Inhibition of heat shock induction of heat shock protein 70 and enhancement of heat shock protein 27 phosphorylation by quercetin derivatives

Article abstract of DOI:10.1021/jm801445c

Inhibitors of heat-induced heat shock protein 70 (HSP70) expression have the potential to enhance the therapeutic effectiveness of heat-induced radiosensitization of tumors. Among known small molecule inhibitors, quercetin has the advantage of being easil

Full text of DOI:10.1021/jm801445c

1912
J. Med. Chem. 2009, 52, 1912–1921
Inhibition of Heat Shock Induction of Heat Shock Protein 70 and Enhancement of Heat Shock
Protein 27 Phosphorylation by Quercetin Derivatives
Rongsheng E. Wang,† Jeffrey L.-F. Kao,† Carolyn A. Hilliard,‡ Raj K. Pandita,‡ Joseph L. Roti Roti,‡ Clayton R. Hunt,‡ and
John-Stephen Taylor*,†
Department of Chemistry, Washington UniVersity, One Brookings DriVe, St. Louis, Missouri 63130, and Radiation Oncology Department,
Washington UniVersity School of Medicine, 4511 Forest Park, St. Louis, Missouri 63108
ReceiVed NoVember 16, 2008
Inhibitors of heat-induced heat shock protein 70 (HSP70) expression have the potential to enhance the
therapeutic effectiveness of heat-induced radiosensitization of tumors. Among known small molecule
inhibitors, quercetin has the advantage of being easily modified for structure-activity studies. Herein, we
report the ability of five monomethyl and five carbomethoxymethyl derivatives of quercetin to inhibit heat-
induced HSP70 expression and enhance HSP27 phosphorylation in human cells. While quercetin and several
derivatives inhibit HSP70 induction and enhance HSP27 phosphorylation at Ser78, other analogues selectively
inhibit HSP70 induction without enhancing HSP27 phosphorylation that would otherwise aid in cell survival.
We also show that good inhibitors of HSP70 induction are also good inhibitors of both CK2 and CamKII,
kinases that are known to activate HSP70 expression by phosphorylation of heat shock transcription factor
1. Derivatives that show poor inhibition of either or both kinases are not good inhibitors of HSP70 induction,
suggesting that quercetin’s effectiveness is due to its ability to inhibit both kinases.
Introduction
Heat shock proteins (HSPsa) are stress proteins that are found
in nearly all living organisms and are highly induced by various
environmental and pathophysiological stimuli.1-5 The heat
shock protein family consists of several different molecular
weight classes, of which HSP60s, HSP70s and HSP90s function
as highly conserved molecular chaperones6 that assist nascent
polypeptide folding, assembly of protein complexes and protein
transport through cellular membranes.7 While HSP90 is known
to stabilize multiple signaling proteins,8 HSP70s are reported
to promote cell survival by inhibiting lysosomal membrane
permeabilization9 and interfering with the cell apoptotic cas-
cade.10 Heat shock proteins are also found to be overexpressed
in cancer cells and participate in oncogenesis and resistance to
chemotherapeutic agents.11-14 Overexpression of HSP90s and
HSP70s in many tumors15 correlates with the proliferation and
survival of oral,16 gastrointestinal,17 colorectal,18 histiocytic
Figure 1. Schematic of the pathway for heat shock induction of HSP70
lymphoma,19 and breast cancers.20,21 Thus heat shock proteins
expression. See text for a discussion.
have recently become of interest as targets for anticancer
17-(dimethylaminoethylamino)-17-demethoxygeldanamy-
therapy.22 Small molecular inhibitors of HSP90 like 17-
cin (17DMAG)25 have been found to enhance tumor killing
allylamino-17-demethoxy-geldanamycin (17AAG)23,24 and
by ionizing radiation. Recently it has been shown that
inhibition of HSP70 in tumor cells either by a gene
knockout26 or by antisense agents27 enhances heat-induced
radiosensitization (HIR). It would therefore be highly desir-
able to have a small molecular inhibitor of heat-induced
HSP70 that could be administered prior to HIR to further
enhance killing.
Heat induction and regulation of HSP70 is a rather compli-
cated process, involving initial release of the heat shock
transcription factor 1 (HSF1) from HSP90, followed by trim-
erization, translocation into the nucleus, and then binding to
the heat shock element (HSE) (Figure 1).28-30 For transcription
to occur the trimer must first be activated by phosphorylation
at serine 230 with calcium/calmodulin kinase II (CamKII)31 and/
or threonine 142 by casein kinase 2 (CK2).32 In contrast,
phosphorylation at other sites down regulates the transcriptional
activity of HSF1. For example, phosphorylation at serine 121
* To whom correspondence should be addressed: Phone: (314) 935-6721.
Fax: (314) 935-4481. E-mail: taylor@wustl.edu.
†
Department of Chemistry, Washington University.
Radiation Oncology Department, Washington University School of
‡
Medicine.
a Abbreviations: CaMKII, Ca2+/calmodulin-dependent protein kinase II;
CK2, casein kinase II; COSY, correlation spectroscopy; DCM, dichlo-
romethane; DMEM, Dulbecco’s modified essential medium; DMF, dimethyl
formamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, eth-
ylenediaminetetraacetic acid; ERK, extracellular signal-regulated protein
kinase; ESI, electrospray ionization; EtOAc, ethyl acetate; EtOH, ethanol;
FAB, fast-atom bombardment; HIR, heat-induced radiosensitization; HMBC,
heteronuclear multiple bond coherence spectroscopy; HMQC, heteronuclear
multiple quantum coherence spectroscopy; HPLC, high pressure liquid
chromatography; HSE, heat shock element; HSF1, heat shock factor 1; HSP,
heat shock protein; IC50, 50% inhibitory concentration; JNK, c-Jun
N-terminal kinase; MAPK, mitogen-activated protein kinase; MeOH,
methanol; MK2 (MAPKAPK2), MAPK-activated protein kinase 2; THF,
tetrahydrofuran; TLC, thin layer chromatography; Tris, tris(hydroxymethyl)-
aminomethane.
10.1021/jm801445c CCC: $40.75
2009 American Chemical Society
Published on Web 03/18/2009

Quercetin DeriVatiVes and Heat Shock Proteins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1913
Scheme 1a
Figure 2. Two known inhibitors of heat-induced HSP70 expression.
by MAPK-activated kinase 2 (MK2) causes unfolding of HSF1
and rebinding to HSP90.33 Likewise, phosphorylation of serine
307 by ERK or serine 363 by JNK inactivates HSF1.34
A number of small molecule inhibitors of heat-induced HSP70
expression have been reported, the most effective of which are
triptolide and quercetin (Figure 2).35 Triptolide is a highly
functionalized molecule that has been found to prevent heat
shock HSF1 from activating transcription in the steps following
trimerization, phosphorylation and DNA binding.36 Quercetin,
on the other hand, is a structurally much simpler natural product
of the flavone family, that has been reported to have a wide
variety of biological activities,37,38 one of which is inhibition
of heat-induced HSP70 expression by as yet unknown mecha-
nisms.39,40 Quercetin inhibits a number of kinases,41 including
CK2, which has been shown to enhance HSP70 induction
through phosphorylation of HSF1 threonine 142.32 It is therefore
possible that quercetin inhibits HSP70 induction by blocking
phosphorylation of HSF1 by CK2 and/or CamKII.31 Quercetin
is also metabolized in ViVo, and it is possible that some of the
metabolites are the active pharmaceutical agents.42
To guide the design and synthesis of biotinylated probes for
identifying the target of quercetin inhibition of heat-induced
HSP70 expression, and to enhance its specificity, we synthesized
all of the monomethyl and selected carbomethoxymethyl
derivatives of quercetin. Systematic methylation (“methyl scan-
ning”) has been previously proposed and validated as a general
method for mapping the interactions of biologically active
molecules with receptors and for designing affinity probes.43,44
The quercetin derivatives were tested for their ability to inhibit
heat-induced HSP70 expression and modulate HSP27 phospho-
rylation in human Jurkat cells and human HeLa cells, respec-
tively, by Western blot analysis. Two classes of active agents
were discovered, one that both inhibits HSP70 induction and
enhances HSP27 phosphorylation, and another that inhibits
HSP70 induction without enhancing HSP27 phosphorylation.
Quercetin and derivatives that inhibited HSP70 induction were
also found to inhibit both CK2 and CamKII kinases. On the
other hand, derivatives that showed poor inhibition of either or
both kinases did not inhibit HSP70 induction, indicating that
quercetin’s effectiveness is due to its ability to inhibit more than
one enzyme.
a Reagents and conditions: (a) BrCH2CO2CH3, K2CO3/DMF; (b)
H2/Pd(OH)2.
synthetic route (Scheme 1). Thus, quercetin was benzylated to
give 20% of the tribenzyl derivative 1 and 60% of the
tetrabenzyl derivative 2. The tribenzyl derivative was selectively
carbomethoxymethylated at the 3′-OH to give 4, whereas the
tetrabenzyl derivative was carbomethoxymethylated at the 5-OH
to give 3. The products were debenzylated with palladium
hydroxide and hydrogen to afford the methyl esters D8 and D1
respectively.
To carbomethoxymethylate positions 3 and 7, we utilized the
3′,4′-diphenylmethylene derivative of quercetin, compound 5
(Scheme 2). This derivative reacted with methylbromoacetate
under basic conditions at the 3-OH to give 6, which then
afforded the carboxymethyl derivative D5 following removal
of the diphenylmethylene group by refluxing with acetic acid,
which also hydrolyzed the ester. To obtain the O7 derivative,
the 3-OH of compound 5 was first benzylated to give 7, which
was then carbomethoxymethylated at the 7 position. Removal
of the benzyl and diphenylmethylene groups was carried out in
two steps by hydrogenolysis with palladium hydroxide followed
by hydrolysis with acetic acid and water, which also unexpect-
edly hydrolyzed the ester to the carboxymethyl product D9.
Many attempts to methylate D9 to produce D10 were
unsuccessful, and so other synthetic routes were investigated.
The best of these involved a previously reported method for
selectively methylating or benzylating the 7-OH of quercetin
by treatment of quercetin pentaacetate with the alkylating agent
in the presence of potassium carbonate and potassium iodide
in acetone.46 Thus treatment of quercetin pentaacetate47 under
these conditions with methyl bromoacetate yielded compound
10, which could be deacetylated in high yield under neutral
conditions with N-methyl-2-dimethylaminoacetohydroxamic
acid48 to give D10 (Scheme 3). This deacetylation method was
chosen as more basic conditions, such as sodium methoxide
and methanol at 0 °C, tend to result in air oxidation of the
product,49 and more acidic conditions lead to hydrolysis of the
methyl ester.
Chemistry
(a) Synthesis of O-Alkylated Quercetin Derivatives. To
determine the importance of individual hydroxyl groups on the
inhibition of heat-induced HSP70 expression and other biologi-
cal activities, we synthesized the monomethylated quercetin
derivatives D2, D3, D4, D6, and D7 according to previously
reported procedures.45 Because we ultimately intend to attach
affinity tags such as biotin to quercetin to help in identifying
the protein target(s) of quercetin activity, we also synthesized
a number of carboxymethylated derivatives by the same
(b) Structural Characterization. The sites of alkylation in
all the compounds were confirmed by analysis of the coupling
1
patterns in the 1D H NMR spectra together with correlations
in the COSY, HMQC and HMBC spectra. As an illustration,

1914 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Wang et al.
Scheme 2a
a Reagents and conditions: (a) BnBr, K2CO3/DMF; (b) BrCH2CO2CH3, K2CO3/DMF; (c) H2/Pd(OH)2; (d) CH3CO2H/reflux.
Scheme 3a
multiple bond correlations to the two meta coupled protons in
the A ring in the HMBC spectrum (crosspeaks b and c in panel
C).
Biological Studies. (a) Inhibition of Heat-Induced
HSP70 Expression by Quercetin Derivatives. To examine the
ability of the quercetin and its derivatives to inhibit heat-induced
HSP70 expression, we chose human Jurkat cells due to their
low basal level of HSP70 expression and the large induction
upon heat shock. Western blot analysis of the cellular proteins
in Jurkat cells following heat shock revealed that preheat
treatment with the quercetin derivatives D1, D2, D7 and D10
all had a similar inhibitory effect on HSP70 induction as did
quercetin, indicating that the 3′ and 7 hydroxyls are important
for activity (Figures 5A, Table 1). Compound D1, which bears
a carbomethoxymethyl group at O3′, is not as good an inhibitor
as D2, which bears a methyl group, indicating that the 3′
hydroxyl group occupies a more sterically constrained site in
the target protein than does the 7 hydroxyl group. Compound
D9, which bears a carboxylic acid group at O7, does not inhibit
HSP70 like D10, which bears an ester group, presumably
because it forms a salt at neutral pH that greatly reduces its
membrane permeability.
(b) Enhancement of HSP27 Phosphorylation. In examining
the effects of quercetin treatment on other heat shock proteins,
we found that quercetin enhances HSP27 phosphorylation at
Ser78 in the absence of heat shock. Phosphorylated HSP27 has
antiapoptotic activity2 which would tend to counteract any lethal
effects achieved from inhibiting HSP70 induction. Thus, it
would be highly desirable to inhibit HSP70 without enhancing
phosphorylation of HSP27. The HSP27 studies were carried out
in HeLa cells, which unlike Jurkat cells, have a high basal level
of HSP27, making it easier to detect drug-induced phosphory-
lation of HSP27 by Western analysis. HeLa cells, however, are
not as suitable for concurrent inhibition studies of HSP70
induction since they have high basal level of HSP70. Only D7
and D10 showed enhancement of HSP27 phosphorylation in
HeLa cells, and both of these compounds also showed good to
a Reagents and conditions: (a) BrCH2CO2CH3, KI, K2CO3/acetone; (b)
N-methyl-2-dimethylamino-acetohydroxamic acid, THF/MeOH, phosphate
buffer.
the B ring hydrogens H5′ and H6′ of D1 (Figure 3) and D10
(Figure 4) were assigned by the large ortho coupling, and the
strong crosspeak in the COSY spectrum (crosspeak a, panel A).
These assignments led to the assignment of H2′ through meta
1
coupling with H6′ that could be detected in the 1D H NMR
spectrum. The carbons directly attached to these protons could
then be assigned through the HMQC spectra (panel B). The
hydrogen signal at 10 ppm in D1 could then be assigned to the
4′-OH by the presence of a long-range correlation to C5′ at 116
ppm (Figure 3, crosspeak e) and to a quaternary carbon signal
at 146 ppm (crosspeak b) together with the absence of a
correlation to C2′ in the HMBC spectrum (panel C). The signal
at 146 ppm could therefore be assigned to C3′ which in turn
showed a correlation to the R hydrogen of the carbomethoxy-
methylene group (crosspeak d, panel C). The position of the
carbomethoxymethylene group in D10 could be assigned to O7
via a correlation between the R-methylene protons and a
quaternary carbon signal at 162 ppm (Figure 4, crosspeak d in
panel C). The signal at 162 ppm could be assigned to C7 via

Quercetin DeriVatiVes and Heat Shock Proteins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1915
Figure 3. Structure assignment of D1. (A) COSY, (B) HMQC and (C) HMBC of D1 in DMSO-d6.
strong inhibition of HSP70 induction in Jurkat cells (Figure 5B).
On the other hand, D1 and D2, which were also good inhibitors
of HSP70 induction, did not activate HSP27 phosphorylation.
(c) Effect of Quercetin on HSF1 Binding to the HSE. In
order to gain insight into the mechanism by which quercetin
inhibits HSP70 induction, gel mobility shift analysis was carried
out to examine binding of heat shock element (HSE) DNA by
heat shock transcription factor 1 (HSF1), the transcription factor
responsible for heat-induced HSP70 gene transcription (Figure
6A upper panel). No HSF1/HSE complex was detected with
nuclear extracts prepared from control, nonheated Jurkat cells
but the complex was readily detected with extracts prepared
immediately following a 43 °C 30 min heat shock. The amount
of complex increased by 1 h postheat then declined by 4 h
postheat and returned to control levels by 8 h postheat. In Jurkat
cells treated 1 h prior to heat shock with quercetin, however,
only a very low level of complex formed in extracts from heated
cells. Since an identical low level of complex was also detected
in extracts from quercetin-treated but nonheated cells, this
response appears to be due to drug induced cell stress not heat.
In contrast to Jurkat cells, HeLa cells displayed an accelerated
lost of HFS1/HSE complex during recovery from heat shock
with nearly undetectable levels by 4 h postheat (Figure 6A,
lower panel). Moreover, quercetin treatment did not inhibit
HSF1/HSE complex formation in heated HeLa cells as it did
in Jurkat cells suggesting a complex interaction between the
drug and possibly multiple cell specific factors.
Further information about the mechanism by which quercetin
inhibits heat-induced HSP70 expression was obtained by
Western blot analysis of the nuclear extracts for the presence
and size of the HSF1 protein (Figure 6B). In nonheated Jurkat
control cells, HSF1 is not detected in nuclear extracts while
only a very low level can be detected in HeLa cells. Surprisingly,
nuclear extracts from quercetin treated Jurkat cells contained
significant levels of HSF1 while similar HeLa cell extract
contained the very low levels detected in untreated extracts. In
both Jurkat and Hela cells, heating increased HSF1 nuclear
extract levels and induced a corresponding decrease in gel
mobility that reflects the increased level of heat-induced
phosphorylation required for transcriptional activity.31,32 While
quercetin treatment did not block the heat-induced increase in
nuclear HSF1 levels in either Jurkat and HeLa cells, the drug
did inhibit the decrease in HSF1 mobility, presumably by
inhibiting the critical phosphorylation modifications required for
transcription activation. Thus in Jurkat cells, quercetin treatment
appears to affect at least two steps in heat-induced HSP70
expression, HSF1 post translational modification and DNA
binding activity. Quercetin treatment of HeLa cells does not
result in loss of HSF1 DNA binding activity but does appear to
inhibit heat-induced phosphorylation.

1916 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Wang et al.
Figure 4. Structure assignment of D10. (A) COSY, (B) HMQC and (C) HMBC of D10 in DMSO-d6.
(d) Kinase Inhibition Assays. We then determined whether
or not the activity of the quercetin analogues correlated with
their ability to inhibit either or both CKII and CamKII kinases
that have previously been shown to phosphorylate HSF1. We
used an enzyme-linked inhibition assay that utilizes luciferase
to quantify the amount of ATP remaining in a kinase reaction.50
The IC50 values for quercetin and D1-D10 are listed in Table 2
in comparison to their relative ability to inhibit HSP70 induction.
Good inhibition of HSP70 induction appears to correlate with
good inhibition of both CK2 and CamKII. Quercetin derivatives
that showed poor inhibition (IC50 > 100 µM) of either or both
kinases showed little to no ability to inhibit heat induction of
HSP70. The only exception was D9 which was a very good
inhibitor of both kinases, but showed no ability to inhibit HSP70
induction, which we attribute to the presence of a carboxylate
which reduces membrane permeability.
Discussion
Figure 5. Effect of quercetin and its derivatives on heat-induced HSP70
expression and phosphorylation of HSP27. Panel A. Inhibition of heat-
induced HSP70 expression. Jurkat cells pretreated 1 h with 50 µg/mL
(approximately 150 µM) of the quercetin derivatives D1-D10 were
heat shocked as indicated then allowed to recover at 37 °C for 8 h
before protein isolation and analysis. HSP70 and actin protein levels
were determined by Western blotting: lane C, nonheated cells; lane N,
heated no drug; lanes D1-D10, quercetin derivatives. Panel B. Drug
induced phosphorylation of human HSP27 Ser78. HeLa cells were
treated for 1 h with drug (50 µg/mL, approximately 150 µM) before
cellular proteins were isolated for Western blot analysis with actin and
HSP27 Ser78P antibodies: lane C, no drug; lane V, DMSO vehicle
only; lane Q, quercetin; lanes D1-D10, quercetin derivatives.
Inhibition of heat-induced HSP70 expression has been shown
to enhance radiosensitization by heat,26,27 and thus small
molecule inhibitors of HSP70 induction have potential thera-
peutic value. Quercetin has long been known to inhibit heat-
induced HSP70 expression, though the exact mechanism is
unclear. The results herein suggest that quercetin treatment
blocks heat-induced phosphorylation of the HSF1 transcription
factor that is responsible for inducing HSP70 expression. In
Jurkat but not HeLa cells, an additional level of inhibition
appears to be loss of HSF1 HSE element binding capacity,

Quercetin DeriVatiVes and Heat Shock Proteins
Table 1. Quercetin Derivativesa
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1917
compd
R3
R5
R7
R3′
R4′
HSP70 inhibition
HSP27 phosphorylation
quercetin
D1
D2
D3
D4
D5
D6
D7
D8
+++
++
+++
+
+++
-
CH2CO2CH3
CH3
-
CH3
-
CH3
CH2CO2H
-
-
-
-
+++
-
-
+++
-
CH3
CH2CO2CH3
-
CH3
+++
-
D9
D10
CH2CO2H
CH2CO2CH3
-
+++
a The substituent is an H unless otherwise noted.
to show that the C7 and C3′ hydroxyls on quercetin can be
derivatized with a bulky carbomethoxymethyl group without
affecting its ability to inhibit heat-induced HSP70, and could
therefore be used to attach a biotin affinity probe.
The finding that the 3′ and 7 hydroxyls of quercetin can be
modified without affecting inhibition of heat-induced HSP70
expression suggested that modification of these hydroxyls might
also increase the selectivity by interfering with binding to other
protein targets. Quercetin has been crystallized with a number
of kinases, and inspection of the crystal structures reveals that
the 7-OH of quercetin is buried in the Hck kinase complex52 as
well as in the F1-ATPase53 and therefore O7 derivatives would
not be expected to inhibit these enzymes. The O7 position of
quercetin is not buried, however, in the phosphoinositide
3-kinase complex,54 but the O3′ position is, suggesting that
quercetin derivatized at both the O7 and O3′ positions would
also not inhibit any of these enzymes, but would still inhibit
HSP70 induction. This class of doubly derivatized quercetin
remains to be synthesized and examined for its ability to inhibit
HSP70 induction.
Given that quercetin is a well-known kinase inhibitor, and
that the pathway for heat induction of HSP70 expression
involves phosphorylation of the heat shock factor 1 (HSF1) by
CK2 and CamKII kinases, we investigated whether the two
might be correlated. It has previously been established that
quercetin is an inhibitor or CK2 kinase, but its ability to inhibit
CamKII kinase has not been reported. We now show that
quercetin is also a good inhibitor of CamKII and that quercetin
derivatives D1, D2, D7 and D10, which are good inhibitors of
HSP70 induction, are also good inhibitors of both CK2 and
CamKII kinases. Quercetin derivatives that were good inhibitors
of only one or neither of the two kinases, however, were not
good inhibitors of HSP70 induction. It would seem that full
activation of HSP70 by HSF1 may only require phosphorylation
by either CK2 or CamKII, both of which quercetin and
derivatives D1, D2, D7 and D10 are able to inhibit due to their
broader specificity.
The ability of quercetin to enhance phosphorylation of HSP27
has not been previously recognized. Phosphorylation of serine
78 has been shown to be carried out by the MK2 kinase,55 which
in turn is activated by MAP kinase (MAPK). It is not clear
how quercetin activates phosphorylation of HSP27, as inhibiting
any of the kinases in the pathway should abolish activation.
Activation of HSP27 has been associated with poor prognosis
Figure 6. Effect of quercetin on HSF1 binding to HSE DNA. Jurkat
(panel A, upper) or HeLa cells (panel A, lower) were heated or
pretreated with quercetin (50 µg/mL, approximately 150 µM) 1 h prior
to heating and allowed to recover at 37 °C for 0-8 h at 37 °C. Cells
were isolated and nuclear extracts prepared for EMSA analysis with a
5′-radiolabeled HSE oligodeoxynucleotide. Position of the HSF1-HSE
complex is indicated by the arrows. Lane O: HSE oligonucleotide
without extract. Lane C: unheated extract. Lane Q: quercetin treated,
no heat. HSF1 content of EMSA nuclear extracts (panel B). Nuclear
extracts utilized in panel A were subjected to Western blot analysis
with anti-HSF1 antibody to determine the HSF1 levels and relative
size. Lane C: nuclear extract from unheated cells. Lane Q: quercetin
treated for 2.5 h. Lane HS: 1 h after a 43 °C/30 min heat shock. Lane
HS+Q: 1 h quercetin treatment, 43 °C/30 min heat shock and 1 h
recovery.
which has also been reported in quercetin/heated human COLO
320DM cells.51 Unfortunately, quercetin is also well-known to
affect many additional biological processes, probably as a result
of its ability to inhibit a number of different kinases,41 which
diminishes its potential as a therapeutic. On the other hand it
seems reasonable that the specificity for inhibiting HSP70
induction could be increased by selective modification of
quercetin. Also identifying the target of quercetin responsible
for inhibiting HSP70 induction could aid in the development
of more selective inhibitors. To these ends we have been able

1918 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Wang et al.
Table 2. IC50 Values (µM) for Inhibition of CK2 and CamKII by Quercetin and Derivatives D1-D10
Q
D1
D2
D3
D4
D5
CK2
CamKII
HSP70
5.6 ( 1.2
3.3 ( 0.6
+++
53 ( 3.7
6.3 ( 1.3
++
0.50 ( 0.01
0.91 ( 0.25
+++
>100
16.4 ( 1.9
+
0.83 ( 0.10
2.45 ( 0.25
>100
>100
-
-
D6
>100
4.0 ( 1.1
-
D7
D8
>100
69 ( 6.0
-
D9
D10
CK2
CamKII
HSP70
9.7 ( 1.4
8.9 ( 0.3
+++
9.7 ( 3.6
9.3 ( 1.8
-
0.85 ( 0.30
9.1 ( 0.1
+++
and resistance to chemotherapy or radiation,56 and so the
quercetin analogues D1 and D2 which retain the ability to inhibit
HSP70 induction without inducing HSP27 phosphorylation may
have superior therapeutic properties to D7 and D10.
1
spectra were recorded using a 0.3 s H-13C nulling period. The
90° H pulse width was 9.8 s, and the 90° 13C pulse width was
1
12.5 s. Phase sensitive 2D spectra were obtained by employing the
Hypercomplex method. A 2 × 256 × 2048 data matrix with 16
scans per t1 value was collected. Gaussian line broadening was used
in weighting both the t2 and the t1 dimension. After two-dimensional
Fourier transform, the spectra resulted in 512 × 2048 data points,
which were phase and baseline, corrected in both dimensions.
Compound 4. A solution of 2 (0.60 g, 1.1 mmol) in DMF (20
mL) was mixed with potassium carbonate (0.19 g, 1.4 mmol) under
nitrogen. Methyl bromoacetate (0.160 g, 1.05 mmol) was then
slowly added. The resulting solution was stirred at room temperature
for 12 h, diluted with 40 mL of water and extracted with 60 mL
ethyl acetate. The organic phase was washed with water, dried over
anhydrous sodium sulfate and concentrated under reduced pressure.
The residue was purified by flash column chromatography using
Conclusion
The original goal of this study was to find derivatives of
quercetin that could be elaborated into affinity probes to identify
the targets responsible for inhibition of heat induction of HSP70.
We were able to identify two such derivatives, which are now
in the process of being converted to biotinylated probes. In the
course of evaluating these derivatives, we discovered that their
ability to inhibit HSP70 induction correlated with their ability
to inhibit both CK2 and CamKII, kinases that are known to
activate HSP70 transcription by phosphorylating HSF1. Deriva-
tives that show poor inhibition of either or both kinases are
poor inhibitors of HSP70 induction, suggesting that HSP70
expression can be activated by either kinase. This would appear
to be a case in which making the drug more specific reduces its
effectiveness, suggesting that future screens for HSP70 inhibitors
be carried out for activity against both kinases. We have also
discovered that quercetin enhances the phosphorylation of
HSP27 required for its antiapoptotic action, which would serve
to diminish the radiosensitizing effects of inhibiting HSP70
induction. Two derivatives of quercetin, D1 and D2, were found,
however, that could inhibit HSP70 induction without enhancing
HSP27 phosphorylation, but the degree to which they may be
able to enhance heat-induced radiosensitization remains to be
determined. In addition to probing the mechanism of inhibition
of HSP70 induction by quercetin, this family of methylated
quercetin derivatives might also be useful for mapping out
interactions with its other targets.
1
DCM/hexane (85:15) to afford 4 in 90% yield (0.58 g). H NMR
(CDCl3, 300 MHz): δ 12.74 (s, 1H, OH), 7.67 (d, J ) 2 Hz, 1H),
7.66 (dd, J ) 9, 2 Hz, 1H), 7.46-7.22 (m, 15H), 6.93 (d, J ) 9
Hz, 1H), 6.44 (d, J ) 2 Hz, 1H), 6.40 (d, J ) 2 Hz, 1H), 5.19 (s,
2H), 5.07 (s, 2H), 5.05 (s, 2H), 4.54 (s, 2H), 3.71 (s, 3H). 13C NMR
(CDCl3, 300 MHz): δ 179.0 (CdO), 169.4 (COO), 164.8, 162.3,
156.9, 156.2, 151.3, 147.6, 137.8, 136.8, 136.6, 136.1, 129.0, 128.9,
128.6, 128.5, 128.4, 127.8, 127.6, 124.0, 123.6, 115.9, 114.0, 106.4,
98.9, 93.3, 74.7, 71.1, 70.7, 66.8 (OCH2), 52.4 (OCH3). MS (FAB)
[M + Na+] m/z 667.2. HRMS (FAB) C39H32O9Na [M + Na+],
calculated m/z 667.1944, found 667.1932.
Compound D1. A suspension of compound 4 (100 mg) was
dissolved in a minimum amount of EtOH/THF (1:1) and treated
with 10% palladium hydroxide (0.01 g). The suspension was stirred
overnight at room temperature under one atmosphere of hydrogen
from a balloon. After filtration with Celite to remove the palladium,
the filtrate was concentrated under reduce pressure, followed by a
recrystallization with methanol and water to give D1 in 75% yield
1
(43 mg). H NMR ([D6]DMSO, 300 MHz): δ 12.45 (s, 1H, OH),
10.77 (s, 1H, OH), 9.96 (s, 1H, OH), 9.45(s, 1H, OH), 7.72 (d, J
) 2 Hz, 1H), 7.68 (dd, J ) 8.5, 2 Hz, 1H), 6.96 (d, J ) 8.5 Hz,
1H), 6.42 (d, J ) 2 Hz, 1H), 6.17 (d, J ) 2 Hz, 1H), 4.81 (s, 2H),
3.72 (s, 3H). 13C NMR ([D6]DMSO, 500 MHz): δ 175.9 (CdO),
169.4 (COO), 163.9, 160.7, 156.1, 149.2, 146.2, 145.6, 135.9, 122.6,
121.9, 116.1, 114.2, 103.0, 98.2, 93.4, 65.9 (OCH2), 51.8 (OCH3).
MS (ESI) [M + Na+] m/z 397.1. HRMS (ESI) C18H14O9Na [M +
Na+], calculated m/z 397.0536, found 397.0543.
Experimental Section
General. Reagents and solvents were obtained from either Sigma
Aldrich or Alfa Aesar. Anhydrous solvents were distilled and then
stored over activated 5 Å molecular sieves. All other commercial
materials were used without further purification unless otherwise
stated. Analytical thin-layer chromatography was performed on
Aldrich silica gel 60 F254 plates (0.25 mm) and compounds were
visualized with a UVG-54 mineral light UV lamp at 254 nm. Flash
column chromatography was conducted using the indicated solvent
on E. Merck silica gel 60 (40-63 µm).
Compound 3. Two equivalents of methyl bromoacetate (0.46
g, 3.0 mmol) and anhydrous potassium carbonate (0.42 g, 3.0 mmol)
were added to a solution of compound 1 (1.0 g, 1.5 mmol) in 35
mL of DMF under nitrogen. After stirring for 24 h, the resulting
mixture was worked up with 50 mL of water and extracted with
100 mL of EtOAc. The organic phase was washed with water, dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. The red crude product was recrystallized with EtOAc to
afford 3 in 92% yield (1.0 g). 1H NMR (CDCl3, 300 MHz): δ 7.75
(d, J ) 2 Hz, 1H), 7.57 (dd, J ) 9, 2 Hz, 1H), 7.48-7.23 (m,
20H), 6.97 (d, J ) 9 Hz, 1H), 6.61 (d, J ) 2 Hz, 1H), 6.36 (d, J
) 2 Hz, 1H), 5.25 (s, 2H), 5.12 (s, 2H), 5.08 (s, 2H), 4.97 (s, 2H),
4.83 (s, 2H), 3.81 (s, 3H). 13C NMR (CDCl3, 300 MHz): δ 174.0
(CdO), 169.0 (COO), 162.8, 159.2, 158.9, 153.5, 150.8, 148.4,
140.0, 137.3, 137.2, 137.0, 135.7, 129.2, 129.0, 128.8, 128.8, 128.7,
128.4, 128.3, 128.2, 128.1, 128.0, 127.6, 127.4, 124.0, 122.4, 115.4,
NMR Spectroscopy. 1H NMR and 13C NMR spectra were
recorded with Mercury-300, Inova-500 and Inova-600 (Varian
Assoc., Palo Alto, CA) spectrometers, and the data were processed
with VNMR software. Proton and carbon chemical shifts were
measured in parts per million (ppm) downfield from an internal
TMS standard. Proton spectra were obtained in DMSO-d6 with a
8200 Hz spectral width collected into 32K data points. Carbon
spectra were obtained with a 34000 Hz spectral width collected
into 64K data points. The gradient COSY experiments were
collected with a spectral width of 8200 Hz and 2048 complex points
in the F2 dimension and 512 real points in the F1 dimension. The
proton-detected heteronuclear multiple quantum coherence (HMQC)

Quercetin DeriVatiVes and Heat Shock Proteins
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1919
114.0, 110.3, 98.9, 95.1, 74.3, 71.3, 71.1, 70.9, 66.8 (OCH2), 52.7
(OCH3). MS (FAB) [M + Li]+ m/z 741.5. HRMS (FAB)
C46H38O9Li [M + Li+], calculated m/z 741.2676, found 741.2677.
Compound D8. Compound 3 (255 mg, 0.35 mmol) was
dissolved in a minimum amount of THF/EtOH (1:1). After addition
of 10% Pd(OH)2 (38 mg), the suspension was stirred for 16 h under
one atmosphere of hydrogen from a balloon. The suspension was
filtered through Celite, diluted with EtOH and concentrated under
vacuum. The residue was purified by recrystallization with MeOH/
H2O to afford D8 as a green solid in 86% yield (111 mg). 1H NMR
([D6]DMSO, 300 MHz): δ 10.77 (s, 1H, OH), 9.50 (s, 1H, OH),
9.28 (s, 1H, OH), 8.76 (s, 1H, OH), 7.64 (d, J ) 2 Hz, 1H), 7.50
(dd, J ) 8.5, 2 Hz, 1H), 6.88 (d, J ) 8.5 Hz, 1H), 6.52 (d, J ) 2
Hz, 1H), 6.25 (d, J ) 2 Hz, 1H), 4.89 (s, 2H), 3.73 (s, 3H). 13C
NMR ([D6]DMSO, 300 MHz): δ 171.5 (CdO), 169.5 (COO),
162.7, 159.2, 158.5, 147.7, 145.7, 142.8, 137.8, 122.9, 119.9, 116.3,
115.2, 106.2, 98.4, 96.3, 66.4 (OCH2), 52.6 (OCH3). MS (FAB)
[M + Na]+ m/z 397.1. HRMS (FAB) C18H14O9Na [M + Na+],
calculated m/z 397.0536, found 397.0518.
Compound 6. A suspension of 5 (0.70 g, 1.50 mmol) was
dissolved in 20 mL of DMF, followed by addition of anhydrous
potassium carbonate (0.311 g, 2.25 mmol) and methyl bromoacetate
(0.23 g, 1.5 mmol). The mixture was stirred overnight at room
temperature, under nitrogen. The solution was diluted with 50 mL
of water, acidified with 1.0 N HCl and extracted with 100 mL of
ethyl acetate. The organic layer was washed with water and brine
and dried over anhydrous sodium sulfate. The residue after
concentration in vacuum was purified by flash column chromatog-
raphy using 5% EtOAc in DCM, to afford 6 in 37% yield (0.30 g).
1H NMR (CDCl3, 300 MHz): δ 7.78 (dd, J ) 8.5, 2 Hz, 1H), 7.71
(d, J ) 2 Hz, 1H), 7.64-7.61 (m, 4H), 7.45-7.41(m, 6H), 7.02
(d, J ) 8.5 Hz, 1H), 6.39 (d, J ) 2 Hz, 1H), 6.28 (d, J ) 2.0 Hz,
1H), 4.80 (s, 2H), 3.73 (s, 3H). 13C NMR (CDCl3, 600 MHz): δ
177.9 (CdO), 169.6 (COO), 162.4, 162.0, 156.6, 155.4, 149.5,
147.4, 139.7, 136.7, 129.3, 128.3, 126.2, 124.2, 123.8, 117.9, 108.9,
108.5, 105.6, 99.2, 93.9, 68.4 (OCH2), 52.0 (OCH3). MS (FAB)
[M + Na+] m/z 561.1. HRMS (FAB) C31H22O9Na [M + Na+],
calculated m/z 561.1162, found 561.1164.
[M + K+]. MS (FAB) [M + Na+] m/z 651.1. HRMS (ESI)
C38H29O9 [M + H+], calculated m/z 629.1830, found 629.1830.
Compound D9. Compound 8 (0.482 g) in 20 mL of EtOH/THF
(1:2) was mixed with 10% Pd(OH)2 (10 mg) and stirred overnight
under hydrogen from a balloon. The suspension was filtered through
Celite, and the clear green filtrate was diluted with 20 mL of EtOH
and concentrated under vacuum. The residue was refluxed with
acetic acid as described in th preparation of D5 to give 0.224 g of
1
D9 in 78% yield. H NMR ([D6]DMSO, 300 MHz) 12.46 (s, 1H,
OH), 9.63 (s, 1H, OH), 9.50 (s, 1H, OH), 9.30 (s, 1H, OH), 7.69
(d, J ) 2 Hz, 1H), 7.55 (dd, J ) 8.5, 2 Hz, 1H), 6.86 (d, J ) 8.5
Hz, 1H), 6.66 (d, J ) 2 Hz, 1H), 6.32 (d, J ) 2 Hz, 1H), 4.80 (s,
2H). 13C NMR ([D6]DMSO, 300 MHz): δ 175.8 (CdO),
169.5(CO2H), 163.2, 160.2, 155.7, 147.8, 147.2, 145.0, 136.0, 121.7,
120,0, 115.5, 115.1, 104.2, 97.7, 92.4, 64.8 (OCH2). MS (ESI) [M
+ H+] m/z 361.1. HRMS (ESI) C17H13O9 [M + H+], calculated
m/z 361.0560, found m/z 361.0555.
Compound 10. Quercetin pentaacetate 9 (1.0 g, 1.95 mmol) in
50 mL of dry acetone was mixed with methyl bromoacetate (1.0
g, 6.55 mmol), anhydrous potassium carbonate (2.0 g, 14.5 mmol)
and 0.168 g of potassium iodide. The suspension was refluxed until
9 was completely consumed as judged by TLC. The suspension
was filtered, and the filtrate was concentrated under vacuum and
flash chromatographed using EtOAc/hexane (4:3). The product was
recrystallized with acetone/hexane to afford 0.577 g of 10 as a white
1
solid in 54% yield. H NMR (CDCl3, 300 MHz): δ 7.74 (dd, J )
8.5, 2 Hz, 1H), 7.70 (d, J ) 2 Hz, 1H), 7.37 (d, J ) 2 Hz, 1H),
6.83 (d, J ) 2 Hz, 1H), 6.20 (d, J ) 2 Hz, 1H), 4.76 (s, 2H), 3.87
(s, 3H), 2.46 (s, 3H), 2.37 (s, 9H). 13C NMR (CDCl3, 300 MHz):
δ 169.8 (CdO), 169.3 (COO), 168.0, 167.9, 167.8, 167.7 (4
CH3CdO), 161.8, 157.8, 153.3, 150.9, 144.2, 142.2, 133.9, 127.9,
126.4, 123.8, 123.7, 111.8, 109.0, 99.6, 65.4 (OCH2), 52.6 (OCH3),
21.0, 20.8, 20.6, 20.5 (4 CH3CdO) MS (ESI) [M + Na+] 565.1,
[M + H+] 543.0, [M - CH2CO + H+] 501.1, [M - 2CH2CO +
H+] 459.1, [M - 3CH2CO + H+] 417.1. HRMS (ESI) C26H22O13Na
[M + Na+], calculated 565.0958, found 565.0948.
Compound D10. Twenty milligrams (0.036 mmol) of 10 was
dissolved in 10 mL of THF/MeOH/pH 7 phosphate buffer (9:2:9),
after which 5 equiv of N-methyl-2-dimethylaminoacetohydroxamic
acid (24 mg, 0.180 mmol) was added. The solution was stirred
under nitrogen atmosphere at room temperature for 12 h, after which
the mixture was worked up with water, acidified to pH 6 by 1 M
HCl and extracted with 50 mL of EtOAc. The organic phase was
washed with water and dried over anhydrous sodium sulfate. The
residue after concentration in vacuum was recrystallized from
MeOH/H2O to afford 11 mg of D10 as a green solid in 83% yield.
1H NMR ([D6]DMSO, 300 MHz): δ 7.71 (d, J ) 2 Hz, 1H), 7.55
(dd, J ) 8.5, 2 Hz, 1H), 6.87 (d, J ) 8.5 Hz, 1H), 6.72 (d, J ) 2
Hz, 1H), 6.37 (d, J ) 2 Hz, 1H), 4.94 (s, 2H), 3.71 (s, 3H). 13C
NMR ([D6]DMSO, 600 MHz): δ 176.4 (CdO), 169.0 (COO),
163.5, 160.8, 156.2, 148.3, 147.9, 145.5, 136.5, 122.2, 120.4, 116.0,
115.7, 104.8, 98.2, 93.0, 65.3 (OCH2), 52.4 (OCH3). MS (ESI) [M
+ Na]+ 397.1. HRMS (ESI) C18H14O9Na [M + Na]+, calculated
397.0536, found 397.0535.
Compound D5. Compound 6 (0.30 g, 0.56 mmol) was mixed
with 50 mL of CH3CO2H/H2O (4:1) and refluxed overnight. The
solution is worked up by addition of 50 mL of water and then
carefully neutralized by dropwise addition of saturated NaHCO3.
After the extraction with 100 mL of EtOAc, the organic layer was
washed with brine, dried over anhydrous sodium sulfate and vacuum
concentrated. The crude residue was recrystallized with methanol
1
to afford 0.156 g of D5 as a red solid in 74% yield. H NMR
([D6]DMSO, 300 MHz): δ 12.61 (s, 1H, OH), 7.63 (d, J ) 2 Hz,
1H), 7.62 (dd, J ) 8, 2 Hz, 1H), 6.93 (d, J ) 8 Hz, 1H), 6.48 (d,
J ) 2 Hz, 1H), 6.26 (d, J ) 2 Hz, 1H), 4.73 (s, 2H). 13C NMR
([D6]DMSO, 300 MHz): δ 177.4 (CdO), 171.5 (CO2H), 169.8,
164.1, 161.0, 156.1, 155.1, 148.6, 145.0, 135.7, 121.0, 120.6, 115.5,
103.9, 98.5, 93.4, 67.7 (OCH2-CO2H). MS (ESI) [M + H+] m/z
361.0. HRMS (ESI) C17H13O9 [M + H+], calculated 361.0560,
found 361.0599.
Compound 8. Compound 7 (0.50 g, 0.90 mmol) in 30 mL of
DMF was mixed with methyl bromoacetate (0.138 g, 0.90 mmol),
followed by addition of anhydrous potassium carbonate (0.152 g,
1.10 mmol). The mixture was stirred under nitrogen for 6 h at room
temperature and then neutralized with 1.0 N HCl. The solution was
extracted with 100 mL of EtOAc, washed with water, and dried
over anhydrous sodium sulfate. The solvent was removed under
vacuum and the residue was flash chromatographed using EtOAc/
petroleum ether (1:1) to afford 0.360 g of the desired product 8 in
HSP70 Inhibition Assays. Exponentially growing Jurkat cells,
grown in RPMI media with 10% fetal bovine serum, were treated
with 50 µg/mL of quercetin (165 µM) or its derivatives (methyl,
158 µM; carboxymethyl, 139 µM; carbomethoxymethyl, 134 µM)
for 1 h prior to being heated at 43 °C for 30 min. The cells were
allowed to recover at 37 °C for 8 h to allow for HSP70 expression
and cells harvested for Western blot analysis with mouse mono-
clonal antibodies against inducible HSP70 (Assay Designs, SPA-
810) and actin (ICN, clone C4). Secondary antibodies were goat
antimouse conjugated horse radish peroxidase (Millipore) or alkaline
phosphatase (Jackson ImmunoResearch). Protein bands were visual-
ized by either chemiluminescence (SuperSignal West Femto
Substrate, Thermo Scientific) or colorimetric (BCIP/NBT, Sigma)
detection.
1
64% yield. H NMR (CDCl3, 300 MHz): δ 12.78 (s, 1H, OH),
7.62-7.15 (m, 17H), 6.92 (d, J ) 8.5 Hz, 1H), 6.40 (d, J ) 2 Hz,
1H), 6.36 (d, J ) 2 Hz, 1H), 5.04 (s, 2H), 4.70 (s, 2H), 3.83 (s,
3H). 13C NMR (CDCl3, 300 MHz): δ 207.3, 179.0 (CdO),
168.7(COO), 163.6, 162.4, 157.1, 156.8, 149.6, 147.5, 140.0, 137.5,
136.4, 126.7, 129.2, 128.7, 128.5, 126.5, 124.4, 124.3, 118.1, 109.3,
108.6, 106.8, 98.5, 93.1, 74.7, 65.4 (OCH2), 52.8 (OCH3). MS (ESI)
m/z 629.2 for [M + H+], m/z 651.2 for [M + Na+], m/z 667.1 for
HSP27 Phosphorylation Assays. Phosphorylation of HSP27 was
detected by Western blot analysis of total cell extracts prepared
from exponentially growing HeLa cells. Cells were grown in

1920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Wang et al.
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Dawra, R.; Lerch, M. M.; Saluja, A. Heat shock protein 70 increases
tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma.
Cancer Res. 2007, 67, 616–625.
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with Janus-like properties in cancer. Int. J. Hyperthermia 2008, 24,
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Heat shock proteins in cancer: chaperones of tumorigenesis. Trends
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(13) Garrido, C.; Brunet, M.; Didelot, C.; Zermati, Y.; Schmitt, E.; Kroemer,
G. Heat shock proteins 27 and 70: anti-apoptotic proteins with
tumorigenic properties. Cell Cycle 2006, 5, 2592–2601.
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Jaattela, M. Members of the heat-shock protein 70 family promote
cancer cell growth by distinct mechanisms. Genes DeV. 2005, 19, 570–
582.
(15) Nanbu, K.; Konishi, I.; Komatsu, T.; Mandai, M.; Yamamoto, S.;
Kuroda, H.; Koshiyama, M.; Mori, T. Expression of heat shock
proteins HSP70 and HSP90 in endometrial carcinomas. Correlation
with clinicopathology, sex steroid receptor status, and p53 protein
expression. Cancer 1996, 77, 330–338.
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expression in human oral cancer cells. Int. J. Cancer 2000, 85, 1–5.
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Y.; Sugimachi, K. Overexpression of the heat shock protein HSP70
family and p53 protein and prognosis for patients with gastric cancer.
Oncology 2000, 58, 144–151.
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D.; Nakopoulou, L.; Kittas, C.; Golematis, B. C. Heat shock protein
70 and HLA-DR molecules tissue expression. Prognostic implications
in colorectal cancer. Dis. Colon Rectum 1995, 38, 739–745.
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human histiocytic lymphoma cell line, U937. Cell Struct. Funct. 1993,
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W. J.; McGuire, W. L. Heat shock protein hsp70 in patients with
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McGuire, W. L. Response of human breast cancer cells to heat shock
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DMEM media with 10% calf serum and treated for 1 h with 50
µg/mL of quercetin or its derivatives before protein isolation. A
rabbit antibody specific for human HSP27 phosphorylated on serine
78 (Assay Designs, SPA-523) coupled with goat antirabbit alkaline
phosphatase secondary antibody (Jackson ImmunoResearch) was
utilized for colorimetric detection.
HSF1 Electrophoretic Mobility Shift Assays (EMSA). Analy-
sis of HSF1 interactions with the HSE DNA promoter element was
carried out by EMSA essentially as described previously57 except
that nuclear extracts were utilized in place of whole cell extracts.
The heat shock element probe (HSE) d(GCGAAACTGCTGGAA-
GATTCCT) was 5′-32P end labeled followed by annealing to the
complementary oligonucleotide to produce the duplex DNA.
Western blot analysis of the same nuclear extracts utilized for
EMSA was carried out with rabbit anti-HSF1(Cell SignalingTech-
nology) followed by goat antirabbit HRP (Millipore) and chemi-
luminescent detection.
Kinase Inhibition Assays. Phosphate kinases CAMKII and CK2,
substrates, and buffers were from New England BioLabs. Inhibition
assays were carried out with the PKlight HTS Protein Kinase Assay
kit (Lonza) according to the manufacturer’s procedure. Briefly, this
assay involves determining in triplicate the extent of phosphory-
lation of a peptide substrate in the presence and absence of various
concentrations of inhibitor by quantifying the amount of ATP
consumed via a luciferase-based bioluminescence assay. A kinase
concentration (EC50) is used that results in 50% consumption of
ATP in the absence of inhibitor under the reaction time and
conditions. The data was then fit via nonlinear least-squares fitting
to a standard sigmoidal dose-response equation. CK2 inhibition
assay: Casein kinase II (CK2) was incubated at 30 °C for 30 min
with 300 µM substrate (RRRADDSDDDDD), 100 µM ATP, in a
buffer containing 20 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2,
with or without the presence of inhibitor. CamKII inhibition assay:
Ca2+/calmodulin-dependent protein kinase II (CaMKII) supple-
mented with 200 µM ATP, 1.2 µM calmodulin and 2 mM CaCl2
was incubated for 10 min at 30 °C to preactivate the enzyme and
then diluted to the required concentration. The activated CaMKII
was then incubated at 30 °C for 30 min with or without the inhibitor,
along with 300 µM autocamtide-2 (KKALRRQETVDAL), 100 µM
ATP, in a buffer containing 50 mM Tris-HCl, 10 mM MgCl2, 2
mM DTT, 0.1 mM Na2EDTA at pH 7.5.
Acknowledgment. This work is supported by NIH PO1 CA
104457 and by grants to the Washington University NIH Mass
Spectrometry Resource (Grant No. P41 RR000954) and Wash-
ington University NMR facility (Grant No. RR1571501).
Supporting Information Available: HPLC purity analysis of
compounds D1-D10. This material is available free of charge via
the Internet at http://pubs.acs.org.
(25) Dote, H.; Cerna, D.; Burgan, W. E.; Camphausen, K.; Tofilon, P. J.
ErbB3 expression predicts tumor cell radiosensitization induced by
Hsp90 inhibition. Cancer Res. 2005, 65, 6967–6975.
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