From Bacteria to Cancer: A Benzothiazole-Based DNA Gyrase B Inhibitor Redesigned for Hsp90 C-Terminal Inhibition

Article abstract of DOI:10.1021/acsmedchemlett.0c00100

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for the folding and maturation of client proteins that are associated with all ten hallmarks of cancer. Hsp90 N-terminal pan inhibitors have experienced unfavorable results in clinical trials due to induction of the heat shock response (HSR), among other concerns. Novobiocin, a well characterized DNA gyrase B inhibitor, was identified as the first Hsp90 C-terminal inhibitor that manifested anticancer effects without induction of the HSR. In this letter, a library of Hsp90 C-terminal inhibitors derived from a benzothiazole-based scaffold, known to inhibit DNA gyrase B, was designed, synthesized, and evaluated. Several compounds were found to manifest low micromolar activity against both MCF-7 and SKBr3 breast cancer cell lines via Hsp90 C-terminal inhibition.

Full text of DOI:10.1021/acsmedchemlett.0c00100

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Letter
From Bacteria to Cancer: A Benzothiazole-based DNA Gyrase
B Inhibitor Redesigned for Hsp90 C-terminal Inhibition
Kyler W. Pugh, zheng zhang, Jian Wang, Xiuzhi Xu, Vitumbiko Munthali, and Brian S.J. Blagg
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00100 • Publication Date (Web): 11 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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From Bacteria to Cancer: A Benzothiazole-based DNA Gyrase B
Inhibitor Redesigned for Hsp90 C-terminal Inhibition
Kyler W. Pugh,a, 1 Zheng Zhang,a, 1 Jian Wang,b Xiuzhi Xu,b Vitumbiko Munthali,a and
Brian S. J. Blagg*a
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a Department of Chemistry and Biochemistry, 305 McCourtney Hall, The University of Notre Dame, IN 46545, United States
b Fujian Medical University, Fuzhou, China
1These authors contributed equally to this work
KEYWORDS: Hsp90 inhibitors, DNA Gyrase B, Hsp90 C-terminal, Benzothiazole.
ABSTRACT: Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for the folding and maturation of client
proteins that are associated with all ten hallmarks of cancer. Hsp90 N-terminal pan inhibitors have experienced unfavorable results
in clinical trials due to induction of the heat shock response (HSR), among other concerns. Novobiocin, a well characterized DNA
gyrase B inhibitor, was identified as the first Hsp90 C-terminal inhibitor that manifested anticancer effects without induction of the
HSR. In this letter, a library of Hsp90 C-terminal inhibitors derived from a benzothiazole-based scaffold, known to inhibit DNA gyrase
B, was designed, synthesized, and evaluated. Several compounds were found to manifest low micromolar activity against both MCF-
7 and SKBr3 breast cancer cell lines via Hsp90 C-terminal inhibition.
The 90 kDa heat shock proteins (Hsp90) remain a sought
after target for the development of novel therapeutics for the
treatment of cancer and/or neurodegenerative diseases.1-2
Hsp90 is a molecular chaperone that plays a pivotal role in
cellular proteostasis and is responsible for the folding and/or
activation of more than 300 Hsp90-depedent client substrates.
Moreover, Hsp90-dependent client proteins such as Her2, Akt,
Raf1, CDK4/6, Src, telomerase, and survivin are directly
associated with the ten hallmarks of cancer, which further
supports the development of Hsp90 inhibitors for the
treatment of cancer. Hsp90 contains an N-terminal ATP-
binding site, a middle domain, and a C-terminus.3-4
only subunit that contains an ATP-binding domain.7, 8 Hsp90
and DNA gyrase B share high homology between their ATP-
binding sites.11 In fact, both proteins have a unique Bergerat
fold in this region that allows for the design of selective
inhibitors.14,15 Novobiocin is a clinically approved DNA Gyrase B
inhibitor used to treat bacterial infection by binding to the
Bergerat fold prese-
OH
OH
H
N
O
O
O
O
Historically, the focus of Hsp90 research centered on the
design of inhibitors that target the N-terminal ATP-binding-site
for the treatment of cancer, and this approach led to 17
therapeutic agents that underwent clinical investigation.
Unfortunately, the Hsp90 N-terminal inhibitors experienced
major challenges in the clinic, as induction of the pro-survival
heat shock response (HSR) led to increased levels of Hsp90 and
consequently, cytostatic activity.5 During the HSR, heat shock
proteins are overexpressed to maintain cell viability under
conditions in which proteins are often denatured.5-6 Therefore,
the discovery of novel Hsp90 inhibitors that manifest anti-
cancer activity without induction of the pro-survival HSR is
highly desired.6,16
O
MeO
O
OH
O
1
H2N
O
H
Br
Br
O
N
N
S
NH
HO
N
H
O
2
Figure 1. Structures of DNA Gyrase B and Hsp90 inhibitors.
(1) Novobiocin, a DNA Gyrase B and Hsp90 C-terminal
inhibitor, (2) Benzothiazole-based DNA Gyrase B inhibitor.
DNA gyrase is a Type II topoisomerase that reduces the
topological strain caused by the breaking and rejoining of
double stranded DNA during replication. DNA gyrase is
composed of two subunits (GyrA and B), whereas GyrB is the
nt in DNA Gyrase (Figure 1). Due to similarities between DNA
gyrase and Hsp90, Neckers and coworkers hypothesized that
novobiocin may also inhibit Hsp90’s Bergerat fold.9-10 Upon
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scientific investigation, novobiocin was found to inhibit Hsp90
Reagents and conditions: (a) Pyridine, rt, 12 h; (b) H2, 10%
Pd/C, MeOH and THF, rt, 12 h; (c) DCM, Et3N, rt, 12 h.
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3
4
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7
8
at a previously unrecognized C-terminal binding region and to
exhibit allosteric control over the N-terminal ATP-binding site.
Moreover, novobiocin was identified as the first Hsp90 C-
terminal inhibitor and remarkably, was capable of Hsp90
inhibition without induction of the HSR.9-10 Unfortunately,
novobiocin manifests an EC50 value of ~700 µM against Hsp90,
and no co-crystal structure of Hsp90 bound to a C-terminal
inhibitor exists, which hinders the development of improved
analogs.12 Consequently, it was proposed that other DNA
Gyrase B inhibitors (eg. compound 2) may also inhibit the
Hsp90 C-terminus.
R1
NH
N
S
R2
N
H
4
H
Br
Br
a
R1
N
S
N
S
O
N
NH2
+
NH
9
Cl
O2N
O2N
10
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9
10
5
R1
NH
N
S
b
R1
NH
N
Scaffold A
O2N
O
O
O
S
H2N
10
N
S
11
O
H
NH
N
N
H
O
Br
R1
NH
c
N
S
R1
NH
N
S
O
3
+
Cl
R2
Br
N
H2N
O
H
11
6
4-4k
H
N
Br
Br
Scaffold B
O
O
N
S
O
NH
N
H
Scheme 2. Synthesis of Scaffold B
O
4
Reagents and conditions: (a) DCM, Et3N, rt, 12 h; (b) H2,
10% Pd/C, MeoH and THF, rt, 12 h; (c) Pyridine, rt, 12 h.
Figure 2. Benzothaizole-based DNA Gyrase B inhibitors
reveal two novel scaffolds for Hsp90 C-terminal inhibition.
breast cancer cell lines, and compound 4 was demonstrated to
inhibit Hsp90 via the C-terminus (see Figure 1S). Therefore,
structural diversification of these scaffolds were sought and
included (1) modifications to the ethyl oxalyl substituent,
and/or (2) modifications/substitutions to the pyrrole.
RESULTS AND DISCUSSION
Two novel Hsp90 C-terminal scaffolds, A and B, were
selected (Figure 2) from a series of benzothiazole-based DNA
Gyrase B inhibitors discovered by Kikelj and coworkers (Figure
1, compound 2).7, 13 Recently, DNA Gyrase B inhibitors were
identified that manifest anti-influenza activity through Hsp90
inhibition, although no data was provided to clarify where the
compounds bound Hsp90.17 In this study, compounds 3 and 4
were shown to manifest anti-proliferative activity against
Replacements for the ethyl oxalyl substituent included an
acetamide, oxopropanamide, and an 2-oxo-phenylacetamide.
The acetamide group was selected to probe the substituents
that are necessary for activity. Whereas, investigation of the
electrophilic properties of the ethyl oxalyl moiety were
investigated via replacement with an oxopropanamide or 2-
oxo-2-phenylacetamide. A variety of aromatic moieties were
also proposed as surrogates for the pyrrole ring and included
indole derivatives, mono and di-bromo pyrroles, dimethyl
pyrrole, phenyl[1,3]dioxole, phenyl, and pyrimidine moieties.
R1
NH
N
S
R2
N
H
3
a
N
S
O
R1
NH
N
S
In this study, two general synthetic schemes were utilized
to prepare these scaffolds (Schemes 1 and 2). Commercially
available 6-nitrobenzo[d]thiazol-2-amine (5) was coupled with
ethyl oxalyl chloride (6) to form intermediate 7. The nitro
group was reduced to the aniline (intermediate 8) via a Pd-
catalyzed hydrogenolysis under a hydrogen atmosphere.
Subsequent amide coupling provided compound 3, as well as
related analogs (Scheme 1). Synthesis of analog 4 and scaffold
B derivatives, followed a similar synthetic scheme (Scheme 2).
Commercially available 6-nitrobenzo[d]thiazol-2-amine (5)
was coupled with acid chloride 9 to form intermediate 10. The
nitro group was reduced to the aniline (intermediate 11) by Pd-
catalyzed hydrogenolysis under a hydrogen atmosphere.
Subsequent amide coupling provided compound 4, as well as
NH2
+
O
Cl
O2N
O2N
O
5
6
7
R1
NH
N
b
R1
NH
N
S
O2N
S
H2N
7
8
R1
NH
N
S
H
Br
c
R1
NH
O
N
S
N
R2
+
N
Cl
H2N
H
Br
3-3d
9
8
Scheme 1. Synthesis of Scaffold A
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cancer cell lines, respectively (Table 2). Using the EC50 data
related analogs.
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2
3
4
5
6
7
8
manifested by 4e and 4d, we proposed that an increase in
electron density on the indole could contribute to an increase
in anti-proliferative activity. Although it is also possible that the
addition of a hydrogen bond acceptor may increase inhibitory
activity. Consequently, compound 4f was synthesized to
include an ethyl moi-
Several of the compounds displayed low µM EC50 values
against breast cancer cell lines. For example, compounds 3 and
4 manifested EC50 values of 3.59 µM and 2.41 µM against the
SKBr3 breast cancer cell line, respectfully (Tables 1 and 2).
Compound 3c was synthesized to contain a 4-bromo-pyrrole
substituent, but activity for this analog was decreased ~8-fold
as compared to lead compound 3. Additional substitutions to
the R1 side chain of Scaffold A were pursued, but those
compounds produced similar results. Compounds 3b and 3d
were also found to decrease anti-proliferative activity,
however, analogs derived from scaffold B continued to show
reasonable activity against both SKBr3 and MCF-7 breast
cancer cells. Based on the data collected, we shifted our focus
to the preparation of scaffold B analogs due to their greater
inhibitory activities. (Table 2).
N
R1
N
Table 2. RScaffold B Analogs
H
2
S
9
N
H
10
11
12
13
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SKBr3
EC50
MCF-7
EC50
Entry
R2
R1
(µM)
(µM)
H
N
O
O
Br
Br
O
O
4
2.57 ± 0.86
>50
>50
>50
O
Replacement of the ethyl oxalyl group with the 2-oxo-2-
phenylacetamide or an acetamide moiety on scaffold B,
resulted in compounds 4a-c, which were devoid of activity
(Table 2). Anti-proliferative data produced by 4d
demonstrated that substitutions on the indole ring led to
increased inhibitory activity when a methyl group was paced
at the 5-position, as
O
4a
4b
4c
4d
4e
4f
N
H
O
O
O
O
>50
>50
N
H
O
O
O
>50
>50
N
N
R1
H
Table 1. Scaffold A AnalNogs
O
O
R2
H
S
N
O
O
O
O
O
O
O
O
O
H
2.81 ± 0.4
0.86 ± 0.03
20.62
10.05 ± 2.0
7.03 ± 1.32
25.34
N
SKBr3
EC50
MCF-7
EC50
H
O
O
O
O
O
O
O
O
O
O
Entry
R2
R1
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O
O
O
O
O
O
O
(µM)
(µM)
N
H
H
O
Br
O
N
3
3.59 ± 1.83
10.05 ± 1.91
O
O
Br
Br
O
N
H
H
O
O
N
O
3a
3b
3c
>50
9.73
>50
>50
4g
4h
4i
6.70
>50
Br
N
H
O
O
O
O
O
N
N
O
O
>50
>50
O
O
H
O
O
N
O
27.90
41.26
5.41 ± 0.40
>50
8.99 ± 1.43
>50
N
Br
H
O
H
O
N
4j
3d
6.94 ± 3.90
11.22 ± 0.13
O
O
O
O
O
4k
10.66
>50
O
compared with the unsubstituted analog, 4i. SAR for the 5-
position of the indole moiety was further explored and
additional analogs prepared. This approach led to the
discovery of lead compound 4e, which exhibited an EC50 value
of 0.86 µM and 7.03 µM against SKBr3 and MCF-7 breast
*Compounds with calculated std. dev tested N=3, w/o N=1.
ety at the 5-position. The activity exhibited by 4f was
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diminished ~20 fold, and supported our previous hypothesis
Financial support of the project was provided by the NIH
(CA120458). We would like to thank Dr. Ang Zuo for his
assistance in preparing the manuscript for this project.
1
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4
5
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that electron donating groups increase potency for these
analogs. Therefore, a compound was synthesized to contain a
methoxy group at the 6-position to identify the optimal
position for inclusion of an electron donating group (4g). The
EC50 manifested by this compound increased to ~7 µM, which
when compared to 4e demonstrated that electron donating or
hydrogen bonding groups at the 5-position are more effective.
The anti-proliferative activities manifested by these
compounds were shown to result from Hsp90 inhibition, as
demonstrated via Western blot analysis of SKBr3 cell lysates
following the administration of compound 4e (Figure 3), as
Hsp90-dependent substrates were degraded without
induction of Hsp70 levels.
ABBREVIATIONS
Hsp90, Heat shock protein 90; HSR, heat shock response.
REFERENCES
9
1. Zhang, Z., You, Z., Dobrowsky, R. T., & Blagg, B. S. (2018).
Synthesis and evaluation of a ring-constrained Hsp90 C-
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Figure 3. Western blot analyses from SKBr3 lysates after
administration of compound 4e after 24 hr. [GDA]= 500 nM.
Image quantification provided in S.I.
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Katsamakas, S., Ilaš, J., Tammela, et. al., (2016).
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CONCLUSION
8. Burlison, J. A., Neckers, L., Smith, A. B., Maxwell, A., &
Blagg, B. S. J. (2006). Novobiocin: Redesigning a DNA
Gyrase Inhibitor for Selective Inhibition of Hsp90. J. Am.
Chem. Soc, 128(48), 15529–15536.
Two novel Hsp90 C-terminal inhibitory scaffolds were
identified from a series of DNA Gyrase B inhibitors. Compound
4 was shown to induce Hsp90 client protein degradation
without induction of the HSR, a hallmark of Hsp90 C-terminal
inhibition. Therefore, a library of analogs was prepared and
evaluated against MCF-7 and SKBr3 breast cancer cell lines.
Several analogs were shown to manifest potent anti-
proliferative activity against these breast cancer cells.
9. Marcu, M. G., Schulte, T. W., & Neckers, L. (2000).
Novobiocin and Related Coumarins and Depletion of
Heat
Shock
Protein
90-Dependent
Signaling
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However, Scaffold
B
compounds demonstrated greater
10. Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M., &
Neckers, L. M. (2000). The Heat Shock Protein 90
inhibitory activity than scaffold A, and therefore, SAR studies
were pursued on scaffold B. It was determined that compound
4e manifested the greatest potency and remains a lead
compound for future studies aimed at elucidating structure-
activity relationships for benzothiazole-based Hsp90 C-
terminal inhibitors. The results from such studies will be
reported in due course.
Antagonist Novobiocin Interacts with
a Previously
Unrecognized ATP-binding Domain in the Carboxyl
Terminus of the Chaperone. J. Biol. Chem, 275(47),
37181–37186.
11. Dutta, R., & Inouye, M. (2000). GHKL, an emergent
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ASSOCIATED CONTENT
12. Byrd, K. M., Subramanian, C., Sanchez, J., Motiwala, H. F.,
Liu, W., Cohen, M. S, et. al., (2016). Synthesis and
Biological Evaluation of Novobiocin Core Analogues as
Hsp90 Inhibitors. Chem.: Eur. J., 22(20), 6921–6931.
Supporting Information. Synthetic experimental details,
characterization of compounds, biological data.
13. Tomašič, T. et al, (2017). Design, synthesis and biological
ACKNOWLEDGEMENTS
evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-
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carboxamide derivatives as novel DNA gyrase
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14. Prodromou, C. et al., (1997). Identification and
Structural Characterization of the ATP/ADP-Binding Site
in the Hsp90 Molecular Chaperone. Cell 90, 65–75
15. Bisacchi, G. S. & Manchester, J. I., (2014). A New-Class
Antibacterial—Almost. Lessons in Drug Discovery and
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of Benzo[d]Thiazoles That Target Heat Shock Protein
90. Bioorg. Chem 98, 103733.
For Table of Contents Only
GRAPHICAL ABSTRACT
Hsp90 C-terminal inhibitor
DNA Gyrase B inhibitor
H
Br
Br
O
O
O
N
N
S
N
S
O
O
N
NH
NH
O
H
HO
N
N
H
H
O
O
2
4e
SKBr3 EC50: 10.50 µM
SKBr3 EC50: 0.86 µM
5
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