chain of
6 methylene groups) was added. After 1 h incubation and elimination of
unbound biotin-20 by three washing steps, 87 μL of Eu-labeled anti-GST
antibody solution (300 ng/mL; 1.9 nM), 2.5 μL DMSO solution containing
test compound, and 10 μL solution of GST-JNK1 for a final protein
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Alternatively, 9 was kept as a 10 mM solution in 10% DMSO. Serial
dilutions containing 1% DMSO were prepared and 9 was added at a ratio
of 1:10 to each kinase reaction to obtain the indicated final concentrations.
JNK kinase assays were performed with 50 ng per reaction of active JNK2R2
from Upstate/Millipore (catalogue no. 14ꢀ329) according to the recom-
mendations of the manufacturer with the following changes: GST-c-Jun
(1ꢀ79) was used as a substrate 1 μg per reaction.46 The kinase reactions
were performed at 30 °C for 20 min without Brij-35 and were stopped by
addition of 2ꢁ Laemmli loading buffer and boiling for 3 min. The proteins
were then separated on a mini gel and transferred to a PVDF membrane by
wet blot. The membranes were dried and exposed to film.
Scale no. K15025B-1, Gaithersburg, MD). Levels of TNF-R and IL-1β in
the cell culture medium were determined using a Meso Scale Discovery
Sector Imager 2400 according to the manufacturer’s instructions.
Molecular Modeling. Computational docking studies were per-
formed with GOLD 2.1 (The Cambridge Crystallographic Data Centre,
Cambridge, UK)48,49 and analyzed with Sybyl (Tripos, St. Louis).
Molecular surfaces were generated with MOLCAD.50 The X-ray co-
ordinates of JNK1/20/21 (PDB-ID 1UKI) were used to dock the
compounds. Peptide and 8 and bidentate 9 poses reported in Figure 1 of
the manuscript correspond to those obtained directly from the X-ray
coordinates.
Phospho-c-Jun Detection and Quantification. All cell culture
media and supplements were from Life Technologies. The B16ꢀF10
murine melanocyte cell line was purchased from ATCC and maintained
according to manufacturers recommendations. At 48 h prior to measur-
ing phospho-c-jun levels, cells were transduced with BacMam GFPꢀ
c-Jun (1ꢀ79). BacMam preparation and transductions were performed
as previously reported.47 Briefly, the cells were grown in 10 cm dishes to
approximately 75% confluence. The transduction was performed by
adding 10% vol/vol BacMam virus stock in combination with Trich-
statin A at a final concentration of 0.5 mM. The cells were incubated for
24 h. BacMam GFP-c-Jun (1ꢀ79) transduction efficiency, as deter-
mined by fluorescence microscopy, exceeded in general 80% of the cell
population. All transductions were performed at a signal saturating MOI
(at least 500 IU/cell). Following the transduction, the cells were
trypsinized and plated in white tissue culture treated 384-well plates at
a density of 25000 cell per well in 32 mL of assay medium (Opti-MEM,
supplemented with 0.1% charcoal/dextran-treated FBS, 100 U/mL
penicillin and 100 mg/mL streptomycin, 0.1 mM nonessential amino
acids, 1 mM sodium pyruvate, 25 mM HEPES pH 7.3, and lacking
phenol red). After overnight incubation, cells were pretreated for 60 min
with compound (indicated concentrations) followed by 30 min of
stimulation with 2 ng/mL of TNF-R. The medium was then removed
by aspiration, and the cells were lysed by adding 20 mL of lysis buffer
(20 mM TRIS-HCl pH 7.6, 5 mM EDTA, 1% NP-40 substitute, 5 mM
NaF, 150 mM NaCl, 1:100 protease and phosphatase inhibitor mix,
SIGMA P8340 and P2850, respectively). The lysis buffer included 2 nM
of the terbium labeled antipc-Jun (pSer73) detection antibody (Life
Technologies). After allowing the assay to equilibrate for 1 h at room
temperature, TR-FRET emission ratios were determined on a BMG
Pherastar fluorescence plate reader (excitation at 340 nm, emission 520
and 490 nm; 100 ms lag time, 200 ms integration time, emission ratio =
Em520/Em 490).
Alternatively, HEK293T cells were maintained in DMEM and
supplements (Invitrogen). Cells were seeded at 4000000 cells per well
in a 12-well plate and incubated with or without compound. After 16 h
cells were treated with anisomycin (50ꢀ100 nM) (MP Biomedicals) for
5ꢀ10 min. Levels of phospho-c-Jun were measured using the phospho-
c-Jun Whole Cell Lysate Kit (Meso Scale K151CGD) from Meso Scale
Discovery (Gaithersburg, MD). The assay was performed according to
the manufacturer’s instructions, with duplicates of 10 μg of total protein
from either HEK293T or C57/B6 liver cells.
THP-1 Cell Assay for Inhibition of LPS-Induced TNF-r and
IL-1β Production. THP-1 cells (ATCC TIB 202, ATCC, Rockville,
MD) were maintained at 37 °C, 5% CO2 in 10% fetal bovine serum
(FBS)/RPMI 1640 medium. The day of the assay, 2 ꢁ 106 cells were
resuspended in 1 mL of 3% FBS/RPMI 1640 medium and plated in a 12-
well plate. 19 and 11, 12.5 μM each, or DMSO vehicle was added to the
cell mixture and allowed to preincubate for 60 min at 37 °C, 5% CO2,
prior to stimulation with LPS (Sigma L6529, from Escherichia coli
serotype 055:B5; 1 μg/mL final). LPS stimulation was allowed to
proceed for 5 h at 37 °C, 5% CO2. TNF-R and IL-1β production was
measured directly from cell culture medium by a commercially available
sandwich immunoassay developed by Meso Scale Discovery (Meso
Glucose Tolerance Test. Male NONcNZO10/LtJ mice, 26 weeks
old, from Harlan (Jackson Laboratories, Bar Harbor, Maine 04609,
United States ) were dosed intraperitoneally (ip) with 25 mg/kg of 19
and 11 daily for five days. Mice were fasted 16 h before ip administration
of 2 g/kg D-glucose. Blood samples were taken at designated time points,
and blood glucose levels were measured using a hand-held glucose meter
(OneTouch Ultra, LifeScan, a Johnson & Johnson company, UK).
Liver Injury. Female C57/B6 mice, 7 weeks old, from Harlan were
dosed ip with 25 mg/kg of 19, while control mice were be treated with
the vehicle only (n = 4). One hour later, mice were injected ip daily for
three days with 0.2 μg/gram Jo-2 antibody (Fas/APO-1; BD Biosciences,
catalogue no. 554255). Serum and liver were collected 4 h hence. AST and
ALT levels in serum are determined using the IDEXX VetTest Chemistry
Analyzer per manufacturer instructions.
Chemistry. All anhydrous solvents were commercially obtained and
stored in Sure-seal bottles under nitrogen. All other reagents and
solvents were purchased as the highest grade available and used without
further purification. Thin-layer chromatography (TLC) analysis of
reaction mixtures was performed using Merck silica gel 60 F254 TLC
plates and visualized using ultraviolet light. 1H NMR data were collected
using a 300 MHz Varian instrument and recorded in deuteron-chloro-
form (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6). Chemical shifts
(δ) are reported in parts per million (ppm) referenced to 1H (Me4Si at
0.00). Mass spectral data were acquired on a Shimadzu LCMS-2010EV for
low resolution, and on an Agilent ESI-TOF for high resolution and low
resolution. List of Abbreviations: equivalent (eqv), high performance liquid
chromatography (HPLC), liquid chromatography/mass spectrometry
(LC/MS), room temperature (rt). Purity of compounds was obtained in
a HPLC Breeze from Waters Co. using an Atlantis T3 3 μm 4.6 mm ꢁ
150 mm reverse phase column. All intermediate compounds were >95% pure.
Following the scheme reported in Figure 1: 1 (indazole) was commercially
available, which was iodinated according to the reported procedures.
Synthesis of 19. 18 was coupled with a peptide of D-amino acids on
resin using standard peptide coupling conditions. After coupling reac-
tion complete, resin was removed with the treatment of TFA. Final
compound was purified by rev phase HPLC. The compound was dried
and checked purity again with HPLC (purity was >93%) and analyzed
with MALDI-mass. Please see Supporting Information file for HPLC
trace and MALDI-Mass.
Synthesis of 3-Iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-indazole (3). To a solution of 2 (1.22 g, 5 mmol) in DMF (10 mL)
was added NaH (220 mg, 5.50 mmol) in three portions at 0 °C under
nitrogen atmosphere. The reaction mixture was stirred at the same
temperature for 30 min, and then SEM-Cl (0.9 mL, 5 mmol) was added
dropwise to it. The resulting reaction mixture was stirred at 0 °C for 1 h
then at room temperature for 4 h. The reaction mixture was quenched
with cold water (100 mL), followed by extraction with ether (3 ꢁ
100 mL). The combined organic layers were washed with water
(100 mL) and brine (100 mL), dried (MgSO4), and concentrated in
vacuo. The residue was chromatographed over silica gel (5% ethyl
acetate in hexane) to afford the colorless oil 3 (1.68 g, 90%), HPLC
purity >95%. 1H NMR (300 MHz, CDCl3) δ ꢀ0.07 (s, 9 H), 0.88 (t, J =
7.2 Hz, 2 H), 3.57 (t, J = 7.5 Hz, 2 H), 5.72 (s, 2 H), 7.27 (d, J = 8.2 Hz,
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1 H), 7.44ꢀ7.58 (m, 3 H). HRMS calcd for C13H19IN2OSi 374.0311,
found 374.0312.
8.52 (t, J = 5.4 Hz, 2 H), EIMS m/z 395 (M + H)+, 339, 295, 221, 83.
HRMS calcd for C22H27N4O3 395.2078 (M + H), found 395.2077
Synthesis of N-(3-Aminopropyl)-4-(1H-indazol-3-yl)ben-
zamide (8). To a solution of 7 (21 mg, 0.053 mmol) in CH2Cl2
(2 mL) was added TFA (0.5 mL). The resulting reaction mixture was
stirred at room temperature for 2 h. TFA and dichloromethane were
removed in vacuum to give 8. This compound was used for the next step
without further purification.
Synthesis of Methyl-4-(1-((2-(trimethylsilyl)ethoxy)-
methyl-1H-indazol-3yl)benzoate (4). A mixture of 3 (374 mg,
1 mmol), 4-methoxycarbonylphenyl boronic acid (271 mg, 1.5 mmol),
Pd(dppf)Cl2 (82 mg, 0.1 mmol), saturated aqueous Na2CO3 solution
(4 mL), in ethanol (1 mL) and toluene (10 mL) was stirred at 80 °C for
12 h. Upon completion of the reaction (TLC), the reaction mixture was
extracted with CH2Cl2 (3 ꢁ 50 mL). The combined organic layers were
washed with water (50 mL) and brine (50 mL), dried (MgSO4), and
concentrated in vacuo. The residue was chromatographed over silica gel
(5ꢀ10% ethyl acetate in hexane) to yield the pure product 4 (295 mg,
77%), HPLC purity >96%. 1H NMR (300 MHz, CDCl3) δ ꢀ0.05 (s, 9
H), 0.88 (t, J = 8.4 Hz, 2 H), 3.60 (t, J = 8.4 Hz, 2 H), 3.95 (s, 3 H), 5.81 (s,
2 H), 7.28 (t, J= 7.2Hz, 1 H), 7.42 (t, J = 7.5 Hz, 1 H), 7.62 (d, J =8.4 Hz, 1
H), 7.85ꢀ8.22 (m, 5 H); EIMS m/z 383 (M + H)+, 325, 267, 265, 149,
121, 83. HRMS calcd for C21H27N2O3Si 383.1785, found 383.1784.
Synthesis of 4-(1-((2-(Trimethylsilyl)ethoxy)methyl)-1H-
indazol-3-yl)benzoic Acid (5). To a solution of 4 (282 mg,
0.738 mmol) in THF (6 mL) and methanol (1 mL) was added LiOH
solution (177 mg, 7.380 mmol) in water (2 mL). The resulting reaction
mixture was stirred at room temperature for 18 h. The reaction mixture
was acidified with 1 N HCl, followed by extraction with CH2Cl2 (3 ꢁ
50 mL). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. The residue was chromatographed over silica
gel (20ꢀ30% ethyl acetate in hexane) to afford the acid 5 (195 mg,
90%), HPLC purity >95%. 1H NMR (300 MHz, CDCl3) δ ꢀ0.06 (s, 9
H), 0.92 (t, J = 8.4 Hz, 2 H), 3.64 (t, J = 8.4 Hz, 5.83 (s, 2 H), 7.32 (t, J =
7.2 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 1 H), 8.06
(d, J = 8.4 Hz, 1 H), 8.12 (d, J = 8.7 Hz, 2 H), 8.27 (d, J = 8.1 Hz, 2 H).
EIMS m/z 369 (M + H)+, 339, 311, 251, 149, 121, 99, 55. HRMS calcd
for C20H25N2O3Si 369.1629 (M + H), found 369.1627.
Synthesis of tert-Butyl-3-(4-(1-((2-(trimethylsilyl)ethoxy)-
methyl-1H-indazol-3-yl)benzamido)propylcarbamate (6).
To a solution of 5(155 mg, 0.421 mmol) in DMF (3 mL) were added EDC
(96 mg, 0.505 mmol), HOBt (68 mg, 0.505 mmol), DIEA (0.19 mL, 1.052
mmol), and mono-Boc-1,3-diamino propane (82 mg, 0.463 mmol). The
reaction mixture was stirred at room temperature for 16 h. Upon comple-
tion, the reaction mixture was diluted with water (40 mL), followed by
extraction with ethyl acetate (3 ꢁ 40 mL). The combined organic layers
were washed with saturated NaHCO3 solution (2 ꢁ 30 mL), water (3 ꢁ
30 mL), and brine (30 mL) successively, dried (MgSO4), and concentrated
in vacuo. The residue was chromatographed over silica gel (50% ethyl
acetate in hexane) to give the pure product 6 (175 mg, 79%), HPLC purity
>95%. 1H NMR (300 MHz, CDCl3) δ ꢀ0.063 (s, 9 H), 0.91 (t, J = 8.7 Hz,
2 H), 1.47 (s, 9 H), 1.74 (quintet, J = 5.7 Hz, 2 H), 3.29 (q, J = 6 Hz, 2 H),
3.55 (q, J = 6 Hz, 2 H), 3.63 (t, J = 8.4 Hz, 2 H), 4.95 (br s, 1 H, NH), 5.80
(s, 2 H), 7.30 (d, J = 7.2 Hz, 1 H), 7.47 (t, J = 7.2 Hz, 1 H), 7.63 (d, J = 8.4
Hz, 1 H), 7.98ꢀ8.12 (m, 5 H). HRMS calcd for C28H40N4O4Si 524.2819,
found 524.2817.
Synthesis of N1-(1-(4-(1HIndazol-3-yl)phenyl-16-methyl-
1,7,10,13-tetraoxo-2,6,9,12-tetraazaheptadecan-14-yl)-
2(2-(2-(2-(1-(2-amino-5-guanidinopentanoyl)pyrolidine-
2-carboxamido)-3-hydroxybutanamido)-3-hydroxybuta-
namido)-4-methylpentanamido)succinamide (9). To a solution
of 8 (15 mg, 0.051 mmol) in DMF (2 mL) were added EDC (10 mg, 0.051
mmol), HOBt (6 mg, 0.051 mmol), DIEA (0.5 mL), and Boc-Arg(Pbf)-
Pro-Thr(otbu)-Thr(Otbu)-Leu-Asn(trt)-Leu-Gly-Gly-OH (70 mg, 0.042
mmol) at room temperature. The reaction mixture was stirred at 50 °C
for 16 h. After completion of the reaction, DMF and DIEA were removed in
vacuo to give the protected compound. The crude residue was directly
treated with TFA (1 mL) and H2O (0.2 mL) for 3 h. The final product was
obtained by the following HPLC purification: Atlantis Preparative T3
column (10 mm ꢁ 250 mm), acetonitrileꢀwater system, RT = 5ꢀ34 min,
yield 35%, purity >95%. 1H NMR (300 MHz, CD3OD) δ 0.74ꢀ0.79 (m,
12 H), 1.25ꢀ2.20 (m, 24 H), 2.42ꢀ2.82 (m, 2 H), 3.01ꢀ3.94 (m, 10 H),
4.10ꢀ4.65 (m, 8 H), 7.15 (br, NH), 7.34ꢀ7.45 (m, 4 H), 7.50 (br, NH),
7.61 (d, J = 8.4 Hz, 2 H), 7.74 (d, J = 7.8 Hz, 2 H), 7.89 (d, J = 8.4 Hz, 1 H),
7.95 (br, NH). EIMS m/z 1204 (M + H)+, 1051, 860, 602, 450, 295, 136,
130, 108. HRMS calculated for C56H86N17O13 1204.6585 (M + H), found
1204.6572.
Similarly, the synthesis of 19 was obtained by coupling methyl-
4-(4-(1H-indazol-3-yl)benzamido) butanoate (analogue to 8 but with a
free carboxylic acid in lieu of the free amine; Supporting Information) was
coupled with a peptide of D-amino acids on resin using standard peptide
coupling conditions. After coupling reaction complete, resin was removed
with the treatment of TFA. Final compound was purified by rev phase HPLC.
The compound was dried and checked purity again with HPLC (purity was
>93%) and analyzed with MALDI-mass (Supporting Information).
’ ASSOCIATED CONTENT
S
Supporting Information. Scheme for the synthesis of
b
and analytical data for 19; analytical data; peptide sequences and
IC50 values relative to their ability to displace 20; R2 values for
various types of inhibition by 19 at either substrate or ATP
binding site. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Synthesis of tert-Butyl-3-(4-(1H-indazol-3-yl)benzamido)-
propylcarbamate (7). To a solution of 6 (76 mg, 0.141 mmol) in
THF (5 mL) was added TBAF (0.7 mL, 1 M solution in THF) at room
temperature. The reaction mixture was refluxed for 10 h. Upon
completion, the reaction mixture was partitioned between dichloro-
methane (40 mL) and water (30 mL). The organic layer was washed
with water (30 mL) and brine (30 mL), dried (MgSO4), and concen-
trated in vacuo. The residue was chromatographed over silica gel (80%
ethyl acetate in hexane) to give the pure product 7 (42 mg, 76%), HPLC
Corresponding Author
*Phone: 858-646-3159. Fax: 858-795-5225. E-mail: mpellecchia@
sanfordburnham.org.
Author Contributions
These authors made equal contributions to this work.
1
’ ACKNOWLEDGMENT
purity >95%. H NMR (300 MHz, DMSO-d6) δ 1.38 (s, 9 H), 1.65
(quintet, J = 6.6 Hz, 2 H), 3.01 (q, J = 6.3 Hz, 2 H), 3.30 (q, J = 6.3 Hz, 2
H), 6.83 (br s. NH), 7.24 (t, J = 7.8 Hz, 1 H), 7.42 (t, J = 6.9 Hz, 1 H),
7.62 (d, J = 8.4 Hz, 1 H),7.99 (d, J = 8.7 Hz, 2 H), 8.05ꢀ8.15 (m, 3 H),
Financial support was obtained by the National Institutes of
Health, NIDDK branch, grant number R24DK080263. Small
amounts of 19 for research purposes are available upon request.
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1
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