Development of a Grp94 inhibitor

Article abstract of DOI:10.1021/ja303477g

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

Full text of DOI:10.1021/ja303477g

Article
pubs.acs.org/JACS
Development of a Grp94 inhibitor
Adam S. Duerfeldt,†,⊥ Laura B. Peterson,†,⊥ Jason C. Maynard,‡ Chun Leung Ng,‡ Davide Eletto,∥
Olga Ostrovsky,∥ Heather E. Shinogle,§ David S. Moore,§ Yair Argon,∥ Christopher V. Nicchitta,‡
and Brian S. J. Blagg*,†
§
†Department of Medicinal Chemistry and Microscopy and Analytical Imaging Laboratories, The University of Kansas, Lawrence,
Kansas, 66047, United States
‡Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, 27710, United States
∥Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia and the University of Pennsylvania,
Philadelphia, Pennsylvania, 19104, United States
S
* Supporting Information
ABSTRACT: Heat shock protein 90 (Hsp90) represents a
promising therapeutic target for the treatment of cancer and
other diseases. Unfortunately, results from clinical trials have been
disappointing as off-target effects and toxicities have been
observed. These detriments may be a consequence of pan-
Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors
simultaneously disrupt all four human Hsp90 isoforms. Using a
structure-based approach, we designed an inhibitor of Grp94, the
ER-resident Hsp90. The effect manifested by compound 2 on
several Grp94 and Hsp90α/β (cytosolic isoforms) clients were
investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the
conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had
no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of
2 are described herein.
pan-Hsp90 inhibition may be the cause for these effects, as
clinical inhibitors are known to target all four human isoforms:
Hsp90α, Hsp90β, Trap-1, and Grp94. Hsp90α (inducible) and
Hsp90β (constitutively active) are the cytosolic isoforms,
whereas tumor necrosis factor receptor associated protein
(Trap-1) is localized to the mitochondria, and glucose-
regulated protein, Grp94, resides in the endoplasmic
reticulum.17 Little is known about the client protein selectivity
manifested by each of the four isoforms, and this gap in
understanding may underlie the toxicity concerns that have
arisen in clinical trials. Despite the clinical significance of Hsp90
inhibition, little investigation toward the development of
isoform-selective inhibitors has been reported to delineate
isoform-dependent substrates or as an opportunity to reduce
the potential side effects that result from pan-inhibition.
Unlike the cytosolic chaperones, Hsp90α and Hsp90β, which
have been well-studied, little is known about Trap-1 and Grp94.
At present, no isoform-specific clients have been described for
Trap-1; in fact, neither the crystal nor the solution structure has
been solved. In contrast, Grp94 co-crystal structures have
recently been determined and demonstrate that it contains a
unique secondary binding pocket that may provide an
opportunity to develop isoform-selective inhibitors.18−24 Unlike
INTRODUCTION
■
Molecular chaperones play a critical role in cellular homeostasis
by modulating the folding, stabilization, activation, and
degradation of protein substrates.1,2 Heat shock proteins
(Hsps) represent a class of molecular chaperones whose
expression is upregulated in response to cellular stress,
including elevated temperatures that disrupt protein folding.3,4
Among the various Hsps, the 90 kDa heat shock proteins
(Hsp90) are considered promising anticancer targets due to the
role they play in the maturation of various signaling
proteins.5−7 Hsp90 is both overexpressed and activated in
transformed cells, which provides high differential selectivities
for Hsp90 inhibitors.3,4,8 In addition, Hsp90-dependent
substrates are directly associated with all six hallmarks of
cancer, and thus, through Hsp90 inhibition, multiple oncogenic
pathways are simultaneously disrupted, resulting in a
combinatorial attack on cancer.8−12
Hsp90 contains an atypical nucleotide binding pocket, which
allows for the development of selective inhibitors.13 Several of
these Hsp90 N-terminal inhibitors, e.g. 17-AAG (Phase I−III),
SNX-5422 (Phase I), CNF2024 (Phase II), and NVP-AUY922
(Phase I/II) have been evaluated in clinical trials for various
indications, including melanoma, multiple myeloma, refractory
solid tumors, and breast cancer (Figure 1).14 Unfortunately,
cardiovascular, ocular, and/or hepatotoxicities have been
observed.14−16
Received: April 11, 2012
Published: May 29, 2012
© 2012 American Chemical Society
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Figure 2. Chimeric approach to Hsp90 inhibition.
domain.41−44 Three chimeric scaffolds were identified as
Hsp90 inhibitors that manifested anti-proliferative activity
against various cancer cell lines. Radamide (RDA) was the
first chimera produced, and the first co-crystallized with
cytosolic Hsp90 from yeast (yHsp82) and Grp94 from canine
(cGrp94NΔ41) by the Gewirth laboratory.21,41,42 Analyses of
the two co-crystal structures (Figure 3A−C) revealed the
Figure 1. Some Hsp90 inhibitors previously or currently under clinical
evaluation.
Trap-1, several substrates dependent upon Grp94 have been
identified and include Toll-like receptors (TLR1, TLR2, TLR4,
and TLR9), integrins (CD11a, CD18, CD49d, α4, β7, αL, and
β2), IGF-I and -II, and immunoglobulins.25−34 Since these
clients play key roles in cell-to-cell communication and
adhesion, Grp94-selective inhibitors may disrupt malignant
progression by preventing metastasis, migration, immunoeva-
sion, and/or cell adhesion.30−33,35−38 Interestingly, many of
these Grp94-dependent clients have also been identified as key
contributors to inflammatory disorders such as rheumatoid
arthritis, diabetes, and asthma.29,32,39,40 Therefore, the ability to
develop a Grp94-selective inhibitor may provide not only a new
paradigm for Hsp90 inhibition but also new opportunities for
the treatment of diseases other than cancer.
The biological roles manifested by Grp94 have been
primarily elucidated through the use of RNAi-induced Grp94
knockdown, immunoprecipitation experiments, or through pan-
inhibition of all four Hsp90 isoforms. A selective small molecule
inhibitor of Grp94 would provide an alternative and potentially
powerful method for further elucidation of the roles manifested
by Grp94, as well as the identity of other Grp94-dependent
processes/substrates. Recently, the co-crystal structures of the
chimeric inhibitor, radamide (RDA), bound to the N-terminal
domain of both the yeast ortholog of cytosolic Hsp90
(yHsp82N, PDB: 2FXS) and the canine ortholog of Grp94
(cGrp94NΔ41, PDB: 2GFD) were described.21 Utilizing a
structure-based approach that relied upon these co-crystal
structures, a new class of inhibitors that target Grp94 has been
developed.
RESULTS AND DISCUSSION
■
Design and Synthesis of Grp94 Inhibitors. Co-crystal
structures of the natural products geldanamycin (GDA) and
radicicol (RDC) bound to the highly conserved N-terminal
region have been solved.18−21,24 Subsequent studies showed
that chimeric inhibitors containing the quinone moiety of GDA
and the resorcinol of RDC (Figure 2) also target this
Figure 3. RDA quinone (green) hydrogen-bonding network
comparison between yHsp82N (A) and cGrp94NΔ41 with RDA cis-
amide (teal, B) and RDA trans-amide (teal, C). Red spheres represent
water molecules, and hashed lines represent a hydrogen-bonding
interaction.
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resorcinol ring to bind similarly to both isoforms, making a
direct hydrogen bond with the conserved aspartic acid residue
(Asp79 in yHsp82 and Asp149 in cGrp94NΔ41) involved in
ATP binding. However, the quinone moiety was found to bind
yHsp82N in a linear, trans-amide conformation, which was
distinct from one conformation observed in the cGrp94NΔ41
co-crystal structure. Upon binding cGrp94NΔ41, two opposing
conformations of RDA were observed (50% occupancy each).
One conformation exhibited a cis-amide orientation and
projected the quinone moiety into a hydrophobic pocket that
exists solely in Grp94 due to a five amino acid insertion into the
primary sequence. The second conformation of RDA observed
in the RDA·cGrp94NΔ41 co-crystal structure presented the
amide in a trans-configuration and projected the quinone
toward the outside of the binding pocket, similar to that
observed for RDA in the yHsp82N co-crystal structure.21
Interestingly, RDA was found to exhibit an approximately 2-
fold higher binding affinity for full-length Grp94 than
yHsp82.21
phenyl ring similar to that observed for the RDA quinone, and
therefore the tether between the imidazole and phenyl moiety
was analyzed by computational examination. Compounds 1−5
were designed as hypothetical Grp94 inhibitors that contained
the three aspects envisioned to be important for inhibition: (1)
a resorcinol ring to ensure N-terminal inhibition and correct
orientation within in the ATP-binding pocket, (2) a
predisposed cis-amide conformation that projected the phenyl
appendage toward the unique Grp94 binding pocket, and (3) a
hydrophobic, π-rich surrogate for the quinone, the latter of
which would be incapable of providing the requisite hydrogen-
bonding interactions with cytosolic Hsp90 and should therefore
facilitate binding to the π-rich region of Grp94.
Utilizing Surflex molecular docking software, analogues 1−5
were docked to the RDA·cGrp94NΔ41 complex (PDB:
2GFD). As shown in Scheme 1, the Surflex binding scores
Scheme 1. Synthesis and Surflex Molecular Docking Scores
for Compounds 1−5
Further analyses of the RDA·yHsp82N co-crystal structure
revealed the quinone to mediate an intricate hydrogen-bonding
network, whereas its interaction with cGrp94NΔ41 was limited
(Figure 3). For example, in the RDA·yHsp82N structure, direct
hydrogen bonds between the RDA quinone and Lys98 and
Lys44 were observed. In contrast, no direct hydrogen bonds
were observed between cGrp94NΔ41 and the cis-amide
quinone (Figure 3B), suggesting that functionalities on the
quinone ring may be dispensable for Grp94 binding but
obligatory for cytosolic Hsp90 binding. In addition, this Grp94
hydrophobic pocket contains aromatic amino acids (Phe199,
Tyr200, and Trp223) that are likely to facilitate π-stacking
interactions and could be utilized for the design of inhibitors
that exhibit increased selectivity and affinity for Grp94 over
cytosolic Hsp90. Although the primary sequences and ATP-
binding pockets are highly homologous (>70% similar, 55%
identical), this minor disparity was exploited for the rational
design of Grp94 inhibitors.17 The design elements were focused
on the conformation of RDA when bound to cGrp94NΔ41
versus yHsp82N, the dispensability of the quinone moiety, and
the hydrophobicity of the Grp94 π-rich pocket. On the basis of
these observations, we hypothesized that inhibitors containing a
more hydrophobic surrogate of the quinone linked to the
resorcinol through a cis-amide bioisostere would provide
compounds that inhibit Grp94 selectively.
Multiple bioisosteres exist for the cis-amide functionality;
however, in this instance, those exhibiting a conformational bias
rather than a specific physical property were considered.
Observation that the cis-amide conformation of RDA bound to
cGrp94NΔ41 projects the quinone moiety into the Grp94
hydrophobic pocket suggested that cis-olefins, carbocycles, or
heterocycles may represent appropriate surrogates. In the end,
imidazole was chosen on the basis of the inclusion of a
hydrogen bond acceptor in the same location as the amide
carbonyl, which could provide complementary interactions with
Asn162 (Figure 3).
for compounds 1 and 2 were 1−2 units higher than that of
RDA, suggesting binding affinities 10- to 100-fold higher for
cGrp94NΔ41, respectively. Furthermore, 1−5 failed to dock to
the RDA·yHsp82N complex (PDB: 2FXS), supporting our
hypothesis that these phenyl imidazole analogues may exhibit
selective inhibition. Although 1 and 2 were the only
compounds predicted to bind cGrp94NΔ41, prior studies
demonstrated the Grp94 lid region to undergo significant
variations that are capable of accommodating various ligand
sizes and chemotypes. Unfortunately, available modeling
programs could not account for this phenomenon, and
therefore all five analogues were constructed. Aldehyde 6
(Scheme 1), which was utilized during the synthesis of
RDA,41,42 was readily available and allowed for the rapid
preparation of analogues. As shown in Scheme 1, a
Radziszewski-like condensation of aldehyde 6 with the requisite
aniline/primary amine in the presence of glyoxal and
ammonium bicarbonate provided the desired compounds as
protected silyl ethers.45,46 Addition of tetrabutylammonium
fluoride to the reaction mixture yielded the desilylated
compounds 1−5 in moderate yields.
Since no direct hydrogen-bonding interactions exist between
the quinone and cGrp94NΔ41, and several π-rich amino acids
(Phe199, Tyr200, and Trp223) reside in this secondary pocket,
the utilization of an aromatic ring in lieu of the quinone was
pursued. A phenyl ring was envisioned to provide the desired π-
interactions with Phe199, Tyr200, and Trp223 while providing
a rational starting point for the development of Grp94-selective
inhibitors. The imidazole linker was expected to project the
Binding of Compounds 1−5 to Grp94. Upon prepara-
tion of compounds 1−5, their ability to bind Grp94 was
investigated. Using fluorescence polarization competition assays
with recombinant cGrp94 and FITC-GDA, the ability of each
compound to bind Grp94 and displace FITC-GDA was
determined (Figure 4).47 As evidenced in Figure 4, compounds
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complex found in cells.48 Therefore, compounds 1−5 were
further investigated in cell-based assays.
Effect on Trafficking of the Toll Receptor. Once
compounds 1−5 were evaluated for Grp94 binding, studies
commenced to validate our hypothesis that imidazoles
containing a phenyl moiety inhibit Grp94 in cells. Unlike
cytosolic Hsp90 inhibitors that exhibit anti-proliferative effects,
RNAi experiments have shown that in culture, cell viability is
unhampered by knockdown of Grp94.49 Thus, a functional
assay was necessary to determine Grp94 inhibition.
Grp94 is required for the functional maturation and
trafficking of select TLRs.34,49 Therefore, TLR dependence
upon Grp94 was utilized to develop an assay to quantify Grp94
inhibition. As proof of concept, HEK293 cells were stably
transfected to express Grp94 directed or scrambled shRNA.
Both cell lines were then transfected with a plasmid encoding
expression of the Toll protein, the Drosophila homologue of
the interleukin 1 receptor and the founding member of the
TLR family. Grp94 knockdown prevented presentation of the
Toll receptor at the cell surface (Figure 5A) as indicated by
immunostaining and fluorescence microscopy. In order to
investigate this inhibition of trafficking, cells were permeabilized
with Triton X to effect intracellular staining for Toll. Results
clearly indicated that the Toll receptor was expressed in the
absence of Grp94 but was unable to be trafficked to the cell
Figure 4. Binding of compounds 1−5 to Grp94. Compounds 1−5 (25
μM) were incubated with cGrp94 and FITC-GDA (tracer) for 5 h
before fluorescence polarization values were determined. DMSO (1%)
served as a negative control (vehicle), and GDA (500 nM) served as
the positive control.
1 and 2 were the only analogues that bound Grp94 and
displaced FITC-GDA. These results are consistent with the
Surflex-generated docking scores shown in Scheme 1. Although
fluorescence polarization can be used to confirm binding
affinity for Grp94, prior studies have shown that Hsp90
inhibitors bind preferentially to the entact heteroprotein
Figure 5. (A) Representative fluorescence confocal microscopy images of HEK293 cells stably transfected to produce either scrambled shRNA or
Grp94-targeted shRNA and transfected to express the Toll receptor (green) (blue = DAPI, 100x, TIRF oil immersion). (B) Western blot analysis of
cells treated as in panel A. (C) Table of activities for compounds 1−5 to inhibit the trafficking of toll (error bars = SEM for at least 100 different
cell populations. (D) Representative epifluorescence microscopy images of HEK293 cells transfected to express the Toll receptor (green) and then
treated with increasing concentration of compound 2 for 24 h prior to staining (blue = DAPI, 60x, air objective). (E) Dose−response curve for Toll-
trafficking inhibition of compound 2.
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membrane. Western blot analyses of lysates from Grp94
knockdown cells indicated a difference in the glycosylation
pattern of the Toll protein, consistent with ER-retention and
providing evidence for impaired trafficking to the cell
membrane (Figure 5B).50−53 This may indicate that Grp94
interacts with a chaperone or partner protein that is involved in
the glycosylation of its clients.
domain.57,58 Anti-Grp94 (9G10) is an antibody that recognizes
the acidic region (residues 290−350) in the second domain of
Grp94.59 Occupation of the ATP binding site causes a
conformational switch in this region and prevents the 9G10
antibody from recognizing Grp94.58 Therefore, lysates of
C2C12 cells treated with increasing concentrations of
compound 2 were immunoprecipitated to assess whether it
induces a conformational switch in Grp94. As observed in
Figure 7, compound 2 induces a conformational switch in
Once functional knockdown of Grp94 was established and a
reduced cell surface expression of Toll was observed, this assay
served as readout for Grp94 inhibition. HEK293 cells were
transfected with the same Toll-expressing plasmid and
subsequently exposed to compounds 1−5 for 24 h prior to
surface staining. The extent of surface expression was then
quantified by measuring fluorescence intensity at the cell
surface with Cell Profiler.54 A dose−response curve for each of
the compounds that inhibited at least 50% of Toll trafficking at
5 μM was generated to obtain IC50 values (Figure 5C).
Representative fluorescent microscopic images and a dose−
response curve are shown for compound 2 in Figure 5.
Interestingly, the observed IC50 values for this series of
compounds correlated well with the increased binding affinities
predicted by Surflex docking scores, supporting our proposed
mode of binding. After determining that compound 2 binds
Grp94 and inhibits the trafficking of the Toll receptor, we were
interested in evaluating the ability of compound 2 to affect
other Grp94-dependent processes.
Inhibition of IGF-II Secretion by 2. IGF-II is a second
well-defined Grp94-dependent client protein, and active Grp94
is required for the secretion of IGF-II.56 It has been previously
demonstrated that pan-Hsp90 inhibitors, such as 17-AAG,
prevent the secretion of IGF-II in serum-starved C2C12
myoblast cells.28,55,56 Accordingly, serum-starved C2C12 cells
were treated with increasing concentrations of compound 2,
and the secretion of IGF-II was measured by ELISA (Figure
6A). Approximately 60% reduction of IGF-II was observed
Figure 7. Effect of compound 2 on Grp94 conformation. C2C12 cells
were treated with the indicated concentrations of 2 or RDC overnight,
and cell lysates were immunoprecipitated with the conformation-
specific antibody 9G10 and subsequently were immunoblotted for
Grp94. Lower panel, immunoblot of whole cell lyates with 9G10; HC
= heavy chain; n = 3.
Grp94, as the 9G10 antibody is unable to recognize and
immunoprecipitate the Grp94 in cells treated with 2. This result
parallels the IGF-II secretion data shown in Figure 6, suggesting
that an alteration in Grp94 conformation is incompatible with
IGF-II secretion. Interestingly, this activity of Grp94 inhibitors
appears to be cell-specific, as analogous experiments performed
in CHO cells failed to show an effect on the conformation of
Grp94 (data not shown).
Hsp90 α/β Inhibitory Activity of Compound 2. As
previously mentioned, it has been shown that Grp94 is not
essential for tissue culture cell viability.28 In contrast, loss of
functional Hsp90α or Hsp90β results in cell death. Therefore,
we investigated the anti-proliferative effects of compounds 1−5
against two breast cancer cells, MCF7 (ER+) and SKBR3
(Her2 overexpressing, ER−), and against the non-transformed
HEK293 cells. None of the compounds evaluated manifested
anti-proliferative activity at 100 μM, indicating these com-
pounds do not target Hsp90α or Hsp90β. To support these
findings, Western blot analyses of Hsp90α/β client proteins
were performed from HEK293 cell lysates. Prototypical pan-
Hsp90 inhibitors induce proteasome-mediated degradation of
Hsp90α/β client substrates.6 As shown in Figure 8, compound
2 does not induce the degradation of Raf or Akt, two well-
documented Hsp90α/β-dependent client proteins until 100
μM concentration (see also Figure 9).60−62 At this concen-
tration, induction of Hsp70, similar to the one induced by
GDA, is presumably mediated by targeting of cytosolic Hsp90.
As shown in Figure 8B, the effect on Akt cannot be attributed
to ablation of Grp94.
Figure 6. Inhibition of IGF-II secretion by 2. (A) C2C12 cells were
induced to differentiate by serum-starvation in the presence of the
indicated concentrations of 2. Supernatants were collected 48 h later,
and IGF-II levels were measured by ELISA. Drug, concentration range
of 2. (B) Toxicity of compound 2 ( ) and RDC ( ) against C2C12
cells. The viability of cells treated as in panel A was measured at each
of the indicated concentrations by the XTT assay.
■
○
already at 10 μM 2, while little effect on cell viability was
observed (Figure 6B). The effect on IGF-II secretion is
consistent with previous observations using pan-Hsp90
inhibitors, while the lack of effect on cell viability by 2
indicates that this compound is working through a Grp94-
dependent mechanism and does not exhibit pan-inhibition.
Effect on Grp94 Conformation. Prior studies have shown
that occupation of the Grp94 N-terminal ATP binding pocket
by inhibitors results in an altered conformation of this
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approximately 30 nM and does not affect cytoplasmic proteins
until 100 μM in HEK293 cells, providing evidence for Grp94-
selective inhibition. To further understand the implications of
Grp94-selective inhibition, compound 2 was analyzed in other
Grp94-dependent processes.
Induction of BiP Expression. Inhibition of Hsp90 is also
known to induce expression of Hsp70, and this response is
useful as a diagnostic tool (Figure 8A, GDA).62−64 A parallel
response exists when Grp94 expression is ablated by RNAi, or
when its activity is inhibited by RDC or 17-AAG: a
transcriptional response is initiated that leads to upregulation
of expression of BiP, the ER member of the Hsp70 family
(Eletto et al., submitted). We therefore assessed the ability of 2
to cause BiP up-regulation, in comparison to pan-Hsp90
inhibitors. As shown in Figure 9, treatment of C2C12 cells with
0−75 μM of compound 2 did not lead to up-regulation of BiP,
while treatment with 10 μM RDC (or 25 μM of 17-AAG, data
not shown) did cause BiP up-regulation. Only at concentrations
above 200 μM did compound 2 resemble RDC and induce BiP
expression. However, at these concentrations, the compound
also destabilized Akt, a hallmark of inhibition of cytosolic
Hsp90 (Figure 9). The inability of 2 to upregulate BiP at the
0−75 μM concentration range was surprising, because this
transcriptional response was shown to be a property of Grp94
ablation and not Hsp90 (Eletto et al., submitted).
Effect on Drosophila Development. Previous studies
have demonstrated that Gp93, the Drosophila ortholog of
Grp94, is an essential gene.26 In the Drosophila model,
maternal Gp93 is sufficient to support embryogenesis in Gp93
homozygous null embryos. In the absence of zygotic expression
of Gp93, however, larvae display a pronounced growth defect,
commensurate with disrupted gut epithelial morphology,
decreased gut nutrient uptake, and marked aberrations in
copper cell structure and function. As a consequence, loss of
Gp93 expression is larval lethal in Drosophila. To determine
the effects of compound 2 on Drosophila larval growth, first
instar wild type (w1118) larvae were placed onto fly food
supplemented with either no supplement (A), 0.1% (B), 0.3%
(C), or 0.5% (D) DMSO (vehicle controls) or fly food
supplemented with 250 μg/mL (E), 500 μg/mL (F), 750 μg/
mL (G), or 1 mg/mL (H) compound 2 (Figure 10A−H). As is
Figure 8. (A) Western blot analysis of HEK293 cell lysates (7.5 μg
total protein) after treatment with indicated concentration of
compound 2 (μM) for 24 h. GDA, a known pan-Hsp90 inhibitor, is
shown as a positive control (500 nM), and actin is shown as a loading
control. (B) Lysates of HeLa cells stably expressing either scramble
shRNA (shCTRL) or Grp94-targeting shRNA (shGrp94) were
analyzed by immunoblotting. Grp94 and BiP were detected by the
anti-KDEL antibody. ∗ designates an unknown KDEL-containing
band. 14-3-3 served as loading control. HeLa cells as in panel B were
exposed for 48 h at the indicated concentration of (C) compound 2 or
(D) RDC, closed squares, GRP94-silenced cells; open circles, cells
treated with control shRNA. Cell survival was measured by XTT assay
(n = 4).
Figure 9. Induction of BiP Expression by treatment with 2. NIH-3T3
cells were treated with 10 μM RDC or 0−50 μM 2. After 18 h, cells
were harvested for SDS-PAGE and analyzed by immunoblotting.
Grp94, BiP, and PDIA6 were detected with the monoclonal anti-
KDEL antibody and AKT by rabbit antiserum. 14-3-3 served as
loading control. Numbers below BiP, Grp94, and AKT bands are the
relative expression levels, determined by densitometry.
We also tested the cytotoxicity of compound 2 in cells that
are either Grp94-sufficient or -deficient and compared it to the
cytotoxicity of RDC. As shown in Figure 8C and D, compound
2 is much less toxic: the IC50 for HeLa cell viability is >250 μM,
while RDC already reaches this level at 8 μM. In either case, the
cytotoxicity is not attributable to inhibition of Grp94, because
cells responded equally regardless of the presence of Grp94
(Figure 8C and D). Similar results were obtained with other
cell lines (e.g., C2C12 in Figure 7).
At the lower concentration range compound 2 inhibits the
presentation of the Grp94-dependent Toll receptor at
Figure 10. Effect of compound 2 on Drosophila larval growth.
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equiv). Glyoxal (1 equiv) was then added dropwise by a syringe, and
the reaction was allowed to stir at 25 °C for 8 h. Upon complete
conversion of the aldehyde, as observed by thin-layer chromatography,
tetrabutylammonium fluoride was added dropwise by syringe, and the
reaction was allowed to stir at 25 °C for 30 min, at which time the
reaction was quenched with satd aq NH4Cl and extracted with EtOAc.
The organic layers were combined, dried over Na2SO4, and
concentrated in vacuo. All compounds were purified via flash
chromatography utilizing 95:5 CH2Cl2/MeOH as the eluent. Yields
and characterization for all compounds are provided in the Supporting
Information.
Cell Culture. HEK293 and C2C12 cells were maintained in
DMEM supplemented with non-essential amino acids, L-glutamine (2
mM), streptomycin (500 μg/mL), penicillin (100 units/mL), and 10%
FBS. Cells were grown to confluence in a humidified atmosphere (37
°C, 5% CO2). Stable Grp94-shRNA knockdown cell lines were
generated as folllows: the shRNA sequence 5′-GGCUCAAGGACA-
GAUGAUGtt-3′ was cloned into the A pSilencer 2.0-U6 vector
(Ambion), and positive clones were confirmed by sequencing. The
pSilencer 2.0-U6-Grp94 shRNA vector and a control, non-targeting
pSilencer 2.0-U6 shRNA vector (scrambled, control), were transfected
into HEK293 cells using Lipofectamine 2000 using the manufacturers
protocol. Cell cultures were selected 36 h post-transfection by the
addition of 1 μg/mL puromycin to the media. Puromycin-resistant
clones (both Grp94 shRNA and non-targeting shRNA) were
subsequently expanded and screened for knockdown efficiency by
immunoblotting, using the Grp94 antibody, DU120. Clones displaying
greater than 90% knockdown were selected. Puromycin-resistant
clones from the nontargeting shRNA were obtained in parallel and
screened for normal Grp94 expression, also by immunoblotting with
DU120. C2C12 Cells were maintained and induced to differentiate
into myoblasts as previously described.73
Fluorescence Polarization. Assay buffer (25 μL, 20 mM HEPES
pH 7.3, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 20 mM Na2MoO4,
0.01% NP-40, and 0.5 mg/mL BSA) containing compounds 1−5 or
GDA were plated in 96-well plates (black well, black bottom) to
provide final concentrations of 25 μM or 500 nM, respectively (1%
final DMSO concentration).47 Recombinant cGrp94 and FITC-GDA
were then added (50 and 25 μL) to give final concentrations of 60 and
5 nM, respectively. Plates were incubated with rocking for 5 h at 4 °C.
Fluorescence polarization values were then read using excitation and
emission filters of 485 and 528 nm, respectively. Percent FITC-GDA
bound was determined by using the DMSO millipolarization unit
(mP) as the 100% bound value, and the mP value of free FITC-GDA
as the 0% bound value.
evident from the micrographs of representative larvae, dietary
uptake of 2 was associated with a dramatic growth phenotype
(Figure 10). In parallel experiments, larval gut tissue was
obtained from each of the feeding conditions, and gut epithelial
morphology was evaluated by fluorescence microscopy.
No grossly discernible effects on copper cell structure were
observed, indicating that under these feeding conditions, the
inhibition of Gp93 function was incomplete (data not shown).
Pharmacokinetic studies of compound absorption and metab-
olism may provide additional insights into this partial
phenotypic behavior.
CONCLUSIONS
■
Hsp90 inhibitors have been the subject of intense pharmaceut-
ical research, not only for cancer but also neurodegenera-
tion.12,65−71 All Hsp90 inhibitors that have reached clinical
trials bind to the Hsp90 N-terminal ATP-binding pocket and
demonstrate pan-Hsp90 inhibition, i.e., they inhibit all human
Hsp90 isoforms simultaneously.14,15,72 Toxicities and off-target
effects resulting from Hsp90 inhibition may be a consequence
of pan-inhibition. Therefore, the design of Hsp90 isoform-
selective inhibitors may provide a valuable pharmacological tool
to dissect the roles of each isoform and may lead to more
clinically useful inhibitors.
Comparing the crystal structures of several known Hsp90
inhibitors bound to either cytosolic Hsp90 or to the ER-
resident Grp94 provided a rational design platform for the
development of Grp94 inhibitors. Using structure-based drug
design, five compounds were identified as potential leads that
contain a phenyl ring appended to an imidazole ring, which
serves as a cis-amide bioisostere. The predisposed orientation of
the phenyl ring was postulated to allow interactions with the
unique Grp94 π-rich pocket. Since Grp94 has previously been
shown to be responsible for the trafficking of TLRs to the cell
membrane,34 this activity was used as a functional assay for
Grp94 inhibition. Of the five compounds evaluated, compound
2 manifested the best activity in this assay (32 nM). In
subsequent, direct readout assays, including an in-cell
conformational assay, compound 2 affected Grp94 itself at
the same concentration as that needed to inhibit chaperone
activity.
Toll-Trafficking Assay. HEK293 cells were plated in 6-well cell
culture treated plates in Dulbecco’s Modified Eagle Medium (1x
DMEM) supplemented with 10% FBS containing no antibiotics and
were maintained at 37 °C, 5% CO2, and 95% relative humidity. After
24 h, the cells (95% confluence) were transfected with pcDNA6B-
Toll-Flag using Lipofectamine2000 according to the manufacturer’s
instructions. Cells were transfected for 16 h and then were trypsinized
and plated in 96-well microscopy-quality, black-walled plates that had
been pretreated with attachment factor. After 3 h of incubation at 37
°C to allow the cells to attach, compound at varying concentrations in
DMSO (1% DMSO final concentration) was added, and cells were
returned to incubate for 24 h. After 24 h, the media was removed, and
cells were fixed in freshly made 4% paraformaldehyde in Dulbecco’s
Phosphate Buffered Saline (DPBS) for 10 min at 25 °C. Cells were
washed twice with DPBS and then stained with Wheat Germ
Agglutinin-Texas Red (5 μg/mL in DPBS, 60 min, 25 °C). Cells were
washed twice with DPBS and blocked in 5% bovine serum albumin
(BSA, 10 min, 25 °C), followed by staining for 16 h with an anti-Toll
antibody (1:200 in 5% BSA/DPBS, 4 °C, Santa Cruz, sc-33741). Cells
were washed twice with DPBS and stained with an anti-rabbit-
AlexaFluor488 antibody (1:300 in DPBS, 25 °C, Invitrogen, A-11008)
for 3 h at 25 °C. Cells were then washed twice with DPBS after which
DAPI was added (1 μM in DPBS). Cells were imaged using an
inverted Olympus IX-81 microscope with a 60X long working distance
air objective using appropriate filter sets for the various tags
Once the Grp94 inhibitory activity of compound 2 was
established by these parameters, we evaluated the isoform
selectivity of the compound. Inhibitors of cytosolic Hsp90
(Hsp90α/β) manifest anti-proliferative activity in cell culture.
At concentrations wherein the assays observed activity for
compound 2, there were no cytotoxic effects against any cell
line tested. In addition, compound 2 exhibited no effect on the
prototypical Hsp90α/β client kinases, Akt or Raf, until
concentrations 100x greater than the IC50 for Grp94 inhibition.
Therefore, compound 2 appears to manifest considerable
selectivity for Grp94 versus Hsp90α/β, perhaps explaining its
low toxicity. Lastly, compound 2 stunted the growth of
Drosophila larvae in a dose-dependent manner, suggesting that
it may be a useful Grp94 inhibitor in vivo. Future studies with 2
will help dissect the roles played by Grp94 and will shed light
into the validity of Grp94 as a therapeutic target.
EXPERIMENTAL SECTION
■
General Method for the Synthesis of Compounds 1−5.
Aldehyde 6 (1 equiv) was dissolved in wet MeOH at 25 °C. The
required aniline/amine (1 equiv) was added dropwise by a syringe to
the reaction flask followed by addition of ammonium bicarbonate (1
9802
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Journal of the American Chemical Society
Article
(AlexaFluor488, Texas Red, DAPI). Images were processed using
SlideBook5.0 and analyzed using CellProfiler and CellProfiler Analyst.
Western Blotting. HEK293 cells were plated in 6-well plates and
treated with various concentrations of compound in DMSO (1%
DMSO final concentration) or vehicle (DMSO) for 24 h. Cells were
harvested in cold PBS and lysed in mammalian protein extraction
reagent (MPER, Pierce) and protease inhibitors (Roche) on ice for 1
h. Lysates were clarified at 14,000g for 10 min at 4 °C. Protein
concentrations were determined with the Pierce BCA assay kit per the
manufacturer’s instructions. Equal amounts of protein (10 μg) were
electrophoresed under reducing conditions, transferred to a PVDF
membrane, and immunoblotted with the corresponding specific
antibodies. Membranes were incubated with an appropriate horse-
radish peroxidase-labeled secondary antibody, developed with
chemiluminescent substrate, and visualized.
Grp94 Immunoprecipitation. Detergent lysates of the indicated
cells were immunoprecipitated with 9G10 monoclonal anti-Grp94
(StressGen, Vancouver, BC) followed by protein G-Sepharose (Sigma
Chemicals or Pierce) as previously described.74
IGF-II Secretion. C2C12 cells (ATCC, Rockville, MD) were
induced to differentiate either by complete withdrawal of serum or by
shifting to medium supplemented with 2% house serum. 17-AAG at
concentrations of 10−15 μM in DMSO was used to inhibit Grp94
activity. Cell growth was measured with the XTT formazan
colorimetric assay (Roche); cells were grown in 3% serum, to limit
the background of the assay.
For IGF-II ELISA, plates were coated with anti-IGF-II (mAb 792,
R&D Systems) and incubated with the test cell media. The bound
IGF-II was detected with a biotinylated anti-IGF-II antibody (BAF792,
R&D Systems) and developed with streptavidin-HRP (R&D Systems)
according to the manufacturer’s recommended procedure. Optical
density units were converted to concentrations of the growth factor
with a standard curve generated with recombinant IGF-II (792-MG)
(R&D Systems). Data were acquired in duplicate on a microtiter-plate
reader (Dynatech Laboratories, Chantilly, VA) at 450 nm.
Drosophila. Compound effects on Drosophila larval growth were
examined as previously described.26 Briefly, w1118 Drosophila
embryos were collected and groups of 20−30 were transferred to
plates containing fly food (molasses, corn meal, yeast extract, and agar)
supplemented with the indicated concentrations of compound 2
diluted in DMSO. Control (no drug) plates contained equivalent
concentrations of DMSO. Feeding/growth experiments were con-
ducted for 96 h (third instar), and larvae were then immobilized by
transferring to PBS supplemented with 5 mM EGTA and imaged on a
Leica MZ FLIII stereomicroscope.
Fellowship (L.B.P.), a Madison and Lila Self Graduate
Fellowship (A.S.D), an American Foundation of Pharmaceut-
ical Education (A.S.D), and the Arthritis Foundation (O.O.).
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