Thermodynamic Dissection of Potency and Selectivity of Cytosolic Hsp90 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01715

The cytosolic Hsp90-selective inhibitor TAS-116 has an acceptable safety profile and promising antitumor activity in clinical trials. We examined the binding characteristics of TAS-116 and its analogs to determine the impact of the ligand binding mode on selectivity for cytosolic Hsp90. Analyses of the co-crystal structure of Hsp90 and inhibitor TAS-116 suggest that TAS-116 interacts with the ATP-binding pocket, the ATP lid region, and the hydrophobic pocket. A competitive isothermal titration calorimetry analysis confirmed that a small fragment of TAS-116 (THS-510) docks into the lid region and hydrophobic pockets without binding to the ATP-binding pocket. THS-510 exhibited enthalpy-driven binding to Hsp90α and selectively inhibited cytosolic Hsp90 activity. The heat capacity change of THS-510 binding was positive, likely due to the induced conformational rearrangement of Hsp90. Thus, we concluded that interactions with the hydrophobic pocket of Hsp90 determine potency and selectivity of TAS-116 and derivatives for the cytosolic Hsp90 isoform.

Full text of DOI:10.1021/acs.jmedchem.0c01715

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Article
Thermodynamic Dissection of Potency and Selectivity of Cytosolic
Hsp90 Inhibitors
Chihoko Yoshimura, Satoru Nagatoishi, Daisuke Kuroda, Yasuo Kodama, Takao Uno, Makoto Kitade,
Khoontee Chong-Takata, Hiromi Oshiumi, Hiromi Muraoka, Satoshi Yamashita, Yuichi Kawai,
Shuichi Ohkubo, and Kouhei Tsumoto*
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ABSTRACT: The cytosolic Hsp90-selective inhibitor TAS-116
has an acceptable safety profile and promising antitumor activity in
clinical trials. We examined the binding characteristics of TAS-116
and its analogs to determine the impact of the ligand binding mode
on selectivity for cytosolic Hsp90. Analyses of the co-crystal
structure of Hsp90 and inhibitor TAS-116 suggest that TAS-116
interacts with the ATP-binding pocket, the ATP lid region, and the
hydrophobic pocket. A competitive isothermal titration calorimetry
analysis confirmed that a small fragment of TAS-116 (THS-510)
docks into the lid region and hydrophobic pockets without binding
to the ATP-binding pocket. THS-510 exhibited enthalpy-driven
binding to Hsp90α and selectively inhibited cytosolic Hsp90
activity. The heat capacity change of THS-510 binding was
positive, likely due to the induced conformational rearrangement of Hsp90. Thus, we concluded that interactions with the
hydrophobic pocket of Hsp90 determine potency and selectivity of TAS-116 and derivatives for the cytosolic Hsp90 isoform.
component analysis of molecular dynamics simulations.16
INTRODUCTION
■
Thus, the structural classes of Hsp90 inhibitors are diverse.
Heat shock protein 90 (Hsp90) is a molecular chaperone
Hsp90 inhibitors that bind to the Hsp90 NTD are classified
essential for the maintenance of three-dimensional (3D)
based on the binding mode of the compounds. Geldanamycin
structure and the localization of proteins.1,2 There are four
analogs, radicicol, AUY922, and other resorcinol-type Hsp90
isoforms of Hsp90: Hsp90α and Hsp90β, which localize to the
inhibitors are classified as type 1 inhibitors, which occupy the
cytoplasm and nucleus, TRAP1 in the mitochondria, and
ATP-binding pocket (the first pocket).12,17 Purine analogs,
Grp94, also known as gp96, in the endoplasmic reticulum.3
such as BIIB021, BIIB028, and SNX-2112, interact with the
Cytosolic Hsp90 mediates the proper folding and functions of
first pocket and also the second pocket, which is formed when
a diverse range of client proteins including the tyrosine kinases
the Hsp90 ATP lid region adopts a helical form.14,18,19
ALK, FLT3, and MET and the growth factor receptors EGFR
Compounds that bind to both the first pocket and the second
and FGFR, which have all been implicated in oncogenesis.4,5
pocket are classified as type 2 inhibitors. Hsp90 inhibitors in
The inhibition of cytosolic Hsp90 impairs the correct folding
clinical development have a distinct pattern of isoform
and maturation of many oncoproteins required for cancer
selectivity. 17-AAG and AUY922 have pan-Hsp90 inhibitory
growth and survival. Therefore, Hsp90 is a promising target for
activity. BIIB021 and SNX-2112 inhibit not only Hsp90α and
drug development in cancer therapy, and Hsp90 inhibitors
Hsp90β but also moderately inhibit Grp94 and TRAP-1. The
have been tested in clinical trials.6−9
functions of TRAP-1 and Grp94 are poorly understood.
Hsp90 requires ATP to drive its chaperone activity.10 ATP
However, the gut-specific deletion of Grp94 results in the loss
hydrolysis causes large and dynamic conformational changes in
of intestinal homeostasis, and the liver-specific Grp94 knockout
full-length Hsp90 dimers, and conformational changes are also
observed in an ATP lid region of the N-terminal domain
(NTD) of Hsp90.11 Several Hsp90 inhibitors bind to the ATP-
Received: October 29, 2020
Published: February 23, 2021
binding site of Hsp90 NTD, as shown by co-crystal structural
analysis.12−14 Due to the flexibility of the structure of Hsp90,
the shape of the pockets is highly dynamic.15 A recent paper
has indicated the dynamics of conformational changes in the
interaction between HSP90 and inhibitors using a principal
© 2021 The Authors. Published by
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Figure 1. (a) Chemical structure of TAS-116 and 16d. (b) Co-crystal structures of Hsp90α NTD with 16d (PDB ID: 5ZR3) and THS-828 (PDB
ID: 3WQ9). (c−f) A model of the complex of Hsp90α and TAS-116 was created using the MOE software. Shown are (c) a cartoon model of (d)
the ATP-binding pocket, (e) the second pocket, and (f) the third pocket. The Hsp90α is shown in brown. TAS-116 is shown in a ball-and-stick
rendering in orange.
in PTEN-null mice accelerates liver tumorigenesis. TRAP-1 is
reported to protect against mitochondrial apoptosis.20 There-
fore, the isoform specificity of Hsp90 inhibitors may impact
both adverse effects and antitumor efficacy of these agents.21,22
The orally available Hsp90 inhibitor TAS-116 (Figure 1a) is
in clinical testing.23,24 In the preclinical antitumor efficacy
studies, TAS-116 showed the potential to be the best-in-class
Hsp90 inhibitor as an antitumor agent. In addition, TAS-116
prolonged survival in fourth-line treatment settings for patients
with metastatic or unresectable gastrointestinal stromal tumors
in a Phase III clinical trial, which is currently ongoing. TAS-
116 is the most selective compound for the cytosolic Hsp90
forms among the known Hsp90 inhibitors.25 As noted by Ernst
et al., the structure of Hsp90 must be considered in design of
isoform-selective inhibitors.26 Recently, Uno et al. reported a
structure−activity relationship analysis of TAS-116 analogs.27
However, a biophysical characterization of the isoform
selectivity of TAS-116 analogs has not been performed. We
previously reported use of biophysical methods, including
calorimetric analyses, for small-molecule inhibitor identifica-
tion.28−31 Biophysical information on specific inhibitors is
helpful for the selection and design of new hit or lead
compounds for other target proteins. In this study, we
examined the characteristics of binding of TAS-116 and its
analogs to Hsp90 using structural, biological, and biophysical
analyses, with particular emphasis on calorimetric and
competitive analyses.
analog, THS-828 (Figure S1a), in complex with human
Hsp90α NTD has also been reported (PDB ID: 3WQ9).
Based on the binding modes of these inhibitors (Figure 1b),
TAS-116 is expected to bind to the first and second pockets
and also to a hydrophobic pocket, which we refer to as the
third pocket, of Hsp90 (Figure 1b,c). When the amido group
of TAS-116 was changed to carbonic acid (THS-2415, Figure
S1b), no binding to Hsp90 was observed (data not shown).
This result indicates that similar to other Hsp90 inhibitors, the
hydrogen-bonding network with D93, T184, N51, and water
molecules in the ATP-binding pocket of the Hsp90 NTD are
critical for binding of TAS-116 to Hsp90. TAS-116 docked
model was constructed from a 16d co-crystal structure (PDB
ID: 5ZR3) by modifying the methyl group to an ethyl group,
and energy minimization was performed using the Molecular
Operating Environment (MOE) software package. In the
model, hydrogen-bonding network in ATP pocket and π−π
interactions with F138 and a hydrogen bond with Y139 in the
second pocket are retained (Figure 1d,e). Water-mediated
interaction with Gln23, which was reported in the literature
about other types of third pocket inhibitor, was not observed in
the co-crystal structures of TAS-116 analogs.32 After
optimization, Gln23 directly interacts with the nitrogen atom
of the imidazole-pyrazole group of TAS-116 unlike co-crystal
structures (Figure 1f). Gln23 seems flexible and might
contribute to the transition state of the interaction between
TAS-116 analogs and Hsp90 NTD.
To investigate the contribution of the amino acids of
Hsp90α to the interaction with TAS-116, a site-directed
mutagenesis study was performed. Four sites (Q23, N105,
F138, and Y139) were selected for mutation based on the
interactions observed in the crystal structure of the THS-828
and Hsp90 complex (PDB ID: 3WQ9) and 16d and Hsp90
complex (PDB ID: 5ZR3). Q23, F138, and Y139 are expected
RESULTS
■
Characterization of Binding Modes of TAS-116. A co-
crystal structure of the very close analog of TAS-116, 16d
(Figure 1), with the human Hsp90α NTD has been reported
recently (PDB ID: 5ZR3).27 The structure of another TAS-116
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Figure 2. (a) SPR analysis of the interaction between Hsp90α and TAS-116. (b) Binding locations of various inhibitors to Hsp90α (gray). The
proteins in complexes with AUY922 (magenta) (PDB: 2VCI) and BIIB021 (green) (PDB: 3QDD) were aligned. TAS-116 (orange) was docked
into the Hsp90α structure. (c) Fold difference in Ki values of inhibitors for mutant relative to wild-type Hsp90.
to interact directly with TAS-116. From the literature about
the third pocket inhibitor,16,32 F138 is expected to have
hydrophobic interaction with TAS-116. N105 is one of the
constituent residues of ATP-lid region, and the local structure
is highly flexible and forms a helix when Hsp90 takes helical
form.33 N105 faces the solvent front in the helical form and
seems not to interact directly with ligand (Figure 2b).
Therefore, N105 was selected as a residue which is expected
not to interact with TAS-116. Although D93 also interacts with
TAS-116, we could not prepare D93A mutant due to low
expression level. We successfully prepared Q23A, N105A,
F138L, and Y139F mutants of Hsp90 and performed a
previously described competitive binding assay25 to evaluate
interaction of Hsp90 inhibitors 17-AAG, AUY922, BIIB021,
SNX-2112, and TAS-116 with wild-type and mutant proteins.
AUY922 and 17-AAG are type 1 inhibitors, and BIIB021 and
SNX-2112 are type 2 inhibitors (Figure S1). The relative IC50
values are summarized in Figure 2c, and plots are shown in
Figure S3. The perturbation effect for helical form specific
inhibitors (TAS-116, SNX-2112, and BIIB-021) by Q23A
mutation is greater than that of type 1 inhibitors (17-AAG and
AUY-922), even though SNX-2112 and BIIB-021 do not
interact with Q23. These results indicate that Q23 affects
formation of the helical form and is not crucial for the
interaction with TAS-116. Unexpectedly, Y139F mutation does
not weaken the inhibitory effects of SNX-2112 and TAS-116,
both were considered to form hydrogen bonds with Y139.
Although the binding of other inhibitors was reduced, TAS-
116 bound to the F138L mutant with affinity similar to its
binding to the wild-type Hsp90. F138 forms a hydrophobic
interaction with type 1 inhibitors.12 In contrast, the type 2
inhibitors, BIIB021 and SNX-2112, interact with F138 through
π−π interaction (PDB: 3QDD and 3R92).19,34,35 From our
modeling study, we expected that the interaction between
TAS-116 and F138 would be critical; however, as shown in the
mutational study, the binding of TAS-116 to human Hsp90α
does not depend strongly on the interaction with F138 (Figure
2b). This low dependence of TAS-116 on F138 may be a
property acquired by TAS-116 through the use of the third
pocket. The shape of the third pocket is not constant in many
crystal structures of the helical form Hsp90 NTD. Therefore,
we hypothesized that the shape of TAS-116 is the key to bind
and stabilize the helical form with the third pocket of Hsp90
NTD.
Kinetic analysis using surface plasmon resonance (SPR)
demonstrated that TAS-116 has a slow off-rate from the
human Hsp90α NTD (Figure 2a). The off-rate of TAS-116
was slower than that of type 1 inhibitor 17-AAG and type 2
inhibitor BIIB-021 (Figure S2). Another type 2 inhibitor SNX-
2112 showed a slower off-rate (Figure S2), which has a similar
structure and binding mode with TAS-116. From the literature,
changes in conformations and internal dynamics of Hsp90 are
reported to be induced by inhibitor bindings, and some
residues are proposed as hot spots.16 As TAS-116 is not the
highest affinity binder among known Hsp90 inhibitors (Figure
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S3), the slow-off rate is likely due to the unique binding mode
of TAS-116.
binding stoichiometry was not 1:1, and the binding enthalpy
change was small. In other words, the pyrazole compound is
not suitable for more quantitative and precise evaluation such
as temperature dependence and competitive assay, as shown in
this study due to unexpected errors. On the other hand, we
quantitatively analyzed the binding of THS-510 to HSP90 in
ITC. As shown in Figure 3f, moderate binding of THS-510 was
observed (KD = 2.7 μM) at 25 °C. The stoichiometry of
binding calculated for THS-510 from the ITC data was 0.72
0.03; therefore, THS-510 likely binds human Hsp90α in a 1:1
molar ratio. The thermodynamic parameters of the interaction
between THS-510 and human Hsp90α NTD were ΔG of −7.6
0.1 kcal/mol, ΔH of −5.7 0.2 kcal/mol, and − TΔS of
−1.9 0.1 kcal/mol, indicating that complex formation was
primarily enthalpy driven. This suggests that hydrogen bonds
or van der Waals stabilize the interaction between the human
Hsp90α NTD and THS-510. It is noteworthy that this
interaction also involved a slight entropic gain.
Characterization of Isoform Selectivity and Thermo-
dynamics of THS-510. The Hsp90 inhibitory activities of
TAS-116 among Hsp90 isoforms have been reported
previously. The Ki values of TAS-116 for Hsp90α, Hsp90β,
Grp94, and TRAP-1 are 34.7 nM, 21.3 nM, >50,000 nM, and
>50,000 nM, respectively, in a competitive binding assay using
FITC-labeled geldanamycin.25 TAS-116 analog 16d is reported
to bind preferentially to the cytosolic isoforms.27 Like TAS-116
and 16d, THS-828 (Hsp90α, 13 μM; Hsp90β, 17 μM; Grp94,
>300 μM; TRAP-1, >300 μM) also binds to the cytosolic
isoforms. To further study the unique binding characteristics of
TAS-116, we prepared a fragment of TAS-116 that includes
only the moiety that binds to the third pocket; this analog was
named THS-975 (Figure 3a). In addition to the THS-975,
Exploration of the Binding Mode of THS-510 Using
Competitive Thermodynamic Profiling. To estimate the
contact region of THS-510 on Hsp90, a competitive binding
analysis with known Hsp90 inhibitors was performed using
ITC. We hypothesized that if a highly potent inhibitor of
Hsp90 occupies the same binding site as THS-510 on Hsp90,
the titration of THS-510 into Hsp90 prebound to the potent
inhibitor will not cause exothermic reactions. In contrast, if the
binding pockets of the potent Hsp90 inhibitor and THS-510
are different, THS-510 will bind to Hsp90 in the presence of
the other inhibitor. We selected Hsp90 inhibitors of different
types for this analysis: The type 1 inhibitors AUY922 and 17-
AAG are expected to occupy the ATP-binding pocket because
geldanamycin binds the ATP-binding pocket (PDB: 1YET).
BIIB021, a type 2 inhibitor, binds to the Hsp90 helical form
and occupies the ATP-binding pocket and the second pocket
(PDB: 3QDD).17,19
When THS-510 was titrated into Hsp90α prebound to
AUY922, an exothermic reaction was observed (Figure 4a).
However, 17-AAG, which binds to the same site as AUY922,
did not cause an exothermic reaction (Figure S4). These
results indicate that THS-510 does not occupy the ATP-
binding pocket of Hsp90α. In contrast, addition of THS-510 to
Hsp90α prebound to BIIB021 did not result in an exothermic
reaction (Figure 4b), suggesting that THS-510 and BIIB021
have overlapping binding sites. As BIIB021 interacts with both
the ATP-binding pocket and the second pocket of Hsp90α,
our data suggest that THS-510 binds to the second pocket of
the helical form of Hsp90α NTD but does not bind to the
ATP-binding pocket.
We hypothesized that the helical conformation of Hsp90
results from a conformational rearrangement of some residues
of the ATP lid region. THS-510 was manually placed on the
second and third pockets, and the complex was minimized
using MOE (Figure S5A,B). In the energy-minimized
structure, the imidazole ring of THS-510 interacts with F138
in the second pocket. A hydrogen bond and van der Waals
interactions were observed between the ligand and Hsp90 in
the third pocket. These noncovalent interactions probably
contribute to the binding enthalpy of THS-510. The structural
alignments of the structure of the complex of THS-510 with
Hsp90 with the structures of BIIB021 and AUY922 in complex
with Hsp90 suggest that the binding site of THS-510 partially
overlaps with that of BIIB021 (Figure S5A) but does not
overlap with the binding site of AUY922 (Figure S5B).
Figure 3. Characteristics of the interaction of THS-975 and THS-510
with Hsp90. (a, d) Chemical structure of THS-975 and THS-510. (b,
e) Isoform selectivity of THS-975 and THS-510. Plotted is the
percent ratio of the intensity of the test and control lines versus
concentration of inhibitor. The data are the means of two
independent experiments. (c, f) Enthalpy change on the binding of
THS-975 and THS-510 to the human Hsp90α NTD.
THS-510 was synthesized (Figure 3d). The binding activity
and isoform selectivity of THS-975 and THS-510 were
analyzed using the competitive binding assay. The IC50 values
of THS-975 for Hsp90α, Hsp90β, Grp94, and TRAP-1 were
around 300 μM (T/C (%) showed 51% at 300 μM), 218 μM,
>300 μM, and >300 μM, respectively (Figure 3b). The IC50
values of THS-510 for Hsp90α, Hsp90β, Grp94, and TRAP-1
were 293 μM, 89 μM, >300 μM, and >300 μM, respectively
(Figure 3e). Although both THS-975 and THS-510 are smaller
and their activity were very weak compared to TAS-116, THS-
975 and THS-510 clearly bound to the cytosolic isoforms
Hsp90α and Hsp90β but not to TRAP-1 or Grp94.
The direct binding of THS-975 and THS-510 to the human
Hsp90α NTD was investigated using isothermal titration
calorimetry (ITC). The calorimetric data were analyzed using
a single-site binding model to obtain the thermodynamic
parameters of the interaction between the human Hsp90α
NTD and these fragment compounds. In the case of THS-975,
although we obtained the exothermic binding reaction, the
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area (ASA) of Hsp90 in each complex. As shown in Table 1,
the nonpolar and polar ASAs of Hsp90 in the THS-510
complex (5809.7 and 4979.4 Å2) were larger than those in the
TAS-116 complex (5785.9 and 4900.9 Å2), respectively. This
difference was derived from the empty ATP-binding pocket of
Hsp90. We speculate that the hydrophobic surface of the ATP-
binding pocket is exposed when the THS-510 binding induces
α-helix formation of the ATP lid region.
Based on the putative binding mode of THS-510, we
proposed that there would be an affinity enhancement if THS-
510 was linked to a compound that filled the ATP-binding
pocket of Hsp90. Actually, THS-762 (Figure 5c), a conjugate
of THS-510 and THS-828, showed the affinity enhancement.
ITC revealed that the reaction of THS-762 with human
Hsp90α NTD was exothermic (Figure 5a). We could not
perform the ITC experiment with THS-828 due to its low
solubility. In comparison with the thermodynamic parameters
of THS-510, THS-762 had a higher binding affinity, with an
entropic gain and an enthalpic loss (Figure 5a). In THS-762,
hydrogen-bond networks between the benzamide moiety,
Asp93, and water molecules are expected to contribute to
the enthalpic gain, such as the case of TAS 116 analog 16d,27
and the cyclohexanol group at the solvent front may contribute
to the entropy gain. The decrease in favorable binding enthalpy
means that enthalpy−entropy compensation occurred in the
binding. Thus, TAS-116 is a favorable inhibitor, possessing the
enthalpic contribution based on THS-510.
DISCUSSION
■
The cytosolic Hsp90-selective inhibitor TAS-116 has a best-in-
class potential as an anticancer drug; clinical trials are
ongoing.23,24 In this study, we investigated the modes of
binding of TAS-116 and its analogs. Co-crystal structures of
Hsp90α with the TAS-116 analogs THS-828 (PDB ID:
3WQ9) and 16d (PDB ID: 5ZR3)27 showed that the
compounds bind to the Hsp90α helical form and interact
with the ATP-binding pocket. Unlike the other tested
inhibitors, TAS-116 bound to the Hsp90α mutant F138L
almost as well as it bound to the wild-type protein. By binding
to the third, hydrophobic pocket on Hsp90, TAS-116 has a
reduced dependency on F138 compared to the other inhibitors
tested, although modeling suggests that TAS-116 forms π−π
interactions with F138. We also analyzed a fragment of TAS-
116, THS-510, which includes only the moiety that binds to
the third pocket. ITC experiments showed that THS-510 binds
Hsp90α in an enthalpy-driven manner. THS-828, 16d, TAS-
116, and THS-510 all show cytosolic Hsp90 selectivity,
implying that inhibitors that bind to the third pocket are
highly selective for cytosolic Hsp90. Tricyclic compounds have
also been reported to bind Hsp90 in the third pocket region.32
SPR analysis showed that TAS-116 has a very slow off rate,
likely because it binds to Hsp90 in a conformation-specific
manner. We hypothesize that TAS-116 functions as a
conformation lock for cytosolic Hsp90. As conformational
change in Hsp90 is needed for its ATPase activity, TAS-116
might block this conformational change. Paralog inhibitors
such as the Grp94 inhibitor PU-H54 and the TRAP1 inhibitor
BIIB021 bind mainly in the ATP-binding pocket (Figure S7).38
In addition, it was reported that XL-888 occupies the ATP-
binding pocket, the second pocket, and the extended space of
the second pocket of Hsp90 in the co-crystal structure,39 but
the helical structure of the ATP lid region was disordered.
Therefore, the large conformational changes of the ATP lid in
Figure 4. (a) Schematic of complex of human Hsp90α NTD with
AUY922 and the competitive binding titration of THS-510 (upper).
The ITC profile of the titration of THS-510 into human Hsp90 NTD
in the presence of AUY922 (lower). (b) Schematic of the complex of
human Hsp90α NTD with BIIB021 and the competitive binding
titration of THS-510 (upper). The ITC profile of the titration of
THS-510 into human Hsp90 NTD in the presence of BIIB021
(lower).
Thermodynamic Characterization of TAS-116 and Its
Analogs. To characterize the biophysical properties of the
binding of TAS-116 to Hsp90, we examined the thermody-
namics of the interaction of Hsp90 with TAS-116 and its
analog THS-510. Binding reactions of both ligands to human
Hsp90α NTD were exothermic. The tight binding of TAS-116
to the human Hsp90α NTD (KD = 0.17 μM) was enthalpy
driven at 25 °C (Figure 5a).
To characterize the molecular mechanisms of the binding of
THS-510 and TAS-116 to Hsp90, we examined the heat
capacity changes (ΔCp) over the temperature range from 20 to
30 °C using ITC (Figure 5b, Figure S6). Unexpectedly,
binding of THS-510 to the human Hsp90α NTD had a
positive ΔCp value (0.56 kcal/mol·K), whereas that of TAS-
116 had a negative ΔCp value (−0.29 kcal/mol·K). The
negative value of ΔCp is suggestive of binding to a hydrophobic
buried region, as previously described for ligands with binding
that involves driving solvent molecules out of a pocket.32
There are previous works that described positive ΔCp
thermodynamics for the protein−small ligand interactions;
the positive heat capacity change is thought to result when the
ligand binds to an exposed nonpolar surface.36,37 Based on the
model structures, we calculated the solvent-accessible surface
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Figure 5. (a) Thermodynamic parameters for the interactions of THS-510, THS-762, and TAS-116 with human Hsp90α NTD. (b) Temperature
dependence of the binding enthalpy change for THS-510 and TAS-116 binding to the human Hsp90α NTD. (c) Chemical structure of THS-762
and predicted regions of Hsp90 bound by each part of the molecule.
Table 1. Solvent Accessible Surface Areas of Hsp90 and
Inhibitors TAS-116 and THS-510
that THS-510 did not interact with the ATP-binding pocket.
Based on this observation, hybrid compounds of THS-510 and
the ATP-binding pocket binders were synthesized, which were
bound with higher affinity to Hsp90 than THS-510. From this
result, it is expected that a hybrid design combining THS-510
and other Hsp90 inhibitors will provide a novel type of Hsp90
inhibitor. In the case of Hsp90, co-crystal structures helped
shape the design of the hybrid compound. However, even in
the absence of co-crystal structures, competitive ITC analysis
can reveal binding locations of lead compounds.
nonpolar (Å2)
polar (Å2)
total (Å2)
Hsp90
TAS-116
total
5785.9
61.7
5847.6
4900.9
1.0
4901.9
10686.8
62.7
10749.5
Hsp90
THS-510
total
5809.7
12.8
5822.5
4979.4
9.1
4988.5
10789.1
21.9
10811
CONCLUSIONS
■
In this study, the cytosolic Hsp90-selective inhibitor TAS-116
and analogs were analyzed using biophysical techniques
including ITC. The combined use of competitive ITC analysis
and molecular modeling enabled the prediction of binding
modes and revealed that compounds that bind to the third
binding pocket of Hsp90 interacted with an enthalpy gain and
induced the rigid conformation of ATP lid region. The third
binding pocket of the compound is thought to determine the
cytosolic Hsp90 binding selectivity and activity. A deeper
understanding of isoform selectivity of Hsp90 will facilitate the
design of additional compounds and will lead to a greater
understanding of the roles of Hsp90 paralogs in diseases.
Hsp90 and the positions of some residues in the second pocket
and the third pocket are crucial for the isoform selectivity of
Hsp90 inhibitors. As the roles of Hsp90 isoforms TRAP-1 and
Grp94 are not fully understood, whether or not cytosolic
selectivity will reduce the risk of adverse effects or influence
antitumor activity are not known. TAS-116 and analogs will
provide the opportunity to study the significance of isoform
selectivity. In situ analyses of the kinetics of isoform-specific
inhibition at the cellular level using fluorescently labeled
derivatives of TAS-116 that retain isoform selectivity may
clarify the functions of Hsp90 isoforms in more detail.
In this study, we utilized a combination of biophysical
methods to analyze the binding modes of the evaluated
inhibitors. Although ITC is a powerful tool for investigation of
label-free investigation of ligand interactions with proteins,
issues with compound solubility or aggregation may limit its
use. We were not able to detect the thermodynamic binding
energy of THS-828 to Hsp90 because THS-828 was not
soluble. This makes it difficult to compare thermodynamic
binding parameters of a series of compounds. However,
thermodynamic analysis does provide information that can
guide drug design. For example, knowledge of whether binding
was enthalpically or entropically driven was suggestive of
modes of action. Moreover, competitive ITC analyses revealed
EXPERIMENTAL SECTION
■
Materials. For the enzyme assay, human Hsp90α and human
Hsp90β were purchased from Enzo Life Sciences International, Inc.
GRP94 was purchased from ATGen Co., Ltd. TRAP-1 was purchased
from Enzo Life Sciences International, Inc. For the mutagenesis
analysis, the human Hsp90α gene was mutated by a polymerase chain
reaction (PCR) using PrimeSTAR Mutagenesis Basal Kit (Takara Bio
Company). For the ITC experiments and crystal structural analysis,
the NTD of human Hsp90α (amino acids 1−236) fused to a 6 × His
tag was expressed in E. coli BL21 and purified over a nickel-affinity
column. For SPR, the full-length human Hsp90α gene was cloned into
the pCR8/GW/TOPO cloning vector (Invitrogen). The gene was
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Article
then Gateway cloned into the destination vector, pEU-GST-DEST
containing a biotin ligation site at the 5′ end of the GST coding
sequence. The biotinylated GST-Hsp90α protein was expressed in the
wheat cell-free synthesis system.40
Chemicals. TAS-116, THS-828, THS-510, THS-762, BIIB021,
and AUY922 were synthesized at Taiho Pharmaceutical Co., Ltd. as
previously described.41,42 17-AAG was purchased from BIOTREND.
FITC-labeled geldanamycin was purchased from Enzo Life Sciences
International, Inc.
Surface Plasmon Resonance. Biotin-GST-Hsp90αNTD was
immobilized on a streptavidin sensor chip (SA-chip, GE Healthcare),
and measurements were obtained using a Biacore T-100 system (GE
Healthcare Life Sciences). For control experiments, the sensor chip
surface was analyzed in the absence of the compound. All experiments
were performed in a running buffer containing PBS and 1 mM DTT.
After the measurement, the sensor chip was washed and regenerated
with a regeneration buffer containing PBS, 1 mM DTT, and 5%
DMSO. All measurements were performed at 25 °C.
AUTHOR INFORMATION
■
Corresponding Author
Kouhei Tsumoto − The Institute of Medical Science and
Department of Bioengineering, Graduate School of
Engineering, The University of Tokyo, Tokyo 108-8639,
Japan; orcid.org/0000-0001-7643-5164; Phone: +81-3-
5841-0399; Email: tsumoto@bioeng.t.u-tokyo.ac.jp;
Fax: +81-3-5841-0397
Authors
Chihoko Yoshimura − Discovery and Preclinical Research
Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki
300-2611, Japan
Satoru Nagatoishi − The Institute of Medical Science, The
University of Tokyo, Tokyo 108-8639, Japan; orcid.org/
0000-0002-0794-3963
Competitive Binding Assay. The interaction of Hsp90
inhibitors with Hsp90 was evaluated using the fluorescence
polarization competitive binding assay with geldanamycin-FITC.43,44
Each compound was incubated with recombinant HSP90α for 2 h.
Geldanamycin-FITC was added, and the mixture was incubated for a
further 5 h. The concentration of each compound required to reduce
by 50% the amount of recombinant proteins bound to geldanamycin-
FITC (IC50) was determined, and the Ki values were calculated from
the IC50 values with the Chen−Prusoff equation.
Thermodynamic Analysis of the Binding of THS-510 to
Human Hsp90α NTD. Thermodynamic parameters for the
interaction of the ligand and human Hsp90α NTD were determined
using a MicroCal iTC200 isothermal titration calorimetry instrument
(Malvern Instruments). The compound solution (100 μM) was
titrated into a sample cell containing a solution of human Hsp90α
NTD (10 μM). These experiments were conducted in 150 mM NaCl,
20 mM Tris-HCl, 5% DMSO, pH 7.5 at a constant temperature over
the range of 15−30 °C. The free energy of ligand binding is described
by the following equation:
Daisuke Kuroda − Department of Bioengineering, Graduate
School of Engineering, The University of Tokyo, Tokyo 113-
8656, Japan; orcid.org/0000-0003-2390-4785
Yasuo Kodama − Discovery and Preclinical Research Division,
Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki 300-2611,
Japan
Takao Uno − Discovery and Preclinical Research Division,
Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki 300-2611,
Japan; orcid.org/0000-0002-3259-4603
Makoto Kitade − Chemical Technology Laboratory, CMC
Division, Taiho Pharmaceutical Co. Ltd., Kodama-gun,
Saitama 367-0241, Japan
Khoontee Chong-Takata − Discovery and Preclinical
Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba,
Ibaraki 300-2611, Japan
Hiromi Oshiumi − Formulation Research, CMC Division,
Taiho Pharmaceutical Co. Ltd., Kawauchi-cho, Tokushima
771-0194, Japan
ΔG = ΔH − TΔS
The heat capacity upon binding was derived from ITC experiments at
Hiromi Muraoka − Discovery and Preclinical Research
Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki
300-2611, Japan
different temperatures:
Satoshi Yamashita − Discovery and Preclinical Research
Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki
300-2611, Japan
Yuichi Kawai − Discovery and Preclinical Research Division,
Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki 300-2611,
Japan
Shuichi Ohkubo − Discovery and Preclinical Research
Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki
300-2611, Japan
d(ΔH)
ΔCp =
dT
Competitive Isothermal Binding Analysis. Competitive
isothermal titration analysis was performed by titrating ligand (600
μM) into the sample cell containing human Hsp90α NTD (20 μM)
and a known Hsp90 inhibitor (100 μM) at 25 °C.
Modeling Study. Using the co-crystal structures of human
Hsp90α NTD in complexes with 16d or THS-828 (PDB ID 5ZR3
and 3WQ9, respectively) as the templates, TAS-116 and THS-510
were manually put onto human Hsp90α NTD using MOE.45 The
protein was kept rigid, and the water molecules were mostly retained.
Energy minimizations were performed to maximize favorable
interactions between protein and ligand. The solvent-accessible
surface area was computed based on the Lee and Richards method
implemented in the NACCESS program.46
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01715
Author Contributions
C.Y. and K.T. conceived the study. All authors designed the
experiments and analyzed data. C.Y., S.N., and K.T. wrote the
manuscript. All authors approved the manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01715.
■
Funding
sı
This work was supported by the Platform Project for
Supporting Drug Discovery and Life Science Research (Basis
for Supporting Innovative Drug Discovery and Life Science
Research (BINDS)) from AMED of Japan under Grant
Number JP19am0101094 (to K.T.) and by JSPS KAKENHI-A
grant 16H02420 (to K.T.).
Scheme for the synthesis of compounds (TAS-116,
THS-510, THS-975, and THS-762), the data of SPR,
competitive binding assay, ITC, molecular model of
complexes (PDF)
Notes
Molecular formula strings (CSV)
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Ms. Chizuru Saruta for collecting ITC data.
ABBREVIATIONS USED
■
Hsp90, heat shock protein 90; NTD, N-terminal domain;
MOE, molecular operation environment; ITC, isothermal
titration calorimetry; SPR, surface plasmon resonance; ASA,
solvent-accessible surface area
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