Structure Based Design of N-(3-((1H-Pyrazolo[3,4-b]pyridin-5-yl)ethynyl)benzenesulfonamides as Selective Leucine-Zipper and Sterile-α Motif Kinase (ZAK) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00572

A series of N-(3-((1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)benzenesulfonamides were designed as the first class of highly selective ZAK inhibitors. The representative compound 3h strongly inhibits the kinase activity of ZAK with an IC₅₀ of 3.3 n

Full text of DOI:10.1021/acs.jmedchem.7b00572

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Brief Article
Structure-Based Design of N-(3-((1H-pyrazolo[3, 4-
b]pyridin-5-yl)ethynyl) Benzenesulfonamides as Selective
Leucine-Zipper and Sterile-# Motif Kinase (ZAK) Inhibitors
Yu Chang, Xiaoyun Lu, Marthandam Asokan Shibu, Yi-Bo Dai, Jinfeng
Luo, Yan Zhang, Yingjun Li, Peng Zhao, Zhang Zhang, Yong Xu, Zheng-
Chao Tu, Qingwen Zhang, Cai-Hong Yun, Chih-Yang Huang, and Ke Ding
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017
Downloaded from http://pubs.acs.org on June 6, 2017
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Structure Based Design of Nꢀ(3ꢀ((1Hꢀpyrazolo[3,4ꢀb]pyridinꢀ5ꢀ
yl)ethynyl) Benzenesulfonamides as Selective LeucineꢀZipper
and Sterileꢀα Motif Kinase (ZAK) Inhibitors
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⊥
⊥
#,
Yu Chang,#,† ,‡ Xiaoyun Lu,#,‡ Marthandam Asokan Shibu,#,§ YiꢀBo Dai, Jinfeng Luo, Yan Zhang,
ǁ
Yingjun Li,⊥ Peng Zhao,ǁ Zhang Zhang,⊥ Yong Xu,⊥ ZhengꢀChao Tu,⊥ QingꢀWen Zhang,*,† CaiꢀHong
*,
Yun, ChihꢀYang Huang,*,§ and Ke Ding*,‡
ǁ
†State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Maꢀ
cau, Macao, China
‡School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
§Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan; Department of Health and Nutriꢀ
tion Biotechnology, Asia University, Taichung, Taiwan, China
ǁ Institute of Systems Biomedicine, Department of Biophysics and Beijing Key Laboratory of Tumor Systems Biology,
School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
⊥Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou
510530, China
KEYWORDS: ZAK, myocardial hypertrophy, selective inhibitors
ABSTRACT: A series of Nꢀ(3ꢀ((1Hꢀpyrazolo[3, 4ꢀb]pyridinꢀ5ꢀyl)ethynyl)benzenesulfonamides were designed as the first class of
highly selective ZAK inhibitors. The representative compound 3h strongly inhibits the kinase activity of ZAK with an IC50 value of
3.3 nM, and doseꢀdependently suppresses the activation of ZAK downstream signals both in vitro and in vivo, while it is signifiꢀ
cantly less potent for the majority of 403 nonꢀmutated kinases evaluated. Compound 3h also exhibits orally therapeutic effects on
cardiac hypertrophy in a spontaneous hypertensive rat model.
7, 20ꢀ23
ZAK.
Given the fact that mechanisms of ZAKꢀinduced
INTRODUCTION
myocardial hypertrophy remain elusive, it is highly desirable
to discover selective small molecule ZAK inhibitors as reꢀ
search probes to further validate the physiological and/or
pathological functions of ZAK in hypertrophic symptom deꢀ
velopment.
Leucineꢀzipper and sterileꢀα motif kinase (ZAK) belongs to
a mixedꢀlineage kinase family (MLKs), and structurally disꢀ
tinguishes itself from the other family members by presence of
1ꢀ4
a sterileꢀα motif (SAM) domain.
ZAK is prominently exꢀ
pressed in heart (especially infarcted heart), skeletal muscle,
2, 5, 6
placenta and liver etc.
Overexpression of ZAK has been
RESULTS AND DISCUSSION
closely related to the development of cardiac hypertrophy and
8
myocardial fibrosis7, by activating downstream cꢀJun Nꢀ
ZAK shares an approximate 36% sequence identity with
Val600 → Glu600 mutant of BꢀRaf serine/threonine protein kiꢀ
nase (BꢀRaf V600E) that functions as a “driving force” for a vaꢀ
riety of human cancers (e.g. melanoma, hairy cell leukemia
and papillary thyroid carcinoma etc.) (Supporting Information,
Figure S1). A superposition of the crystal structure of ZAK
(PDB: 5HES)20 with BꢀRafV600E (PDB: 4XV1) suggested that
the hinge regions and βꢀsheets at Nꢀlobes of the two proteins
almost totally overlapped (Figure 2B). It is also noteworthy
that several well characterized BꢀRafV600E inhibitors (e.g.
vemurafenib (1)20 and PLX4720 (2)20, 24, Figure 1) display
strong “off target” potencies against ZAK kinase. 7, 21, 25
terminal protein kinase (JNK) and p38 mitogen activated proꢀ
9ꢀ13
tein kinase (MAPK) pathways etc.
For instance, ZAK
overexpression induced characteristic hypertrophic features in
H9c2 cardio myoblast cells, including increasing cell sizes,
expression of hypertrophic marker proteins (i.e. atrial natriuꢀ
retic factor (ANF) and brain natriuretic peptide (BNP)), and
14ꢀ19
actin fiber organization etc.
Conversely, cardio myoblast
cells harboring dominant negative (DN) ZAK exhibited reꢀ
sistance against doxycycline induced hypertrophic feature
15,
transition and suppressed the expression of ANF and BNP.
16
These data collectively suggest that ZAK may serve as a
novel potential target to attenuate myocardial hypertrophy
We recently discovered a series of Nꢀ(3ꢀ((1Hꢀpyrazolo[3, 4ꢀ
b]pyridinꢀ5ꢀyl)ethynyl)benzenesulfonamides as selective Bꢀ
Although Nꢀ(3, 5ꢀdifluoroꢀ4ꢀ((3ꢀ
4ꢀb]pyridinꢀ5ꢀyl)ethynyl)phenyl)
benzenesulfonaꢀmide (3a) exhibited moderate inhibitory
which is one of the most frequent causes of heart failure. 6
26
RafV600E inhibitors.
There is no selective ZAK inhibitor reported to date, altꢀ
hough a number of kinase inhibitors have been identified to
exhibit unspecific suppression on the enzymatic function of
methoxyꢀ1Hꢀpyrazolo[3,
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activity against BꢀRafV600E with an IC50 value of 169.2 nM, it
potently suppressed ZAK with an IC50 value of 19.2 nM in an
(two steps); (h) Pd(dba)2, CuI, K2CO3, tꢀ(Bu)3P, THF, Ar2,
120 °C, overnight, 50%; (i) TFA, reflux, overnight, 87%.
27, 28
ADPꢀGloTM kinase assay.
A preliminary computational
Table 1. In Vitro kinase inhibition of 3a-3o.
study suggested that 3a could fit nicely into the ATP binding
site of ZAK and capture several key interactions with the
protein. The 1Hꢀpyrazolo[3, 4ꢀb]pyridine moiety formed two
hydrogen bonds with the backbone NHꢀ and C=O of Ala85,
respectively, in the hinge region, and it might also capture a
potential πꢀπ stacking interaction with Tyr84. The 2ꢀethynylꢀ1,
3ꢀdifluorophenyl linker could form a cationꢀπ interaction with
Lys45, while the sulfonamide group was crucial for the
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8
Kinase inhibition (IC50 nM)
Cpds
R1
R2
Bꢀ
9
ZAKa
BꢀRafV600E b
169.2±11.1
>1000
RafWT b
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19.2±0.66
5.1±0.31
4.2±0.31
16.8±0.40
>10000
>1000
>6000
>1000
3a
3b
3c
3d
terminal phenyl group to adopt
a
backwardꢀbending
orientation and form a πꢀπ interaction with Phe152 in the back
pocket of the protein (Figure 2C). Investigation also implied
that 3a could bind with BꢀRafV600E with a similar mode to that
of ZAK (supporting Information, Figure S2C).
901.8±9.8
>10000
12.1±0.40
7.5±0.65
5.0±0.62
>10000
>7000
>1000
>10000
>1000
>1000
3e
3f
Figure 1. Chemical structures of nonꢀselective ZAK inhibitors
Br
3g
3.3±0.29
9.6±0.08
>3000
>1000
>2000
3h
3i
>10000
22.6±0.86
994.1±84.7
5.1±0.63
>30000
>10000
>10000
>10000
>10000
>10000
>10000
>5000
3j
3k
3l
Figure 2. Design of selective ZAK inhibitors. (A) Molecular
design of new ZAK inhibitors. (B) Superposition of ZAK (PDB:
5HES, orange) and BꢀRafV600E (PDB: 4XV1, purple). (C)
Predicted binding model of 3a (blue) with ZAK (PDB: 5HES,
orange).
>10000
3m
42.7±4.2
>2000
>10000
>10000
3n
Scheme 1. Synthesis of Compounds 3a-3o.
143.7±14.4
>10000
3o
1
31.4±5.2
(23c)
84.12±
27.8
ꢀ
ꢀ
41.2±13.6
aZAK kinase inhibition was detected using ADPꢀGloTM kinase
assay according to the manufacturer’s instructions.27,28 bKinase
inhibitions against BꢀRaf wildꢀtype and V600E mutant were
performed using FRETꢀbased Z'ꢀLyte kinase assay according to
the manufacturer’s instructions as previously descripted.26 The
data are means from 3 independent experiments. cReported
data.20,25
Reagents and conditions: (a) con. H2SO4, KNO3, rt, 2 hrs,
91%; (b) SnCl2, con. HCl, EtOH, reflux, 93%; (c)
benzenesulfonyl chloride, pyridine, dry DCM, rt, 2 hrs, 93%;
(d) NH2NH2·H2O, EtOH, 80 °C, overnight, 93%; (e) pꢀ
methoxybenzyl chloride, NaOH, DMSO, 0 °C to rt, 1 hr, 62%;
(f) NaH, CH3I, DMF, rt, overnight, 47%ꢀ53%; (g) (i)
trimethylsilylacetylene, Pd(dba)2, CuI, K2CO3, tꢀ(Bu)3P, THF,
Ar2, 120 °C, overnight, 51%; (ii) TBAF, THF, rt, 1 hr, 88%
ZAK possesses a slightly larger back pocket than BꢀRafV600E
because of its outward shifting αCꢀhelix (Figure 2B). A small
hydrophobic substituent might be introduced at the terminal
phenyl group in 3a to improve the ZAK inhibitory potency
and selectivity. Based on this hypothesis, compounds 3b, 3c
and 3d, in which a chloride was introduced at orthoꢀ, metaꢀ or
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paraꢀ position of the corresponding phenyl moiety,
that observed in the 1ꢀZAK structure. This is likely because
the Pꢀloop in the 1ꢀZAK complex was tethered by the
hydrophobic interactions between the chlorobenzene moiety
and Cys22/Phe27, while in 3g the partially hydrophilic
methoxyl moiety could not tether the Pꢀloop in the same way.
respectively, were first designed and synthesized (Scheme 1).
It was shown that orthoꢀ (3b) and metaꢀ (3c) substituted
molecules indeed exhibited obviously improved ZAK
inhibitory activities with IC50 values of 5.1 and 4.2 nM,
respectively. The compounds also exhibited significantly
increased target selectivity by abolishing the inhibition against
BꢀRafV600E. Although a paraꢀchloro substitution (3d) barely
affected the ZAK suppressive function, it induced obvious
improvement on ZAK selectivity. These data also suggested
that metaꢀ position of the phenyl ring is optimal for further
optimization, and this position was well tolerated to a variety
of substituents, such as methoxylꢀ (3f), bromoꢀ (3g) and
cyanoꢀ (3h) groups to exhibit strong and selective ZAK
inhibitory potencies. For instance, compound 3h strongly
suppressed the kinase activity of ZAK with an IC50 value of
3.3 nM, but had no activity against wildꢀtype BꢀRaf and its
V600E variant. However, when the metaꢀchloride was
replaced with a methyl group, the resulting compound 3e
displayed a 3ꢀfolds potency loss. It was also noteworthy that
the orthoꢀ, metaꢀ dichloride substituted derivative 3i exhibited
an IC50 value of 9.6 nM against ZAK, which is approximately
2 fold less potent than the corresponding monoꢀsubstituted
compounds 3b and 3c.
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Figure 3. The coꢀcrystal structure of inhibitor 3g with ZAK. (A)
Coꢀcrystal structure of compound 3g and ZAK. (PDB: 5X5O) (B)
Superposition of previous ZAK (PDB: 5HES, orange) and 3gꢀ
ZAK complex crystal structure (PDB: 5X5O, bule).
Taking 3h as an example, its binding affinity with ZAK was
further determined by using an activeꢀsiteꢀdependent
competition binding assay (conducted by Ambit Bioscience,
San Diego, CA). It was shown that compound 3h tightly
bound to ZAK with a binding constant (Kd) value of 2.7 nM,
but was obviously less potent for the majority of 468 kinases
evaluated (including 403 nonmutated kinases and 65 mutated
kinases). Its kinase selectivity S(10) and S(1)30 scores are
0.017 and 0.002, respectively, at a concentration of 1.0 ꢀM,
which is approximately 370 fold higher than its Kd value with
ZAK. The potential “offꢀtarget” kinases included aurora
kinase B (AURKB), BꢀRafV600E, fynꢀrelated kinase (FRK),
KIT protoꢀoncogene receptor tyrosine kinase (KIT), mitogen
extracellularꢀsignalꢀregulated kinase kinase 5 (MEK5), saltꢀ
inducible kinase (SIK) and Srcꢀrelated tyrosine kinase lacking
Cꢀterminal regulatory tyrosine and Nꢀterminal myristylation
sites (SRMS) etc. Further evaluation revealed that 3h
exhibited approximately 17ꢀ230ꢀfold less potency against the
majority of the “off target” kinases, with an exception of FRK,
which displayed a Kd values of 22 nM (Figure 4B). These
results collectively supported the extraordinary target
specificity of 3h.
The investigation further suggested the 3ꢀmethoxyl group in
3c could be removed (3l) to display a similar potency and
selectivity against ZAK with an IC50 value of 5.1 nM.
However, the introduction of a large hydrophobic substituent,
e.g. an isoꢀpropoxyl (3n) or a cyclicꢀpentyloxyl (3o) group,
caused approximately 10ꢀ34 folds potency loss. The
corresponding compounds 3n and 3o exhibited IC50 values of
42.7 and 143.7 nM, respectively. Our modeling suggested that
the potential dual hydrogen bond network between the 1Hꢀ
pyrazolo[3, 4ꢀb] pyridine moiety with Ala85 is critical for the
binding of the compounds with ZAK protein. The replacement
of the nitrogen atom (N) with a carbon at 7ꢀ position (3k) or
methylation of the 1ꢀNH moiety (3m) indeed resulted in
almost total abolishment of ZAK inhibitory potency. The
replacement of the 1Hꢀpyrazolo[3, 4ꢀb] pyridine moiety in 3l
with a 1Hꢀpyrrolo[2, 3ꢀb]pyridine group also caused a 5ꢀfold
potency loss. The resulting compound 3j displaced an IC50
value of 22.6 nM against ZAK kinase.
Compound 3h also exhibited reasonable pharmacokinetic
properties in SpragurꢀDawley (SD) rats, with an oral
bioavailability of 20%, a high exposure value (areas under
curve (AUC)) of 253794 ꢀg/L·h and a T1/2 value of 34.6 hrs at
an oral dose of 10 mg/kg/day (Supporting Information, Table
S5). Further determination revealed that the compound
exhibited a hign plasma protein binding rate of 99.8%
(Supporting Information, Table S6).
Although the metaꢀsubstituted compounds 3c, 3g, 3h and 3l
demonstrated the strongest ZAK inhibitory potencies, they
failed to be docked into the ATP binding pocket of ZAK by
utilizing the crystal structure previously disclosed (PDB:
5HES) because of a steric hindrance with Leu57. In order to
elucidate detailed interaction between the inhibitors and ZAK,
a 1.87 Å Xꢀray crystallographic structure of 3gꢀZAK complex
was determined. In accordance with our prediction, the 1Hꢀ
pyrazolo[3, 4ꢀb]pyridine moiety indeed formed two critical
hydrogen bonds and captured a πꢀπ stacking interaction with
Ala85 and Tyr84, respectively (Figure 3A). A cationꢀπ
interaction was also found between the difluorophenyl linker
and Lys45. Additionally, two hydrogen bonds were formed
between the sulfonamide and His158 and Gly153, respectively.
Interestingly, the metaꢀ bromine phenyl group in 3g induced
an outward rearrangement of the αC helix and a reorientation
of Leu57 residue to avoid the potential steric hindrance with
the molecule. The Pꢀloop in the 3gꢀZAK complex crystal
structure adopted a conformation dramatically different from
The potential therapeutic effect of 3h on hypertrophy was
first evaluated in an AngII induced myocardial hypertrophic
cell model. Treatment of AngII obviously elevated the level of
hypertrophic biomarker BNP and induced activation
(phosphorylation) of JNK and P38 in H9c2 cardio myoblast
cells. Whereas, compound 3h doseꢀdependently suppressed
elevation of the protein levels of BNP, phosphorylated JNK
and phosphorylated p38, indicating its lockage on the AngII
induced myocardial hypertrophy process (Figure 5).
The in vivo therapeutic efficacy of 3h on myocardial
hypertrophy was further investigated in spontaneous
hypertensive rats (SHRs) which were previously validated to
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harbor high level of ZAK kinase (Figure 6D). The
compound 3h strongly inhibits the kinase activity of ZAK
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normotensive WistarꢀKyoto (WKY) rats were utilized for
direct comparison. The SHRs were randomly divided into 3
groups and treated with 3h with oral dosages of 0, 10 or 20
mg/kg/day, respectively, for 4 continuous weeks based on PK
profiles of the inhibitor. The experimental rats were sacrificed
and the left ventricles were collected for biological
determination. It was shown that the SHRs displayed greater
left ventricular masse at diastole (LVd mass) and systole (LVs
mass) than that of the normotensive WKY rats, indicating the
hypertrophic symptom of the animals. Compound 3h doseꢀ
dependently suppressed the LVd and LVs mass elevation in
SHRs. For instance, the average LVd mass value was 1.20
grams for the vehicle control SHRs, while the values were
decreased to 0.97 and 0.95 grams, which were close to the
value in normotensive WKY rats, after 10.0 (SHR L) and 20.0
(SHR H) mg/kg/day treatments of 3h, respectively (Figure 6A,
6B). The treatment also induced LVs mass decrease by 3.0ꢀ
8.0%. Hematoxylin and eosin (H&E) biopsy staining images
showed that the cardiomyocytes in normotensive control
tissues displayed normal arrangement, whereas the tissue
sections from SHRs exhibited more interstitial spaces (Figure
6C). Treatments of 3h successfully restored the cardiomyocyte
disarrangement to normal phenomena in a dose dependent
manner. Consistent to the observation in H9c2 cells,
compound 3h also evidently interrupted the phosphorylation
of JNK and p38 and decreased the level of BNP in the isolated
left ventricles (Figure 6D). These data collectively
demonstrated the therapeutic effect of 3h on spontaneous
hypertensionꢀinduced pathological cardiomyocyte hypertrophy.
with an IC50 value of 3.3 nM, and doseꢀdependently
suppresses the activation of ZAK downstream signals both in
vitro and in vivo. However, the compound is significantly less
potent for a panel of 403 nonꢀmutated kinases. A high
resolution complex crystal structure was further determined to
elucidate the detail interaction between an inhibitor and ZAK
protein.
Moreover,
compound
3h
exhibits
good
pharmacokinetic properties and promising orally therapeutic
effects on cardiac hypertrophy in a spontaneous hypertensive
rat model. To the best of our knowledge, this molecule
represents the first selective ZAK inhibitor demonstrating in
vivo efficacy. The potent ZAK inhibitory activity and
extraordinary target specificity make 3h a valuable research
probe for further biological investigation on ZAK.
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Figure 6. Compound 3h inhibits myocardial hypertrophy in vivo.
(A) Compound 3h doseꢀdependently inhibits pathological
increasing of left ventricular mass at diastole (LVd mass, bule)
and systole (LVs mass, red). (B) Quantitative data for LVd and
LVs masses. ***P<0.05 significant increase compared to WKY
control rats. ##P<0.01, ###P<0.001 significant decrease compared
to SHR rats (n = 6 per group). (C) Hematoxylin and eosin (H&E)
staining images of the heart tissue sections. H&Eꢀstained heart
sections from normal rats treated with saline show normal
architecture of cardiac myocytes with centrally placed nuclei.
Heart sections from SHR rats show disorganization of the
myofibrils with large inter spaces between the myocytes (long
arrow) and abnormal nuclear orientation (arrow head). Treatment
with 3h ameliorate these effects noted in SHR rat hearts. (D)
Compound 3h doseꢀdependently inhibits the expression and
phosphorylation of ZAK downstream signaling in vivo.
Figure 4. KinomeScan kinase selectivity profiles of 3h. (A)
Compound 3h was profiled at a concentration of 1.0 ꢀM against a
diverse panel of 468 kinases by DiscoveRx. (B) Binding constants
(Kd values) of compound 3h against the “top hits”
EXPERIMENTAL SECTION
General Chemistry. Reagents and solvents were obtained
from commercial suppliers and used without further
purification. Flash chromatography was performed using silica
gel (200ꢀ300 mesh). All reactions were monitored by TLC,
using silica gel plates with fluorescence F254 and UV light
1
visualization. H and 13C NMR spectra were recorded on a
Bruker AVꢀ400 spectrometer at 400 MHz and Bruker AVꢀ500
spectrometer at 125 MHz, respectively. Coupling constants (J)
are expressed in hertz (Hz). Chemical shifts (δ) of NMR are
reported in parts per million (ppm) units relative to internal
control (TMS). The firstꢀorder peak patterns are indicated as s
(singlet), d (doublet), t (triplet), q (quadruplet). Complex
nonfirstꢀorder signals are indicated as m (multiplet). The lowꢀ
or highꢀresolution of ESIꢀMS was recorded on an Agilent
1200 HPLCꢀMSD mass spectrometer or Applied Biosystems
QꢀSTAR Elite ESIꢀLCꢀMS/MS mass spectrometer,
respectively. The purity of compounds was determined by
reverseꢀphase highꢀperformance liquid chromatography
Figure 5. Compound 3h doseꢀdependently inhibits the expression
and phosphorylation of ZAK downstream signaling in AngIIꢀ
induced H9c2 cardiomyoblast cells. The bands are representatives
from 3 independent experiments.
CONCLUSION
In summary, a series of Nꢀ(3ꢀ((1Hꢀpyrazolo[3, 4ꢁb]pyridinꢀ
5ꢀyl)ethynyl)benzenesulfonamides were discovered as novel
selective ZAK inhibitors. One of the most promising
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(HPLC) analysis confirming to be over 95% (>95%).
control. The plate was measured on an EnVision Multilabel
Reader (PerkinꢀElmer). Curve fitting and data presentations
were performed using Graph Pad Prism, version 5.0. Every
experiment was repeated at least for 3 times.
Analytical HPLC analyses were conducted using an Agilent
1260 system (G1310B Iso pump and G1365D MWD VL
detector) with an YMCꢀTriart C18 reversedꢀphase column
(250×4.6 mm, 5 ꢀm) at 254 nm. Elution was MeOH in water
and flow rate was 1.0 mL/min. Preparative HPLC (PHPLC)
purifications were performed using Agilent 218 Solvent
Delivery Module and an Agilent 325 dule wavelength UVꢀVIS
detector with an YMCꢀTriart C18 reversedꢀphase column
(250×20 mm, 5 ꢀm) at 254 nm. The LC column was
maintained at room temperature.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and compound characterization, procedures,
and in vitro enzymatic activity assay, amino acid sequence alignꢀ
ment, computational study, KINOMEscan, animal expriments,
western blot analysis, hematoxylin and eosin staining, crystallizaꢀ
tion and structure determination, pharmacokinetics study. 1H
NMR, 13C NMR and HR MS spectra of compounds 3aꢀ3o (PDF)
and Molecular Formula Strings (CSV).
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In Vitro Enzymatic Activity Assay. The functional assays
of compounds on the kinase activities of ZAK were deterꢀ
mined using the ADPꢀGloTM kinase assay according to the
manufacturer’s instructions (Promega, Madison, WI).27, 28 The
reaction was carried out in a 384ꢀwell plate in a volume of 10
ꢀL solvent containing 50 ꢀM ATP, 200 ꢀg/mL substrate (myeꢀ
lin basic protein, MBP) and serials of diluted compound with
appropriate amount of kinase ZAK. Reactions were started
immediately by adding ATP. After 30 min of incubation at
room temperature, 10 ꢀL of ADPꢀGlo reagent was added into
each well to stop the reaction and consumed the remaining
ADP within 40 min. At the end, 20 ꢀL of kinase detection
reagent was added into the well and incubated for 30 minutes
to produce a luminescence signal and the light generated was
examined with EnVision Multilabel Reader (Perkin Elmer,
Inc.). Data analysis and curve fitting were performed using
Graphpad Prism5 (Graphpad Software, Inc). Every experiment
was repeated at least 3 times.
BꢀRaf, BꢀRafV600E (as BꢀRafV600E in supplier’s catalogue)
and the Z’ꢀLyte Kinase Assay Kit were purchased from
Invitrogen. The experiments were performed according to the
instructions of the manufacturer as previuosly descripted.26
The concentrations of kinases were determined by
optimization experiments and the respective concentration was:
BRAFV600E (PV4173, Invitrogen) 0.22 ꢀg/ꢀL. First, the
compounds were diluted threeꢀfold from 10ꢀ10 M to 1x10ꢀ4 M
in DMSO and a 400 ꢀM compound solution was prepared (4
ꢀL compound dissolved in 96 ꢀL water). Second, a 100 ꢀM
ATP solution in1.33×Kinase Buffer was prepared. Third, a
kinase/peptide mixture containing 2×kinase and 4 ꢀM
Ser/Thr3 peptide (Invitrogen, PV3193) was prepared right
before use. Kinase/peptide mixture was prepared by diluting
Z’ꢀLYTE Ser/Thr3 peptide (Invitrogen, PV3176 ) and kinase
in 1×Kinase Buffer, and 0.2 ꢀM Ser/Thr3 phosphoꢀpeptide
solution was made by adding Z’ꢀLYTE Ser/Thr3 phosphoꢀ
peptide to 1×Kinase Buffer. The final 10 ꢀL reaction consists
of 0.002 ng of BꢀRaf, 2 ꢀM Ser/Thr3 peptide in 1×kinase
buffer. For each assay, 10 ꢀL kinase reactions were made at
AUTHOR INFORMATION
Corresponding Authors
*dingke@jnu.edu.cn (K. D.)
*cyhuang@mail.cmu.edu.tw (C.Y. H.)
*yunch@hsc.pku.edu.cn (C.H. Y.)
*qwzhang@umac.mo (Q.W. Z.)
Author Contributions
#Yu Chang, Xiaoyun Lu, Marthandam Asokan Shibu and YiꢀBo
Dai contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors appreciate the financial support from National Natuꢀ
ral Science Foundation of China (81425021, 81673285 and
31270769),
Guangdong
Province
(2013A022100038,
2015A030312014, 2015A030306042, and 2016A050502041),
Guangzhou City (201508030036), Jinan University and Taiwan
Department of Health Clinical Trial and Research Center of Exꢀ
cellence (MOHW106ꢀTDUꢀBꢀ212ꢀ113004).
ABBREVIATIONS
ZAK, leucineꢀzipper and sterileꢀα motif kinase; MLK, mixedꢀ
lineage kinase; SAM, sterileꢀα motif; JNK, cꢀJun Nꢀterminal proꢀ
tein kinase; MAPK, mitogen activated protein kinase; ANF, atrial
natriuretic factor; BNP, brain natriuretic peptide; DN, dominant
negative; AURKB, aurora kinase B; FRK, fynꢀrelated kinase;
KIT, KIT protoꢀoncogene receptor tyrosine kinase; MEK5, mitoꢀ
gen extracellularꢀsignalꢀregulated kinase kinase 5; SIK, saltꢀ
inducible kinase; SRMS, srcꢀrelated tyrosine kinase lacking Cꢀ
terminal regulatory tyrosine and Nꢀterminal myristylation sites;
SD, SpragurꢀDawley; AUC, areas under curve; SHRs, spontaneꢀ
ous hypertensive rats; WKY, WistarꢀKyoto; LVd mass, left venꢀ
tricular mass at diastole; LVs mass, left ventricular mass at systoꢀ
le; H&E, hematoxylin and eosin; SDSꢀPAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene
difluoride.
first (including 2.5 ꢀL compound solution,
5
ꢀL
Kinase/Peptide Mixture, and 2.5 ꢀL ATP solution). Mixed the
plate thoroughly and incubated for one hour at room
temperature. Then 5 ꢀL development solution was added to
each well and the plate was incubated for 1h at room
temperature; the nonphosphopeptides were cleaved at this time.
In the end, 5 ꢀL stop reagent was loaded to stop the reaction.
For the control setting, 5 ꢀL phosphoꢀpeptide solution instead
of kinaseꢀpeptide mixture was used as 100% phosphorylation
control. 2.5 ꢀL 1.33×Kinase Buffer instead of ATP solution
was used as 100% inhibition control, and 2.5 ꢀL 4% DMSO
instead of compound solution was used as the 0% inhibitor
ANCILLARY INFORMATION
PDB code of ZAKꢀ3g coꢀcrystal structure: 5X5O.
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