Synthesis and MEK1 inhibitory activities of imido-substituted 2-chloro-1,4-naphthoquinones

Article abstract of DOI:10.1016/S0968-0896(03)00267-0

Mitogen activated protein kinases are of interest as research tools and as therapeutic target for certain physiological disorders. In this study, we found 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone 6 to be a selective inhibitor of MEK1 with an IC₅₀ of 0.38 μM. An open-chain homologue, 10, showed selective cytotoxicity against renal cancer in the NCI in vitro tumor screening. Structure-activity relationship study of eight compounds showed the cyclic imido-substituted chloro-1,4-naphthoquinone as more potent and selective MEK1 inhibitors than the open chain homologues. The imido-substituted chloro-1,4-naphthoquinones were synthesized in a straightforward fashion by refluxing 2-amino-3-chloro-1,4-naphthoquinone with the appropriate acid chloride or diacyl dichloride.

Full text of DOI:10.1016/S0968-0896(03)00267-0

Bioorganic & Medicinal Chemistry 11 (2003) 3165–3170
Synthesis and MEK1 Inhibitory Activities of Imido-Substituted
2-Chloro-1,4-naphthoquinones
Oladapo Bakare,a,* Curtis L. Ashendel,b Hairuo Peng,c Leon H. Zalkowc
and Edward M. Burgessc
aDepartment of Chemistry, Howard University, 525 College Street, Washington DC 20059, USA
bDepartment of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy, Purdue University,
West Lafayette, IN 47907, USA
cSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
Received 13 July 2001; accepted 3 April 2003
Abstract—Mitogen activated protein kinases are of interest as research tools and as therapeutic target for certain physiological
disorders. In this study, we found 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone 6 to be a selective inhibitor of MEK1 with an
IC50 of 0.38 mM. An open-chain homologue, 10, showed selective cytotoxicity against renal cancer in the NCI in vitro tumor
screening. Structure–activity relationship study of eight compounds showed the cyclic imido-substituted chloro-1,4-naphtho-
quinone as more potent and selective MEK1 inhibitors than the open chain homologues. The imido-substituted chloro-1,4-
naphthoquinones were synthesized in a straightforward fashion by refluxing 2-amino-3-chloro-1,4-naphthoquinone with the
appropriate acid chloride or diacyl dichloride.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Significantly increased expression and functional activ-
ities of MEK1, MEK2, and ERK1, ERK2 was found in
human hepatocellular carcinoma.6 In addition, mam-
mary tumors initiated by neu, v-Ha-ras and c-Myc
showed high levels of active MAPK, whose activation
may be correlated with MEK activation. The anchorage
independent growth of these tumor cells are strongly
inhibited by MEK inhibitor 1 (PD098059).7,8 In addi-
tion to the Park Davis inhibitor PD098059,8 Favata and
his co-workers9 have identified the selective inhibition of
MEK1 by UO126, 2, while Williams et al.10 reported
the inhibitor RO 09-2210 (3). Compound 3 was able to
block anti-CD3 induced peripheral blood T-cell acti-
vation and antigen induced IL-2 secretion by the inhi-
bition of MEK1. This suggests that in addition to being
useful in the control of tumor growth, MEK1 inhibitors
could be important in controlling the progression of
other diseases such as rheumatoid arthritis and multiple
sclerosis which are linked to T-cell proliferation.
Recently, Taher et al.11 reported that cell signaling in
response to g-radiation is transduced through the
MEK1/2/p42/44 MAP kinase pathway to increase HIV
gene expression in a HeLa cell clone. Hence, apart from
being useful in the understanding of cellular activities,
all these compounds serve as leads to the development
Over the past few years, the mitogen activated protein
kinases (MAPKs) have been at the forefront of a rapid
advance in understanding of cellular events in growth
factors and cytokine receptor signaling. The MAPKs
are a group of protein serine/threonine kinases that are
activated in response to a variety of extracellular stimuli
and mediate signal transduction from cell surface to
nucleus.1 In combination with several other signaling
pathways, they can differentially alter the phosphoryla-
tion status of transcription factors. A controlled reg-
ulation of MAP kinase cascades is thus involved in cell
proliferation, differentiation and apoptosis, whereas an
unregulated activation can result in oncogenesis.1À4
Several lines of evidence support the critical role for the
activation of the Raf-1/MEK/MAPK pathway in onco-
genic transformation. For example, constitutive activa-
tion of the MAPK cascade was found to be associated
with the carcinogenesis of human renal cell carcinomas.5
*Corresponding author. Tel.: +1-202-806-6882; fax: +1-202-806-
5442; e-mail: obakare@howard.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00267-0

3166
O. Bakare et al. / Bioorg. Med. Chem. 11 (2003) 3165–3170
of therapeutically useful candidates. In our in-house
screening of a number of compounds, we identified the
chloro-1,4-naphthoquinone derivative 4, as a MEK1
inhibitor. This led to the design and synthesis of ana-
logues of 4 as potential inhibitors of the MAP kinase
cascade. Some compounds with the chloro-1,4-
naphthoquinone skeleton were recently reported to
possess antineoplastic,12 antihypoxic,13 antiischemic,13
anti-platelet, anti-inflammatory and anti-allergic activ-
ities;14,15 while others have been shown to inhibit the
human cytomegalovirus (HCMV) protease.16 We here
report a novel MEK1 selective inhibitor, 2-chloro-3-(N-
succinimidyl)-1,4-naphthoquinone (6), and various
homologues with a chloro-1,4-naphthoquinone skeleton.
These compounds provide new leads for the develop-
ment of potent and pharmacologically useful MEK
inhibitors.
Chemistry
The synthesis of compounds 4 and 6–12 was accom-
plished in a straightforward way as shown in Scheme 1.
Compound 4 was obtained by refluxing acetic anhy-
dride and 2-amino-3-chloro-1,4-naphthoquinone with a
catalytic amount of concd H2SO4. Compounds 6–12
were made by refluxing 2-amino-3-chloro-1,4-naphtho-
quinone in the appropriate acid chloride or diacyl
dichloride for 30–60 min. In the case of 9–12, the mono-
substituted amide- derivatives were also isolated and
separation was achieved by silica gel chromatography.
2-Amino-3-chloro-1,4-naphthoquinone 5 is commer-
cially available, but could easily be obtained from
2,3-dichloro-1,4-naphthoquinone and ammonia in a
mixture of concd ammonium hydroxide and ethanol.
Results and Discussion
The inhibitory activities of the compounds studied are
listed in Table 1. Structure–activity relationship study
showed the derivatives with cyclic substituents 6, 7, and
8 to have higher potency than the open chain sub-
stituted derivatives. These compounds also show better
MEK1 activity than PD098059, 1, which displayed an
IC50 of 3.8 mM in our assay. The succinimydyl deriva-
tive 6 with IC50 of 0.38 mM was found to be the most
potent inhibitor of MEK1 in this study. This shows a
considerable selectivity over the inhibition of ERK1
with IC50 of 82.9 mM and raf1 at 34.5 mM. Inhibition of
bovine heart PKA and PKC by this compound was
Scheme 1.

O. Bakare et al. / Bioorg. Med. Chem. 11 (2003) 3165–3170
3167
Table 1. IC50 (mM) of the 2-chloro-1,4-naphthoquinone derivatives
(4 and 6–12) on PKA, PKC, ERK1, MEK1 and raf1
group of renal cancer cell lines was least on SN12C with
log TGI of À4.01 where the mean value for all the 50
cancer cell lines screened was À4.28. Most of the
remaining 42 cell lines screened in this study displayed
less sensitivity to 10. The selectivity of 10 for renal car-
cinoma cell lines is consistent with findings that MAPKs
are constitutively activated in some renal cell carcino-
mas,5 suggesting that 10 has potential for significant
clinical impact on this disease.
Compd
PKA
(bovine heart) (mouse rec.) (rat brain)
PKA
PKC
ERK1 MEK1 Raf1
4
6
7
8
8.1
16.3%*
9.2
14.3%*
—
6.2
7.6
—
8.9
—
—
10.6
26.0%*
44.7%*
60.3
—
>50
—
—
>60
99.4 7.5 54.9
82.9 0.38 34.5
112
>296
1.1 62.6
3.6 41.5
9
106.3 10.3 71.9
126.5 6.6 31.63
130.4 15.7 71.8
98.5 10.9 61.2
10
11
12
7.7
7.9
4.7%*
3.3%*
Conclusion
Positive controls were used in all the studies: For MEK this was
PD098059 with IC50 of 3.8 mM. For raf1, it was staurosporine, which
was tested at 20 mg/mL but failed to inhibit at this concentration.
Staurosporine also served as control for ERK1, PKA and PKC with
IC50 of 6.2, 0.37 and 0.01 mM, respectively.
We have synthesized and tested eight imido-substituted
2-chloro-1,4-naphthoquinones as potent selective
MEK1 inhibitors. The cyclic substituted derivatives
were generally more potent and selective that the open
chain homologues. All the three cyclic imido- sub-
stituted derivatives 6, 7, and 8 in this study were selec-
tive and more potent MEK1 inhibitor than PD098059,
with 6 being the most potent and selective MEK1 inhi-
bitor in the series. An open-chain homologue 10 was
found to exhibit selective cytotoxicity on a panel of
renal cancer lines. These compounds provide new leads
for the development of potent and pharmacologically
useful selective inhibitor of the MAP kinase cascade.
*% Inhibition obtained at 10 mM.
negligible. The glutarimydyl 6-membered ring sub-
stituted derivative 7, inhibited MEK1 with an IC50 of
1.1 mM and also showed slightly less selectivity than 6.
Likewise, the phthalimydyl substituted derivative 8
showed lower potency and selectivity than 6 as shown in
Table 1. The open chain imides generally showed lower
inhibitory activity for MEK1 and higher inhibitory
activity for bovine heart PKA making them less selec-
tive than those with cyclic substituent. The most potent
open chain substituted compound in this series was the
dibutylamino derivative 10 with an IC50 of 6.6 mM for
MEK1. This compound is also the best inhibitor of
PKA in the whole series displaying an activity with IC50
of 6.2 mM but was an order of magnitude less potent for
PKC (IC50 greater than 60 mM). While the imido-sub-
stituted chloronaphthoquinones in this study showed
reasonable activities on bovine heart PKA, they dis-
played little or no activities on PKC.
Experimental
Melting points were determined on a Koffler hot-stage
equipped with a digital thermometer and are uncor-
rected. IRspectra were recorded on a Nicolet 520
FT-IRspectrometer using KBr disc for solids and NaCl
plate for liquids. 1H NMRspectra were recorded in
CDCl3 or DMSO-d6 with
a Varian Gemini-300
(300 MHz) spectrometer and chemical shifts are repor-
ted (d) relative to TMS (0 ppm) as internal standard.
Mass spectra were recorded on a VG analytical 70-SE
spectrometer equipped with 11-250J data system, and
all exact mass determinations were recorded at 5000
resolution. Microanalysis were performed by Atlantic
Microlab, Norcross, GA, USA.
Evaluation of 10 against 50 human cancer cell lines in
the NCI in vitro screening revealed a selective cytotoxi-
city against the renal cancer cell lines. ACHN and
CAXI-1 cell lines were the most sensitive to 10 with
total growth inhibitory potency (log TGI) of À5.59 and
À5.46, respectively (Table 2). The effect of 10 in this
2-Chloro-3-diacetylamino-1,4-naphthoquinone (4). Con-
centrated sulfuric acid (0.5 mL) was added dropwise
(over 10 min) to a mixture of 2-amino-3-chloro-1,4-
naphthoquinone 5 (1.5g, 7.23mmol) and acetic anhydride
(200 mL) at À5 ꢀC. The resulting mixture was stirred at
room temperature for 10 min and heated at 100–110 ꢀC
for 50 min after which it was concentrated in vacuo and
the residue added to ice water. The resulting mixture was
filtered, washed with small amount of acetic acid followed
by water to obtain a yellow solid. The crude product was
re-crystallized from ethanol to give yellow crystallÀin1e
Table 2. Inhibitory potencies [Log GI50 (M)a, Log TGI (M)b and
Log LC50 (M)c] of 2-chloro-3-dibutyrylamino-1,4-naphthoquinone
(10) on Renal Cancer Cell Lines from the NCI in vitro screening
Cell line
Log GI50
Log TGI
Log LC50
786-0
A498
À5.04
À4.97
À5.97
À5.76
À5.64
À4.78
À4.39
À5.79
À5.37
2.02
À4.40
À4.41
À5.59
À5.46
À5.33
À4.01
À4.82
À5.22
À4.28
1.59
>À4.00
À4.06
À5.21
À5.26
À5.02
>À4.00
À4.39
À4.61
À4.15
1.22
ACHN
CAXI-1
RXF393
SN12C
TK-10
UO-11
Meand
Rangee
material (1.29 g, 83.7%). Mp 155–158 ꢀC; IR(cm
)
3445, 3332, 3102, 3078, 2936, 1739, 1708, 1676, 1609,
1589, 1420, 1368, 1320, 1231, 1122, 1067, 1037, 974,
aGI50, drug molar concentration causing 50% cell growth inhibition.
bTGI, drug molar concentration causing total growth inhibition.
cLC50, drug molar concentration causing 50% cell death.
dMean values over all cell lines tested.
eThe difference in value of the least sensitive cell and the most sensitive
cell of all the cells tested.
1
865, 714, 627, 598. H NMR(CDCl ) d 2.39 (s, 6H),
3
7.27–8.20 (m, 2H), 8.23–8.28 (m, 2H); MS m/e
293.043991
(293.0454858 M+2
expected
for
C14H10NO4Cl). Anal. calcd for C14H10NO4Cl: C, 57.65;
H, 3.46; N, 4.80; found C, 57.73; H, 3.47; N, 4.73.

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O. Bakare et al. / Bioorg. Med. Chem. 11 (2003) 3165–3170
General synthesis of imido-substituted 2-chloro-1,4-naph-
thoquinones 6-12 as exemplified by the synthesis of
2-Chloro-3-diisovalerylamino-1,4-naphthoquinone (12).
Melting point 118–121 ꢀC. IR(cm À1) 3440, 2962, 2881,
1731, 1709, 1683, 1611, 1591, 1450, 1375, 1368, 1258,
2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone (6).
mixture of 2-amino-3-chloro-1,4-naphthoquinone
A
5
1
1163, 1091, 873, 736, 636. H NMR(CDCl ) d 0.95,
3
(280mg, 1.35mmol) and succinyl chloride (10 mL) was
refluxed gently for 35min. The resulting solution was fro-
zen at À15ꢀC and treated with diethyl ether (100mL) to
precipitate a yellow solid. The mixture was filtered under
suction and the precipitate washed with diethyl ether. The
product was re-crystallized from methanol to obtain 6
as a yellow solid (155 mg, 59.6%). Mp 199–202 ꢀC. IR
(cmÀ1) 3493, 3012, 2947, 1760, 1722, 1689, 1617, 1387,
0.96 (d, 6H, J=2.8 Hz), 0.97, 0.98 (d, 6H, J=2.8 Hz),
2.14–2.30 (m, 2H), 2.44–2.62 (m, 4H), 7.83–7.88 (m,
2H), 8.17–8.28 (m, 2H). MS m/e 375.1217 (375.1237
expected M+ for C20H22NO4Cl). Anal. calcd for
C20H22NO4Cl: C, 63.91; H, 5.90; N, 3.73; found C,
63.79; H, 5.97; N, 3.75.
Materials and methods for bioassays
1
1275, 1163, 870, 730, 623, 426. H NMR(DMSO- d6) d
3.03 (s, 4H), 7.92–7.99 (m, 2H), 8.05–8.10 (m, 1H),
Reagents. Catalytic subunit of cAMP dependent protein
kinase (PKA) isolated from bovine heart and protein
kinase C isolated from rat brain were purchased from
Pierce. Mouse recombinant cAMP dependent protein
kinase catalytic subunit was purchased from Calbio-
chem. The dye-labeled substrates, Kemptide (Lissamine
Rhodamine B-LRRASLG) and Myelin basic protein
(MBP 4-14) (Lissamine Rhodamine B-EKRPSQRS-
KYL), were purchased from Pierce as a lyophilized
powder with 1% bovine serum albumin in amber vials.
8.12–8.16 (m, 1H). MS m/e 289.0118 (289.0142 expected
M
+ for C14H8NO4Cl). Anal. calcd for C14H8NO4Cl: C,
58.05; H, 2.78; N, 4.84; found C, 58.05; H, 2.85; N, 4.83.
2 - Chloro - 3 - (N - glutarimidyl) - 1,4 - naphthoquinone (7).
Melting point dec. >160 ꢀC. IR(cm À1) 3434, 2960,
2624, 1738, 1689, 1673, 1640, 1620, 1594, 1404, 1365,
1280, 1247, 1150, 1120, 1005, 832, 711. 1H NMR
(CDCl3) d 2.15–2.24 (m, 2H), 2.85 (t, 4H, J=7.0 Hz),
7.78–7.85 (m, 2H), 8.12–8.17 (m, 1H), 8.21–8.27 (m,
1H). MS m/e 304.0335 (304.0377 expected M+1 for
C15H10NO4Cl). Anal. calcd for C15H10NO4Cl: C, 59.32;
H, 3.32; N, 4.61; found C, 59.27; H, 3.30; N, 4.63.
Mitogen activated protein kinase assay
Inhibitors were dissolved in DMSO to give 10 mg/mL
solution and successive 10-fold dilutions were prepared.
The test solution (1.0 mL) was placed in a plastic test
tube and 30 mL enzyme solution (8 mg GST-ERK1,
0.32 mg/mL bovine serum albumin, 16 mg/mL leupeptin,
20 mM Tris pH 7.4 buffer) was added and the tube
placed in ice. The reactions were started by adding to
each tube 20 mL of substrate solution (ATP to give
50 mM final concentration of 1000–2000 cpm per pmol,
porcine myelin basic protein to give 1.0 mg/mL final
concentration, magnesium chloride to give 10 mM final
concentration in the assay, and p-nitrophenylphosphate
to give 2.5 mM in the assay) and the placement of the
tubes at 30 ꢀC for 30 min. Each reaction was terminated
by removing a 5-mL portion and applying it to P81
phosphocellulose paper and immediately washing the
paper with distilled water. The radioactivity on the
washed and dried papers was determined by counting
Cerenkov radiation in a liquid scintillation counter.
Mouse ERK1 (as a recombinant glutathione S-trans-
ferase fusion protein) was prepared by glutathione-
agarose affinity chromatography from extracts of
JM109 Escherichia coli containing the plasmid pGEX-
3X:ERK1. Porcine myelin basic protein was prepared
from pig brain.
2-Chloro - 3- (N -phthalimidyl)-1,4-naphthoquinone (8).
Melting point 243–245 ꢀC. IR(cm À1) 3435, 1792, 1730,
1681, 1617, 1374, 1275, 1102, 1084, 888, 862, 718, 710,
1
628. H NMR(CDCl ) d 7.82–7.89 (m, 4H), 7.97–8.02
3
(m, 2H), 8.17–8.22 (m, 1H), 8.25–8.30 (m, 1H). MS m/e
337.0123 (337.0142 expected M+ for C18H8NO4Cl);
Anal calcd for C18H8NO4Cl: C, 64.01; H, 2.39; N, 4.15;
found C, 63.95; H, 2.42; N, 4.18.
2-Chloro -3- dipropionylamino -1,4 -naphthoquinone (9).
Melting point 139–143 ꢀC. IR(cm À1) 3457, 3341, 2983,
2944, 2881, 1742, 1708, 1677, 1610, 1591, 1462, 1354,
1313, 1284, 1258, 1180, 1152, 1111, 1075, 1033, 1013,
914, 859, 797, 720, 632. 1H NMR(CDCl 3) d 1.16 (t, 6H,
J=7.2 Hz), 2.69 (m, 4H), 7.85 (m, 2H), 8.19 (m, 1H),
8.25 (m, 1H). MS m/e 319.0595 (319.0611 expected M+
for C16H14NO4Cl)
2 - Chloro - 3 - dibutyrylamino - 1,4 - naphthoquinone (10).
Melting point 111–113 ꢀC. IR(cm À1) 3466, 3335, 2966,
2933, 2874. 1742, 1709, 1683, 1611, 1591, 1255, 1183,
1
1156, 1123, 1097, 860, 722, 630. H NMR(CDCl ) d
3
0.95 (t, 6H, J=7.4 Hz), 1.64–1.76 (m, 4H). MS m/e
348.0974 (348.1003 expected M+1 for C18H18NO4Cl).
Anal. calcd for C18H18NO4Cl: C, 62.16; H, 5.22; N,
4.03; found C, 61.98; H, 5.16; N, 3.95.
MEK1 and Raf1 assays
Inhibitors were dissolved and diluted in DMSO and
1 mL of dilution to give the indicated final concen-
tration, was placed in a plastic test tube. 30 mL enzyme
solution was added. To assay for raf-1, partially purified
raf-1 (expressed in Sf9 insect cells infected with baculo-
virus directing expression of wt human c-raf-1) was
supplied as the enzyme and a kinase-inactive mutant of
GST-MEK1 was included as substrate. To assay
MEK1, GST-MEK1 expressed in bacteria and purified
2-Chloro-3-divalerylamino-1,4-naphthoquinone (11). Vis-
cous liquid (gum). IR(cm À1) 2959, 2933, 2881, 1730,
1683, 1610, 1600, 1472, 1318, 1286, 1259, 1180, 1146,
1089, 1040, 870, 797, 715, 636. 1H NMR(CDCl 3) d
0.88–0.93 (m, 6H), 1.28–1.40 (m, 4H), 1.57–1.70 (m,
4H), 2.50–2.72 (m, 4H), 7.80–7.88 (m, 2H), 8.15–8.20
(m, 1H), 8.22–8.28 (m, 1H). MS m/e 378.1472 (378.1472
expected M+3 for C20H22N04Cl).

O. Bakare et al. / Bioorg. Med. Chem. 11 (2003) 3165–3170
3169
on glutathione agarose was used as the enzyme and a
kinase-inactive mutant of His-6-ERK1 (expressed in
bacteria and purified on Ni-NTA-agarose) was included
as substrate. All enzyme solutions additionally included
leupeptin at 10 mg/mL (final concentration in the assay).
The reactions were started with the addition of 20 mL of
substrate solution (ATP to give 50 mM final concen-
tration of 1000–2000 cpm per pmol, magnesium chloride
to give 10 mM final concentration in the assay, and
p-nitrophenylphosphate to give 2.5 mM in the assay)
and the placement of the tubes at 30 ꢀC for 20 min. Each
reaction was terminated by the addition of 50 mL of stop
solution (10 mg/mL sodium dodecylsulfate, 10% 2-mer-
captoethanol, 20 mM EDTA) and heating at 95 ꢀC for
10 min. The phosphoproteins in the reactions were ana-
lyzed by separation on 10% polyacrylamide-SDS gels,
electrophoretic transfer onto nitrocellulose membranes,
and autoradiography with X-ray film. The radioactivity
present in the substrate protein bands was determined
by densitometry and the concentration of inhibitor nee-
ded for 50% inhibition of the enzyme was determined
from the dose-response curve for each compound.
Reactions containing solvent (DMSO) only were run as
uninhibited controls. Each compound but 6 and 7 were
tested for inhibition of MEK1 at 1 and 5 mg/mL, while 6
and 7 were tested at 0.2 and 1.0 mg/mL. All compounds
were tested for inhibition of raf-1 at 10 and 30 mg/mL.
enzyme (1.7 U for bovine heart PKA, 0.17 U for mouse
recombinant PKA) with a pH of 7.4. Positive control
contained 1 mL of DMSO instead of the inhibitor solu-
tion. Negative control contained the same volume of
dilution buffer instead of the enzyme solution. The
reaction mixtures were vortexed and incubated at 30 ꢀC
for 30 min. Then, timed aliquots (30 mL every 30 s) were
removed from each assay tube and applied to the affi-
nity membrane of an individual SpinZymeTM Affinity
Separation Unit. Phosphopeptide binding buffer (2 Â
250 mL, pH 5.0) that contained 0.1 M sodium acetate,
0.5 M sodium chloride, and 0.02% sodium azide, were
added to each separation unit and incubated for 3 min.
The buffer, which eluted non-phosphorylated peptide,
was washed through the membrane by centrifugation at
5500–5800 rmp for 1 min. The membrane-containing
bucket was moved to a new receptacle. Phosphopeptide
elution buffer (2 Â 250 mL, pH 8.0) that contained
0.1 mM ammonia bicarbonate and 0.02% sodium azide,
was added to the bucket, and incubated for 3 min and
centrifuged at 5500–5800 rmp for 1 min. The resulted
500 mL eluted solution of phosphorylated substrate was
measured with a Shimadzu UV spectrometer at 570 nm
against a blank containing 500 mL of elution buffer.
Averaged negative control absorbance values were sub-
tracted from all inhibitor sample absorbance and the posi-
tive control absorbance before averaging their triplicate
absorbance values. The% inhibition value for an inhibitor
sample was calculated from the following equation:
Protein kinase A and protein kinase C assays
The PKA assay and protein kinase C assay were per-
formed using the colorimetric PKA assay kit and
colorimetric PKC assay kit (SpinZymeTM Kit, Pierce),
respectively. Standard curves were generated for both
assays according to the instruction booklets that came
with the kits. The best enzyme concentration was cho-
sen from the standard curve to ensure that the amount
of enzyme added to each assay tube phosphorylated the
peptide substrate in a linear fashion over a 30-min
incubation, and the UV absorbance of the phosphory-
lated substrate in the control is larger than 0.5 AU. For
bovine heart PKA, 1.7 unit of PKA (a unit of activity is
the amount of enzyme required to catalyze the transfer
of 1 pmol of phosphate to casein in 1 min at pH 7.4)
added to each tube. For mouse recombinant PKA, 0.17
unit of the enzyme (a unit is defined as the amount of
enzyme that will transfer 1 pmol of phosphate to kemp-
tide per min at 30 ꢀC, pH 7.5) was chosen. For rat brain
PKC, 0.05 unit of PKC (a unit is defined as the enzyme
activity that catalyzes the transfer of 1 nmol of phos-
phate to histone H1 in one min at 30 ꢀC) was chosen
and added to each tube.
% Inhibition ¼ ð1 À Inhibitor Absorbance Value=
Positive Control Absorbance valueÞ ꢀ 100%
Protein kinase C assay
The procedure of protein kinase C assay is similar to
that of PKA assay. The 36 mL of final reaction mixture
contained 2 mM ATP, 10 mM MgCl2, 0.002% Triton
X-100, 20 mM Tris, 0.1 mM CaCl2, 0.2 mg/mL phos-
phatidyl-l-serine, 350 mM Lissamine Rhodamine B
labeled Myelin basic protein, and 0.05 unit of the
enzyme PKC with a pH of 7.4. The phosphorylated
peptide substrate was isolated from the unpho-
sphorylated one using the SpinZymeTM Affinity
Separation membrane. The% inhibition value for inhi-
bitor samples were calculated with the same equation
used for PKA assay.
Acknowledgements
Cyclic AMP dependent protein kinase assay
The compounds were dissolved and diluted in DMSO to
appropriate concentration and 1 mL of the solution was
added to each tube followed by addition of buffers and
substrate solutions. Finally timed additions (every 30 s)
of enzyme solutions to each assay tube initiated the
reaction. The 36 mL of final reaction mixture contained
2 mM ATP, 10 mM MgCl2, 0.002% Triton X-100,
20 mM Tris, 100 mM cAMP, 310 mM Kemptide, and the
We are grateful to the National Cancer Institute of the
National Institute of Health for financial support.
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