A novel class of small molecule inhibitors of Hsp90.

Abstract

Unregulated cellular proliferation, caused by mutation or dysregulation of growth-promoting proteins, is an underlying cause of cancer. Many such growth-promoting proteins exhibit an increased dependence on the activity of the chaperone heat-shock protein 90 (Hsp90) for correct folding and maturation in the cell. One can therefore envision that inhibition of Hsp90 would be an effective and broadly applicable strategy for the development of anticancer agents. Hsp90 functions in multichaperone complexes driven by the binding and hydrolysis of ATP. Encouraging results have been obtained by inhibiting Hsp90 with 17-AAG, an active-site binding ATP analog. Here we present the results of a different approach to inhibiting Hsp90 by disrupting its interaction with a cochaperone named Hsp organizing protein (HOP). We have used an AlphaScreen technology based high-throughput in vitro screen to identify compounds that inhibit this interaction. In addition, we demonstrate that these compounds are active in vivo. Treatment of human breast cancer cell lines BT474 and SKBR3 with these compounds decreases the levels of the Hsp90-dependent client protein HER2, with associated cell death.

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ANovelClassofSmallMoleculeInhibitorsof
Hsp90
ARTICLE
Fang Yi†and Lynne Regan†,‡,*
†Department of Molecular Biophysics & Biochemistry and‡Department of Chemistry, Yale University, 266 Whitney Avenue,
New Haven, Connecticut 06520
D
the growth and progression of many tumors. For in-
stance, approximately one-third of all breast cancer
cells overproduce the cell surface receptor HER2 (desig-
nated HER2 positive) (1). The more HER2 a cell pro-
duces, the more aggressive its growth and the poorer
the patient prognosis (2, 3). One of the most effective
developments in the treatment of breast cancer in the
past20yearsisahumanizedmonoclonalantibody(Her-
ceptin (trastuzumab)) that binds to the extracellular do-
main of HER2 on the surface of breast cancer cells. Her-
ceptin has been proposed to work via several different
mechanisms, among which are blockade of down-
stream HER2 signaling pathways (4) and targeting of
the cancer cells for destruction by the immune system
(5).
Analternativeandmoregeneralapproachtoantican-
certherapeuticswouldbetopreventHER2andotheron-
cogenic proteins from ever folding and maturing in the
cell. Certain proteins are dependent on the activity of
other proteins, known as chaperones, for correct fold-
ing and maturation. About half of all proteins whose
folding depends on the activity of the chaperone Hsp90
are proteins whose mutation or overproduction pro-
motes cancer. Examples include but are not limited to
pp60v-src, Bcr-Abl, p53, Akt, Flt3, HIF-1?, B-Raf, EGFR,
and HER2. Inhibition of Hsp90 function therefore pre-
sents an effective route by which to disrupt multiple
growth-promoting signaling pathways simultaneously
(6).
Hsp90 activity is ATP-dependent, and inhibitors that
bind at the ATP active site, for example, geldanamycin
(GA) and its derivative 17-AAG, have already shown
promise as anticancer agents (7, 8). 17-AAG has anti-
ysregulation of signal transduction pathways,
for example, by the overexpression of growth
factor receptors, is a well-known contributor to
*Corresponding author,
lynne.regan@yale.edu.
Received for review June 3, 2008
and accepted July 28, 2008.
Published online September 12, 2008
10.1021/cb800162x CCC: $40.75
© 2008AmericanChemicalSociety
ABSTRACT Unregulated cellular proliferation, caused by mutation or dysregula-
tion of growth-promoting proteins, is an underlying cause of cancer. Many such
growth-promoting proteins exhibit an increased dependence on the activity of the
chaperone heat-shock protein 90 (Hsp90) for correct folding and maturation in the
cell. One can therefore envision that inhibition of Hsp90 would be an effective
and broadly applicable strategy for the development of anticancer agents. Hsp90
functions in multichaperone complexes driven by the binding and hydrolysis of
ATP. Encouraging results have been obtained by inhibiting Hsp90 with 17-AAG,
an active-site binding ATP analog. Here we present the results of a different ap-
proach to inhibiting Hsp90 by disrupting its interaction with a cochaperone named
Hsp organizing protein (HOP). We have used an AlphaScreen technology based
high-throughput in vitro screen to identify compounds that inhibit this interac-
tion. In addition, we demonstrate that these compounds are active in vivo. Treat-
ment of human breast cancer cell lines BT474 and SKBR3 with these compounds
decreases the levels of the Hsp90-dependent client protein HER2, with associated
cell death.
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tumoractivityinseveralhumanxenograftmodels.More-
over, 17-AAG is currently being tested in clinical trials
for use alone or in combination with other anticancer
agents against melanoma, breast, prostate, and thyroid
cancer (9).
Although 17-AAG illustrates the potential of inhibit-
ing Hsp90 as a route to novel anticancer therapeutics,
it is not a “magic bullet”. The most common dose-
limitingsideeffectsof17-AAGincancerpatientsarean-
orexia,nausea,diarrhea,hepatoxicity,fatigue,thrombo-
cytopenia, and anemia. 17-AAG has low solubility in
aqueous solution, which is also limiting for its clinical
applications(10,11).Althoughtheprecedentof17-AAG
is encouraging, there is clearly a need for novel Hsp90
inhibitors with higher solubility and fewer side effects.
Here we present an entirely new approach to inhibit-
ing Hsp90 activity. Hsp90 does not function in isolation
butratherispartofamultiproteincomplex(12).Thecur-
rent consensus model for Hsp90-dependent protein
folding assumes that a newly synthesized unfolded
polypeptide first binds to Hsp40 and is then passed
from Hsp40 to Hsp70. Hsp70 and Hsp90 are brought
into close proximity by their C-terminal peptide interac-
tions with two independent tetratricopeptide repeat
(TPR) domains of HOP, TPR1, and TPR2A, respectively.
Substrates are transferred from Hsp70 to
Hsp90 to complete the final stage of matu-
ration. The interaction of Hsp90 with HOP,
via its TPR2A domain, is absolutely essen-
tial for Hsp90 activity (13), with a dissocia-
tionconstant(Kd)of11?M(14).Weseekto
inhibit this interaction as a novel route by
which to inhibit Hsp90 activity (Figure 1).
In previous studies we have demon-
strated the potential of the proposed ap-
proach.WehaveshownthatadesignedTPR
domain, which binds to the C-terminus of
Hsp90 more tightly and more specifically
than TPR2A, can compete for binding to
Hsp90 after being transfected into breast
cancer cells and thereby prevent formation
ofthefunctionalHsp90?HOP?Hsp70com-
plex. Hsp90 is thus inhibited, the levels of
HER2 are reduced, and the cells die (15).
Here we present the identification, via a
high-throughput screen (HTS), of small mol-
ecules that inhibit the Hsp90?TPR2A inter-
action.Moreover,weshowthatsuchcompoundsareef-
fective in vivo, causing a decrease in HER2 levels in
human breast cancer cells (BT474 and SKBR3), with as-
sociated cell death. Because our compounds act by a
mechanism completely different from those of 17-AAG,
Herceptin, and traditional nonspecific chemotherapeu-
tics,theyhavehighpotentialtobecombinedsynergisti-
cally with these and other anticancer agents.
RESULTSANDDISCUSSION
Hit Identification Using the High-Throughput
AlphaScreen Assay. Small molecule inhibitors of the
Hsp90?HOP interaction, rather than peptides, are re-
quired for the development of this approach as a
therapeutic strategy. We therefore developed an
AlphaScreen technology based HTS to identify novel in-
hibitors of the Hsp90?TPR2A interaction. A schematic
of the AlphaScreen is shown in Figure 2. We demon-
strated the suitability of this assay using free Hsp90
peptide. Free Hsp90 peptide effectively competes with
biotinylated Hsp90 peptide for its binding to TPR2A, re-
sulting in decreased fluorescence signals. Measuring
the concentration-dependent inhibition of free Hsp90
peptide gives an IC50of about 5 ?M, which is consis-
tent with the known Kdof the Hsp90-TPR2A interaction
(14).
TPR1 TPR2A TPR1 TPR2A
Folded
protein
Degraded
protein
TPR1 TPR2ATPR1 TPR2A
(Compounds)
Unfolded
peptide
Unfolded
peptide
17-AAG
a
b
Figure 1. Schematic illustration of Hsp90-mediated protein folding and proposed mechanism of
identified compounds. a) For Hsp90-dependent proteins, the newly synthesized polypeptide
chain (black line) is chaperoned through the initial steps of folding by Hsp70 (yellow rectangle).
Hsp70 and Hsp90 (orange dimer) are brought together in a complex, via their interactions with
the TPR1 (blue) and TPR2A (purple) domains of HOP, respectively. Partially folded polypeptide is
passed from Hsp70 to Hsp90, where the final steps of folding occur. If Hsp90 activity is inhib-
ited, for example, by 17-AAG, the Hsp90-dependent protein, e.g., HER2, dissociates from the
multichaperone complexes, does not complete its folding, and is targeted for degradation by the
proteasome. b) Proposed mode of action of molecules that inhibit the Hsp90?HOP interaction.
The first, Hsp70-dependent step of folding will initiate as normal. The inhibitory molecule will
prevent the interaction of Hsp90 with HOP. Thus Hsp90 cannot form the HOP-mediated complex
with Hsp70 and consequently cannot receive the partially folded substrate. The incompletely
folded protein is targeted for degradation by the proteasome.
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Using the AlphaScreen in a 384-well format, we
screened the MicroSource Discovery Systems GenPlus
library (960 compounds) and the Maybridge diversity li-
brary (20,000 compounds) at ?10 ?M concentration
of each compound. The NCGC compound library
(76,174 compounds) was screened in a 1536-well as-
say format at a titration series of seven concentrations
with0.1%BSAand0.01%Tween-20includedintheas-
saybuffertodecreasethelikelihoodofcompoundsact-
ing by aggregate formation for promiscuous inhibition
(16). The average Z= factor value for all assays in both
formats is about 0.7, indicating the robustness. Al-
though we identified early “hits” in each of the librar-
ies, the compounds shown to have the desired proper-
ties by follow-up assays were all from the NCGC
compound library. For clarity, therefore, we present the
results only from this library.
For each of the compounds, a concentration-
dependent inhibition curve was determined, and 149
compoundswithIC50valuesof10?Morlesswereiden-
tified as primary hits. Figure 3 is a flowchart showing
thecharacterizationofthehitcompoundsusingsecond-
ary in vitro and in vivo assays. Following structure clus-
teringanalysis,41representativeswereselectedforfur-
ther competition confirmation and subjected to a
counter screen for false-positive identification. In the
counter-screen, compounds were tested for their inhibi-
tion in a version of the AlphaScreen in which the donor
and acceptor beads are brought together by a covalent
biotin-His6linker. Compounds that inhibit fluorescence,
mimic either biotin or NTA, or inhibit by any nonspecific
mechanism will be eliminated by the counter-screen.
Only three compounds remained as true positive
hits after these steps. An examination of the structures
of these three compounds revealed that they have a
ab
AlphaScreen-based assay for the
Hsp90-TPR2A interaction
Competition assay for small molecule inhibitors
of the Hsp90-TPR2A interaction
680 nm680 nm
DDAA
1O2
1O2
Small molecule
inhibitors
Biotin-Hsp90Biotin-Hsp90 His6-tagged
TPR2A
His6-tagged
TPR2A
520∼620 nm
No signal
Figure 2. Schematic illustration of the AlphaScreen. The donor bead (D, red) is coated with streptavidin (dark blue), which
binds N-terminally biotinylated (gray square) Hsp90 peptide (gray oval). The acceptor bead (A, orange) is coated with Ni-
NTA (black Y), which binds the C-terminal His6tag (blue diamond) of TPR2A (green oval). a) In the absence of inhibitors,
the Hsp90?TPR2A interaction brings the donor and acceptor beads together. Excitation of the donor beads at 680 nm pro-
duces excited singlet state oxygen species, which can diffuse up to 200 nm and react with a chemiluminescor in the ac-
ceptor bead, further activating flurophores in the acceptor bead, which emit light at 520?620nm. b) In the presence of
small molecule inhibitors (black circles) that inhibit the Hsp90?TPR2A interaction, the donor and acceptor beads are no
longer brought to within 200 nm of each other, and no acceptor bead fluorescence is observed upon excitation of the do-
nor bead.
NCGC
(76,741 cpds)
149 hits
41 hits
41 hits
3 hits
6 hits
6 hits
AlphaScreen-detected competition curve
(IC50 < 10 µM)
Structure-based clustering analysis
2 representatives from each of 13 clusters,
plus 15 singletons were chosen
Confirmation of competition curves
Counter screen to identify false positives
5 commercial homologues
Fluorescence polarization assay
Cell proliferation and Western Blot assay
+
Figure 3. Flowchart of the steps in hit identification and verification.
ARTICLE
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common core. We therefore purchased and tested five
additional structurally related compounds that were not
presentintheoriginallibrary.Onlythreeofthefivecom-
pounds tested positive in the AlphaScreen (Figure 4).
All of the concentration?response competition curves
for the six active compounds had Hill slopes close to
?1, indicating no cooperative binding of multiple mol-
eculesorcompoundaggregation.Alsoitisreassuringto
see that none of this class of compounds was identi-
fied as “promiscuous inhibitors” in an aggregate profil-
ingscreenagainstthesameNCGCcompoundsbymoni-
toring, with or without 0.01% Triton X-100, their
inhibition of the activity of AmpC ?-lactamase, a pro-
tein target completely unrelated to our study (see Pub-
Chem AID ? 584, 585). At this point we continued with
further characterization of the six positive hit com-
pounds and one of the inactive compounds, to serve
as a negative control.
Confirmation of Compound Binding Using a
Fluorescence Polarization Assay. We tested these six
hit compounds using an independent in vitro assay, to
rule out any false positives that may have been missed
in the AlphaScreen counter-screen. We therefore devel-
oped a fluorescence polarization (FP) assay by which to
monitor the fluorescein-labeled C-terminal Hsp90
peptide?TPR2A interaction and its inhibition. Com-
pounds were screened for their ability to inhibit the
Hsp90?TPR2A interaction, as monitored by decreases
in the polarization of the bound fluorescein-labeled
Hsp90 peptide. The intensities from both the perpen-
dicular and horizontal orientations were checked to
make sure that the FP value (ratio between these two)
decreases were not due to abnormal intensity changes
that might come from artificial compound interference.
Noabnormalintensitychangeswereobserved.Also,we
measured the intrinsic fluorescence of compounds and
observed very low background signal in the detecton
range for both FP and AlphaScreen assays. Thus the
datasupportsthehypothesisthatthechangesinFPob-
served when the active compounds are added are be-
cause of differences in the tumbling of the fluorescent
Hsp90 peptide when free as compared to when bound
to TPR2A.
All six compounds tested positive in the FP assay,
with Kdvalues in the micromolar range, consistent with
those measured from the AlphaScreen competition as-
say.Moreover,thecompoundthatwasinactiveintheAl-
phaScreen,whichwebroughtforwardasanegativecon-
trol,wasalsoinactiveintheFPassay(Figure5,panela).
Demonstration of Direct Binding between
Compound 8 and TPR2A. The data presented to date
show that we have identified compounds that compete
fortheTPR2A?Hsp90interaction.Todemonstratedirect
binding between compounds and protein or peptide,
we performed isothermal titration calorimetry (ITC) mea-
surements with the representative compound 8 and ei-
ther the TPR2A protein or Hsp90 peptide. As we might
have anticipated, because it is the larger molecule and
hasa“bindingcleft”,ITCshowedadirectinteractionbe-
tween TPR2A and compound 8, with a Kdof 16 ?M
and a 1:1 binding stoichiometry (Figure 5, panel b). No
interaction between the compound and Hsp90 peptide
was detected (data not shown).
−9
−4
−5
−6
−7
−8
120
0
20
40
60
80
100
Percent of control binding
log[compound] (M)
−9
−4
−5
−6
−7
−8
120
0
20
40
60
80
100
Percent of control binding
log[compound] (M)
8
9
10
11
12
13
Inactive
analogue
12
N
R1
3
4
4a
5
6
7
8
8a
a
b
c
8
9
10
11
12
13
Inactive
analogue
N
N
N
N
O
O
R2
Figure 4. Competition curves for selected compounds in the Al-
phaScreen assay. a) Competition curves for different compounds
using the AlphaScreen with the donor and acceptor beads brought
together by Hsp90 peptide and TPR2A protein. Compounds 8?13
all show typical dose-dependent inhibition of the acceptor fluores-
cence. The inactive structural analogue compound does not.
b) AlphaScreen in counter-screen format. A covalent linker peptide
(biotin-His6peptide) directly brings the donor and acceptor beads
together. Any compounds that inhibit in this assay are consid-
ered to be false positives. Percent of control binding (no inhibition
? 100%) is plotted against log[compound]. Error values were
calculated from multiple experiments. c) Core structure represen-
tative of the hit compounds.
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In Vivo Activity of Selected Compounds. Activity in
vitro does not necessarily mean that the compounds
will be active in vivo. Not least of the requirements for
in vivo activity is that the compounds must be able to
enter cells and function in a cellular context. We there-
fore tested the activity of the 6 hit compounds and the
one inactive compound in cell based assays to assess
the overall capacity of the compounds to inhibit cancer
cellgrowth.Forcomparison,wealsotestedtheeffectsof
17-AAG as a positive control.
Figure 6, panel a shows examples of concentration-
dependent killing of BT474 cells by the compounds as
measured by a WST-1 cell proliferation assay. Only the
compoundsthatshowedactivityinvitroshowedcellkill-
ing activity. Although we do not know the exact concen-
trationsinthecellsofthecompoundsandtheirdifferen-
tial partition into each cellular compartment, the
observationthattherangeofconcentrationsoverwhich
the compounds are active coincides with the Kdvalues
measured in vitro suggests that these compounds per-
meate the cell membrane well and are likely inhibiting
the targeted interaction in cells. Because all six hit com-
pounds behaved similarly in the in vitro binding assays
and the BT474 cell proliferation assay, we chose just
one representative (compound 8) for more detailed
follow-up assays.
Cell-Line Specificity. The molecular basis for the cell-
line specificity of different anticancer agents is not well
understood and cannot be predicted a priori. To deter-
mine whether our hit compounds can selectively kill
breast cancer over normal breast cells, we compared
the cell-killing activity of 17-AAG and compound 8 with
that of an inactive structural homologue in two different
HER2 positive human breast cancer cell lines (BT474,
SKBR3) alongside MCF-12F (a non-tumorigenic mam-
maryepithelialcellline).Figure6,panelsbandcshows
the concentration-dependent cell killing of these three
cell lines by 17-AAG and by compound 8. 17-AAG was
significantly more potent in killing BT474 cells than
SKBR3 and normal cells. Compound 8 also showed
some selectivity in preferentially killing BT474 cells, but
this effect was less pronounced. The inactive structural
homologue was inactive in all three cell lines (data not
shown). Many factors can contribute to the differences
in cell-line specificity of a given compound, including
cellular uptake and metabolism or “off target” interac-
tions with other cellular components. Optimization of
−10
−4
−5
−6
−7
−8
110
−10
10
30
50
70
90
Percent of control binding
log[compound] (M)
8
9
10
11
12
13
Inactive
analogue
a
−3
−9
0.02
−0.12
−0.08
−0.06
−0.04
−0.02
0.00
µcal s−1
b
−0.10
−8
−4
−2
0
−6
−1060 40302010
Time (min)
800705090
1.5 0.50.0
Molar ratio
2.52.0 1.03.0
kcal (mol injectant)−1
Figure 5. Characterization of the binding between compounds and
TPR2A protein using secondary in vitro binding assays. a) Examples
of inhibition curves for selected compounds in the fluorescence
polarization assay. A peptide corresponding to the C-terminus of
Hsp90 (10-mer peptide, N-terminally labeled with fluorescein) and
His6-tagged TPR2A protein were used in these experiments. Per-
cent of control binding (peptide in complex with TPR2A, FP is high
? 100%) is plotted versus log[compound]. Data are reported as
mean and standard deviation of two experiments. b) Binding con-
stant and stoichiometry for the interaction of TPR2A with com-
pound 8 were determined by ITC measurements. Top: Incremental
heat effect upon titration of compound 8 (0.5 mM) into TPR2A
(40 ?M). Bottom: integrated heat effects normalized to the amount
of injected compound 8 and fitted curve based on a 1:1 binding
model. Constants determined were Kd? 16 ?M, stoichiometry N ?
1.07.
ARTICLE
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compound 8 to improve its tumor cell specificity will be
the topic of further studies.
Effect of Active Compounds on the Levels of HER2
and Hsp70. The cell-killing effects suggest that the
compounds act in the hypothesized fashion. However,
to more explicitly explore their mechanism of action, it
wasimportanttostudytheireffectsonHsp90clientpro-
teins. BT474 and SKBR3 cells are both HER2 positive
and have been extensively used in HER2 and Hsp90 re-
search. We therefore investigated the concentration-
and time-dependence of the compounds’ effect on
HER2 levels in these cell lines. Again, we compared the
activity of our compounds with that of 17-AAG. We per-
formedWesternBlotsontotalcellextracts,usingappro-
priate antibodies. Examples of the data obtained in
these experiments are shown in Figure 7. Shown on
the left is a time-dependent decrease in HER2 levels in
BT474cellsaftertreatmentwithcompound8or17-AAG.
Also compared are the levels of the chaperone Hsp70
and the enzyme glyceraldehyde-3-phosphate dehydro-
genase (GAPDH), which serves as a non-Hsp90-
dependent control. On the right are the same experi-
ments performed in SKBR3 cells.
We observed a very reproducible behavior in BT474
cells: HER2 levels rapidly decrease after compound
treatment for 6 h and then increase at 12 h. Similar re-
sults have been reported by others for cells treated with
17-AAG. Although compound cell entry, metabolism, or
unknown features of HER2 regulation could all contrib-
ute to this effect, the underlying mechanism has not yet
been elucidated and will be a topic of future studies.
It has long been observed that treatment of cells, by
GA or other ATPase inhibitors of Hsp90, induces the ex-
pression of the chaperone Hsp70 (17, 18). Induction of
Hsp70 is undesirable, because Hsp70 has potent anti-
apoptotic activity (19, 20), which to some extent can
counteract the desired cell killing. In our experiments,
we also observed a significant increase in Hsp70 levels
when cells were treated with 17-AAG (Figure 7). By con-
trast, none of our compounds induced Hsp70 overpro-
duction,ascanbeclearlyseenintheWesternblotswith
compound 8 as an example. This result is consistent
with our hypothesis that these compounds act via a dif-
ferent mechanism from that of 17-AAG and suggests a
potential advantage of these compounds over the
ATPase inhibitors of Hsp90.
−10
−4
−5
−6
−7
−8
log[17-AAG] (M)
115
−10
15
40
65
90
Percent of control growth
BT474 (17-AAG)
SKBR3 (17-AAG)
Normal (17-AAG)
b
−9
7.05e-9
1.61e-7
6.09e-7
IC50 (M)
−9
−4
−5
−6
−7
log[8] (M)
−8
115
−10
15
40
65
90
Percent of control growth
BT474 (8)
SKBR3 (8)
Normal (8)
c
9.46e-7
1.25e-6
1.21e-6
IC50 (M)
−4
−5
−6
−7
−8
log[compound] (M)
110
−30
90
−10
10
30
50
70
Percent of control growth
a
8
9
10
11
12
13
Inactive
analogue
9.46e-7
9.17e-7
6.90e-7
1.21e-6
1.72e-6
4.27e-6
IC50 (M)
Figure 6. Effects of compounds on the growth of various breast cells. a) Effects
of compounds on the growth of BT474 breast cancer cells. b) Effect of 17AAG
on the growth of BT474, SKBR3, and MCF-12F cells. c) Effect of compound 8 on
the growth of BT474, SKBR3, and MCF-12F cells. Data are reported as mean
and standard deviation of six replicates.
(h)612 24 24Untreated
8
(1 µM)
17-AAG
(178 nM)
BT474
6 12 24 24Untreated
8
(5 µM)
17-AAG
(178 nM)
SKBR3
3
WB
HER2
Hsp70
GAPDH
Figure 7. Effect of compounds on the levels of HER2 and
Hsp70 in BT474, SKBR3 breast cancer cells. Cells treated
with compounds at different concentrations or untreated
were harvested at indicated time points and lysates were
prepared and immunoblotted as described in Methods.
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Conclusion. We have de-
scribed the identification of a
class of small molecules that in-
hibit the interaction of the
C-terminal peptide of Hsp90 and
the TPR2A domain of HOP. We
have demonstrated their inhibi-
tory activity in vitro, using three
different assays, the Alpha-
Screen, a fluorescence polariza-
tion assay, and isothermal titra-
tion calorimetry. We have also
demonstrated that the com-
pounds are active in vivo, in re-
ducing the levels of the cancer-
promoting, Hsp90-dependent
clientproteinHER2andincellkill-
ing. Not only do these com-
poundsinhibitHsp90byaunique
mechanism, but they also have
the desirable property of not in-
ducing overexpression of Hsp70.
Thecompoundsshowmodestse-
lectivityforcancerversusnoncan-
cer cell lines.
All six active compounds have
in common a 7-azapteridine ring
system (pyrimido[5,4-e]
[1,2,4]triazine-5,7-dione)
(Table 1). In the in vitro and
in vivo assays performed to date,
the six related compounds show
similaractivities,withnosubstitu-
tion causing a dramatic change
in activity. We speculate that the
relatively flat structure?activity
relationship (SAR) is because
most of the compounds were dif-
ferent by substitutions at a single
position, presumably not a key position. It is interest-
ing to note, however, that the active compound 8 and
the inactive analogue differ by only a single methyl
group at position 1. Although this subtle difference
mightaccountforthelackofactivityoftheinactiveana-
logue in cell based assays, as a result of its increased
polarity and consequent decreased cell membrane per-
meability,theobservationthatthiscompoundisalsoin-
active in the in vitro binding assays prompts us to
speculate that this methyl group could be involved in a
key hydrophobic interaction in the TPR binding pocket.
Clearly, however, future extensive SAR studies, com-
bined with structural characterizations of the
TPR2A?compound complexes, will provide a more de-
tailed picture. More extensive characterization of the
invivoeffectsofthecompounds,e.g.,exploringcelltype
specificity and the range of Hsp90 client proteins
against which the compounds are effective, is under-
TABLE 1. Summary of hits information
HitSource
IC50 (M)
(WST-1)
Kd (M)
(FP)
Kd (M)
(ALPHA)
Structure
N
N
NN
N
O
O
N
NNN
N
O
O
N
NNN
N
O
O
N
NNN
N
O
O
S
N
N NN
N
O
O
N
NNN
N
O
O
N
NN
H
N
N
O
O
Inactive Commercial
Commercial
Commercial
Commercial
NCGC00046775
NCGC00034880
NCGC00034018
8
9
10
11
12
13
N/A
N/AN/A
1.82e-75.66e-74.27e-6
3.60e-7
3.65e-7
5.85e-7
4.14e-7
4.92e-7
9.28e-7
3.08e-7
1.04e-6
2.02e-7
3.65e-7
1.72e-6
1.21e-6
6.90e-7
9.17e-7
9.46e-7
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way. It is also of our interest to test the synergistic ef-
fects of the compounds with 17-AAG, Herceptin, and
nonspecific chemotherapeutic agents such as
Paclitaxol.
Althoughsomehaveconsidereditachallengetodis-
rupt protein?protein interactions using small mol-
ecules, recent experimental and statistical approaches
have shown that not all residues at a protein?protein
interface contribute equally to the binding free energy
(22). Recent reports of small molecule inhibitors of
protein?protein interactions attest to the idea that in-
hibitors of protein?protein interactions do not have to
belarge,theyjusthavetoinhibitkeyinteractions.Inthe
context of the TPR2A?Hsp90 peptide interaction, ami-
dation of the free carboxy-terminus of the peptide
MEEVD-COO- is sufficient to abolish detectable bind-
ing, a “hot spot” at the interaction interface. We specu-
late that the small molecules we have identified inhibit
theHOP?HSP90interactionbybindingatakeyposition
on the TPR2A interaction interface. Future structural
characterizations will reveal the details of this interac-
tion and may suggest how the compounds can be
chemically modified to achieve higher potency and
specificity.Relativelystraightforwardmethodsfortheto-
tal synthesis of the structural class of compounds iden-
tified in this study have been described (23, 24), which
would make it feasible to test a greater diversity of ana-
logues and to fine-tune their properties.
The compounds identified in this study serve as
the first case of a novel class of small molecule inhibi-
tors of Hsp90 by disrupting its cochaperone interac-
tionandprovideastrongrationaleforpursuingthede-
velopment of small molecule compounds that
specifically interrupt the interaction between Hsp90
and cochaperones. The current generation of com-
pounds show moderate (micromolar) activity in vari-
ous in vitro and in vivo assays. Future generations of
compounds with high affinity and specificity for differ-
ent cochaperone proteins can also be used as chemi-
cal probes to dissect the complex Hsp90-cochaperone
interactions, with the potential to be developed into
novel anticancer drugs.
METHODS
Materials. AlphaScreen Histag fusion detection 10k assay
point kit and Opti-384 plates used in the screening were pur-
chased from PerkinElmer (PerkinElmer). His6-tagged TPR2A was
produced using a bacteria expression system and purified using
Ni-NTA superflow resin from Qiagen. Hsp90 C-terminal peptide
with or without an N-terminal biotin group was synthesized us-
ing automated solid-phase synthesis by the Yale Keck facility
(TEEMPPLEGDDDTSRMEEVD). 17-Allylamino-17-demethoxygeld-
anamycin (17-AAG) was purchased from Assay Designs. The 17-
AAG stock was dissolved in 100% DMSO at a stock concentra-
tion of 1.7 mM and kept at ?20 °C protected from light. It was
further diluted in sterile PBS buffer for the various experiments
in cell culture. The cell proliferation agent WST-1 salt was pur-
chased from Roche Diagnostics Corp. The six toxoflavin struc-
tural class compounds identified through the screening and two
inactive structural homologues were provided by the National
Chemical Genomical Center (NCGC) as 20 mM stock solution in
100%DMSO.TheywerefurtherdilutedinsterilePBSforthevari-
ous experiments in cell culture. HEPES, NaCl, BSA, PBS, DMSO,
and DTT were purchased from Sigma.
Cells and Antibodies. BT474 (ATCC HTB-20) and SBKR3 (ATCC
HTB-30)breastcancercellsandMCF-12F(ATCCCRL-10783)non-
carcinogenic epithelial breast cells were purchased from Ameri-
can Type Culture Collection (ATCC). BT474 and SKBR3 are hu-
man breast cancer cells that overexpress HER2. BT474 cells
were cultured in RPMI 1640 supplemented with 10% heat-
inactivated fetal bovine serum, 2 mM glutamine, 10 ?g mL?1in-
sulin, penicillin, and streptamycin. SKBR3 cells were cultured
in McCoy’s media with 10% heat-inactiveated fetal bovine se-
rum, 2 mM glutamine, penicillin and streptomycin. MCF-12F nor-
mal breast cells were grown in a medium containing a 1:1 mix-
ture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12
medium, with 20 ng mL?1epidermal growth factor (EGF), 0.01
mg mL?1insulin, 500 ng mL?1hydrocortisone, and 5% horse
serum. All cells were grown in a humidified atmosphere of 5%
CO2at 37 °C. Western Blot antibody for rabbit polyclonal anti-
HER2 (sc-284) was purchased from Santa Cruz Biotechnology.
Mouse anti-Hsp70 monoclonal (SPA-810) antibody was pur-
chased from Stressgen/Assay Designs. Secondary HRP conju-
gatedantimouseandantirabbitantibodieswerefromSantaCruz
Biotechnology, Inc. and Amersham, respectively. Horseradish-
peroxidase conjugated anti-GAPDH (ab9482) was from abCam.
Compounds Libraries. Three libraries with a total of 97,134
compounds have been screened using this assay. A library of
960 compounds with known biological functions was purchased
from Microsource Discovery Inc. A library of 20,000 compounds
with chemically diverse structures was purchased from May-
bridge (ACROS Janssen Pharmaceuticalaan 3A). These com-
pounds were stored at a stock concentration of 10 mM in DMSO
in 384-well plates. A third set of 76,174 compounds from differ-
ent sources were screened at NCGC at a series of diluted com-
pound concentrations ranging from 0.3 to 40 ?M in the assay
buffer of 25 mM HEPES, 100 mM KCl, 2 mM MgCl2, 5 mM DTT,
and 0.01% Tween-20 with 0.1% bovine serum albumin (BSA).
PrimaryHitsIdentificationusingAlphaScreenTechnology
Based HTS and Counter Screen. We have screened the above-
mentioned three libraries for compounds that interrupt the inter-
action between TPR2A domain and Hsp90 peptide. A counter
screen using a covalently linked biotin-His6peptide was per-
formed to identify false positives that interfere with the assay
by mechanisms unrelated to the interaction, e.g., singlet oxy-
gen quenchers, biotin mimetics, fluorescence quenchers, and
light scatterers. The ratio between the IC50values for the compe-
tition assay and counter-screen was used to define specific in-
hibitors. Any compounds having similar activities in both the
competition and counter screen assays (IC50ratio ?10) were
considered nonspecific (e.g., inactive structural analogue). Com-
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pounds 8?13, with IC50ratios of ?500, were thus confirmed
as specific inhibitors and subjected to further secondary assay
characterization. All of the detailed technical information regard-
ing the assay development, optimization and implementation
is described elsewhere (Yi, F.; Zhu, P.J.; Southall, N.; Ingelse, J.;
Zheng, W.; Regan, L. Unpublished results).
Fluorescence Polarization Assay. A fluorescence polarization
assay was performed to confirm the binding between the hit
compounds identified from the primary AlphaScreen. Purified
His6-tagged TPR2A protein and fluorescein-labeled 10 mer
C-terminal Hsp90 peptide (DDTSRMEEVD) were used in this as-
say. Assays were performed in a total volume of 25 ?L in the
buffer of 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM DTT. For com-
petition studies, 20 ?L of premixed 20 nM fluorescein-labeled
Hsp90 peptide and 5 ?M His6-tagged TPR2A protein were added
into each well of a 384-well black assay plate with a nonbind-
ing surface coating (Corning). These concentrations were cho-
sen to allow for approximately 50% peptide bound in the pres-
ence of a protein concentration close to the Kd(5 ?M) of the
interaction of interest, to also yield a reasonable polarization
shift upon protein binding by the labeled peptide. Approxi-
mately 250 nL of each compound was transferred from a stock
(10 mM in DMSO) into two wells of the assay plate using an au-
tomatic liquid handler (Tecan EVO) outfitted with a pin tool
(V&P Scientific). Fixed tips on the liquid handler performed 12
three-fold serial dilutions, resulting in final compound concen-
trations ranging from 100 ?M to 0.56 nM, and then transferred
5 ?L of the diluted compounds into the wells with assay mixture
in the assay plate. Results from duplicate dilution series were
averaged for each compound. Each plate included 48 wells of
free fluorescein-labeled Hsp90 peptide and the same peptide in
the presence of TPR2A protein. Plates were incubated at RT for
2 h prior to measurement to ensure binding equilibrium.
Measurements were performed on a PerkinElmer Envision
plate reader using a 480 nm (30 nm bandwidth) excitation fil-
ter and two polarized 535 nm emission filters (40 nm band-
widths) integrating signals over 100 flashes. Upon excitation,
fluorescence emission intensities from both the parallel (S) and
perpendicular (P) orientation were measured, and polarization
values were expressed in milliploarization units (mP), calculated
using the formula mP ?1000(Is? GIp)/(Is? GIp), where Isand
Ipare the parallel and perpendicular emission intensity for the
sample respectively, G is the G factor (? Is/Ip, measured as 0.94
for this study). The fraction of specific binding is defined as the
contribution to bound peptide signal to the total signal, calcu-
lated using the equation b ? (mP ? mPf)/(mPb? mPf), where
b is the fraction of bound peptide, mP is the total mP value re-
corded, mPbis the mP value of the bound peptide control, and
mPfis the mP value for the free fluorescein-Hsp90 peptide. The
assay window was defined as mPb?mPf.
Isothermal Titration Calorimetry. Binding of TPR2A protein to
a representative compound 8 was measured by ITC using a Mi-
crocaliTC200calorimeter(MicrocalInc.).Thesyringewasloaded
with 40 ?L of compounds diluted using buffer from a 20 mM
DMSO stock solution to 0.5 mM in a final 2.5% DMSO. About
200 ?L of purified His6-tagged protein, at 40 ?M in 20 mM Tris,
50 mM NaCl, 10 mM ?-mercaptoethanol, pH 8.0, 2.5% DMSO
wasloadedintothecell.Allexperimentswereperformedwithan
initial injection of 0.5 ?L, followed by 19 2-?L injections with
200 s spacing. The syringe was stirred at 1000 rpm with thermo-
statting at 25 °C. The binding isotherms were fit to a one-site
bindingmodeltoobtainthethermodynamicparameterswiththe
initial point discarded. Data analysis was conducted using Ori-
gin 7 software with the ITC200 add-on package.
WST-1ColorimetricCellProliferationAssay.BT474, SBKR3,
and MCF-12F cells were seeded in 96-well plates at a density
of 2 ? 104cells (100 ?L of culture)?1well?1except for the first
and last columns to which only growth media was added. After
a 24-h adhering period, the cells were either left untreated or
treated with different concentrations of the hit compounds or
17-AAG or DMSO vehicle control. After a 4-day incubation pe-
riod, the WST-1 tetrazolium salt colorimetric proliferation assay
wasperformedbyadding10?LofdissolvedWST-1solutioninto
each well and incubating for 3 h at 37 °C in a 5% CO2incuba-
tor. Absorbance at 440 nm was measured using a PerkinElmer
Envisionplatereader.Thewellsinthefirstandlastrowsforeach
columnwerediscardedtoavoidpossiblevariationduetoevapo-
ration. Each data point represents an average of 6 replicate
wells.
Western Blotting. BT474 and SKBR3 breast cancer cells were
seeded at 2.5 ? 106cells (100-mm dish)?1and allowed to ad-
here overnight. Cells were treated with 17-AAG (0.178 ?M) or
compound 8 (5 ?M in SKBR3 cells, 1 ?M in BT474 cells) or left
untreated. BT474 cells were harvested at 6, 12, and 24 h after
treatment with 1 ?M compound 8 and 24 h after treatment with
0.178 ?M 17AAG. SKBR3 cells were harvested at 3, 6, 12, and
24 h after treatment with 5 ?M compound 8 and 24 h after treat-
ment with 0.178 ?M 17-AAG. Cells treated with compounds
were washed using ice-cold PBS three times and then scraped
into ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
1 mM EDTA and EGTA, 0.1% SDS, 1% NP-40, 1 mM Na3VO4,
25 mM NaF, 25 mM ?-glycerol-phosphate), supplemented with
Complete proteinase inhibitors (Roche Molecular Biochemicals).
After incubating on ice for 30 min, the lysates were centrifuged
at14,000rpm(4°C)for15mintoremoveinsolublematerial.To-
tal protein concentration was determined using the BCA assay
(Pierce). Cell lysates of 25 ?g of total protein were separated on
a 4?12% gradient SDS-polyacrylamide gel (Biorad) followed
by transfer to a 0.45 ?m nitrocellulose PVDF membrane. Mem-
brane was blocked in 5% nonfat milk in Tris-buffered saline con-
taining 0.1% Tween 20 (TBST) with 1 mM Na3VO4for 1 h at RT
on shaker. Primary antibodies were diluted in 5% BSA at a con-
centration of 1:2000 (HER2, antirabbit) or 1:1000 (Hsp70, anti-
mouse). Membrane was incubated in the primary antibodies at
4 °C overnight and washed five times at RT. Secondary HRP con-
jugated antibodies (antimouse antibody from Santa Cruz Bio-
technology, Inc., antirabbit from Amersham) were diluted at
1:2000. Membrane was incubated in the secondary antibodies
at RT for 1 h followed by washing 4 times in TBST and one last
wash in TBS. Membrane was then processed using enhanced
chemiluminescence (ECL, Amersham) development.
Data Analysis. Data were analyzed using Graphpad Prism 4
(GraphPad Software). A sigmoidal dose?response model with
variable slope was used to fit the in vitro competition curves and
growth inhibition curves. The IC50values and the Hillslopes
were generated from the curve fitting. All data were expressed
as mean ? SD.
Assuming a one-site competition model, the IC50’s from the
in vitro assays were converted to Kd’s using the Cheng and Pru-
soff equation (25):
Kd(cpd)?
IC50
1?[pep]
KD(Pep)
where Kd(cpd)is the binding affinity for the compound-TPR2A in-
teraction; [pep] is the Hsp90 peptide concentration used in the
premixed complexes, which is 10 nM for the AlphaScreen assay
and 20 nM for the FP assay; and Kd(pep)is the binding affinity
for the Hsp90-TPR2A interaction, 10 ?M is used in the
calculation.
Acknowledgment: We thank Debbie Koay for her valuable
help with the first cell proliferation assays; Dr. Elizabeth
ARTICLE
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Rhoades for allowing us to use her cell culture facilities; Wei
Zheng and Pingjun Zhu for their help with the NCGC screen; and
Janie Merkel and Michael Salcius for help with the FP assay.
We thank Aitziber Cortajarena, Clarence Chen, Tijana Grove,
Meredith Jackrel, and Lenka Kundrat for helpful comments on
the manuscript. We are grateful for the support of the Breast
Cancer Alliance (L.R.) and for a Concept Award from the
Department of Defense, Breast Cancer Research Program
(W91ZSQ7167N696) and a NIH Molecular Libraries Screening
Centers Network grant (XO1-MH077625-01) (F.Y.).
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