Diverging Novobiocin Anti-Cancer Activity from Neuroprotective Activity through Modification of the Amide Tail

Article abstract of DOI:10.1021/acsmedchemlett.6b00224

Novobiocin is a natural product that binds the Hsp90 C-terminus and manifests Hsp90 inhibitory activity. Structural investigations on novobiocin led to the development of both anti-cancer and neuroprotective agents. The varied pharmacological activity man

Full text of DOI:10.1021/acsmedchemlett.6b00224

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Letter
Diverging Novobiocin Anti-cancer Activity from
Neuroprotective Activity through Modification of the Amide Tail
Suman Ghosh, Yang Liu, Gaurav Garg, Mercy Anyika, Nolan T.
McPherson, Jiacheng Ma, Rick T. Dobrowsky, and Brian S.J. Blagg
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00224 • Publication Date (Web): 05 Jul 2016
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Diverging Novobiocin Antiꢀcancer Activity from Neuroprotective
Activity through Modification of the Amide Tail
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Suman Ghosh1#, Yang Liu3#, Gaurav Garg1, Mercy Anyika1, Nolan T. McPherson1, Jiacheng Ma2, Rick
T. Dobrowsky2, and Brian S. J. Blagg1*
2
1Department of Medicinal Chemistry; Department of Pharmacology and Toxicology, The University of Kansas, Lawrence,
3
#
Kansas 66045; Department of Medicinal Chemistry, Fujian Medical University, Fuzhou China 350004. These authors
contributed equally. *To whom correspondence should be addressed: Brian S. J. Blagg. The University of Kansas,
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, 4070 Malott Hall, Lawrence, KS 66045ꢀ7563. Tel: 785ꢀ864ꢀ
4495. Fax: 785ꢀ864ꢀ5326. Eꢀmail: bblagg@ku.edu.
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Key Words: Hsp90, Hsp90α, Aha1, antiꢀcancer, neuroprotection, novobiocin, structureꢀactivity
relationship.
____________________________________________________________________________________
Abstract: Novobiocin is a natural product that binds the Hsp90 Cꢀterminus and manifests Hsp90
inhibitory activity. Structural investigations on novobiocin led to the development of both antiꢀcancer
and neuroprotective agents. The varied pharmacological activity manifested by these novobiocin analogs
prompted the investigation of structureꢀfunction studies to identify these contradictory effects, which
revealed that modifications to the amide side chain produce either antiꢀcancer or neuroprotective
activity. Compounds that exhibit neuroprotective activity contain a short alkyl or cycloalkyl amide side
chain. In contrast, antiꢀcancer agents contain five or more carbons, disrupt interactions between Hsp90α
and Aha1, and induce the degradation of Hsp90ꢀdependent client proteins.
____________________________________________________________________________________
and Hsp90, and often results in cytostatic
activity.4 Although difficult to overcome for the
treatment of cancer, increased chaperone levels
are beneficial for the treatment of
neurodegenerative disorders that result from the
accumulation of aggregated or misfolded
proteins, such as Alzheimer’s and Huntington’s
disease.5 Thus, Hsp90 is considered a target for
Introduction:
Hsp90 represents a highly sought after
target for the treatment of cancer as substrates
dependent upon Hsp90 are associated with all
ten hallmarks of cancer.1ꢀ3 Hsp90 is
overexpressed in cancer to fold oncogenic
substrates in the presence of various coꢀ
chaperones. While inhibition of Hsp90 can
induce the degradation of oncogenic clients via
the ubiquitinꢀproteasome pathway, it also
induces the proꢀsurvival heat shock response,
which leads to concomitant induction of the heat
shock proteins, including Hsp27, Hsp40, Hsp70
both
cancer
and
neurodegeneration.6ꢀ9
Therefore, methods to segregate these activities
represent a novel paradigm for which Hsp90
modulators can be developed to treat cancer or
neurodegeneration.
Figure 1. Structures of novobiocin based Hsp90 Cꢀterminal inhibitors
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KUꢀ32 (Figure 1) is a neuroprotective
Scheme 1. Synthesis of KUꢀ32 Analogs.
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novobiocin derivative that binds the Hsp90 Cꢀ
terminal dimerization domain10ꢀ12 and induces a
robust heat shock response without concomitant
client protein degradation. KUꢀ32 manifests
efficacy in attenuating the death of cortical
neurons induced by βꢀamyloid peptides12 and
can improve multiple physiological indices of
diabetic peripheral neuropathy.9,13ꢀ15 The
efficacy exhibited by KUꢀ32 to protect against
glucotoxicity correlates directly with an
increase in mitochondrial bioenergetics.9 KUꢀ32
does not induce the degradation of Hsp90ꢀ
dependent client proteins such as Akt and Raf
until much higher concentrations than that
needed to induce the heat shock response and
promote neuroprotection.13,14
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In contrast to the acetamide side chain
on KUꢀ32, KUꢀ174 is a novobiocin derivative
that contains an aryl amide side chain and
exhibits potent antiꢀcancer activity by inducing
the degradation of Hsp90ꢀdependent client
proteins without concomitant induction of the
heat shock response.16 Unfortunately, the
mechanisms for distinguishing between these
opposing activities remain unclear. Since Hsp90
forms a complex with several proteins to assist
in the protein folding process, interactions
between Hsp90 and its coꢀchaperones were
investigated, which ultimately revealed the
subtle nuances manifested by these two distinct
classes of novobiocin analogs.
Activator of Hsp90 ATPase Activity
(Aha1) is a coꢀchaperone that binds to and
facilitates the ATPase activity of Hsp90, which
is required during the protein folding process.17ꢀ
23 We have shown previously that a novobiocinꢀ
derived, Hsp90 Cꢀterminal inhibitor could
disrupt the Hsp90α/Aha1 complex.22 Those
studies indicated that the noviose sugar was
responsible for binding Hsp90 while the
benzamide side chain present in KUꢀ174 (Figure
1) interacted with Aha1, and when combined,
manifested antiꢀcancer activity.22 In contrast,
replacement of the benzamide with an
acetamide chain as in the case of KUꢀ32, did not
disrupt the Hsp90α/Aha1 complex and
consequently, did not exhibit antiꢀcancer
activity.22
In an effort to systematically investigate
the differences manifested by the alkyl and aryl
containing amide side chains, structureꢀfunction
studies were investigated to identify the point of
divergence in which a neuroprotective agent is
transformed into an antiꢀcancer agent.
Results and Discussion:
Analogs containing increasingly larger
alkyl and cycloalkyl groups on the amide side
chain were pursued (Scheme 1) to identify the
point at which KUꢀ32 is transformed from a
neuroprotective agent into an antiꢀcancer agent.
As shown in scheme 1, synthesis of these
analogs began by the noviosylation of phenol
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resulting benzyl carbonate furnished aniline 4,
Table 1. Antiꢀproliferative activity of KUꢀ32 analogs.
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which underwent amide coupling with acids
containing sequentially larger alkyl substituents,
followed by solvolysis of the carbonate to afford
the desired diols, 5AꢀI, in moderate to good
yields.
The antiꢀproliferative activity manifested
by these compounds was evaluated against the
highly metastatic, Her2 overꢀexpressing SkBr3
breast and the androgenꢀindependent PC3ꢀMM2
prostate cancer cell lines. Increasing the alkyl
chain length or the inclusion of a cycloalkyl ring
onto the amide side chain resulted in a sizeꢀ
dependent increase in antiꢀproliferative activity
as shown in Table 1, which was dependent upon
chain length (R2=0.8732) or bulk (R2=0.8626)
(Figure 2).
Entry
R
SKBr3
(IC50, µM)
PC3ꢀMM2
(IC50, µM)
>900
>500
504
KU32
5A
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643 ± 50a
313 ± 9
466
5B
118 ± 43
84 ± 38
317
5C
53.7 ± 0.35
34.7 ± 1.82
>500
5D
58.3 ± 2.1
517 ± 29
220 ± 16
67.0 ± 1.4
5E
Since rematuration of firefly luciferase is
dependent upon the Hsp90 protein folding
machinery, the refolding of denatured firefly
5F
284 ± 99.85
144.5 ± 10.6
5G
5H
A.
46.8 ± 0.7
54.55 ± 4.92
5I
aValues represent mean ± standard deviation for at
least two separate experiments performed in
triplicate.
A.
600
R2=0.8732
5A
KU-32
5B
400
B.
5C
200
5E
5D
0
0
2
4
6
Chain Length
600
R2=0.8626
B.
KU-32
5F
400
200
0
5G
Figure 3. KUꢀ32 analogs inhibit Hsp90ꢀ
dependent luciferase refolding. Luciferase
activity fold change compared to DMSO (1.0
fold) after incubation of heatꢀdenatured
luciferase with (A) KUꢀ32, 5A, 5B, 5C, 5D,
5E, or (B) KUꢀ32, 5F, 5G, 5H or 5I. 17ꢀAAG
5H
5I
0
2
4
6
Ring Size
Figure 2. The IC50 (µM) values of PC3ꢀMM2
cells were plotted against the chain length (A) or
the ring size (B).
124 with activated noviose25ꢀ29 (2) in the
presence of catalytic boron trifluoride etherate
to give 3 in good yield. Hydrogenolysis of the
was used as
a
positive control. The
concentrations of KUꢀ32 and analogs used
during this assay ranged from 0.02 to 20 ꢁM.
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whereas compounds containing longer chains
A.
1
(5D and 5E) or carbocycles (5H and 5I)
inhibited the reꢀmaturation of firefly luciferase
(Figure 3). In exciting contrast, analogs with
shorter chains (KUꢀ32, 5Aꢀ5C) or small
carbocycles (KUꢀ32, 5Fꢀ5G) stimulated the reꢀ
maturation of firefly luciferase. In fact, there
was a clear point of divergence for the inhibition
of luciferase refolding. These data suggest that
the antiꢀproliferative activities manifested by
these compounds (Table 1) result from
modulation of the Hsp90 protein folding
machinery (Figure 3).
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B.
Increased chaperone activity can
produce neuroprotection that can be readily
assessed through measurement of mitochondrial
function via the seahorse bioassay. Previous
studies revealed that KUꢀ32 improved
mitochondrial bioenergetics and could reverse
sensory neuropathy in vivo.9,12 Since the
preliminary studies demonstrated that
a
systematic increase in chain length or the larger
cycloalkyl rings resulted in antiꢀproliferative
activity, analogs with larger side chains were
expected to reduce the maximal respiratory
capacity in the neuronal cells. As shown in
Figure 4, the inclusion of propyl to heptyl alkyl
chains led to a progressive decrease in maximal
respiratory capacity, compared to KUꢀ32.
C.
Figure
4.
KUꢀ32
analogs
worsen
mitochondrial bioenergetics in 50B11
neuronal cell line. AꢀB. 50B11 cells were
treated for 24h with KUꢀ32 or its analogs (5
ꢁM each) for mitochondrial bioenergetic
analysis. Basal oxygen consumption rate
(OCR) was measured prior to the addition of
oligomycin (a) to assess ATPꢀcoupled
respiration, FCCP (b) to measure uncoupled
respiration, and (c) rotenone+antimycin A to
assess nonꢀmitochondrial respiration. C. The
OCR values at the 88.3 min were plotted and
compared to the DMSO value.
HSF1
1.0 2.0 1.2 1.1 1.5 0.6 0.6
Actin
HSF1
1.0
4.0 2.0
0.1
3.7 3.7
Actin
Figure 5. The expression of HSF1 upon
treatment with the KUꢀ32 analogs. PC3ꢀ
MM2 cells were treated with KUꢀ32 or its
analogs (10 ꢁM each) for 24 h and the levels of
HSF1 were measured compared to the levels of
βꢀactin.
luciferase was assessed to confirm whether
these analogs retained the ability to inhibit
Hsp90.10,29ꢀ31 As expected, KUꢀ32 did not
inhibit the ability of Hsp90 to refold luciferase,
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Similarly, substitution of propyl to hexyl
not induce the heat shock response (Figure 5),
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carbocycles also produced a decline in maximal
respiration. Thus, the decrease in maximal
respiratory capacity correlated directly with the
antiꢀproliferative activity manifested by these
analogs (Table 1).
Heat Shock Factor 1 (HSF1) is the
transcription factor that serves to regulate the
heat shock response, and is required to manifest
neuroprotective activity.32 Hsp90 binds HSF1 to
but did exhibit decreased mitochondrial
respiration (Figure 4), increased antiꢀ
proliferative activity (Table 1), and Hsp90
inhibitory activity (Figure 3). Consequently,
these compounds were further evaluated to
determine whether they could induce the
degradation of known Hsp90ꢀdependent client
proteins. As shown in figure 6, the degradation
of Hsp90ꢀdependent client proteins such as
Her2, Akt, Rafꢀ1 occurred in the presence of
5D, 5E, 5H, and 5I without induction of Hsp90
levels, which is a hallmark of Hsp90 Cꢀterminal
antiꢀcancer agents.26,33ꢀ35 Compounds 5Aꢀ5C, 5F
and 5G were unable to induce Hsp90ꢀclient
protein degradation at 100 µM.
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form
a
stable complex, which prevents
induction of the heat shock response under
normal conditions. However, upon exposure to
high temperature or Hsp90 inhibitors, this
complex can disassemble and induce the heat
shock response, which is necessary to refold
proteins that become denatured at high
temperature. Neuroprotective agents are sought
that activate HSF1 and refold protein aggregates
that occur in many neurodegenerative
disorders.32 Therefore, the levels of HSF1 were
evaluated in PC3ꢀMM2 cells treated with KUꢀ
32 and its analogs. The results clearly show that
treatment with KUꢀ32 activated HSF1 ~ 2ꢀ3.7
fold (Figure 5). However, as the size of the alkyl
or cycloalkyl substitution increased, the levels
of HSF1 decreased (Figure 5), once again
suggesting that smaller groups (KUꢀ32, 5Aꢀ5C,
5Fꢀ5H) induce the proꢀsurvival response,
whereas larger groups (5D, 5E, 5I) prevent
induction of the heat shock response.
Since an increase in luciferase
maturation was observed in Figure 3 with
shorter amide side chains, it was hypothesized
that Aha1 remained bound to Hsp90 under these
conditions, whereas in the presence of larger
side chains, Aha1 association was disrupted.
Therefore, Hsp90α/Aha1 interactions were
investigated in PC3ꢀMM2 cells treated with
compounds 5AꢀI for 24 h, before Aha1 was coꢀ
immunoprecipitated and the percent of Hsp90α
remained bound to Aha1 determined. As
expected, Hsp90α/Aha1 disruption occurred
more readily for analogs with large alkyl and
cycloalkyl amide side chains (Figure 7). The
percent of Hsp90α remained bound to Aha1 was
plotted against the IC50 values obtained from
Table 1, which once again demonstrated a
Compounds containing larger alkyl or
cycloalkyl substitutions 5D, 5E, 5H, and 5I did
5D
5E
5H
5I
L
H
L
H
L
H
L
H
Her2
Akt
Raf1
Hsp90
Actin
Figure 6. Cytotoxic KUꢀ32 analogs downꢀregulate the expression of Hsp90 client proteins. PC3ꢀ
MM2 cells were treated with the cytotoxic analogs of KUꢀ32 (5D, 5E, 5H, and 5I) with the
concentrations of H: 3xIC50 and L: 0.5xIC50 for 24 h and the levels of known Hsp90 client proteins
were measured. Actin was used as the loading control.
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chain is sufficient for the disruption of
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Hsp90α/Aha1 interactions and defines a point of
divergence that transforms a neuroprotective
agent into an anticancer agent.
-
Hsp90α
Aha1
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Supporting Information
The supporting information is available free of
charge on the ACS publication website at
http://pubs.acs.org
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Acknowledgements
This work was supported by National Institutes
of Health grants [CA109265] to BSJB,
[DK095911] to RTD and [NS075311] to BSJB
and RTD.
Aha1IP
Figure 7. KUꢀ32 analogs disrupt the
Hsp90α/Aha1 complex. AꢀB. PC3ꢀMM2 cells
were treated with nothing (NT), DMSO (0.1%),
KUꢀ32 (100 µM), 5A (100 µM), 5B (100 µM), 5C
(100 µM), 5D (50 µM), 5E (35 µM), or 5F (100
µM), 5G (100 µM), 5H (100 µM), 5I (50 µM) for
24 h . Aha1 was immunoprecipitated and Hsp90α
and Aha1 were analyzed by western blotting. Aha1
bound Hsp90α was quantified using Image J
software and expressed as percent bound compared
to the cells treated with 0.1% DMSO (control).
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Lay Summary:
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In this study, it was determined that the size of
the amide side chain on novobiocin elicits a
direct effect on several properties that are
mediated by the Hsp90 molecular chaperone.
The shorter amide side chains exhibited proꢀ
survival activity that required assembly of the
Hsp90/Aha1 complex, whereas longer side
chains disassembled the Hsp90/Aha1 complex
and led to more potent antiꢀproliferative
activities.
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