Chemical Features Important for Activity in a Class of Inhibitors Targeting the Wip1 Flap Subdomain

Article abstract of DOI:10.1002/cmdc.201700779

The wild-type p53 induced phosphatase 1, Wip1 (PP2Cδ), is a protein phosphatase 2C (PP2C) family serine/threonine phosphatase that negatively regulates the function of the tumor suppressor p53 and several of its positive regulators such as ATM, Chk1, Chk2

Full text of DOI:10.1002/cmdc.201700779

Accepted Article
Title: Chemical features important for activity in a class of inhibitors
targeting the Wip1 flap subdomain
Authors: Harichandra D Tagad, Subrata Debnath, Victor Clausse,
Mrinmoy Saha, Sharlyn Mazur, Ettore Appella, and Daniel H.
Appella
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ChemMedChem
Chemical features important for activity in a class of
inhibitors targeting the Wip1 flap subdomain
Harichandra D. Tagad,[a]† Subrata Debnath,[a]† Victor Clausse,[b] Mrinmoy Saha,[b] Sharlyn J.
Mazur,[a] Ettore Appella,[a] Daniel H. Appella*[b]
[a] Dr. H. D. Tagad, Dr. S. Debnath, Dr. S. Mazur, Dr. E. Appella
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA
†These authors contributed equally to this work
[b] Dr. V. Clausse, Dr. M. Saha, Dr. D. H. Appella
Synthetic Bioactive Molecules Section, LBC, NIDDK, NIH, 8 Center Drive, Room 404, Bethesda, MD 20892 USA
E-mail: appellad@niddk.nih.gov
Abstract
The wild-type p53 induced phosphatase 1, Wip1 (PP2Cδ), is a PP2C family Ser/Thr phosphatase that negatively regulates the function
of multiple proteins such as p53, ATM, Chk1, Chk2, Mdm2 and p38 MAPK involved in a DNA damage response. Wip1
dephosphorylates and inactivates its protein targets which are critical for cellular stress responses. Additionally, Wip1 frequently
amplified and overexpressed in several human cancer types. Because of its negative role in regulating the function of essential
proteins, Wip1 has been identified as a potential therapeutic target in various types of cancers. Based on a recently reported Wip1
inhibitor (G-1), we performed an extensive structure-activity relationship (SAR) analysis. This led us to interesting findings in SAR
trends and to the discovery of new chemical analogues with good specificity and bioavailability.
biochemical high throughput screen (HTS) reported by
Introduction
Gilmartin et al. [17]. Very limited SAR is available for these
classes of compounds as the manuscript of Gilmartin et
Wip1 (PPM1D or PP2Cδ), a member of the protein
phosphatase 2C (PP2C) family, was first identified as the
al. only discloses a small number of molecules. We
product of a gene induced by wild-type p53 after DNA
therefore performed a more thorough SAR study and
damage.[1] Similar to the other members of the PP2C
developed analogues that exhibit good affinity and
family, Wip1 is monomeric, insensitive to oakadaic acid,
bioavailability. Our study demonstrates interesting SAR
and dependent on millimolar concentrations of Mn2+ or
trends and also provides critical insights into designing
Mg2+ for activity in vitro.[2] Wip1 inactivates a wide range of
new compounds that target the Flap subdomain of Wip1.
proteins, such as p38 MAPK,[3] Chk1,[4] Chk2,[5] ATM,[6]
and p53,[4] by dephosphorylating phosphothreonine (pT)
Results and Discussion
or phosphoserine (pS) residues. Wip1 is amplified or
overexpressed in numerous human cancers including
breast cancer,[7] ovarian clear cell carcinoma,[8] gastric
We performed detailed biochemical studies of analogs
based on the initial hit of the biochemical screen described
by Gilmartin et al.,[17] hereafter referred to as compound
G-1, (Figure 1A). To facilitate the SAR study, we
synthesized variants of G-1 with alterations in functional
groups at either end as well as a central linker between
the ends that could be altered to adjust the distance.
cancer,[9]
pancreatic
adenocarcinoma,[10]
medulloblastoma,[11] and neuroblastoma.[12] Moreover,
Ppm1d-null mice show a tumor-resistant phenotype,
suggesting Wip1 has a critical role in controlling cell cycle
checkpoints in response to DNA damage.[13] Thus,
developing new strategies for inhibiting Wip1 activity may
be beneficial in the treatment of a number of human
cancers.[14]
The Wip1 inhibitors identified to date have been
discovered through the use of extensive chemical library
[15]
screening approaches
or substrate-based design of
phosphopeptides.[16] Based on biochemical and
biophysical screening of small molecule and DNA-
encoded compound libraries, an allosteric inhibitor of
Wip1, GSK2830371, was recently reported.[17] It was
proposed that the basis for the selectivity of this inhibitor
for Wip1 over other PP2C family members was due to its
interaction with the Flap of Wip1. TheFlap is a sub-domain
located near the catalytic site that is important for
substrate recognition and exhibits substantial amino acid
sequence variability among PP2C family members.
Although GSK2830371 is a selective and potent inhibitor
of Wip1 activity in cells, it does not exhibit favorable
pharmacokinetics.[12a, 17-18]
As the Flap subdomain provides the key structural linkage
between the activity and the substrate selectivity of PP2C
phosphatases, we believe it is important to understand the
structure-activity relationships (SAR) in Flap subdomain-
based inhibitors. As the starting point of our investigation,
we chose one of the initial compounds identified in the
Figure 1. Initial Wip1 inhibitors. (A) Structures of compound G-1 and
1. (B) Inhibition of Wip1 phosphatase activity toward the ATM (1981pS)
phosphopeptide by compounds G-1 and 1.
At the outset, the central 3-cyclohexyl-L-alanine moiety of
G-1 was retained and 2-carboxythiophene was used to
form the N-terminal amide. To introduce a linker, an
amide was formed with the ε amine sidechain of Lysine.
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ChemMedChem
The α-amine of Lysine was attached to
a
3-
The loss in inhibition observed upon substitution at
position X1 indicates that the 3-cyclohexyl-L-alanine is
highly constrained, plays a vital role in binding to the Wip1
protein and hence cannot be changed.
Next, we synthesized compounds 11-23 with variations at
the X2 and X3 positions using various carboxy acids and
amines, respectively, while retaining the central
cyclohexyl-L-alanine according to route-A as mentioned in
hydroxybenzoyl group that serves as the C-terminal cap
(Figure 1A). Compound 1 was synthesized via amino acid
coupling reactions using 2-thiophenecarboxylic acid, 3-
cyclohexyl-L-alanine, L-Lysine and 3-hydroxybenzoic acid
as shown in Scheme 1. Compounds 2-10 were
synthesized in a similar manner as compound 1 with
variation of the central X1 positioned 3-cyclohexyl-L-
alanine moiety. Importantly, we chose to test the activity
of these analogs to inhibit the phosphatase activity of Wip1
a
supporting information. On the other hand,
phosphoserine containing compounds 24-26 were
synthesized by the solid-phase peptide synthesis utilizing
9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl chemistry.
All synthesized compounds were tested for inhibitory
activity as shown in Table 2. Similarly, compounds 27-39
were synthesized by varying the amine at position X3 (as
shown in Table 3) while keeping X1 and X2 positioned 3-
cyclohexyl-L-alanine and carboxy thiophene moiety
respectively. (Route-B: supporting information.)
using the ATM (1891pS) phosphopeptide, which is a
19]
physiological substrate,[6,
rather than fluorescein
diphosphate which is an artificial substrate. When using
the ATM phosphopeptide substrate in assays, compound
G-1 had an IC50 of 4.9 μM, which is about ten times weaker
compared to inhibition tests using fluorescein diphosphate
as the substrate. Under the same testing conditions, 1 had
an IC50 of 0.92 μM, approximately five-fold better that G-1
(Figure 1B). To better understand the SAR of these types
of molecules, we identified three regions of 1 for further
modifications (Figure 2).
Replacement of the 2-thiophenecarboxyl moiety with 3-
aminobenzoyl at the X2 position and varying X3 with
differently substituted aromatic moieties produced
moderate effects on the activity (11-14). However,
reducing the distance between 3-cyclohexyl-L-alanine and
a 3-chloro substituted aromatic ring at X3 (compounds 11
and 13) resulted in a five-fold improvement in activity.
Within position X2, N-terminal extension of the 3-amino
benzoyl moiety with acetyl (15), orotyl (16), 4-
hydroxyphenylacetyl (17), or 2-nitrophenyl sulfonyl (18)
(with reduced distance between X1 and X3 positioned
substituted aromatic ring) led to activities similar to those
of compounds 11-14. We next explored various
substitutions at X2, such as N-acetyl proline (19), 1H-
tetrazole-5-acetyl (21), Cbz-Lysine (22), and orotyl (23),
all of which led to complete loss of activity except for weak
activity exhibited by compound 20 (IC50 =167 µM). We
also tested some phosphorylated serine analogues (24-
26) at position X2, but these compounds exhibited little to
no inhibitory activity. Furthermore, we synthesized several
compounds (data not shown) with di- and tri- substituted
thiophenecarboxyl derivatives at the X2 position of
compound 1, and these showed no improvement in
activity or resulted into very weak inhibitors.
Figure 2. X1, X2, and X3 positions of compound 1.
We initially explored variation of the 3-cyclohexyl-L-
alanine moiety at position X1 as well as variations at X3
(Table 1). Replacement of the 3-cyclohexyl-L-alanine with
L-leucine (2) dropped IC50 by more than a factor of 250.
The corresponding L-Tyrosine (3) and L-Serine (4)
analogs showed only 25% and 20% inhibition
respectively, at 50 µM concentration, respectively.
Modification with L-Phenylalanine (5) or (S)-2-(4-
pentenyl)alanine (6) resulted in a complete loss of activity.
Notably, changing the stereochemical configuration of 3-
cyclohexyl-L-alanine to the D isomer (7) also led to the
complete loss of inhibitory activity. Furthermore,
substitution with L-statine (8), substitution with racemic 1-
aminocyclohexanecarboxyl (9), or addition of an amide
linkage to the cyclohexyl moiety (10) did not improved
activity, suggesting critical roles of both the stereogenic
center and the cyclohexyl ring.
These results suggest that SAR at position X2 is highly
constrained. Most of the modifications we tested at
position X2 led to significant or complete loss of inhibitory
potency for Wip1. Only, inhibitor 18 showed a potency
similar to that of the inhibitor 1, with an IC50 of 1 µM.
Although some modifications at position X2 resulted in
moderate improvements (11-26), the SAR appears to be
largely
constrained
to
the
original
2-
thiophenecarboxamide. Based on the overall results, any
modification at X1 is highly disfavored and only slight
alterations at X2 are tolerated.
To evaluate the effects of modifying position X3, we
retained the L-cyclohexyl alanine and carboxy thiophene
moieties at positions X1 and X2, respectively. We modified
X3 as depicted in Table 3. Initially we acetylated the α-
amino group of L-lysine with free carboxy acid (27) or
methyl ester (28), each of which showed moderate
activity. Further extension of these acetyl analogous with
substituted-benzene derivatives (compounds 29 and 30)
resulted in 3.5 and 7-fold improved activity, respectively,
compared with compound 28. Furthermore, compound 30
showed the same activity as compound 18. On the other
hand, replacing the 3-hydroxybenzoyl group with 4-
hydroxyphenylacetyl (31), 6-bromo-3-carboxy pyridine
Scheme 1. General synthesis protocol for Compound 1; Reagents
and conditions: (a) EDC, HOBt, NMM, DMF, RT, overnight, (b)
H2/Pd/C, MeOH, RT, 6 h; (c) LiOH, MeOH:H2O (4:1), 5 h.
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Table 1. SAR at position X1
Compound
Structure
IC50 (µM)
inhibition†
1
0.92
2
3
>100
25%
20%
4
5
ND
ND
6
7
8
9
ND
ND
ND
10
ND
†
* Racemic compound, Inhibition at 50 µM compared with DMSO; ND-not detectable
(32), or 4-Bromo-α-hydroxyphenylacetyl (33) resulted in ≥
3-fold reduced activity compared with compound 30,
suggesting that substitution with electron donating or
electron withdrawing groups (compound 29-33) at X3 has
very little or no effect on the activity. In addition, replacing
the aromatic ring (compound 29-33) with the aliphatic
diethylphosphonoacetyl moiety (34) led to an 18-fold drop
in activity. Substitution of the aromatic ring of X3 position,
with 3-hydroxybenzyol at the α-amino group of L-lysine,
restored inhibition (35), indicating that proper substitution
of the six-membered aromatic ring in X3 is essential for
activity. Improvements in activity were observed with
compounds 36, 37, and 38 with modest changes in the
arrangement of functional groups and carbon chain
lengths. Interestingly, we found a moderate effect of the
carbon chain length between the X1 and X3 positions in
comparing compounds 38 and 39. We found that
compound 39, which consists of 2-carboxythiophene, 3-
cyclohexyl-L-alanine, and glycine-linked 3-chloroaniline at
X2, X1, and X3 positions respectively, was the most active
inhibitor in this study.
In summary, slight modifications at X1 led to complete
losses in activity. At position X1, it is important to maintain
the correct type of cyclic ring (cyclohexane) as well as the
correct chirality (S). Slight modifications are tolerated at
positions X2 and X3. For instance, compounds 18 (with
modification at X2) and 38 (with modification at X3)
retained activities similar to that of compound 1. A small
reduction in carbon chain length between positions X1 and
X3 led to slightly improved activity (compounds 38 and 39).
Taken together, these results suggest that the SAR for the
class of compound is not permissive for a wide range of
substitutions. Compound 39 was identified as the best
analog from the series of molecules, and was therefore
selected for additional studies.
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Table 2. Structural modifications at positions X2 and X3
Compound
X2
X3
IC50 (µM)†
inhibition‡
11
11
12
13
4.3
2.2
14
15
16
1.7
12.9
4.5
17
18
3.3
1.0
19
20
ND
>100
21
22
23
ND
ND
ND
24
25
ND
7%†
8%†
26
* Racemic compound. † ND: not detectable. ‡ Inhibition at 50 µM compared with DMSO.
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As specificity within the same family of PP2C
complex, ES, inhibiting catalytic activity. The dependence
of the initial rates of reaction on the concentrations of
substrate and compound 39 were globally fitted to the
Mixed Inhibition Model (Figure 3C). The data were well
described by the model, indicating that compound 39
inhibits Wip1 activity toward phosphopeptide substrates
by an allosteric mechanism with parameter estimates of
kcat = 7.45 ± 0.25 s-1, Ki = 2.2 ± 0.7 µM and Km = 76 ± 7 µM
with α = 1.41. As the value of α is greater than unity, the
binding of the inhibitor is stronger to the free enzyme than
to the enzyme-substrate complex.
phosphatases is a critical issue in drug development, we
assessed whether compound 39 exhibited inhibitory
activity toward human PPM1A, a closely related PP2C
phosphatase. As shown in Figure 3A, compound 39
potently inhibited Wip1 phosphatase activity while
remaining inactive with PPM1A. This result is consistent
with the high specificity of the inhibitors reported by
Gilmartin et al. [17]
Next, we investigated the mechanism of inhibition. In the
Mixed Inhibition Model (Figure 3B), the inhibitor can bind
to both the free enzyme, E and the enzyme-substrate
Table 3. SAR at X3 position
Compound
X3
IC50 (µM)
27
9.0
28
29
30
31
6.9
1.9
1.0
3.8
32
3.9
33
34
35
36
37
3.0
17.9
5.4
2.6
1.5
38
39
1.0
0.65
* Racemic compound
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Figure 3. Selectivity and mechanism of inhibition of compound 39 over PPM1A.(A) Inhibition of Wip1 activity toward the ATM (1981pS) phosphopeptide
by compound 39. (B) Mixed Mode Inhibition Scheme. (C) Dependence of Wip1 phosphatase activity on concentrations of [S1981pS] human ATM (1976-
1986)-GY peptide in absence (
) and presence of 0.4 µM (
) and 2.0 µM (
) compound 39.
We chose to test the bioavailability and inhibitory activity
of compounds 1 and 39 on Wip1 phosphatase activity in
the human breast adenocarcinoma cell line MCF-7. The
S139-phosphorylated form of the histone variant H2AX
(γH2AX) is an important component of the DNA double
strand break-induced DNA damage response (DDR)
signaling pathway. Phosphopeptides containing H2AX
pS139 are directly dephosphorylated by Wip1 and
dephosphorylation of γH2AX by Wip1 is important for the
recovery of cells following the induction of DNA damage
In agreement with previously reported qualitative
results,[17] pre-treatment of MCF7 cells with GSK2830371
resulted in a dose-dependent increase in γH2AX levels 75’
min after exposure to 10 Gy ionizing radiation (IR) (Figure
S1, EC50 = 0.20 ± 0.12 μM, n = 3). Similarly, pre-treatment
of MCF7 cells with compound 1 or compound 39 resulted
in a dose-dependent increase in γH2AX levels 75’ min
after exposure to 10 Gy IR (Figure 4). These results show
that compounds 1 and 39 are bioavailable in MCF7 cells
and suppress Wip1 enzymatic activity towards IR-induced
H2AX phospho-Ser139. Interestingly, treatment of MCF7
cells with GSK2830371, compound 1 or compound 39
produced small, dose dependent increases in γH2AX
levels in the absence of exposure to IR (Figure S2). In
response to the formation of endogenous DNA double-
stranded breaks, particularly during S phase, cells lacking
.
[19-20] γH2AX levels following the induction of DNA double-
strand breaks by ionizing radiation or chemicals have
been used as an indicator of Wip1 activity in human
cells.[21]
To assess the bioavailability of our Wip1 inhibitors in
MCF7 cells, we adapted a recently reported ELISA
method for quantitative determination of ƴH2AX levels.[22]
Wip1 activity exhibit higher γH2AX levels [23]
.
Figure 4. Dose-dependent inhibition of Wip1-induced dephosphorylation of γH2AX in human breast cancer cells during recovery from following exposure
to ionizing radiation (IR). MCF7 cells were incubated with various concentrations of compound 1 (left panel) or compound 39 (right panel) for 1 h prior to
exposure to 10 Gy IR and continuing through a 75-min recovery period. γH2AX levels were determined by quantitative ELISA. The dose response was
fitted by a four-parameter logistical model. Curves shown are representative. Compound 1: EC50 = 56 ± 5μM, n = 3. Compound 39: EC50 = 24 ± 6 μM, n
= 3.
central position X1. All tested modifications at this position
Conclusions
resulted in complete loss of inhibitory activity. Some
In summary, we performed an extensive SAR study based
modifications at positions X2 and X3 positions were
on compound G-1, an initial hit from a screen that was
used for development of a novel Wip1 inhibitor.[17] In the
present study, we found that the inhibitory activity is highly
dependent on the carbocyclic ring and chirality at the
tolerated, but most did not lead to significant
improvements in activity. All three parts of the scaffold
investigated in the SAR study are crucial for activity.
These studies led to the development of compound 39,
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10.1002/cmdc.201700779
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which exhibited improved inhibitory compared with that of
4 N HCl in 1,4-dioxane was added to the afforded Boc-
protected peptide/amine (ca. 1.0 mmol) at 0 °C and stirred
for 1 h at room temperature. The solvent was removed
under vacuum to afford the product as the corresponding
HCl salt and used directly to the next step without
purification.
compound G-1 in assays that are designed to test activity
in the presence of physiologically-relevant substrates.
Furthermore compounds 1 and 39 were found to suppress
Wip1 enzymatic activity towards H2AX phospho-Ser139 in
MCF-7 cells, demonstrating their bioavailability to cells.
Further work to identify new classes of Wip1 Flap-targeted
inhibitors will continue, using the assays described in this
study.
1.3. General procedure for ester hydrolysis
To a solution of methyl ester (1 mmol) in 1:1 (THF: H2O, 5
mL) was added lithium hydroxide monohydrate (1.5–2
mmol) at 0 °C and stirred for 6 h at room temperature. The
solvent was evaporated under vacuum, the residue was
taken into water (2–3 mL) and acidified to pH = 2 with 1N
HCl and extracted with ethyl acetate (3 × 20 mL). The
organic layer was washed with brine, dried over Na2SO4
and filtered. The filtrate was evaporated under vacuum to
afford corresponding acid.
Experimental Section
Fmoc or Boc-protected amino acids were purchased from
Novabiochem (San Diego CA). All chemicals and solvents
were purchased from Sigma-Aldrich (St. Louis, MO) and
AnaSpec (Fremont, CA). All reagents and solvents were
used as received from commercial sources without further
purification.
Final compounds were purified using reversed-phase
1.4. General procedure for hydrogenation
high-performance liquid chromatography (RP-HPLC) on a
preparative C4 column (BioAdvantage Pro 300, Thomson
Liquid Chromatography) with a binary solvent system: a
linear gradient of CH3CN in 0.04 % trifluoroacetic acid
(TFA) and water in 0.05% TFA with a flow rate 7 mL/min
and detected at 220 nm. Analytical HPLC was performed
using a C4 reverse phase column with a binary solvent
system: linear gradient of 0.04 % TFA:CH3CN 10–900%
in 0.05% aqueous TFA in 30 min at a flow rate of 1 mL/min,
detected at 230 nm. The purity of the compounds was
found to be >94% using analytical HPLC based on peak
area percentage. Mass spectra were obtained using a
Waters MALDI micro MX (MALDI-TOF) with α-cyano-4-
hydroxy cinnamic acid a matrix. Proton NMR spectra were
recorded at 400 MHz on a Bruker 400 spectrometer. For
NMR spectra, chemical shifts are expressed in parts per
million (ppm) downfield from internal tetramethylsilane (δ
0) in DMSO-d6 as a solvent
To a solution of Cbz-protected amine (1 mmol) in MeOH
(10 mL) was added 10% Pd/C (35 mg). The suspension
was stirred under 1 atm of H2 for 5–6 h and then vacuum
filtered through a bed of Celite, which was washed with
Methanol. Evaporation under vacuum to afford the free
amine which was directly used for next step without
purification.
2. In vitro phosphatase assay:
Phosphatase activity was measured using the biomol
green-based assay as described previously. [16, 17] In brief,
reactions were carried out in 50 mM Tris-HCl, pH 7.5, 0.1
mM EGTA, 1 mM DTT, 1 mM CHAPS, 0.1 mg/ml BSA, 30
mM MgCl2 for 7 min at 30 ˚C. Amounts of phosphate
released were calculated using a phosphate standard
curve. Inhibition of phosphatase activity was determined
using 100 µM ATM (1981pS) substrate peptide in 50 mM
Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mM DTT, 1 mM
CHAPS, 0.1 mg/ml BSA, and 30 mM MgCl2 for 7 min at 30
˚C in the presence of various concentrations of inhibitor.
IC50 values were determined by fitting a standard logistical
curve to log(concentration) vs. inhibition using GraphPad
Prism, with the upper plateau being determined from the
extent of reaction in the absence of inhibitor (negative
control) and the lower plateau being determined from
readings in the absence of enzyme. GSK2830371 (Sigma-
Aldrich) was used as the positive control.
1. General procedure for synthesis of compounds 1–23 and
27–39:
1.1 General procedure for peptide coupling:
The acid (1 mmol) was dissolved in DMF (2 mL) and
cooled to 0 °C with an ice bath. HOBt. H2O (1.2 mmol),
EDC.HCl (1.2 mmol), and N-methylmorpholine (3 mmol)
were added sequentially. The resulted mixture was stirred
for additional 5 min before amine (1 mmol) was added.
After stirring at 0 °C for 30 min, the reaction mixture was
allowed to attain room temperature and stirred overnight.
The reaction mixture was diluted with ethyl acetate (15
mL) and 10% citric acid (10 mL), the aqueous layer was
further extracted with ethyl acetate (2 × 10 mL). The
combined organic extracts were washed with water, sat.
NaHCO3, brine, dried over anhydrous Na2SO4 and
concentrated in vacuo to give the crude product which was
used in the next step without purification. In case of final
compounds, a small portion of the product was purified by
preparative HPLC. The desired fractions were collected
and immediately lyophilized to afford amorphous powders.
3. γ-H2AX ELISA assay
3.1. Cell culture
MCF7 cells were cultivated in DMEM (Gibco)
supplemented with 10% fetal bovine serum (Atlanta
Biologicals) and 2 mM L-Glutamine (Gibco) at 37 °C in a
humidified atmosphere containing 5% CO2 at 37 °C.
GSK2830371 (Sigma-Aldrich), Compound 1, or
Compound 39 dissolved in DMSO were added in culture
medium at different concentrations to inhibit Wip1.
3.2 γH2AX ELISA
To assess γH2AX levels in MCF7 cells, we modified the
γH2AX ELISA assay protocol described by Ji et al.22
Briefly, cells were pre-incubated with Wip1 inhibitors for 60
1.2. General procedure for N-Boc-deprotection with 4N HCl in
dioxane
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returned to the incubator at 37 °C for 75 minutes. Cells
were then harvested and pellets were lysed for 30 minutes
on ice with Cell Extraction Buffer (Invitrogen)
supplemented freshly with a protease inhibitor cocktail
(Roche Diagnostics GmbH), phosphatase inhibitors
(Roche Applied Science) and 1 mM phenylmethylsulfonyl
fluoride (Sigma-Aldrich). After lysis, SDS (Sigma-Aldrich)
was added to a final concentration of 1% and lysates were
sonicated, then boiled for 5 minutes. Lysates were clarified
by centrifugation (12000 × g, 4 °C, 10 min), and used to
perform the ELISA assay. High-capacity antibody-binding
white opaque 96-well plates (Thermo Scientific) were
coated overnight at 4 °C with phospho-H2AX(Ser139)
mouse monoclonal antibody, clone JBW301 (Millipore) in
a carbonate-bicarbonate buffer and blocked for 1.5 hours
at 37 °C with SuperBlock (TBS) blocking buffer (Thermo
Scientific Pierce). The γH2AX standard was a synthetic
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[7]
peptide
from
Invitrogen
(AVLLPKKTSATVGPKAPSGGKKATQA[PS]QEY).
Samples, standards and controls were loaded onto the
plate and incubated for 18 h at 4 °C. After 4 washes with
PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich),
anti-histone H2AX rabbit polyclonal antibody (Abcam,
Cat#: ab10475) diluted in PBS with 2% BSA (Affymetrix)
supplemented with 10 µl of mouse serum (Sigma-Aldrich)
was added to a final concentration of 0.3 µg/ml and
incubated for 2.5 h at room temperature. After 4 washes
with PBS-0.1% Tween, Goat anti-rabbit HRP-conjugated
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polyclonal antibody (KPL) was added at
a final
concentration of 0.5 µg/ml in PBS with 2% BSA and
incubated in the dark for 1.5h at room temperature. Plate
was then washed again 4 times with PBS with 0.1%
Tween and SuperSignal® ELISA Pico Chemiluminescent
Substrate (Thermo Scientific) was added. Signal
measurement was then performed immediately with a
Victor3 V1420 Multilabel plate reader (Perkin Elmer), and
analysis was performed with GraphPad Prism 7 software.
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Acknowledgements
This research was supported by the Intramural Research
Programs of the Center for Cancer Research
(CCR), National Cancer Institute (NCI) and the National
Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK), National Institutes of Health (NIH).
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Keywords: Wip1, Inhibitors, Enzyme kinetics, ELISA
assay.
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