Discovery of novel anti-breast cancer agents derived from deguelin as inhibitors of heat shock protein 90 (HSP90)

Article abstract of DOI:10.1016/j.bmcl.2020.127374

A series of O-substituted analogues of the B,C-ring truncated scaffold of deguelin were designed as C-terminal inhibitors of heat shock protein 90 (HSP90) and investigated as novel antiproliferative agents against HER2-positive breast cancer. Among the synthesized compounds, compound 80 exhibited significant inhibition in both trastuzumab-sensitive and trastuzumab-resistant breast cancer cells, whereas compound 80 did not show any cytotoxicity in normal cells. Compound 80 markedly downregulated the expression of the major client proteins of HSP90 in both cell types, indicating that the cytotoxicity of 80 in breast cancer cells is attributed to the destabilization and inactivation of HSP90 client proteins and that HSP90 inhibition represents a promising strategy to overcome trastuzumab resistance. A molecular docking study of 80 with the homology model of a HSP90 homodimer showed that 80 fit nicely in the C-terminal domain with a higher electrostatic complementary score than that of ATP.

Full text of DOI:10.1016/j.bmcl.2020.127374

Journal Pre-proofs
Discovery of Novel Anti-Breast Cancer Agents Derived from Deguelin as In‐
hibitors of Heat Shock Protein 90 (HSP90)
Cong-Truong Nguyen, Jihyae Ann, Raghaba Sahu, Woong Sub Byun,
Sangkook Lee, Gibeom Nam, Hyun-Ju Park, Soeun Park, Yoon-Jae Kim, Ji
Young Kim, Jae Hong Seo, Jeewoo Lee
PII:
S0960-894X(20)30485-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127374
BMCL 127374
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 June 2020
22 June 2020
29 June 2020
Please cite this article as: Nguyen, C-T., Ann, J., Sahu, R., Sub Byun, W., Lee, S., Nam, G., Park, H-J., Park, S.,
Kim, Y-J., Young Kim, J., Hong Seo, J., Lee, J., Discovery of Novel Anti-Breast Cancer Agents Derived from
Deguelin as Inhibitors of Heat Shock Protein 90 (HSP90), Bioorganic & Medicinal Chemistry Letters (2020),
doi: https://doi.org/10.1016/j.bmcl.2020.127374
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Graphical Abstract
Discovery of Novel Anti-Breast Cancer Agents Derived from Deguelin as Inhibitors of Heat
Shock Protein 90 (HSP90)
Cong-Truong Nguyen a, Jihyae Ann a, Raghaba Sahu a, Woong Sub Byun a, Sangkook Lee a, Gibeom Nam b,
Hyun-Ju Park b, Soeun Park c,d, Yoon-Jae Kim c,e, Ji Young Kim c, Jae Hong Seo c,d, Jeewoo Lee a,*

Bioorganic & Medicinal Chemistry Letters

Discovery of Novel Anti-Breast Cancer Agents Derived from Deguelin as
Inhibitors of Heat Shock Protein 90 (HSP90)
Cong-Truong Nguyen a, Jihyae Ann a, Raghaba Sahu a, Woong Sub Byun a, Sangkook Lee a, Gibeom Nam
b, Hyun-Ju Park b, Soeun Park c,d, Yoon-Jae Kim c,e, Ji Young Kim c, Jae Hong Seo c,d, Jeewoo Lee a,*
a College of Pharmacy, Seoul National University, Seoul 08826, Korea
b School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea
c Division of Medical Oncology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 152-703, Republic of Korea.
d Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea University, Seoul 152-703, Republic of Korea.
e Department of Biomedical Research Center, Korea University Guro Hospital, Korea University, Seoul 152-703, Republic of Korea.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of O-substituted analogues of the B,C-ring truncated scaffold of deguelin
were designed as C-terminal inhibitors of heat shock protein 90 (HSP90) and
investigated as novel antiproliferative agents against HER2-positive breast cancer.
Among the synthesized compounds, compound 80 exhibited significant inhibition in
both trastuzumab-sensitive and trastuzumab-resistant breast cancer cells, whereas
compound 80 did not show any cytotoxicity in normal cells. Compound 80 markedly
downregulated the expression of the major client proteins of HSP90 in both cell types,
indicating that the cytotoxicity of 80 in breast cancer cells is attributed to the
destabilization and inactivation of HSP90 client proteins and that HSP90 inhibition
represents a promising strategy to overcome trastuzumab resistance. A molecular
docking study of 80 with the homology model of a HSP90 homodimer showed that 80
fit nicely in the C-terminal domain with a higher electrostatic complementary score
than that of ATP.
Keywords:
HER2-positive breast cancer
Human epidermal growth factor receptor 2
Heat shock protein 90
Deguelin
2017 Elsevier Ltd. All rights reserved.
Human epidermal growth factor receptor 2 (HER2) is
amplified in 20-30% of all breast cancers, and amplification of
the gene correlates with a poorer prognosis.1-3 HER2 is a
member of the HER receptor tyrosine kinase family, which also
includes EGFR (HER1), HER3 and HER4.4 The interactions
between the intracellular domains of the HER receptors lead to
the activation of the kinase domains and the subsequent
transphosphorylation of several tyrosine residues. The
phosphorylation event promotes the expression of genes that act
together to regulate cell survival, proliferation and
differentiation, which drives tumor progression.5-7 Trastuzumab,
a humanized anti-HER2 monoclonal antibody, has significant
clinical benefits for disease-free patients and improves the
overall survival of patients with HER2-amplified metastatic and
early breast cancer.8-9 However, nearly 70% of patients do not
respond to trastuzumab when given as a single agent therapy due
to either primary or acquired resistance, suggesting that
overcoming this issue remains a large unmet clinical need.10
Since HER2 is one of the most sensitive client proteins of
HSP90, the inhibition of HSP90 could be an effective strategy
for the treatment of HER2-positive breast cancer. HSP90 is a
homodimeric protein approximately 90 kDa in size, and each
monomer contains an N-terminal ATP-binding domain, a middle
cochaperone and client-binding domain, and a C-terminal
dimerization domain. Most HSP90 inhibitors that have been
clinically studied were identified to bind to the ATP-binding
domain in the N-terminus.13-14 However, although these N-
terminal inhibitors have shown encouraging efficacy in clinical
trials, these inhibitors induce a heat shock response (HSR) that
ultimately leads to an increase in the production of HSP90 and
anti-apoptotic proteins, such as HSP70 and HSP27. Therefore,
the inhibition of a different ATP-binding site in the C-terminus
provides an alternative strategy to overcome these side effects.15-
18
The extensive efforts directed at the development of the C-
terminal inhibitors as well as isoform-selective inhibitors have
been reported based on natural products such as novobiocin and
deguelin.19-20
Heat shock protein 90 (HSP90) is a molecular chaperone that
is responsible for the functional competence of HER2 by
regulating the stability, maturation and activity of HER2.11-12
Deguelin (1) is a naturally occurring rotenoid that exhibited
potent antitumor activity by inhibiting ATP binding to the C-

terminal inhibition of HSP90.21 To discover a novel scaffold for
a C-terminal HSP90 inhibitor based on deguelin, we22 and other
group23 have investigated simplified deguelin analogues through
a ring-truncation approach. The simplified deguelin surrogates
displayed more potent antiproliferative and antiangiogenic
activities compared to that of deguelin. The mechanistic studies
revealed that the ring-truncated analogues also disrupted HSP90
function by binding to its C-terminal ATP-binding pocket and
interfering with the interaction with its cochaperones and client
proteins, triggering their degradation.22
Figure 1. Deguelin and its B,C-ring-truncated analogue
In this study, we have investigated O-substituted
analogues of the B,C-ring truncated template with an amide
linker (e.g., 2 for R=Me) as anti-breast cancer agents in which a
variety of polar side chains as solubilizing moieties were
incorporated to improve the pharmacodynamic and
pharmacokinetic properties of the analogues. The synthesized
compounds were screened and evaluated for their cytotoxicity
towards two types of HER-2-positive breast cancer cells,
trastuzumab-sensitive and trastuzumab-resistant cells, along with
normal human cells. Once a potent inhibitor was found in the
series, we sought to explore its concentration-dependent
cytotoxicity in breast cancer cell lines. In addition, we explored
the effects of the inhibitor on the major clients of HSP90 in a
mechanistic study and performed molecular modeling to identify
the binding mode of the inhibitor in the C-terminus of HSP90.
Scheme 1. Reagents and conditions: (a) RBr or RI, K2CO3, DMF, 70 °C, 3 h;
(b) ROH, PPh3, DEAD, CH2Cl2, 0 °C-r.t., overnight; (c) BrCH2(CH2)nBr,
K2CO3, DMF, 70 °C,
3 h; (d) amine (piperidine, morpholine, 1-
methylpiperazine, or N-Boc piperazine), DMF, 70 °C, overnight
Generally, the final compounds were synthesized by
coupling between the A-ring amines, which were prepared from
4-nitroguaiacol derivatives, and 5-methoxy-2,2-dimethyl-2H-
chromene-6-carboxylic acid acted as the D-E rings. The acyclic
side chains were incorporated into 4-nitroguaiacol by either the
Williamson reaction with the corresponding halides to give 4-5
or the Mitsunobu reaction with the corresponding alcohols to
provide 6-10. The cyclic side chains were also incorporated
using these two methods. First, 4-nitroguaiacol was alkylated
with 1,2-dibromoethane or 1,3-dibromopropane to produce O-
bromoalkyl derivatives (11, 12), which were then reacted with
the corresponding amines to yield 13-17. Using the Mitsunobu
reaction with compound 3 and the corresponding alcohols
provided 18-29 (Scheme 1).
For the synthesis of the final compounds (Scheme 2), the 4-
nitroguaiacol derivatives (4-10, 13-29) were reduced to their
corresponding amines and then coupled with chromene acid
under 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
conditions to yield the final compounds (65, 68, 71, 72, 75, 77,
78, 82-84, 86, 89, and 90) or the penultimate products (54-64).
The Boc or TBS deprotection of 54-64 resulted in the final
compounds (66, 67, 69, 70, 73, 76, 79, 80, 85, 87, and 88). The
N-methyl derivatives 74 and 81 were obtained from 73 and 80 by
reductive amination, respectively.
Scheme 2. Reagents and conditions: (a) H2, Pd/C, MeOH, 2 h; (b) 5-
methoxy-2,2-dimethyl-2H-chromene-6-carboxylic acid, EDC.HCl, HOBt,
TEA, CH2Cl2, reflux, overnight.; (c) TFA, CH2Cl2, r.t., overnight; (d) TBAF,
CH2Cl2, r.t., overnight; (e) (HCHO)n, ZnCl2, CH2Cl2, r.t., 1 h; then NaBH4,
reflux, overnight

Table 1. Synthesized HSP90 inhibitors
Figure 2. Primary screening of NCT analogues (2, 65-90) (a-c) Effects of
the synthesized 27 compounds on cell viability in HER2-positive breast
cancer cells and normal human embryonic kidney HEK293 cells.
Trastuzumab-sensitive BT474 (a), trastuzumab-resistant JIMT-1 (b) and
normal HEK293 (c) cells were treated with each of the compounds at 10 µM
for 72 h, and the cell viability was assessed using a MTS assay. The red
dotted boxes indicate the antiproliferative activity of compound 80 in cancer
cells (a-b) and normal cells (c). The results are presented as the mean ± SEM
of at least three independent experiments and analyzed using Student’s t-test
(* p<0.05, versus DMSO control).
To examine the primary antiproliferative activities of the
synthesized compounds (Table 1) in HER2-positive breast
cancer cells, we evaluated the cytotoxicity of the 27 compounds
in trastuzumab-sensitive BT474 cells, trastuzumab-resistant
JIMT-1 cells (Figure 2) and normal human embryonic kidney
(HEK293) cells at a concentration of 10 μM for 72 h. Among the
tested compounds, nine compounds (73, 74, 80, 81, 84, 85, 86,
87, and 88) suppressed cell viability by levels below 50% in both
the BT474 and JIMT-1 cells (Figure 2A and 2B). In particular,
compound 80 exhibited significant inhibition of cell proliferation
in HER2-positive breast cancer cells, while it had minimal
cytotoxicity in normal HEK293 cells (Figure 2C).
Among the compounds tested in the primary screening,
compound 80 showed the most promising cytotoxicity in HER2-
positive breast cancer cells. To further examine the
concentration-dependent cytotoxicity of 80, BT474 and JIMT-1
cells were treated with various concentrations of 80 (0.1, 0.5, 1,
5, 10 or 20 μM) for 72 h. MTS assays revealed that 80 (1-20 μM)
significantly suppressed the cell viability in both BT474 and
JIMT-1 cells in a dose-dependent manner. The inhibitory
concentrations (IC50) of 80 in BT474 (a) and JIMT1 (b) cells
were 8.53 μM (95% CI: 6.429 - 11.33 μM)] and 4.45 μM (95%
CI: 2.959 - 6.691 μM), respectively (Figure 3).
Figure 3. Effect of compound of 80 on cell viability in trastuzumab-
sensitive and trastuzumab-resistant HER2-positive breast cancer cells.
(a-b) Effect of 80 on the cell viability in BT474 (a) and JIMT-1 (b) cells. The
cells were treated with various concentrations of 80 (0.1, 0.5, 1, 5, 10 or 20
µM) for and 72 h. The cell viability was determined by MTS assay. The
results are presented as the mean ± SEM of at least three independent
experiments and analyzed by one-way ANOVA followed by Bonferroni’s
post hoc test (* p<0.05, versus DMSO control). IC50 values and 95%
confidence intervals (CI) were calculated with GraphPad software (version 5;
GraphPad Prism, San Diego, CA).
We examined the effect of 80 on the expression and
activation of the major clients of HSP90, including HER2,
EGFR and survivin, in BT474 and JIMT-1 cells in vitro.
Treatment with 80 (IC50 values of approximately 5-10 µM)
markedly downregulated the expression of the total HER2 and
total EGFR proteins, concomitant with decreases in their
phosphorylation in both trastuzumab-sensitive and trastuzumab-
resistant cells (Figure 4A), suggesting that HSP90 inhibition
represents
resistance.
a potential strategy to overcome trastuzumab

A molecular docking study of compound 80 was conducted
to determine the binding mode of compound 80 in the C-terminal
domain of HSP90. The flexible docking of 80 was analyzed
using the homology model of the previously prepared open form
of a HSP90 homodimer.26,27 As illustrated in Figure 5A,
compound 80 fits well in the C-terminal domain, and the 2-
piperidine moiety occupies the ATP binding pocket where the
deoxyribose moiety is located. The protonated amine group
forms a strong hydrogen bond with the carboxy side chain of
Glu611. Two aromatic groups, from the central benzene and
chromene rings, create π-cation interactions with Lys615 of each
HSP90 chain. The methoxy and amide carbonyl oxygen groups
of compound 80 also form hydrogen bonds with Asn622 and
Lys615 (A), respectively. These various interactions allow
compound 80 to be in contact with both chains of HSP90 and
stabilize the open form of the HSP90 homodimer (Figure 5B).
We also conducted docking analysis for both the R and S form of
compound 80 and obtained similar poses and docking scores,
suggesting that stereochemistry is only a minor factor in the
compound’s activity (data not shown). Last, we explored the
electrostatic complementarity (EC) surface and score of
compound 80 by using the Flare program.28 By using XED force
field calculations, the complementarity of the ligand and protein
were visualized. As shown in Figure 5C, the overall structure of
compound 80 is electrostatically favored by HSP90. Both the
electrostatic complementarity (EC) surface and score indicate
that compound 80 binds well to HSP90, and since compound 80
has a higher EC score than ATP, these results support the idea
that compound 80 is an effective C-terminal HSP90 inhibitor.
Figure 4. Effect of compound 80 on the expression of HSP90 client
proteins and HSP70 in HER2-positive breast cancer cells. (a) BT474 and
JIMT-1 cells were treated with the indicated concentrations of 80 for 72 h.
Equal amounts (30 µg) of protein from the total cell lysates were separated
by SDS-PAGE and immunoblotted using HER2, phospho-HER2
(Tyr1221/1222), EGFR and phospho-EGFR (Tyr1068), survivin antibodies.
Actin was used as a loading control for the total cell lysates. (b) Immunoblot
analyses of HSP90 and HSP70 protein expression in BT474 and JIMT-1 cells
following exposure to 80 for 72 h. (c) Representative confocal images of
immunocytochemistry for HSP70 expression. Cells were immunostained for
HSP70 (green) and nuclei were counter-stained with DAPI (blue) after
treatment of geldanamycin (Gelda; 0.3 µM) or 80 (10 µM) for 24 h.
Microphotographs were taken using confocal microscopy (original
magnification: ×500).
In summary, to discover C-terminal terminus inhibitor of
HSP90 as novel anti-breast cancer agents, we investigated a
series of O-substituted analogues of the B,C-ring truncated
template of deguelin with an amide linker as anti-breast cancer
agents in which a variety of polar side chain were incorporated.
The synthesized compounds were evaluated for their
cytotoxicities towards two types of HER2-positive breast cancer
cells, trastuzumab-sensitive (BT4747) and trastuzumab-resistant
(JIMT-1) cells, and normal human cells in the primary screening.
Among the synthesized compounds, compound 80 exhibited
significant inhibition of cell proliferation in HER2-positive
breast cancer cells, with an IC50 = 8.53 and 4.45 μM in BT474
and JIMT1, respectively, whereas compound 80 exhibited
minimal cytotoxicity in normal HEK293 cells. A mechanistic
study of 80 indicated that it markedly downregulated the
expression of the total HER2 and total EGFR proteins,
concomitant with decreasing their phosphorylation, and
additionally considerably decreased the content of the anti-
apoptotic protein survivin in both BT474 and JIMT-1 cells. The
immunocytochemical analysis for HSP70 in the presence of 80
or N-terminal HSP90 inhibitor indicated that the inhibitory effect
of 80 on cancer cell proliferation was caused by the
destabilization and inactivation of HSP90 client proteins without
the induction of HSR as evidence of the C-termial inhibitor.
These results indicate that the cytotoxicity of compound 80 in
breast cancer cells is attributed to the destabilization and
inactivation of HSP90 client proteins and that HSP90 inhibition
is a promising strategy to overcome trastuzumab resistance. A
molecular docking study of compound 80 with the homology
model of the open form of the HSP90 homodimer indicated that
compound 80 fit well in the C-terminal domain through key
interactions with the domain, and the electrostatic
complementary score of compound 80 was higher than that of
ATP.
The anti-apoptotic protein survivin, a member of the
inhibitor of apoptosis protein (IAP) family, is an important
determinant in the regulation of cell division and proliferation
and in the evasion of apoptosis.24,25 Exposure to 80 considerably
decreased the survivin protein content in both BT474 and JIMT-
1 cells (Figure 4A). We next examined whether 80 treatment is
associated with the induction of HSR in HER2-positive breast
cancer cells, expression levels of HSP90 and HSP70 in the
presence or absence of 80 were determined by Western blot
analysis. There were no differences in protein expression of
HSP90 and HSP70 between the control vehicle- and 80-treated
cells (Figure 4B). To confirm this observation,
immunocytochemical analysis for HSP70 in the presence of 80
or N-terminal HSP90 inhibitor, geldanamycin was performed.
Further evidence supported the notion that exposure to 80 did not
affect HSP70 expression, while N-terminal HSP90 inhibitor,
geldanamycin treatment resulted in a marked upregulation of
HSP70 (Figure 4C). These observations suggest that the
compound 80-induced inhibitory effect on cancer cell
proliferation could be caused by the destabilization and
inactivation of HSP90 client proteins without the induction of
HSR.

Figure 5. Molecular docking model of compound 80 with HSP90. (A) Compound 80 represented as a green ball-and-stick model (Surflex-Dock score: 8.3236,
CScore: 5). The two HSP90 chains are indicated in orange (Chain A) and sky-blue (Chain B). The hydrogen bonds, π-cation interactions and salt bridges are
represented as yellow, green and magenta dashed lines, respectively. (B) The binding site of 80 in the dimerization interface. Overall, the HSP90 homodimer is
represented as a ribbon, and the binding site is represented with an electrostatic potential surface. The top view of the 80 docking model is highlighted in the
upper black box. (C) Lipophilic potential surface (brown color: hydrophobic; blue color: hydrophilic) of the HSP90 homodimer and 80. The Connolly surface of
80 is represented by a green mesh. (D) The electrostatic complementarity (EC) surface and score. Green = perfect electrostatic complementarity, red = perfect
electrostatic clash.
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Acknowledgments
This work was supported by a grant (HI12C1852, 1720340) from
the Korea Health Technology R&D Project through the Korea
Health Industry Development Institute (KHIDI), funded by the
Ministry of Health & Welfare, Republic of Korea.
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Graphical Abstract

Discovery of Novel Anti-Breast Cancer Agents Derived from Deguelin as Inhibitors of Heat
Shock Protein 90 (HSP90)
Cong-Truong Nguyen a, Jihyae Ann a, Raghaba Sahu a, Woong Sub Byun a, Sangkook Lee a, Gibeom Nam b,
Hyun-Ju Park b, Soeun Park c,d, Yoon-Jae Kim c,e, Ji Young Kim c, Jae Hong Seo c,d, Jeewoo Lee a,*