Transformation of the non-selective aminocyclohexanol-based Hsp90 inhibitor into a Grp94-seletive scaffold

Article abstract of DOI:10.1021/acschembio.6b00747

Glucose regulated protein 94 kDa, Grp94, is the endoplasmic reticulum (ER) localized isoform of heat shock protein 90 (Hsp90) that is responsible for the trafficking and maturation of toll-like receptors, immunoglobulins, and integrins. As a result, Grp94 has emerged as a therapeutic target to disrupt cellular communication, adhesion, and tumor proliferation, potentially with fewer side effects compared to pan-inhibitors of all Hsp90 isoforms. Although, the N-terminal ATP binding site is highly conserved among all four Hsp90 isoforms, recent cocrystal structures of Grp94 have revealed subtle differences between Grp94 and other Hsp90 isoforms that has been exploited for the development of Grp94-selective inhibitors. In the current study, a structure-based approach has been applied to a Grp94 nonselective compound, SNX 2112, which led to the development of 8j (ACO1), a Grp94-selective inhibitor that manifests -440 nM affinity and ≥200-fold selectivity against cytosolic Hsp90 isoforms.

Full text of DOI:10.1021/acschembio.6b00747

Articles
pubs.acs.org/acschemicalbiology
Transformation of the Non-Selective Aminocyclohexanol-Based
Hsp90 Inhibitor into a Grp94-Seletive Scaffold
Sanket J. Mishra,† Suman Ghosh,† Andrew R. Stothert,‡ Chad A. Dickey,‡ and Brian S. J. Blagg*,†
†Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas, United States
‡Department of Molecular Medicine and Byrd Alzheiemer’s Research Institute, University of South Florida, Tampa, Florida 33613,
United States
S
* Supporting Information
ABSTRACT: Glucose regulated protein 94 kDa, Grp94, is the endoplasmic reticulum
(ER) localized isoform of heat shock protein 90 (Hsp90) that is responsible for the
trafficking and maturation of toll-like receptors, immunoglobulins, and integrins. As a
result, Grp94 has emerged as a therapeutic target to disrupt cellular communication,
adhesion, and tumor proliferation, potentially with fewer side effects compared to pan-
inhibitors of all Hsp90 isoforms. Although, the N-terminal ATP binding site is highly
conserved among all four Hsp90 isoforms, recent cocrystal structures of Grp94 have
revealed subtle differences between Grp94 and other Hsp90 isoforms that has been
exploited for the development of Grp94-selective inhibitors. In the current study, a
structure-based approach has been applied to a Grp94 nonselective compound, SNX
2112, which led to the development of 8j (ACO1), a Grp94-selective inhibitor that
manifests ∼440 nM affinity and >200-fold selectivity against cytosolic Hsp90 isoforms.
he heat shock protein 90 kDa (Hsp90) family of proteins
that many of these side effects result from pan-inhibition of all
Tis responsible for the conformational maturation of four Hsp90 isoforms.15 Therefore, the development of isoform-
selective Hsp90 inhibitors can provide an opportunity to fine-
tune the drug discovery process while simultaneously
identifying isoform-dependent clients.
nascent polypeptides into their biologically active three-
dimensional structures.1 Hsp90 has generated great interest as
a chemotherapeutic target due to its role in the modulation of
cancer, neurodegenerative disorders, and infectious diseases.
There are >200 protein substrates that require Hsp90 for their
folding, maturation, and/or activation, termed Hsp90 clients.2
During cellular stress, including elevated temperature, Hsp90 is
induced to refold proteins that have undergone denaturation.3,4
Similarly, Hsp90 is also upregulated in cancer cells, wherein it is
required for the maturation of clients that drive the
proliferation and growth of tumors.4−7 Structurally, Hsp90
exists as a homodimer, and each monomer contains a C-
terminus, N-terminus, and a highly charged middle domain.
The N-terminus contains an ATP-binding site, which is
responsible for ATP hydrolysis and provides the energy
necessary for the folding of client protein substrates.8,9
Hsp90 exists as four isoforms: Hsp90α and Hsp90β reside in
cytoplasm, whereas Grp94 localizes in the endoplasmic
reticulum, and Trap1 is found in the mitochondria. The
natural products, geldanamycin and radicicol, were among the
first Hsp90 inhibitors identified and have served as useful tools
to study Hsp90 biology, validating Hsp90 as a druggable target
for the development of new anticancer agents.10−12 Con-
sequently, 17 Hsp90 inhibitors have entered clinical trials for
the treatment of cancer, however, all of these compounds
exhibit pan inhibition of all four Hsp90 isoforms.13,14
Unfortunately, some Hsp90 inhibitors have manifested
undesired activity during these investigations, which is likely
to hinder subsequent evaluation. It has also been hypothesized
Glucose regulated protein 94 kDa (Grp94), also known as
gp96 or endoplasmin, is the endoplasmic reticulum (ER)
localized Hsp90 isoform. Grp94 is the most abundant protein
in the ER lumen, where it is responsible for the maturation of
secreted proteins that modulate immunity, cellular communi-
cation, and/or cell adhesion.16 Grp94 is also a regulator of the
unfolded protein response (UPR), a proteostatic mechanism
triggered by the accumulation of misfolded proteins in the
ER.17,18 Client proteins that require Grp94 for their maturation
include integrins, which are important for cell adhesion and
metastasis, supporting Grp94 as a potential target for the
development of antimetastatic agents.19 Grp94 knockdown
experiments in the highly metastatic breast cancer cell line,
MDA-MB-231, and the reactive oxygen species (ROS) resistant
MCF-7 cell line resulted in the inhibition of cell migration and
metastasis.20 In addition, myocilin represents another Grp94-
dependent protein, which upon its aggregation leads to
increased ocular pressure that results in primary open angle
glaucoma (POAG), supporting Grp94 inhibition as a viable
approach for the treatment of glaucoma.21 Recently, maturation
of the GARP and Wnt coreceptor, LRP6, was shown to be
Grp94-dependent.24 Since LRP6 is overexpressed in multiple
Received: August 25, 2016
Accepted: November 10, 2016
© XXXX American Chemical Society
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myeloma, Grp94 inhibition may be a useful for the treatment of
such cancers.22−24 As a consequence of these prior studies, the
development of Grp94-selective inhibitors was sought for the
treatment of various diseases, including cancer and glaucoma,
while avoiding the potential side effects that result from
inhibition of all four Hsp90 isoforms.
The N-terminal ATP-binding pocket of Grp94 is ∼85%
identical to other Hsp90 isoforms, which presents a significant
challenge for the design of isoform-selective inhibitors.25
However, a five amino acid (QEDGQ) insertion into the
Grp94 primary sequence results in a conformational change
within the ATP-binding pocket that produces a small
hydrophobic cleft that can be utilized to develop selective
inhibitors.26 Although, 5′-N-ethylcarboxamidoadenosine
(NECA, Figure 1; II) was the first selective inhibitor of
RATIONAL DESIGN
■
Upon examination of the binding modes for both III and IV
bound to Grp94, it was revealed that Grp94 undergoes a
conformational change when interacting with IV about helix 5
to form a binding pocket (site 2, Figure 2A) that is distinct
from the structure of III bound to Grp94. The rotation about
helix 5 brings Phe-195 in close proximity to the purine ligand
and results in a closed conformation. Since the purine scaffold
is not amenable to modifications that increase π interactions
with Phe-195, a novel chemotype was designed to interact with
Phe-195 while simultaneously accessing site 2. Recent work
from our lab has explored the II binding pocket (Figure 2A) via
derivatives of III;30,31 however, access to site 2 remains
underinvestigated. In an effort to design new analogs that
provide access to these regions, the binding modes of Hsp90
inhibitors under clinical evaluation were investigated. In
particular, SNX 2112 (I), a novel benzamide-containing
compound was shown to bind both cytosolic Hsp90 isoforms
(Hsp90 α/β Kd: 4/6 nM) with high affinity, but manifested
lower affinity (Grp94 Kd: 484 nM) against Grp94 and TRAP1
(Figure 3A).32
Molecular modeling studies were then used to compare the
binding mode of I with other scaffolds, including III and IV,
which revealed I to occupy site 1 with the bulky
tetrahydroindazolone fragment (Figure 3B). When bound to
Hsp90, the tetrahydroindazolone fragment rotates and attains a
near perpendicular geometry with the benzamide ring. Upon
overlay of I bound to Hsp90 and IV bound to Grp94, it was
observed that the tetrahydroindazolone fragment produces a
steric clash in the closed conformation of Grp94 as illustrated in
Figure 3C. In particular, the ketone, dimethyl, and trifluor-
omethyl groups of I appeared unable to bind the closed
conformation of Grp94. Therefore, the tetrahydroindazolone
fragment was computationally replaced with a pyrrole, which
can also attain a coplanar geometry with the benzamide ring
while simultaneously occupying site 2 to impart Grp94
selectivity. In fact, a substituted phenyl ring attached to the
2-position of the pyrrole appeared to preferentially bind the
closed Grp94 conformation with high selectivity when
compared to other Hsp90 isoforms (Figure 4). In addition,
rotation of the pyrrole about the benzamide ring appeared to be
disfavored as eclipsing interactions occur between the phenyl
and aminocyclohexanol appendages (Figure 4A). The pyrrole
also appeared to produce π interactions with Phe-195 in the
Grp94 closed conformation (Figure 4B), and substitutions at
the pyrrolic 2-position could lead to increased hydrogen
bonding interactions with Asn-107, a residue that resides on the
perimeter of the hydrophobic cleft. On the basis of these
computational studies and observations, a new class of Grp94-
selective inhibitors was synthesized and evaluated.
Figure 1. Structures of nonspecific Hsp90 inhibitors (GDA and SNX
2112) and Grp94-selective inhibitors (RDA, BnIm and PU-WS13, and
NECA).
Grp94 identified, it manifests nonspecific agonistic activity
against adenosine receptors.27 However, the cocrystal structure
of II bound to Grp94 revealed the ethyl amide to project into a
small hydrophobic cleft within Grp94, which resulted in
isoform-selective inhibition.26 In an effort to identify other
Grp94-selective inhibitors, radamide (RDA), a radicicol/
geldanamycin chimeric inhibitor, was cocrystallized with both
Grp94 and Hsp90 to probe binding interactions.28 The
cocrystal structure of RDA bound to Grp94 presented two
modes of binding in which the amide bond existed in the trans
or cis configuration, which was in contrast to the Hsp90
cocrystal structure, wherein the amide existed solely as the trans
isomer. Upon further inspection, the structures suggested that
the cis-amide selectively binds Grp94, whereas the trans isomer
binds both homologues. Therefore, cis-amide bioisosteres of
RDA were pursued and ultimately led to the discovery of BnIm
(III),30 which incorporates an imidazole ring in lieu of the cis-
amide. Recently, Chiosis and co-workers reported another
Grp94-selective inhibitor, PU-WS13 (IV), which binds to an
alternative conformation of Grp94 than that reported for both
II and RDA (Figure 2).29
RESULTS AND DISCUSSION
■
Chemistry and Structure−Activity Relationship Stud-
ies. Synthesis of the proposed compounds began by treatment
of pyrrole 1 with N-bromosuccinimide (NBS), followed by in
situ protection of the nitrogen utilizing di-tert-butyl dicarbonate
to form the corresponding N-boc-2-bromopyrrole, 2 (Scheme
1).33,34 The brominated pyrrole (2) was then subjected to a
Suzuki cross-coupling reaction with substituted aryl boronic
acids using Pd(dppf)Cl2 as a catalyst and potassium carbonate
as the base. Subsequent removal of the Boc protecting group
under basic conditions led to intermediates 3a−3j.35 Inter-
B
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Figure 2. (A) Docking pose of III in Grp94 cocrystal structure with RDA (PDB code: 2GFD), showing III binding site 3 and site 1. (B) Binding
pose of IV in Grp94 (PU-H54 in PDB code: 3O2F) in which the protein undergoes a conformational change and reveals site 2, bringing Phe-195
closer to the ligand. Site 3 and site 1 are not observed in this conformation.
Figure 3. (A) I and its steric interactions with Grp94 (indicated in red). (B) I occupies site 1 when bound to Hsp90 (PDB code: 4NH7),
establishing a hydrogen bond with Tyr-139, which might be lost during the conformational change of Grp94. (C) Overlay of the I Hsp90 binding
mode with IV bound to Grp94. Steric clash manifested by I are depicted in red circles.
mediates 4a−4j were obtained via a nucleophilic substitution
reaction between 3a−3j and 4-fluoro-2-bromobenzonitrile
utilizing sodium hydride as the base in a solution of
dimethylformamide. In the final step, amination of 4a−4j was
accomplished by microwave irradiation using trans-amino-
cyclohexanol in the presence of potassium tert-butoxide and
Pd(OAc)2/DPPF. The crude material was then subjected to
hydrolysis to convert the nitriles into the corresponding
benzamides, 5a−5j, in the presence of hydrogen peroxide
and sodium hydroxide.
was determined that compound 5a bound Grp94 (Kd ∼ 9 μM)
with greater affinity than Hsp90α (Kd > 100 μM), supporting
our hypothesis that these analogs represent a new class of
Grp94-selective inhibitors (Figure 4B, Table 1). Following
these encouraging results, structure−activity relationship (SAR)
studies were explored for substituents on the phenyl ring to
investigate both spatial and electronic requirements of the
Grp94 binding pocket. The incorporation of methyl sub-
stituents onto the phenyl ring was pursued to explore steric
demands at the 2-, 3-, and 4-positions (5b, 5c, and 5d). These
compounds exhibited comparable affinity to 5a, suggesting that
additional space was available in this hydrophobic cleft.
Compound 5a, which contains an unsubstituted phenyl ring,
was evaluated for binding affinity toward Grp94 and Hsp90. It
C
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a
Table 1
compound
R =
Kd, Grp94 (μM)
Kd, Hsp90α (μM)
5a
H
9.05 0.31
9.9 0.54
> 100
> 100
5b
2-Me
3-Me
4-Me
2-Cl
5c
9.43 0.26
8.35 0.14
2.94 0.27
15.8 0.31
10.62 0.54
Kd, Grp94 (μM)
> 100
5d
> 100
5e
5f
> 100
3-Cl
> 100
5g
4-Cl
> 100
compound
N Scan
Kd, Hsp90α (μM)
5h
5i
C2 = N
C3 = N
C4 = N
13.98 0.27
5.5 0.15
> 100
> 100
> 100
5j
13.2 0.12
a
Figure 4. (A) Designed series of ACO inhibitors predicted to bind
Grp94 in the desired conformation (left). (B) Docked pose of
compound 5a in the binding site of Grp94 in the closed conformation.
The phenyl appendage at the 2-position of the pyrrole occupies site 2.
Apparent Kd values determined using fluorescence polarization (FP)
assay. Compounds were incubated with cGrp94/Hsp90α and FITC-
GDA in triplicate, and SEM was measured.
a
Scheme 1
in the binding site or that the rigidity of the scaffold may
produce unfavorable steric interactions with the protein. In an
effort to identify detrimental interactions within site 2, we
performed additional molecular modeling studies that
suggested the rigidity of the scaffold was detrimental to
potency, as substituents at the 3-postion of the phenyl ring
were too close to the Grp94 surface (Figure 4B). However, it
appeared that a linker between the pyrrole and phenyl ring
could provide the flexibility needed to overcome these
unfavorable interactions, while projecting the phenyl ring
deeper into the hydrophobic pocket (site 2).
Synthesis of the linker-containing compounds, 8 and 9, is
outlined in Scheme 2. The first synthetic process required
preparation of the 2-benzylpyrrole fragments, 6a−6q, via
nucleophilic attack of the pyrrole onto the corresponding
benzyl bromide/toluene sulfonate esters in the presence of
methyl magnesium bromide. In the subsequent step, fragments
6a−6q were subjected to the same sequence of reactions as
required for the synthesis of 4a−4j in Scheme 1. Toluene
sulfonate esters 11a−11c and 13 (Scheme 3) were prepared via
a reaction between p-toluene sulfonyl chloride and the
corresponding alcohols, which were utilized for the preparation
of compounds 8j−8l and 9.
The two-carbon linker containing compound 9 appeared too
long for accommodation in site 2, but was prepared to validate
our binding model. Binding data confirmed that the benzyl
substituted compound, 8a, manifested greater affinity than the
phenethyl derivative, 9 (Table 2). In fact, 8a produced a Kd of
∼1.3 μM, an improvement over 5a. Encouraged by the
increased affinity and selectivity manifested by 8a, SAR studies
were initiated about the benzyl side chain via the incorporation
of chlorine at all three locations on the phenyl ring, 8b−8d.
The 4-chloro derivative (8d) exhibited lower affinity for Grp94
than 8a, suggesting limited space at the 4-position. Similarly,
the 3-chloro analog, 8c, also produced decreased affinity when
a
Reagents and conditions: (a) NBS, −78 °C, THF. (b) TEA, DMAP,
Boc2O. (c) Pd(dppf)Cl2, aryl boronic acid, toluene/ethanol/water
(6:1:1), K2CO3,100 °C. (d) Methanol, K2CO3. (e) 4-Fluoro-2-
bromobenzonitrile, NaH, DMF. (f) Pd(OAc)2, DPPF, KOtBu, trans-
cyclohexanolamine, MW, 110 °C. (g) DMSO, ethanol, NaOH, H2O2.
Subsequently, chlorine containing compounds (5e−5g) were
synthesized to investigate the electronic effects within site 2.
Both the 3- and 4-chloro substituted compounds (5f and 5g)
manifested decreased affinity; however, the 2-chloro derivative,
5e, exhibited a Kd of ∼3 μM. The enhanced binding affinity
exhibited by 5e is not likely to result from steric effects, since
the analog containing a methyl group (5b) at this location was
less active. In addition, the phenyl ring in 5a was replaced with
a pyridine nitrogen at the 2-, 3-, and 4-positions (5h−5j) to
explore the potential for hydrogen bonds with ASN107 (Figure
4). Unfortunately, 2- and 4-pyridine analogues resulted in
decreased affinity for Grp94; however, 5i, which contains a 3-
pyridine ring, exhibited higher affinity than 5a. Although 5i did
lead to increased affinity, it did not reflect strong interactions
with Grp94 as supported by the computational studies,
suggesting the pyridine nitrogen atom may not align properly
D
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a
the 2-position was not tolerated. Therefore, substituents that
exhibit various electronic features were incorporated into the 2-
position (8e−8g). For example, the 2-fluorine analog, 8e,
resulted in improved affinity (∼724 nM). However, the 3-
fluorine (8f) and 4-fluorine (8g) analogs produced decreased
affinity. Compound 8h contained a bromide, but it too
exhibited decreased affinity. On the basis of the data observed,
we proposed that fluorine interacts with ASN107, which is also
supported by prior studies that have demonstrated that the
fluorine atom can interact with asparagine as a hydrogen bond
acceptor.36 Building on the knowledge that the 2-position of
the benzyl ring is important for affinity and that ASN107 in
Grp94 may produce hydrogen bonding interactions, phenol
containing compounds, 8j−8l, were synthesized. Upon their
preparation, 8j−8l were evaluated for binding affinity.
Interestingly, the 2-phenol containing derivative 8j exhibited
an apparent Kd of ∼446 nM toward Grp94 while maintaining
>100 μM Kd for binding Hsp90 (>200 fold selectivity). The
increased potency of compound 8j could once again be
explained via hydrogen bonding interactions with ASN107 via
the phenol. Furthermore, the 3-phenol compound, 8k, and the
4-phenol, 8l, bound Grp94 with lower affinity (Table 3). The
Scheme 2
a
Reagents and conditions: (a) MeMgBr, benzyl bromide/11a−11c/
13, THF/DCM (1:1). (b) NaH, DMF. (c) Pd(OAc)2, DPPF,KOtBu,
trans-cyclohexanolamine, MW, 15 min, 110 °C. (d) DMSO, ethanol,
NaOH, H2O2 RT (at 65 °C for 8j−8l).
a
Table 3
a
Scheme 3
compound
R =
2-Cl
Kd, Grp94 (μM)
Kd, Hsp90α (μM)
8b
0.83 0.12
2.45 0.45
> 25
> 100
> 100
a
Reagents and conditions: (a) NaH, TsCl (2.2 equiv), THF. (b) NaH,
8c
3-Cl
TsCl (1.1 equiv), THF.
8d
4-Cl
> 100
8e
2-F
0.72 0.14
4.53 0.12
8.35 0.14
12.9 0.11
4.2 0.23
0.44 0.09
> 25
> 100
a
Table 2
8f
3-F
> 100
8g
4-F
> 100
8h
2-Br
> 100
8i
2-Me
2-OH
3-OH
4-OH
2-OMe
2, 6-di-F
N scan
> 100
8j
> 100
8k
8l
> 100
20.55 0.74
1.15 0.02
> 25
> 100
8m
> 100
8n
> 100
compound
Kd, Grp94 (μM)
Kd, Hsp90α (μM)
8o
8p
8q
C2 = N
C3 = N
C4 = N
13.98 0.27
5.5 0.15
> 100
> 100
> 100
compound
Kd, Grp94 (μM)
Kd, Hsp90α (μM)
8a
9
1.13 0.1
> 25
> 100
> 100
13.2 0.12
a
Apparent Kd values determined using fluorescence polarization (FP)
assay. Compounds were incubated with cGrp94/Hsp90α and FITC-
GDA in triplicates and SEM were measured.
a
Apparent Kd values determined using fluorescence polarization (FP)
assay. Compounds were incubated with cGrp94/Hsp90α and FITC-
GDA in triplicate and SEM were measured.
compared to 8a. In contrast, the 2-chloro derivative, 8b,
produced increased affinity toward Grp94, while lacking affinity
for Hsp90. Therefore, additional investigations at the 2-position
were pursued. For this purpose, the 2-methyl substituted
analog, 8i, was synthesized, and upon evaluation, a loss of
affinity was observed (∼3 fold), suggesting that steric bulk at
loss of affinity for the 3- and 4-phenols was in agreement with
the affinities observed for substituents at the 3-and 4-positions.
However, a hydroxyl group could serve as a hydrogen bond
donor or an acceptor; therefore, the 2-methoxy containing
compound, 8m, was synthesized to differentiate the role of the
2-phenol with regard to ASN107. Compound 8m bound Grp94
E
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Figure 5. Biological evaluation of Grp94 inhibitors. (A) DMSO, 8e (10 μM), and 8j (10 μM) treated PC3-MM2 cells for 24 h were divided into
microsomal (MIC), mitochondrial (Mito), and cytoplasmic (Cyto) fractions, and Western blot analysis was performed. Representative Western blots
show the levels of integrin α2, integrin αL, Syne2, VAMP2, Rab10, and actin. (B) PC3-MM2 cells were treated with DMSO, 8e (10 μM), or 8j (10
μM) for 24 h and were fixed and stained with integrin α2 (red), phalloidin (gray), and DAPI (blue). Fluorescent images are representative of three
independent biological replicates.
Figure 6. Wound-healing scratch assay performed with compound 8j and 8e. Top MDA-MB-231 cells and bottom PC-3 MM-2 cells. A camera
mounted microscope was then used to record migration at 0, 16, and 24 h. Compounds 8j and 8e were evaluated in this assay at 10 μM and 5 μM
concentration. Representative live cell images at the corresponding time points of three independent biological replicates of cell migration assay are
shown.
with a Kd of 1.15 μM, which was comparable with 8a, despite
the hydrophobic constraints at the 2-position, which led us to
conclude that the hydrogen bond acceptor role of the phenol is
beneficial. Subsequently, the 2,6-difluorine substituted com-
pound, 8n, was synthesized to simultaneously occupy the
binding pocket at both the 2- and 6-positions. Compound 8n
exhibited decreased affinity for Grp94, suggesting that the
binding pockets surrounding the 2- and 6-positions are
mutually exclusive (Figure 4). A nitrogen scan was also
performed with compounds 8o−8q to seek potential hydrogen
bond interactions with protein, but a loss of binding affinity was
observed compared to 8a.
Grp94 Inhibitors Exhibit Antimigratory Activity. Grp94
is a pro-oncogenic chaperone that is overexpressed in tumors to
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modulate cancer cell migration and metastasis.37,38 Using
Grp94 knockdown cells, it was reported that Grp94 affects
intracellular transport pathways during the metastatic process.
Although the total levels of proteins did not alter, levels in the
microsomal fraction (MIC) were significantly lower in Grp94
knockdown cells compared to control cells, suggesting that
Grp94 affects protein localization/trafficking. Specifically,
Grp94 knockdown cells down-regulated VAMP2 and Rab10,
which are critical for intracellular transport; Syne2, which is
necessary for f-actin attachment to the nucleus; and integrin α2
and αL, which are critical for cell−cell and cell−matrix
adhesion.37 As shown in Figure 5A, the cytoplasmic (Cyto)
fraction of cells treated with 8j and to some extent 8e at 30 μM
resulted in reduced levels of integrin α2, integrin αL, Syne2,
VAMP2, and Rab10 compared to DMSO, supporting that
compound 8j targets Grp94 more effectively than 8e.
lead to abnormal myocilin that readily accumulates and
aggregates, leading to reduced aqueous humor outflow facility
through the TM in the anterior chamber of the eye. As a result,
intraocular pressure (IOP) is increased and contributes to
primary open angle glaucoma (POAG), a degenerative eye
disease.41 Previous studies have found a link between the
accumulation of aggregated mutant myocilin and the ER-
associated chaperone, Grp94, and that the inhibition of Grp94
with small molecules is a viable therapeutic approach that can
potentially lead to the alleviation of known POAG pathologies
via increased clearance of myocilin aggregates from the ER.21
Therefore, varying doses of inhibitors were evaluated in an
assay that measured the levels of I477N mutant myocilin levels
expressed in tetracycline inducible HEK cells that conditionally
overexpress the I477N mutant form of myocilin. In the
Western blot analysis shown in Figure 7, compound 8j
The cell migration process requires cell polarization,
protrusion (filopodia, lamellipodia formation), adhesion, and
retraction of the rear. Cell protrusion is produced by local actin
polymerization causing filamentous actin (f-actin) formation at
the filopodia and lamellipodia.39 Adhesion is facilitated by
formation of the focal adhesion complex at the filopodia tip.
Focal adhesion occurs via integrin receptors that bind the
extracellular matrix.37 Different aspects of cell migration
processes, such as f-actin localization and integrin trafficking,
were therefore investigated by confocal microscopy using
Grp94 inhibitors 8e and 8j. The microscopy data revealed that
f-actin was enriched at the cellular cortex region in DMSO
treated cells, which correlates with normal f-actin distribution.
In the presence of 8j and to some extent 8e, f-actin formed
sporadic patches in the cytoplasm, which further supported the
role of Grp94 inhibitors to modulate cell protrusion via f-actin
reorganization (Figure 5B).40 Immunofluorescence analysis
showed that integrin α2 is localized to the cell surface in
untreated cells; however, in the presence of Grp94 inhibitors
(8e and 8j), integrin α2 was localized as patches in the
cytoplasm, supporting the role of Grp94 to traffic integrins to
the cell membrane (Figure 5B). As shown in Figure 6, Grp94
inhibitors, 8e and 8j, inhibited cell motility. When combined,
these results show that both 8j and to some extent 8e prevent
cancer cell migration by affecting f-actin polymerization and
integrin trafficking and thus provide a clear mechanism by
which Grp94 inhibitors inhibit the metastatic process. Recently,
Grp94 inhibition was shown to diminish cell migration and
metastasis in the highly metastatic breast cancer cell lines,
MDA-MB-231 and ROS-resistant MCF-7.20 The migratory
activity of these cells was therefore determined by employing a
wound healing scratch assay. It was observed that compounds
8j and 8e inhibited migration of the highly metastatic breast
cancer cell line, MDA-MB-231, as well as the highly metastatic
prostate cancer cell line, PC-3 MM-2, at 10 μM and 5 μM,
respectively. The antiproliferative activity manifested by these
compounds did not affect the proliferation of MDA-MB-231 or
PC-3 MM-2 cells (>95% viability at 25 μM 8j and 8e),
suggesting that the antimigratory activity manifested by these
compounds does not result from antiproliferative activity.
Taken together, the results show that these Grp94 inhibitors
inhibit cell migration by disrupting integrin trafficking and
filamentous actin rearrangement.
Figure 7. Western blot analysis of compound 8j on the levels of (a)
mutant myocilin and (b) Hsp70 and Akt with 30 μM 8j and 5 μM of
17-AAG.
exhibited a significant decrease in mutatnt myocilin levels at
30 μM. Effect of drug on cytosolic Hsp90 client protein
maturation was also determined. As can be seen in Figure 7b,
compound 8j did not affect Akt maturation at the evaluated
concentration; Akt is a well-established Hsp90-dependent
client. Furthermore, Hsp70 levels remained unaffected with 8j
when compared to 17-AAG, which is a pan-inhibitor of all four
Hsp90 isoforms.
CONCLUSION
■
Herein, we report the discovery of a novel Grp94-selective
scaffold that binds Grp94 in a conformation that is disfavored
for binding other Hsp90 isoforms. The design and develop-
ment of this new scaffold required the evaluation of existing
inhibitors of the N-terminal ATP-binding site of Hsp90, which
were screened in silico for their ability to bind the Grp94
hydrophobic cleft (site 2). It was determined that the
benzamide-containing compound, SNX 2112 (I), could be
modified to occupy the Grp94 hydrophobic cleft and could be
used for the development of Grp94 selective inhibitors. For this
purpose, the tetrahydroindazolone fragment of I was replaced
with a pyrrole, and subsequent incorporation of a phenyl
appendage led to 5a, which exhibited good selectivity for Grp94
but lacked high affinity. Subsequent SAR studies led to analogs
that incorporated a benzyl moiety in lieu of the phenyl and
resulted in derivatives that exhibited both increased affinity and
Disaggregation of Mutant Myocilin with Grp94
Inhibitors. Myocilin is a secreted protein found in trabecular
meshwork (TM) tissue in the anterior anatomical segment of
the eye. Gain of toxic function mutations in the MYOC gene
G
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Articles
ing specific antibodies. Membranes were incubated with an appropriate
horseradish peroxidase-labeled secondary antibody, developed with a
chemiluminescent substrate and visualized.
selectivity. Additional investigations led to identification of 8j
(ACO1), which contains a phenol at the 2-position of the
benzyl side chain. Biological investigations with 8j determined
that Grp94 was inhibited in cells and ultimately led to
inhibition of cancer cell migration and induced the degradation
of mutant myocilin. As a result, 8j represents a new Grp94-
selective inhibitor that may be useful for the treatment of
metastatic cancer and/or glaucoma.
Immunofluorescence Analysis. For cell imaging, 1 μm-Slide eight-
well ibidiTreat IBIDI glass slides were used. PC3-MM2 cells were fixed
with freshly made 4% (w/w) paraformaldehyde in PBS for 15 min,
permeabilized with 0.1% (w/w) Tween 20 in PBS for 5 min, and
quenched with 0.1% (w/w) sodium borohydride for 5 min. The
sections were blocked with 3% (w/w) BSA in PBS for 1 h and
incubated with the primary antibody at a 1:100 concentration in 1%
BSA in PBS overnight, prior to incubation with secondary antibody
conjugated with Alexa Fluor 568 for 3 h. The sections were
counterstained with DAPI and/or with phalloidin to visualize DNA
and F-actin, respectively. The wells were washed three times with PBS
after each step.
Confocal images were acquired using a custom epifluorescent/
confocal microscope composed of the following components: an
Olympus IX81 inverted spinning disc confocal microscope base
(Olympus America), a Prior microscope stage for automated image
acquisition (Prior Scientific), an Olympus 60× oil immersion objective
for confocal images, and a Hamamatsu Electron Multiplying Charge-
Coupled Device (EMCCD) camera (Hamamatsu). Images were
captured using the SlideBook acquisition and analysis software
(Intelligent Imaging Innovations (3i)).
EXPERIMENTAL PROCEDURES
Chemistry. Synthetic procedures and full characterization of new
compounds is provided in the Supporting Information.
■
Fluorescence Polarization. Assay buffer (25 μL, 20 mM HEPES
at pH 7.3, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 20 mM Na2MoO4,
0.01% NP-40, and 0.5 mg mL−1 BGG) was added to a 96-well plate
(black well, black bottom) followed by the desired compound at the
indicated final concentrations in DMSO (1% DMSO final
concentration). Subsequently, 10 nM recombinant cGrp94/Hsp90α
and 6 nM FITC-GDA were added in 50 and 25 μL assay buffer
respectively, resulting in a 100 μL final volume. Plates were incubated
for 5 h at 4 °C on a rocker. Fluorescence was determined using
excitation and emission filters of 485 and 528 nm, respectively. Percent
FITC-GDA bound was determined by assigning the DMSO
millipolarization unit (mP) value as the 100% bound value and 0%
for FITC-GDA in assay buffer without any protein. Kd values were
calculated from separate experiments performed in triplicate using
GraphPad Prism.
Molecular Modeling. The Surflex-Docking module in Sybyl v8.0
was used for molecular modeling and docking studies. The cocrystal
structures of RDA bound to Grp94 (PDB code: 2GFD), SNX 2112
bound to Hsp90 (PDB code: 4NH7), and PU-H54 bound to Grp94
(PDB code: 3O2F) were utilized for modeling experiments. Pymol
was used for further visualization and figure preparation.
Images were collected with 8−10 image stacks with a 0.3 μm step
size through the cells. Images were processed using ImageJ software
(NIH).
Wound Healing Scratch Assay. The cells were seeded in a 24-well
plate in complete media and allowed to form a monolayer. After
monolayer formation, a scratch was introduced with a sterile 0.1−10
μL pipet tip. The medium was replaced with fresh medium in the
absence or presence of the indicated drug concentrations. Photo-
micrographs were taken at different time points with an Olympus IX71
microscope using a 10× air lens and CellSans Dimensions software.
The images were processed with ImageJ software.
Antiproliferation Assays. Cells were seeded (2000/well, 100 μL) in
96-well plates and incubated overnight. Following incubation,
compounds with varying concentrations in DMSO (1% DMSO final
concentration) were added, and cells were returned to the incubator
for 72 h. After 72 h, the cell viability was determined using an MTS/
PMS cell proliferation kit (Promega) per the manufacturer’s
instructions. Absorption values from 1% DMSO wells were used as
100% proliferation, and values were adjusted accordingly.
Effect of Grp94-Selective Inhibitors on Myocilin Levels. Cell
Culture. Cell culture was performed as previously described.21
Tetracycline-inducible Hek cells stably overexpressing I477N mutant
myocilin were grown and maintained in Dulbecco’s Modified Eagle’s
Medium supplemented with 10% fetal bovine serum (Invitrogen,
penicillin (100 units/mL), streptomycin (100 mg mL−1), and 1%
GlutaMAX (Invitrogen) at 37 °C and 5% CO2). Stably overexpressing
cells were selected for using hygromycin B (200 mg mL−1; InvivoGen)
and G418 (100 mg mL−1; Gibco). I477N myocilin expression was
induced with tetracycline (5 mg mL−1) for 48 h prior to drug
treatment.
Inhibitor Treatment. Inhibitors were solubilized in DMSO (Sigma-
Aldrich) and diluted to documented concentrations. Cells were treated
dropwise with inhibitors 24 h prior to cell harvest. DMSO
concentration was diluted to 1% total cell medium volume.
Cell Harvest. Cell harvest was performed as previously described.21
Twenty-four hours after inhibitor treatment, culture medium was
removed, and cells were washed two times with ice cold PBS.
Mammalian Protein Extraction Reagent (M-PER) buffer (Pierce)
containing protease inhibitor cocktail III (Calbiochem), 100 mM
phenylmethylsulfonyl fluoride (PMSF), and phosphatase inhibitor II
and III mixtures (Sigma) were added to washed cells at a 1:100
dilution and scraped. Scraped cells in lysis buffer were incubated for 10
min on ice to allow for lysis to occur. Cell lysates were then
centrifuged at 16 000g for 10 min to separate cell lysates from nuclear
debris. Bicinchoninic acid assay (BCA) was performed to determine
Protein Trafficking and Anti-Migratory Effects of Grp94
Inhibitors. Chemicals and Cell Culture. Compounds 8e and 8j were
dissolved in DMSO and stored at −20 °C. The PC3-MM2 and MDA-
MB-231 cells were maintained in DMEM (Cellgro) media
supplemented with streptomycin (500 μg/mL), penicillin (100
units/mL), and 10% FBS at 37 °C and 5% CO2.
Antibodies. The following antibodies were used for Western
blotting and/or immunofluorescence: goat anti-integrin αL #sc6609,
rabbit anti-SYNE2 #sc99066, and rabbit antiactin #sc1616-R (Santa
Cruz Biotechnology); rabbit anti-Integrin α 2 #ab181548 and rabbit
anti-VAMP2 #ab3347 (Abcam); Phalloidin 647 #A22287 (Invitro-
gen); and rabbit anti-Rab10 #4262S (Cell Signaling Technology).
Cell Fractionation and Western Blot Analysis. The PC3-MM2
cells treated with DMSO, or compounds dissolved in DMSO at
specified concentrations for 24 h, were trypsinized, washed twice with
ice-cold phosphate-buffered saline (PBS), and resuspended in 10 mL
of isolation buffer containing 10 mM Tris-HCl at pH 7.4, 1 mM
EDTA, 0.2 M D-mannitol, 0.05 M sucrose, 0.5 mM sodium
orthovanadate, 1 mM sodium fluoride, and protease inhibitor
cocktail.42 The cells were homogenized with the aid of a Teflon
pestle, and lysis was confirmed microscopically. After sedimenting the
cell debris, the protein concentration of each lysate was measured in
quadruplicate using the BCA protein assay and bovine serum albumin
as the standard. The coefficient of variation was determined for each
set of quadruplicate measures, and if the variability exceeded 5%, the
protein assay was repeated for that set of samples. The cell lysates
containing equal amounts of protein were centrifuged at 8000g for 10
min, and the crude mitochondrial pellet (Mito) was washed two times
in isolation buffer and frozen. The reserved supernatant was centrifued
at 14 000g to isolate a microsomal fraction (MIC) and the remaining
supernatant (Cyto) was concentrated overnight by TCA precipitation
and dissolved in a minimum amount of isolation buffer and subjected
to SDS-PAGE.
Equal volumes of samples were electrophoresed under reducing
conditions (8% polyacrylamide gel), transferred to a polyvinylidene
fluoride membrane (PVDF), and immunoblotted with the correspond-
H
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Articles
protein concentration in cell lysates, and samples were prepared for
Western blot analysis.
(3) Khandelwal, A., Crowley, V. M., and Blagg, B. S. (2016) Natural
product inspired N-terminal Hsp90 inhibitors: from bench to bedside?
Med. Res. Rev. 36, 92−118.
(4) Jarosz, D. (2016) Hsp90: A Global Regulator of the Genotype-to-
Phenotype Map in Cancers. Adv. Cancer Res. 129, 225−47.
(5) Whitesell, L., Bagatell, R., and Falsey, R. (2003) The stress
response: implications for the clinical development of Hsp90
inhibitors. Curr. Cancer Drug Targets 3, 349−358.
(6) Calderwood, S. K., and Gong, J. (2016) Heat Shock Proteins
Promote Cancer: It’s a Protection Racket. Trends Biochem. Sci. 41,
311−23.
(7) Zhang, H., and Burrows, F. (2004) Targeting multiple signal
transduction pathways through inhibition of Hsp90. J. Mol. Med. 82,
488−499.
(8) Pearl, L. H., and Prodromou, C. (2000) Structure and in vivo
function of Hsp90. Curr. Opin. Struct. Biol. 10, 46−51.
(9) Prodromou, C., and Pearl, L. H. (2003) Structure and functional
relationships of Hsp90. Curr. Cancer Drug Targets 3, 301−323.
(10) DeBoer, C., Meulman, P. A., Wnuk, R. J., and Peterson, D. H.
(1970) Geldanamycin, a new antibiotic. J. Antibiot. 23, 442−7.
(11) Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and
Neckers, L. M. (1994) Inhibition of heat shock protein HSP90-pp60v-
src heteroprotein complex formation by benzoquinone ansamycins:
essential role for stress proteins in oncogenic transformation. Proc.
Natl. Acad. Sci. U. S. A. 91, 8324−8.
(12) Bhat, R., Tummalapalli, S. R., and Rotella, D. P. (2014) Progress
in the discovery and development of heat shock protein 90 (Hsp90)
inhibitors. J. Med. Chem. 57, 8718−28.
(13) Sidera, K., and Patsavoudi, E. (2014) HSP90 inhibitors: current
development and potential in cancer therapy. Recent Pat. Anti-Cancer
Drug Discovery 9, 1−20.
Western Blotting. Western blot analysis was performed as
previously described.41 Cell lysates were prepared at identical protein
concentrations, as determined by BCA analysis, in 2× Laemmli sample
buffer (Bio-Rad) and denatured by boiling for 5 min at 100 °C.
Denatured samples were then loaded onto a 10-well 10% gel (Bio-
Rad). Gels were run at 125 V until the dye front reached the bottom of
the gel cassette. Gels were then transferred onto a PVDF membrane
(Millipore) at 100 V for 1 h. After transfer, blots were blocked in 7%
milk in TBS for 1 h at RT prior to primary antibody incubation.
Statistical analysis of imaged blots was performed with ImageJ analysis
software (NIH).
Antibodies. Myocilin antirabbit primary antibody was provided by
Dan Stamer at Duke University. Actin antimouse was purchased from
Sigma-Aldrich. Secondary antibodies were purchased from Southern
Biotech. All antibodies were diluted at 1:1000 in 7% milk in TBS.
Primary antibodies were incubated shaking overnight at 4 °C.
Secondary antibodies were incubated shaking at RT for 1 h.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.6b00747.
1
Synthetic procedures, characterization data, H and 13C
NMR spectra for 5a and 8j (PDF)
AUTHOR INFORMATION
■
(14) Jhaveri, K., Taldone, T., Modi, S., and Chiosis, G. (2012)
Advances in the clinical development of heat shock protein 90
(Hsp90) inhibitors in cancers. Biochim. Biophys. Acta, Mol. Cell Res.
1823, 742−755.
Corresponding Author
*E-mail: bblagg@ku.edu.
ORCID
(15) Peterson, L. B., Eskew, J. D., Vielhauer, G. A., and Blagg, B. S.
(2012) The hERG channel is dependent upon the Hsp90α isoform for
maturation and trafficking. Mol. Pharmaceutics 9, 1841−1846.
(16) Subbarao Sreedhar, A., Kalmar, E., Csermely, P., and Shen, Y. F.
(2004) Hsp90 isoforms: functions, expression and clinical importance.
FEBS Lett. 562, 11−15.
Brian S. J. Blagg: 0000-0002-6200-3480
Notes
The authors declare the following competing financial
interest(s): The authors, with the exception of Suman Gosh,
are coinventors of a patent based on this work.
(17) Kozutsumi, Y., Segal, M., Normington, K., Gething, M., and
Sambrook, J. (1988) The presence of malfolded proteins in the
endoplasmic reticulum signals the induction of glucose-regulated
proteins. Nature 332, 462−464.
ACKNOWLEDGMENTS
■
We would like to thank Heather Shinogle from the Microscopy
and Analytical Imaging Laboratory at The University of Kansas
for assistance with confocal imaging. This research was
supported by grants CA109265 (B.S.J.B.) and EY024232
(B.S.J.B. and C.A.D.) from The National Institutes of Health
(NIH). NMR instrumentation was supported by NIH Shared
Instrumentation Grants (S10OD016360, S10RR024664) and
an NSF Major Research Instrumentation Grant (0320648).
(18) Li, Z., and Srivastava, P. K. (1993) Tumor rejection antigen
gp96/grp94 is an ATPase: implications for protein folding and antigen
presentation. EMBO J. 12, 3143−51.
(19) Goodman, S. L., and Picard, M. (2012) Integrins as therapeutic
targets. Trends Pharmacol. Sci. 33, 405−412.
(20) Dejeans, N., Glorieux, C., Guenin, S., Beck, R., Sid, B., Rousseau,
R., Bisig, B., Delvenne, P., Buc Calderon, P., and Verrax, J. (2012)
Overexpression of Grp94 in breast cancer cells resistant to oxidative
stress promotes high levels of cancer cell proliferation and migration:
implications for tumor recurrence. Free Radical Biol. Med. 52, 993−
1002.
ABBRIVIATIONS USED
■
SBDD, structure-based drug design; GARP, glycoprotein-A
repetitions predominant protein; DPPF, 1,10-bis-
(diphenylphosphino)ferrocene; LRP6, low-density lipoprotein
receptor-related protein 6; LG, leaving group; RT, room
temperature
(21) Suntharalingam, A., Abisambra, J. F., O’Leary, J. C., III, Koren,
J., III, Zhang, B., Joe, M. K., Blair, L. J., Hill, S. E., Jinwal, U. K.,
Cockman, M., Duerfeldt, A. S., Tomarev, S., Blagg, B. S., Lieberman, R.
L., and Dickey, C. A. (2012) Glucose-regulated protein 94 triage of
mutant myocilin through endoplasmic reticulum-associated degrada-
tion subverts a more efficient autophagic clearance mechanism. J. Biol.
Chem. 287, 40661−40669.
(22) Gullo, C. A., and Teoh, G. (2004) Heat shock protein: to
present or not, that is the question. Immunol. Lett. 94, 1−10.
(23) Obeng, E. A., Carlson, L. M., Gutman, D. M., Harrington, W. J.,
Lee, K. P., and Boise, L. H. (2006) Proteasome inhibitors induce a
terminal unfolded protein response in multiple myeloma cells. Blood
107, 4907−4916.
REFERENCES
■
(1) Taipale, M., Jarosz, D. F., and Lindquist, S. (2010) HSP90 at the
hub of protein homeostasis: emerging mechanistic insights. Nat. Rev.
Mol. Cell Biol. 11, 515−528.
(2) Trepel, J., Mollapour, M., Giaccone, G., and Neckers, L. (2010)
Targeting the dynamic HSP90 complex in cancer. Nat. Rev. Cancer 10,
537−549.
I
DOI: 10.1021/acschembio.6b00747
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
(24) Hua, Y., White-Gilbertson, S., Kellner, J., Rachidi, S., Usmani, S.
Z., Chiosis, G., DePinho, R., Li, Z., and Liu, B. (2013) Molecular
chaperone gp96 is a novel therapeutic target for multiple myeloma.
Clin. Cancer Res. 19, 6242−6251.
(42) Okado-Matsumoto, A., and Fridovich, I. (2001) Subcellular
distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD
in mitochondria. J. Biol. Chem. 276, 38388−93.
(25) Chen, B., Piel, W. H., Gui, L., Bruford, E., and Monteiro, A.
(2005) The HSP90 family of genes in the human genome: insights
into their divergence and evolution. Genomics 86, 627−637.
(26) Soldano, K. L., Jivan, A., Nicchitta, C. V., and Gewirth, D. T.
(2003) Structure of the N-terminal domain of Grp94. Basis for ligand
specificity and regulation. J. Biol. Chem. 278, 48330−48338.
(27) Cristalli, G., Lambertucci, C., Taffi, S., Vittori, S., and Volpini, R.
(2003) Medicinal chemistry of adenosine A2A receptor agonists. Curr.
Top. Med. Chem. 3, 387−401.
(28) Immormino, R. M., Metzger, L. E., Reardon, P. N., Dollins, D.
E., Blagg, B. S., and Gewirth, D. T. (2009) Different poses for ligand
and chaperone in inhibitor-bound Hsp90 and Grp94: implications for
paralog-specific drug design. J. Mol. Biol. 388, 1033−1042.
(29) Patel, P. D., Yan, P., Seidler, P. M., Patel, H. J., Sun, W., Yang,
C., Que, N. S., Taldone, T., Finotti, P., Stephani, R. A., Gewirth, D. T.,
and Chiosis, G. (2013) Paralog-selective Hsp90 inhibitors define
tumor-specific regulation of HER2. Nat. Chem. Biol. 9, 677−684.
(30) Duerfeldt, A. S., Peterson, L. B., Maynard, J. C., Ng, C. L.,
Eletto, D., Ostrovsky, O., Shinogle, H. E., Moore, D. S., Argon, Y.,
Nicchitta, C. V., and Blagg, B. S. J. (2012) Development of a Grp94
inhibitor. J. Am. Chem. Soc. 134, 9796−9804.
(31) Crowley, V. M., Khandelwal, A., Mishra, S., Stothert, A. R.,
Huard, D. J. E., Zhao, J., Muth, A., Duerfeldt, A. S., Kizziah, J. L.,
Lieberman, R. L., Dickey, C. A., and Blagg, B. S. J. (2016)
Development of Glucose Regulated Protein 94-Selective Inhibitors
Based on the BnIm and Radamide Scaffold. J. Med. Chem. 59, 3471−
3488.
(32) Ernst, J. T., Liu, M., Zuccola, H., Neubert, T., Beaumont, K.,
Turnbull, A., Kallel, A., Vought, B., and Stamos, D. (2014) Correlation
between chemotype-dependent binding conformations of HSP90α/β
and isoform selectivityImplications for the structure-based design of
HSP90α/β selective inhibitors for treating neurodegenerative diseases.
Bioorg. Med. Chem. Lett. 24, 204−208.
(33) Gilow, H., and Burton, D. (1981) Bromination and chlorination
of pyrrole and some reactive 1-substituted pyrroles. J. Org. Chem. 46,
2221−2225.
(34) Chen, W., Stephenson, E. K., Cava, M. P., and Jackson, Y. A.
(1992) 2-Substituted Pyrroles from N-tert-Butoxycarbonyl-2-Bromo-
pyrrole: N-tert-Butoxycarbonyl-2-Trimethylsilylpyrrole. Org. Synth. 70,
151.
(35) Chakrabarty, M., Kundu, T., and Harigaya, Y. (2006) Mild
Deprotection Of Tert -Butyl Carbamates Of NH-Heteroarenes Under
Basic Conditions. Synth. Commun. 36, 2069−2077.
(36) Bohm, H. J., Banner, D., Bendels, S., Kansy, M., Kuhn, B.,
Muller, K., Obst-Sander, U., and Stahl, M. (2004) Fluorine in
medicinal chemistry. ChemBioChem 5, 637−43.
(37) Ghosh, S., Shinogle, H. E., Galeva, N. A., Dobrowsky, R. T., and
Blagg, B. S. J. (2016) Endoplasmic Reticulum Resident Heat Shock
Protein 90 (Hsp90) Isoform Glucose-Regulated Protein 94 (Grp94)
Regulates Cell Polarity and Cancer Cell Migration by Affecting
Intracellular Transport. J. Biol. Chem. 291, 8309−8323.
(38) Rachidi, S., Sun, S., and Li, Z. (2015) Endoplasmic reticulum
heat shock protein gp96/grp94 is a pro-oncogenic chaperone, not a
tumor suppressor. Hepatology 61, 1766−1767.
(39) Mattila, P. K., and Lappalainen, P. (2008) Filopodia: molecular
architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 9, 446−454.
(40) Wang, N., Feng, Y., Lau, E. P., Tsang, C., Ching, Y., Man, K.,
Tong, Y., Nagamatsu, T., Su, W., and Tsao, S. (2010) F-actin
reorganization and inactivation of rho signaling pathway involved in
the inhibitory effect of Coptidis Rhizoma on hepatoma cell migration.
Integr. Cancer Ther. 9, 354−64.
(41) Stothert, A., Suntharalingam, A., Huard, D., Fontaine, S.,
Crowley, V., Mishra, S., Blagg, B., Lieberman, R., and Dickey, C.
(2014) Exploiting the interaction between Grp94 and aggregated
myocilin to treat glaucoma. Hum. Mol. Genet. 23, 6470−80.
J
DOI: 10.1021/acschembio.6b00747
ACS Chem. Biol. XXXX, XXX, XXX−XXX