Identification of HSP60 as a primary target of o - carboranylphenoxyacetanilide, an HIF-1α Inhibitor

Article abstract of DOI:10.1021/ja104739t

We succeeded in the design and synthesis of multifunctional chemical probes of the HIF-1α inhibitor carboranylphenoxyacetanilide (1) that combine photoaffinity labeling and click reaction to identify the target protein. HSP60 was identified as a primary target protein of 1 using the chemical probes 2 and 3. Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF activation, indicating that HSP60 affects HIF-1α accumulation directly or indirectly.

Full text of DOI:10.1021/ja104739t

Published on Web 08/09/2010
Identification of HSP60 as a Primary Target of
o-Carboranylphenoxyacetanilide, an HIF-1r Inhibitor
Hyun Seung Ban, Kazuki Shimizu, Hidemitsu Minegishi, and Hiroyuki Nakamura*
Department of Chemistry, Faculty of Science, Gakushuin UniVersity, Mejiro, Tokyo 171-8588, Japan
Received May 31, 2010; E-mail: hiroyuki.nakamura@gakushuin.ac.jp
Abstract: We succeeded in the design and synthesis of multi-
functional chemical probes of the HIF-1R inhibitor carboranylphe-
noxyacetanilide (1) that combine photoaffinity labeling and click
reaction to identify the target protein. HSP60 was identified as a
primary target protein of 1 using the chemical probes 2 and 3.
Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF
activation, indicating that HSP60 affects HIF-1R accumulation
directly or indirectly.
The hypoxia-inducible factor (HIF) is a basic helix-loop-helix
heterodimeric transcription factor composed of one of three subunits
(HIF-1R, -2R, or -3R) and a HIF-1ꢀ subunit.1-3 Under aerobic
conditions, HIF-1R is rapidly degraded via a 26S proteasome-
dependent pathway, whereas under hypoxic conditions, HIF-1R is
stabilized and translocated into the nucleus, where it dimerizes with
the constitutively expressed HIF-1ꢀ.4-7 The HIF-1R/ꢀ dimer binds
to specific nucleotide sequences [hypoxia-responsive elements
(HREs)] in the promoter of hypoxia-responsive genes such as
vascular endothelial growth factor (VEGF), insulin-like growth
factor, heme oxygenase-1, and inducible nitric oxide synthase.8,9
Among the HIF-regulated genes, VEGF plays a pivotal role in
pathological angiogenesis and tumor growth;10,11 therefore, the
inhibition of the VEGF inducer HIF is recognized as an attractive
strategy for cancer therapy.8,12 Our efforts have been focused on
the development of HIF inhibitors13-17 as antiangiogenesis agents.
Recently, we reported that o-carboranylphenoxyacetanilide (1)
induced a potent inhibitory effect on HIF-1R activation under
hypoxic conditions.18 In the present study, we clarified the action
mechanism of 1 against HIF inhibition.
Figure 1. Chemical probes of o-carboranylphenoxyacetanilide 1 and their
HIF inhibitory activities under hypoxic conditions. (A) Chemical structures
of 1 and the chemical probes 2 and 3. (B) Inhibitory effects of 1 and the
probes 2 and 3 on transcriptional activity of HIF were determined by HeLa
cell-based HRE reporter gene assay. (C) Effects of 1 (10 µM) and probes
2 and 3 (30 µM) on the hypoxia-induced increase in HIF-1R protein were
determined by immunoblot analysis in HeLa cells.
Standard chemical biology techniques, including photoaffinity
labeling, click conjugation, and biotinylation, are very useful tools
for detecting target proteins of biologically active molecules having
undefined action mechanisms.19-21 On the basis of this approach,
we designed and synthesized multifunctional chemical probes of 1
substituted with benzophenone to covalently bond with a target
protein by UV (photoaffinity labeling) and an acetylene moiety to
conjugate with azide-linked fluorescence by click reaction (Figure
1A). The chemical probes of 1 (2 and 3) were synthesized from
4-ethynylphenoxyacetic acid (5) and aminobenzophenones 6a-c
(Scheme S1 in the Supporting Information). To confirm whether
the synthesized probes exhibit HIF inhibitory activity, we examined
the effects of the probes on the hypoxia-induced activation of HIF
in HeLa human cervical cancer cells by HRE reporter gene assay
and immunoblot analysis. As shown in Figure 1B,C, although the
inhibitory effects of probes 2 and 3 against HIF transcriptional
activity were decreased almost 10-fold relative to 1 (Figure 1B),
the reducing property of the probes on the HIF-1R protein level
were parallel to that of 1 (Figure 1C). These results indicate that
Figure 2. Fluorescence imaging of target protein bound to the probe. (A)
HeLa cell lysate was irradiated for 30 min at 360 nm with various
concentrations (30, 100, and 300 µM) of each probe. The conjugation of
probe and Alexa Fluor 488 azide was performed by click reaction. (B) Total
cell lysates from HeLa cells were photoaffinity-labeled with 3 (100 µM) in
the presence or absence of 1 (500 µM).
the boronic acid in 1 is an efficient functional group for HIF
inhibition, a finding similar to the results of our previous study.18
Using these chemical probes, we performed photoaffinity labeling
in HeLa cell lysate and click conjugation with Alexa Fluor 488
azide to visualize the target protein (see the Supporting Information).
After sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), proteins bound to the probe Alexa Fluor 488 were
visualized by direct in-gel fluorescence detection. As shown in
Figure 2A, a major fluorescent band above the 55 kDa molecular
weight marker was detected by both probes in a concentration-
dependent manner, and binding of 3 with the protein was slightly
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10.1021/ja104739t  2010 American Chemical Society

C O M M U N I C A T I O N S
that stimulates HIF-1R degradation by affecting folding and
maturation.23-25 Unlike HSP90, the interaction of HSP60 and HIF-
1R has not been established. To our knowledge, this is the first
report demonstrating the implication of HSP60 in HIF activation.
However, details of the functional association between HSP60 and
HIF-1R remain to be elucidated.
In conclusion, we have succeeded in the design and synthesis
of multifunctional molecular probes of the HIF-1 inhibitor o-
carboranylphenoxyacetanilide 1 that combine photoaffinity labeling
and click reaction to identify the target protein. Using the chemical
probes 2 and 3, we identified that HSP60 is the target protein of 1.
Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF
activation, indicating that HSP60 affects HIF-1R accumulation
directly or indirectly.
Acknowledgment. This work was supported in part by a Grant-
in-Aid for Scientific Research on Priority Areas “Cancer Therapy”
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
Figure 3. Fluorescence imaging of probe-labeled recombinant HSP60 and
HIF inhibition by 4. (A) Recombinant human HSP60 (2 µg) was irradiated
with 3 (10 µM) in the presence or absence of 1 (100 µM) and click-
conjugated with Alexa Fluor 488 azide. (B) Structure of HSP60 inhibitor
4. (C) Inhibition of HIF transcriptional activity by 4 in HeLa cells.
Supporting Information Available: Detailed experimental proce-
dures for syntheses and biological assays, analytical data for compounds,
and results of biological experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
more sensitive than that of 2. To confirm the specificity of probe
binding, we performed competition assays with 1. As shown in
Figure 2B, competition with 1 resulted in almost complete
abrogation of the level of the fluorescent band at ∼55 kDa. Similar
results were also observed in a competition assay with 8c, an
acetylene-free analogue of the chemical probes 2 and 3 (Figure S1
in the Supporting Information). These results raise the possibility
that the band at ∼55 kDa may be the main target protein of 1 and
its probes.
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It has been reported that HIF-1R is one of the client proteins of
molecular chaperon HSP90 and geldanamycin, an HSP90 inhibitor
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