A cyclic peptide inhibitor of HIF-1 heterodimerization that inhibits hypoxia signaling in cancer cells

Article abstract of DOI:10.1021/ja402993u

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1β protein-protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein-protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

Full text of DOI:10.1021/ja402993u

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A Cyclic Peptide Inhibitor of HIF‑1 Heterodimerization That Inhibits
Hypoxia Signaling in Cancer Cells
Elena Miranda,†,# Ida K. Nordgren,†,# Abigail L. Male,† Charlotte E. Lawrence,† Franciane Hoakwie,†
Francesco Cuda,† William Court,∥ Keith R. Fox,‡,§ Paul A. Townsend,§,⊥,∇ Graham K. Packham,§,⊥
Suzanne A. Eccles,∥ and Ali Tavassoli*,†,§,⊥
‡
§
†Chemistry, Centre for Biological Sciences, Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ,
United Kingdom
∥Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton SM2 5NG, United Kingdom
⊥Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom
S
* Supporting Information
ABSTRACT: Hypoxia inducible factor-1 (HIF-1) is a
heterodimeric transcription factor that acts as the master
regulator of cellular response to reduced oxygen levels, thus
playing a key role in the adaptation, survival, and progression
of tumors. Here we report cyclo-CLLFVY, identified from a
library of 3.2 million cyclic hexapeptides using a genetically
encoded high-throughput screening platform, as an inhibitor of
the HIF-1α/HIF-1β protein−protein interaction in vitro and
in cells. The identified compound inhibits HIF-1 dimerization
and transcription activity by binding to the PAS-B domain of
HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the
closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for
the identification of protein−protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted
cancer therapeutics.
binds HIF-1β to form the HIF-1 transcription factor complex.
HIF-1 rapidly mounts a transcriptional response to hypoxia1,10
by directing the expression of a wide variety of hypoxia response
genes.11,12 By utilizing changes in the substrate concentration of
a continuously occurring enzymatic reaction (hydroxylation of
HIF-1α), the cellular response to hypoxia is near instanta-
neous,13 with HIF-1α acting as both the sensor and a key
component of the hypoxia response machinery.
Inhibition of HIF-1 has long been known to hold much
potential for cancer therapy;14 there are multiple possible
points for therapeutic intervention in the hypoxia response
network, and molecules that inhibit various components of this
diverse pathway have been reported,15−22 but the absolute
requirement for the dimerization of HIF-1α and HIF-1β for
DNA binding and transcription activity of the HIF-1 complex
makes this protein−protein interaction a seemingly optimal
point of interception. Several high-throughput screens have
been conducted in the effort to identify HIF-1 inhib-
itors,15,19,21,22 but there are currently no selective inhibitors
of the HIF-1α/HIF-1β protein−protein interaction. The only
reported inhibitor of HIF-1 dimerization is the heteroaromatic
INTRODUCTION
■
Homeostasis of oxygen, a key metabolite, is critical for
mammalian cell survival. This necessitates a robust network that
senses and rapidly responds to hypoxia (low oxygen levels). The
key component of this hypoxia response network is hypoxia-
inducible factor (HIF), a heterodimeric transcription factor
composed of an oxygen-regulated α-subunit and a ubiquitously
expressed β-subunit (also known as the aryl nuclear tran-
scription factor or ARNT). Mammals possess three isoforms
of HIF-α; the ubiquitously expressed HIF-1α mounts the
immediate response to reductions in cellular oxygen,1 while
HIF-2α (also known as EPAS1) and HIF-3α are thought to
regulate the response to prolonged hypoxia. While the intricate
interplay between HIF-α isoforms in cancer is complicated and
yet to be fully deciphered,2 the role of HIF-1 activity in angio-
genesis, tumor growth, and metastasis is well established.3,4
HIF-1α is overexpressed in many cancers,5 and oncogene activa-
tion and loss of tumor suppressor function is shown to be
associated with HIF-1 activation.6
HIF-1α is negatively regulated at the protein level by oxygen
via prolyl hydroxylase enzymes that use oxygen as a substrate
for the hydroxylation of residues 402 and 564 of HIF-1α,
marking it for ubiquitination by an E3 ubiquitin ligase complex
and rapid proteolysis.7,8 Reduced oxygen levels lead to the
stabilization and nuclear translocation of HIF-1α,9 where it
Received: March 25, 2013
Published: June 24, 2013
© 2013 American Chemical Society
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acridine derivative acriflavine, which nonselectively inhibits
both HIF-1 and HIF-2.19
P2, and Tat-cyclo-CLLRMY as P3 (Figure 1, R = CGRKKR-
RQRRRPPQ).
A compound that specifically inhibits HIF-1 dimerization in
cells will not only serve as a chemical tool to decipher the mecha-
nism of hypoxia response, but would also form the starting
point for the development of HIF-1-directed therapeutic agents.
Here we report an inhibitor of the HIF-1α/HIF-1β protein−
protein interaction, identified from a genetically encoded library
of 3.2 million cyclic peptides. The most potent identified cyclic
peptide (cyclo-CLLFVY) inhibits HIF-1 dimerization in vitro
and in cells by binding to the PAS-B domain of HIF-1α, and
prevents HIF-1- but not HIF-2-mediated hypoxia signaling in a
variety of cell lines.
P1 Inhibits HIF-1 Activity in a Mammalian Cell Lucif-
erase Reporter Assay. The ability of P1−P3 to disrupt
HIF-1 function in cells was assessed using a HIF-1-dependent
luciferase reporter assay.15,21 The assay uses human osteosar-
coma U2OS cells, stably transfected with a HIF-dependent
luciferase reporter construct (U2OS-HRE-luc), where activa-
tion of HIF results in an increase in luciferase expression.15
Hypoxia (1% O2) results in a ∼12-fold increase in the
luciferase signal, which is inhibited in a dose-dependent manner
by P1 (IC50 of 19 2 μM); P1 did not alter basal luciferase
activity in normoxia (Figure 2A). P2 and P3 did not show an
effect on the luciferase reporter in hypoxia or normoxia. P1−P3
RESULTS AND DISCUSSION
■
Identification of HIF-1 Heterodimerization Inhibitors
Using a Genetically Encoded High-Throughput Screen-
ing Platform. We used our genetically encoded high-
throughput screening platform23−25 to identify inhibitors of
the HIF-1α/HIF-1β protein−protein interaction. We built and
verified the function of a HIF-1 bacterial reverse two-hybrid
system (RTHS, for a detailed description see the Supporting
Information and Figures S1−S3). The HIF-1 RTHS was used
to screen a plasmid-encoded SICLOPPS (split intein circular
ligation of peptides and proteins)26,27 library of 3.2 million
cyclic hexapeptides for HIF-1 dimerization inhibitors.
After the first round of screening, 120 surviving colonies were
observed and subjected to several rounds of secondary
screening to eliminate false positives and nonselective
inhibitors, leaving 12 potential HIF-1 dimerization inhibitors
that were ranked by drop-spotting. Four of these peptides were
significantly more active than the others; the SICLOPPS
plasmids encoding these 4 peptides were sequenced to reveal
the identity of the HIF-1 inhibitors as cyclo-CLLFVY (encoded
by two SICLOPPS plasmids), cyclo-CRLMVL, and cyclo-
CLLRMY (Figure 1, R = H). Interestingly, the two isolated
Figure 2. Effect of P1−P3 in a HIF-1 luciferase-reporter assay. Data
shows fold-increase of the luciferase signal compared to untreated cells
in normoxia. (A) P1 causes a dose-dependent reduction in the HIF-1-
mediated luciferase signal in hypoxic U2OS cells. (B) Assay in (A)
repeated in MCF-7 cells. (C) P1 inhibits the HIF-1-mediated
luciferase signal in a normoxic reporter assay in U2OS cells. (D)
Assay in (C) repeated in MCF-7 cells. (E) Representative blots
showing that 100 μM P1 does not alter HIF-1α protein levels in the
cells in (C) and (D).
Figure 1. Cyclic peptide HIF-1 inhibitors. Cyclic peptide HIF-1
inhibitors identified from a SICLOPPS library of 3.2 million cyclic
hexapeptides (R = H).
cyclo-CLLFVY plasmids encoded the peptide via different codons.
To ensure that the cyclic peptides, not their unspliced peptide
aptamers, were responsible for the observed inhibition of
HIF-1, we utilized mutant SICLOPPS dnaE C-terminal inteins
(H24A, F26A) that do not splice.28 The unspliced peptides lost
the ability of their parent cyclic equivalents to disrupt HIF-1α/
HIF-1β dimerization (Figure S3B), demonstrating that HIF-1
inhibition by these peptides is dependent on their cyclic form.
The three identified HIF-1 inhibitors were synthesized and
tagged with Tat peptide (via a disulfide bond between the set
cysteine of the cyclic peptide and a cysteine introduced to the
start of Tat)25 to aid the translocation across the plasma mem-
brane of mammalian cells. In the following experiments,
Tat-cyclo-CLLFVY is referred to as P1, Tat-cyclo-CRLMVL as
had no effect in the U2OS-luc control cell line15 stably express-
ing luciferase (Figure S4), indicating that P1 does not inhibit
endogenous cellular processes such as transcription or
translation. To assess the cell-specificity of P1, the experiment
was repeated in MCF-7 breast cancer cells with similar results
(P1 IC50 of 16 1 μM) (Figure 2B). Tat-tag alone (100 μM)
did not affect the luciferase signal in these assays (Figure 2A
and 2B).
HIF-1 has been shown to directly promote the expression of
HIF-1α in hypoxia by binding to a hypoxia-response element
(HRE) upstream of the HIF-1α.29 This has been shown to be
dependent on the methylation state of a cytosine in the HIF-1
binding site (ACGTG) upstream of HIF-1α; a methylated
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cytosine prevents HIF-1 binding and inhibits the autotransacti-
vation of HIF-1α in hypoxia.29 Bisulfite sequencing of this region
of MCF-7 and U2OS cells revealed this HRE to be unmethylated,
and thus, an inhibitor of HIF-1α/HIF-1β dimerization would
potentially reduce the HIF-1-promoted upregulation of HIF-1α
mRNA and protein levels in these cell lines. The reduction of
luciferase signal observed in P1-treated U2OS and MCF-7 cells
(Figure 2A and B) could therefore be partially a result of a
reduction in hypoxic HIF-1α levels (this would be the case for any
inhibitor of HIF-1 dimerization in these cell lines). To decouple
P1’s effect on HIF-1α transactivation from its effect on HIF-1α/
HIF-1β dimerization, a normoxic luciferease-reporter assay was
devised where HIF-1α is expressed from a transiently transfected
vector, resulting in continuously elevated levels of HIF-1α in
normoxic cells. P1 continued to inhibit the luciferase signal in both
U2OS and MCF-7 cells in this assay (Figure 2C and D), with
HIF-1α protein levels not being altered by P1 (Figure 2E).
The observed discrepancy between the activity of cyclo-
CRLMVL and cyclo-CLLRMY in the HIF-1 RTHS (Figure S3B),
and the lack of activity of P2 and P3 in the luciferase assay
(Figure 2) is likely due to the 10-fold range in each step of the
drop-spotting assay; P2 and P3 may be up to 10-fold less active
than P1 and still result in the same drop-spotting pattern. P1
was therefore taken forward for further assessment of its
activity. P2, a Tat-tagged cyclic hexapeptide that differs from P1
by two amino acids, was used as a negative control in the
following experiments.
Figure 3. Assessing the activity of cyclo-CLLFVY in vitro. (A) Effect of
10 nM to 500 μM P1 on the heterodimerization of His-HIF-1α1−350 with
GST-HIF-1β1−474 analyzed by ELISA; P1 disrupts this interaction with an
IC50 of 1.3
0.5 μM. (B) Effect of 10 nM to 500 μM P1 on the
cyclo-CLLFVY Disrupts HIF-1, but Not HIF-2 Dimeriza-
tion in vitro by Binding to the PAS-B Domain of HIF-1α.
We next probed the effect of cyclo-CLLFVY on the interaction
of HIF-1α with HIF-1β in vitro. Recombinant His-HIF-1α1−350
and GST-HIF-1β1−474 were produced and purified. Electro-
phoretic mobility shift assay (EMSA) was used to demonstrate
that the recombinant HIF-1 proteins form functional
heterodimers (Figure S5A). Unfortunately, the positively
charged Tat-tag of P1 is incompatible with EMSA (interferes
with the bandshift of DNA). We therefore assessed the ability
of P1 to disrupt HIF-1 dimerization by enzyme-linked
immunosorbent assay (ELISA); P1 was found to disrupt the
protein−protein interaction of His-HIF-1α1−350 and GST-HIF-
1β1−474 with an IC50 of 1.3 μM (Figure 3A). The control
compound P2 had no effect on HIF-1 dimerization in this assay
(Figure S6). To determine the HIF-1-specificity of cyclo-
CLLFVY, we assessed its ability to disrupt HIF-2 dimerization
by ELISA. Recombinant His-HIF-2α1−351 was produced and
purified, and shown to form functional heterodimers with GST-
HIF-1β1−474 by EMSA (Figure S5B). P1 had no effect on the
dimerization of His-HIF-2α1−351 with GST-HIF-1β1−474 (Figure 3B).
To identify the target (HIF-1α or HIF-1β) of our HIF-1
inhibitor, we synthesized a biotinylated derivative of cyclo-
CLLFVY for use as bait in pull-down assays by replacing the
cysteine residue (present in all members of the SICLOPPS
library to allow intein splicing) with propargylalanine. This
compound was linked to biotin-PEG-azide by click-chemistry
(copper-catalyzed alkyne azide reaction) to give biotin-PEG-
triazole-cyclo-ALLFVY as the bait molecule. The bait was
immobilized onto streptavidin-coated beads and mixed with
recombinant His-HIF-1α1−350 and GST-HIF-1β1−474. The pulled-
down protein(s) were analyzed by Western blot, revealing His-
HIF-1α1−350 as the target of cyclo-CLLFVY (Figure 3C, lane 3).
Streptavidin beads did not pull down either HIF-1 subunit in
the absence of the bait molecule (Figure 3C, lane 2), and
propargylalanine click-linked to biotin-PEG-azide did not pull
interaction of His-HIF-2α1−351 with GST-HIF-1β1−474; P1 does not affect
HIF-2 heterodimerization. (C) His-HIF-1α1−350 is selectively pulled-down
by streptavidin beads coated with biotin-PEG-triazole-cyclo-ALLFVY
(lane 3); lane 1 is the loading control. Neither protein is pulled-down
by streptavidin beads alone (lane 2), or streptavidin beads coated with a
biotin-linked control (lane 4). (D) Fluorescent binding assay showing a
Megastoke 673-labeled derivative of cyclo-CLLFVY binding to His-HIF-
1α1−350, while its binding to His-HIF-2α1−351 is close to background levels.
The fluorescent derivative of cyclo-CLLFVY binds the PAS-B domain of
HIF-1α, whereas binding to bHLH and PAS-A domains are close to
background level. (E) ITC shows P1 binding to the PAS-B domain of
HIF-1α with 1:1 stoichiometry and 124 23 nM affinity. Red line shows
control P1 injection into buffer only.
down either HIF-1 subunit (Figure 3C, lane 4). To verify
binding of cyclo-CLLFVY to HIF-1α, we used the propargyla-
lanine derivative of this molecule and azide-Megastoke dye
673 to synthesize a fluorescent derivative by click-chemistry.
His-HIF-1α1−350 and His-HIF-2α1−351 were immobilized onto
Ni2+-coated 96-well plates, and the fluorescent analogue of
cyclo-CLLFVY was washed over these proteins. Binding of
the fluorescent derivative to these proteins was monitored via
increased fluorescence at 680 nm. We observed binding of the
fluorescent derivative of cyclo-CLLFVY to His-HIF-1α1−350
,
with binding to His-HIF-2α1−351 at close to background levels
(Figure 3D).
We next sought to identify the domain of HIF-1α bound by
cyclo-CLLFVY. The HIF-1α protein used in our RTHS is com-
posed of the DNA-binding basic-helix−loop−helix domain (bHLH,
amino acids 1−80) and the protein−protein interaction Per-
ARNT-SIM-A (PAS-A, amino acids 90−155) and Per-ARNT-
Sim-B (PAS-B, amino acids 235−350) domains. Recombinant
His-bHLH, His-PAS-A, and His-PAS-B domains were immo-
bilized onto Ni2+-coated 96-well plates, and the fluorescent derivative
of cyclo-CLLFVY was washed over the bound proteins. We observed
an increase in fluorescence (at 680 nm) of the PAS-B domain,
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whereas bHLH and PAS-A remained close to background,
indicating binding of our inhibitor to the PAS-B domain of
HIF-1α (Figure 3D). We next used P1 in isothermal titration
calorimetry (ITC) to verify these observations and quantify
binding affinities; P1 bound the PAS-B domain of HIF-1α in
1:1 stoichiometry and with a KD of 124 nM (Figure 3E). P1 did
not bind to the bHLH or PAS-A domain of HIF-1α (Figures
S7A and B), and P2 did not bind the HIF-1α PAS-B domain
(Figure S7C). P1 did not bind HIF-1β1−474 (Figure S7D). The
observed selectivity of P1 for HIF-1 over HIF-2 in vitro (Figure
3A versus B, and 3D) suggests the possibility of selectivity for
HIF-1 over HIF-2 in cells.
P1 Disrupts HIF-1 Dimerization in MCF-7 and U2OS
Cells. The effect of P1 on the endogenous HIF-1α/HIF-1β
interaction in intact cells was directly probed using an in situ
proximity ligation assay (PLA);30 primary antibodies (HIF-1α
and HIF-1β here) raised in different species are bound by
specific secondary antibodies that are tagged with a short DNA
strand. The DNA on the interacting PLA probes forms a mini-
plasmid that is amplified and bound by a red fluorescent dye.
The HIF-1α/HIF-1β interaction is thus visualized as red dots in
the DAPI-stained nuclei of MCF-7 and U2OS cells (Figure 4).
P1 Inhibits HIF-1-Mediated Cellular Hypoxia Response.
The effect of P1’s disruption of the HIF-1α/HIF-1β
heterodimer on cellular hypoxia response was next charac-
terized. We measured the effect of P1 on vascular endothelial
growth factor (VEGF), a HIF-1 regulated gene that stimulates
vasculogenesis and angiogenesis in hypoxia.31 The transcription
of VEGF increased ∼3-fold after incubation for 16 h in hypoxia
in untreated MCF-7 and U2OS cells; P1-treated cells showed a
dose-dependent reduction in VEGF mRNA in both cell
lines, while the control peptide P2 (100 μM) had no effect
(Figure 5A). VEGF protein levels increased 3- to 5-fold in both
MCF-7 and U2OS cells after 16 h of incubation in hypoxia,
with P1-treatment resulting in a dose-dependent reduction of
VEGF protein (measured by a quantitative immunoassay) in
both cell lines; pretreatment with 50 μM of P1 fully inhibited
the hypoxic induction of VEGF protein, resulting in normoxic
VEGF levels in hypoxic MCF-7 and U2OS cells (Figures 5B).
As VEGF is a regulator of angiogenesis, we next probed the
effect of P1 on HIF-1 mediated tubule formation in hypoxic
human umbilical vein endothelial cells (HUVEC),32 and
observed a dose-dependent reduction of HUVEC tubulariza-
tion in P1-treated cells, with no effect from P2 (Figure 5C; for
representative images, see Figure S8). The effect of P1 on
HIF-1 signaling was further assessed via carbonic anhydrase IX
(CAIX), an extracellular metalloenzyme whose expression is
significantly upregulated (∼25-fold) by HIF-1 after 16 h
incubation in hypoxia.33 Treatment of MCF-7 and U2OS cells
with 50 μM P1 resulted in a 5-fold reduction of CAIX mRNA
in hypoxia, with no effect from 100 μM P2 (Figure 5D). A
sulforhodamine B (SRB) cytotoxicity assay34 was used to
demonstrate that the observed effects are not due to toxicity of
P1 (Figure 5E). The cytotoxicity and effect of P1 on cell
proliferation was further probed by measuring the effect of
increasing doses of P1 (1−100 μM) on MCF-7 cells over 72 h,
with 1 μM 4-hydroxytamoxifen used as a positive control. P1 at
100 μM did not affect the viability of MCF-7 cells, whereas
1 μM 4-hydroxytamoxifen inhibited proliferation as expected
(Figure 5F).
P1 Does Not Affect HIF-2-Mediated Hypoxia Signal-
ing. The cellular HIF-1-specificity of P1 was probed using
786-O cells, a VHL-defective renal cell carcinomal line that
does not express detectable levels of HIF-1α, but instead
expresses HIF-2α at a high constitutive level.35 This results in
regulation of hypoxia response genes such as VEGF and lysyl
oxidase (LOX) in 786-O cells by HIF-2 instead of HIF-1.36 We
reasoned that if P1 also inhibits the dimerization of HIF-2 in
cells, a dose-dependent reduction in the mRNA and protein
products of these hypoxia response genes would be observed in
hypoxic 786-O cells, whereas a specific HIF-1 inhibitor would
not affect hypoxia-response in this cell line. In contrast to
MCF-7 and U2OS cells, P1 at concentrations up to 100 μM
had no effect on a variety of HIF-2-promoted genes in 786-O
cells (Figure 6A). In addition, P1 had no effect on VEGF
protein levels in hypoxic 786-O cells (Figure 6B). To further
probe the effect of P1 on HIF-2, a luciferase reporter assay was
developed and used. The assay was derived from the HIF-1
luciferase reporter assay detailed above (Figure 2C and D) and
adapted for HIF-2 by replacing the plasmid encoding HIF-1α
with the equivalent plasmid encoding HIF-2α. P1 did not
affect the luciferase reporter signal in MCF-7 or U2OS cells
(Figure 6C and D), providing additional evidence that P1
inhibits HIF-1, but not HIF-2 signaling in cells. P2 and P3 also
had no effect on HIF-2 in this assay (Figure S9).
Figure 4. Inhibition of HIF-1 dimerization by cyclo-CLLFVY assessed
by immunofluorescence detection of endogenous HIF-1α/HIF-1β
dimerization by in situ PLA in MCF-7 cells. The PLA signal is absent
in normoxia (panel 1) but readily observed in hypoxia (panel 2). P1-
treatment (25 or 50 μM) of hypoxic cells results in a loss of the PLA
signal (panels 3 and 4, respectively), whereas 100 μM P2 shows no
effect (panel 5). Inset: Western blot analysis of cell lysates show that
P1 does not affect HIF-1α levels in this assay.
We observed an increase in PLA signal in MCF-7 cells incubated
in hypoxia (1% O2) for 4 h, indicating the expected formation of
the HIF-1 dimer in hypoxia (Figure 4, panel 1 versus panel 2).
Hypoxic MCF-7 cells dosed with 25 and 50 μM P1 showed a
reduction in the HIF-1 PLA signal (Figure 4, panels 3 and 4).
P2 at 100 μM did not have any effect on the PLA signal (Figure 4,
panel 5). The observed effect is not due to a reduction in HIF-1α
by P1 (Western blot inset in Figure 4) and thus may be solely
attributed to disruption of HIF-1α/HIF-1β dimers.
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Figure 5. Inhibition of hypoxia response in MCF-7 and U2OS cells by P1. (A) qPCR analysis shows the dose-dependent reduction of VEGF mRNA,
elevated by ∼3-fold in hypoxia (1 = normoxic levels), by P1 in hypoxic MCF-7 and U2OS cells; 100 μM P2 (control) has no effect in either cell line.
(B) VEGF protein level, elevated by 3- to 5-fold in hypoxia (1 = normoxic levels), is also reduced by P1 in hypoxic MCF-7 and U2OS cells; 100 μM
P2 (control) has no effect in either cell line. (C) P1 pretreatment causes the dose-dependent reduction of tubule length in hypoxic HUVECs; 250
μM P2 (control) has no effect. For representative images, see Figure S8. (D) qPCR analysis shows 50 μM P1 significantly reduces CAIX mRNA,
which is elevated by ∼25-fold in hypoxia (1 = normoxic levels); 100 μM P2 (control) has no effect. (E) SRB assay results reveal that 100 μM P1 is
not cytotoxic to MCF-7 or U2OS cells. (F) Cell proliferation assays show that 100 μM P1 has no effect on MCF-7 proliferation over 72 h, whereas
1 μM 4-hydroxytamoxifen (control) arrests cell division.
radiation therapy38 provides additional evidence for its signifi-
cance as a target. But the challenges of identifying protein−pro-
tein interaction inhibitors in the absence of structural data,39
combined with the difficulties associated with uncovering transcrip-
tion factor inhibitors, are substantial. Peptides and macro-
molecules are increasingly viewed as the optimal scaffold for
protein−protein interaction inhibitors,40 and the high-through-
out screening platform employed here has previously proven
robust for the identification of cyclic peptide inhibitors of a
variety of protein−protein interactions.24,25,41 When employed
for the identification of HIF-1 inhibitors, the platform identified
cyclo-CLLFVY from a plasmid encoded library of 3.2 million
cyclic peptides, via two independent plasmids. Verification of
the function of this compound in vitro and in cells revealed that
it disrupts HIF-1 dimerization by binding the PAS-B domain of
HIF-1α, without affecting HIF-2.
Interestingly, the PAS-B domain of HIF-1α (and HIF-2α) is
also targeted by acriflavine.19 Furthermore, a recently reported
heteroaromatic HIF-2 dimerization inhibitor also functions by
binding a cavity on HIF-2α PAS-B;42 although this compound
was specifically developed to target HIF-2α PAS-B, the screen
for acriflavine and our screen were not biased toward a single
Figure 6. P1 does not affect HIF-2 mediated hypoxia signaling. (A)
100 μM P1 does not affect the transcription of HIF-2α, or HIF-2-
promoted VEGF, LOX, CITED2 in hypoxic 786-O cells. (B) 100 μM
domain, yet both identified compounds binding to HIF-α PAS-
P1 does not affect VEGF protein levels in hypoxic 786-O cells. (C) P1
B. This suggests the PAS-B domain of HIF-α as the optimal
point of intervention for a HIF dimerization inhibitor. A
homology model of HIF-1α PAS-B mapped onto the NMR
structure of HIF-2α PAS-B suggests that the PAS-B cavity is
substantially smaller in HIF-1α than HIF-2α.42 This postulated
difference in the cavity size between the two isoforms may be
the source of the HIF-1 selectivity observed with cyclo-CLLFVY.
As well as its potential for cancer therapy, a specific inhibitor
of HIF-1 serves as a chemical tool to verify hypotheses about
the unique and sometimes opposing cellular function of HIF-1
does not affect the reporter signal in a HIF-2 luciferase reporter assay
in MCF-7 cells. (D) P1 does not affect the reporter signal in a HIF-2
luciferase reporter assay in U2OS cells.
CONCLUSIONS
■
There is extensive evidence verifying HIF-1 as key in multiple
processes critical to cancer progression, and thus a target of
significant potential for cancer therapy.3−5,37 Recent findings
such as HIF-1 regulating the survival of tumor cells that escape
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and HIF-2.2 The compound identified here will also serve to
further illuminate the recently reported nontranscriptional
function of HIF-1α.43 A key challenge is the availability of tools
to separate the transcriptional function of HIF-1α (or any other
transcription factor) from its nontranscriptional function in
cells.41,44 Current approaches that knockdown or knockout the
target protein (e.g., siRNA) will not suffice, as they will equally
deplete both functions of the protein. In contrast, a HIF-1
dimerization inhibitor only affects the transcriptional function
of HIF-1α.
We are currently conducting structural and SAR studies to
guide the design of our second-generation HIF-1 dimerization
inhibitors. We have previously demonstrated the development
of potent, cell-permeable, small molecule protein−protein
interaction inhibitors from similarly identified cyclic peptides.45
The evolution of cyclo-CLLFVY, the first example of a molecule
that selectively inhibits HIF-1 dimerization in cells, to a small
molecule by a similar approach is currently underway in our
laboratory.
growth advantage in the presence of arabinose were isolated and
retransformed into the original selection strain and reassessed for
IPTG-dependent inhibition of growth, and arabinose growth rescue.
SICLOPPS plasmids from colonies demonstrating the expected
phenotypes were assessed for their HIF-1 specificity by transformation
into two identical RTHS, except for the replacement of HIF-1 with
unrelated proteins (ATIC, a homodimeric enzyme used in purine
biosynthesis, and P6/UEV, a heterodimeric interaction required for
the budding of HIV from infected cells).24,25 Plasmids that caused a
growth-advantage in the ATIC or P6/UEV RTHS were discarded for
being nonspecific. The activity of the cyclic peptides encoded by the
remaining SICLOPPS plasmids was ranked by retransforming into the
HIF-1 RTHS and drop spotting of 10-fold dilutions. The identity of
the variable insert regions encoding the active cyclic peptides was
revealed by DNA sequencing.
Peptide Synthesis. Cyclic peptides were synthesized and
characterized as detailed in the Supporting Information.
HIF Luciferase Reporter Assays. Endogenous HIF-1 luciferase
reporter assays were conducted as previously reported in U2OS-HRE-
luc15 and MCF-7 cells.46 For plasmid-expressed HIF-α luciferase reporter
assays, MCF-7 and U2OS cells were transiently transfected with plasmids
expressing HIF-1α, HIF-2α, or a blank control (pcDNA3.1-HIF-1α,
pcDNA3.1-HIF-2α, or pcDNA3.1), a renilla-encoding control (phRL-
TK), and a HIF-dependent firefly luciferase reporter construct (pGL2-
TK-HRE), using Transfast (Promega) according to the manufacturer’s
instructions. After 24 h, cells were recovered and plated (4000 cells/well)
in 96-well plates (Perkin-Elmer) and incubated for 5 h before either
hypoxic or aerobic incubation in presence or absence of cyclic peptide
inhibitors. Firefly and renilla activities were determined using Dual-
Glo Reagent (Promega) according to the manufacturer’s instructions.
The luciferase signal was normalized using the corresponding renilla
values.
Recombinant Production of HIF-1α and HIF-1β. HIF-1, HIF-2,
HIF-1, bHLH, PAS-A, PAS-B, and PAS-B were expressed in E. coli
(BL21.DE3) as detailed in the Supporting Information.
In Vitro Assays. Pull downs, ELISA, fluorescent binding assays,
and ITC were conducted as detailed in the Supporting Information.
Dosing Cells with Inhibitors. Cells were treated with the stated
concentrations of inhibitor (P1, P2, or P3) and incubated in normoxia
for 4 h, followed by incubation in a hypoxic environment. All
manipulation of cell pellets (e.g., lysis, mRNA, and protein extraction)
was conducted in a hypoxic environment.
Duolink Proximity Ligation Assay. Duolink proximity ligation
assay was conducted using the in situ PLA Kit (O-Link Bioscience,
Uppsala, Sweden) according to the manufacturer’s instructions. The
antibodies used were rabbit monoclonal anti-HIF-1α (NB100-449,
Novus Biologicals) and mouse monoclonal anti-HIF-1β (H00000405-
B01P, Abnova). Cells were dosed with inhibitors as above and
incubated in a hypoxic environment for 4 h, after which they were
fixed with cold methanol for 10 min and permeabilized with 0.2%
Triton (diluted in PBS) for 10 min. After preincubation with the
Duolink Blocking Reagent for 1 h, samples were incubated overnight
with the primary antibodies to HIF-1α (1:500) and HIF-1β (1:500).
Duolink PLA probes and reagents were added as recommended by the
manufacture’s instructions.
Quantitative Reverse-Transcription Polymerase Chain Re-
action. Total RNA was extracted from MCF-7 and U2OS cells using
RNeasy Mini Kit (74104, QIAGEN) and quantified using a Nanodrop
ND-1000 spectrophotometer. Complementary DNA was synthesized
in a 20 μL reaction containing 1 μg of total RNA, using qScript cDNA
SuperMix (95048-100, Quanta Biosciences) according to the manu-
facturer’s instructions. Quantitative real-time PCRs were performed
using Universal Taqman PCR master mix (Applied Biosystems) >and
the Taqman gene expression assay of interest (Applied Biosystems) on
an ABI StepOnePlus Real-Time PCR system (Applied Biosystems).
Expression assays used in this study were HIF-1α (00936376_m1),
VEGF-A (Hs00173626_m1), CAIX (Hs00154208_m1), LOX
(Hs00942480_m1), HIF2α (Hs01026149_m1), CITED2
(Hs01897804_s1), and 18S (Hs99999901_m1). All expression values
were normalized using expression of 18S.
EXPERIMENTAL SECTION
■
Oligonucleotides used in this study are detailed in Table S1.
DNA synthesis and sequencing was carried out by Eurofins MWG
Operon (Germany). All restriction endonucleases were purchased
from New England Biolabs; all other molecular biology reagents were
purchased from New England Biolabs, Fisher Scientific, or Promega
and were used as directed by the manufacturer. Chemical reagents
were purchased from Sigma Aldrich, Fisher Scientific, or Merck and
were used as received. Amino acids and peptide coupling reagents
were obtained from Novabiochem or Matrix Innovations. DNA
purification carried out using QIAquick PCR Purification Kit, and
plasmid purification was carried out using QIAGEN Plasmid Mini Kit.
The CRIM plasmids pAH68 and pAH69 were obtained from the E.
coli Genetic Stock Centre, Yale University. All peptides were
synthesized using a Liberty 1 microwave peptide synthesizer (CEM),
and purified on a Waters HPLC system using a Waters C18 Atlantis
T3, or a Waters C18 Atlantis Prep OBD column. ITCs were con-
ducted using a MicroCal iTC200 (GE Healthcare). All cell lines were
maintained in DMEM (Life Technologies) containing 10% FBS; for
aerobic incubation, cells were cultured at 37 °C in 5% CO2. For
hypoxic treatment, cells were cultured and manipulated (DNA, RNA,
and protein extraction) in a H35 hypoxia workstation (Don Whitley
Scientific) in 1% O2, 5% CO2 and 94% N2. Luminecence was mea-
sured in a GloMAX-96 microplate luminometer (Promega). All assays
were conducted in triplicate. Data was analyzed in Excel (Microsoft)
or Prism (GraphPad Software).
SICLOPPS Screening for HIF-1 Dimerization Inhibitors. The
HIF-1 RTHS, associated control RTHS, and SICLOPPS library were
constructed as detailed in the Supporting Information. Electro-
competent cells of the HIF-1 RTHS were prepared and transformed
with the C+5 SICLOPPS plasmid library. Transformation efficiency,
assessed by plating 10-fold serial dilutions of the recovery solution
on LB agar supplemented with chloramphenicol (35 μg/mL), was
consistently found to be ∼5 × 107, thus ensuring adequate coverage
of the 3.2 × 106 member cyclic peptide library. Transformants were
washed with minimal media and plated onto minimal media agar
plates supplemented with ampicillin (50 μg/mL), spectinomycin
(25 μg/mL), kanamycin (50 μg/mL), 3-AT (7.5 μM), IPTG (100 μM),
L-arabinose (6.5 μM), and chloramphenicol (35 μg/mL). The plates
were incubated for 2−3 days at 37 °C until individual colonies were
visible. Colonies were picked and restreaked onto LB agar plates
containing ampicillin (50 μg/mL), spectinomycin (25 μg/mL), and
chloramphenicol (35 μg/mL) and incubated overnight at 37 °C.
Surviving colonies from these plates were grown overnight and
assessed by drop-spotting 10-fold serial dilutions onto minimal media
plates, supplemented with antibiotics, IPTG and 3-AT as above, with
and without 6.5 μM L-arabinose. Plasmids from strains showing a
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Western Immunoblotting. Cells were washed with ice-cold PBS
and lysed by incubation on ice in radioimmunoprecipitation assay
buffer (50 mM Tris (pH 7.4), 150 μM NaCl, 1 mM EDTA, 1% v/v
Triton X-100), and protease inhibitor cocktail (Sigma) for 20 min.
Lysates were centrifuged at 14 500 rpm for 15 min at 4 °C, and the
protein concentration in the supernatant quantified by Bradford assay.
Proteins were separated on precast NuPAGE 4% to 12%
polyacrylamide gradient Bis−Tris gels (Invitrogen) under denaturing
conditions, transferred to PVDF membranes (Invitrogen), and
subjected to immunoblot analysis. Mouse monoclonal anti-HIF-1α
(610958, BD Biosciences) and rabbit anti-β-actin (ab8226, Abcam)
antibodies were diluted (1:250 and 1:10 000, respectively) in PBS
containing 5% nonfat powdered milk and 0.1% Tween-20 and then
incubated with the membrane overnight at 4 °C. Horseradish
peroxidase conjugated secondary antibodies (Cell Signaling) were
used. Bound immunocomplexes were detected using ECL prime
Western blot detection reagent (RPN2232, GE Healthcare).
(8) Ivan, M.; Kondo, K.; Yang, H.; Kim, W.; Valiando, J.; Ohh, M.;
Salic, A.; Asara, J. M.; Lane, W. S.; Kaelin, W. G., Jr. Science 2001, 292,
464.
(9) Chilov, D.; Camenisch, G.; Kvietikova, I.; Ziegler, U.; Gassmann,
M.; Wenger, R. H. J. Cell Sci. 1999, 112 (Pt 8), 1203.
(10) Ebert, B. L.; Bunn, H. F. Mol. Cell. Biol. 1998, 18, 4089.
(11) Manalo, D. J.; Rowan, A.; Lavoie, T.; Natarajan, L.; Kelly, B. D.;
Ye, S. Q.; Garcia, J. G.; Semenza, G. L. Blood 2005, 105, 659.
(12) Schodel, J.; Oikonomopoulos, S.; Ragoussis, J.; Pugh, C. W.;
Ratcliffe, P. J.; Mole, D. R. Blood 2011, 117, e207.
(13) Jewell, U. R.; Kvietikova, I.; Scheid, A.; Bauer, C.; Wenger, R.
H.; Gassmann, M. FASEB J. 2001, 15, 1312.
(14) Kung, A. L.; Wang, S.; Klco, J. M.; Kaelin, W. G.; Livingston, D.
M. Nat. Med. 2000, 6, 1335.
(15) Chau, N. M.; Rogers, P.; Aherne, W.; Carroll, V.; Collins, I.;
McDonald, E.; Workman, P.; Ashcroft, M. Cancer Res. 2005, 65, 4918.
(16) Escuin, D.; Simons, J. W.; Giannakakou, P. Cancer Biol. Ther.
2004, 3, 608.
(17) Jones, D. T.; Harris, A. L. Mol. Cancer Ther. 2006, 5, 2193.
(18) Kung, A. L.; Zabludoff, S. D.; France, D. S.; Freedman, S. J.;
Tanner, E. A.; Vieira, A.; Cornell-Kennon, S.; Lee, J.; Wang, B.; Wang,
J.; Memmert, K.; Naegeli, H. U.; Petersen, F.; Eck, M. J.; Bair, K. W.;
Wood, A. W.; Livingston, D. M. Cancer Cell 2004, 6, 33.
(19) Lee, K.; Zhang, H.; Qian, D. Z.; Rey, S.; Liu, J. O.; Semenza, G.
L. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 17910.
(20) Nordgren, I. K.; Tavassoli, A. Chem Soc Rev 2011, 40, 4307.
(21) Rapisarda, A.; Uranchimeg, B.; Scudiero, D. A.; Selby, M.;
Sausville, E. A.; Shoemaker, R. H.; Melillo, G. Cancer Res. 2002, 62,
4316.
ASSOCIATED CONTENT
* Supporting Information
Supporting Information includes Figures S1−S9, Tables S1 and
S2, and supplemental methods. This material is available free of
charge via the Internet at http://pubs.acs.org
■
S
AUTHOR INFORMATION
Corresponding Author
a.tavassoli@soton.ac.uk
■
Present Address
∇P.A.T.: Institute for Cancer Sciences, University of Manchester,
St. Mary’s Hospital, Manchester M13 9WL, UK.
(22) Welsh, S.; Williams, R.; Kirkpatrick, L.; Paine-Murrieta, G.;
Powis, G. Mol. Cancer Ther. 2004, 3, 233.
(23) Horswill, A. R.; Savinov, S. N.; Benkovic, S. J. Proc. Natl. Acad.
Sci. U.S..A 2004, 101, 15591.
(24) Tavassoli, A.; Benkovic, S. J. Angew. Chem., Int. Ed. 2005, 44,
Author Contributions
#E.M. and I.K.N. contributed equally to this work.
Notes
2760.
The authors declare no competing financial interest.
(25) Tavassoli, A.; Lu, Q.; Gam, J.; Pan, H.; Benkovic, S. J.; Cohen, S.
N. ACS Chem. Biol. 2008, 3, 757.
(26) Scott, C. P.; Abel-Santos, E.; Wall, M.; Wahnon, D. C.;
Benkovic, S. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13638.
(27) Tavassoli, A.; Benkovic, S. J. Nat Protoc 2007, 2, 1126.
(28) Ding, Y.; Xu, M. Q.; Ghosh, I.; Chen, X.; Ferrandon, S.; Lesage,
G.; Rao, Z. J. Biol. Chem. 2003, 278, 39133.
ACKNOWLEDGMENTS
■
We gratefully acknowledge funding of this work by Cancer
Research UK (Career Establishment Award A10263 to A.T.,
and Drug Discovery Grant A9984 to A.T.), the Gerald Kerkut
Charitable Trust (Ph.D. studentship for C.E.L. to A.T. and
P.A.T.), and the Biotechnology and Biological Sciences
Research Council (Doctoral Training Award for I.K.N. to
A.T.). We thank Peter Gimeson and GE Healthcare for
assistance with ITC, and Prof. Christopher Pugh, Dr.
Christopher Bradfield, and Dr. Oliver Hankinson for providing
HIF-1β cDNA. We are grateful to Prof. Margaret Ashcroft for
U2OS-HRE-luc and U2OS-luc control cells. We also thank Dr.
Daniel Asby, Alex Hoose, Dr. David Rusling, Dr. Alison
Donlevy, Dr Samantha Larkin, and Dr. Josephine Corsi for
helpful discussions and technical assistance.
(29) Koslowski, M.; Luxemburger, U.; Tureci, O.; Sahin, U. Oncogene
2011, 30, 876.
(30) Soderberg, O.; Gullberg, M.; Jarvius, M.; Ridderstrale, K.;
Leuchowius, K. J.; Jarvius, J.; Wester, K.; Hydbring, P.; Bahram, F.;
Larsson, L. G.; Landegren, U. Nat. Methods 2006, 3, 995.
(31) Forsythe, J. A.; Jiang, B. H.; Iyer, N. V.; Agani, F.; Leung, S. W.;
Koos, R. D.; Semenza, G. L. Mol. Cell. Biol. 1996, 16, 4604.
(32) Calvani, M.; Rapisarda, A.; Uranchimeg, B.; Shoemaker, R. H.;
Melillo, G. Blood 2006, 107, 2705.
(33) Wykoff, C. C.; Beasley, N. J.; Watson, P. H.; Turner, K. J.;
Pastorek, J.; Sibtain, A.; Wilson, G. D.; Turley, H.; Talks, K. L.;
Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P. J.; Harris, A. L. Cancer Res.
2000, 60, 7075.
(34) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. JNCI,
J. Natl. Cancer Inst. 1990, 82, 1107.
(35) Maxwell, P. H.; Wiesener, M. S.; Chang, G. W.; Clifford, S. C.;
Vaux, E. C.; Cockman, M. E.; Wykoff, C. C.; Pugh, C. W.; Maher, E.
R.; Ratcliffe, P. J. Nature 1999, 399, 271.
REFERENCES
■
(1) Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 5510.
(2) Keith, B.; Johnson, R. S.; Simon, M. C. Nat. Rev. Cancer 2012, 12,
9.
(3) Harris, A. L. Nat. Rev. Cancer 2002, 2, 38.
(4) Semenza, G. L. Nat. Rev. Cancer 2003, 3, 721.
(5) Giaccia, A.; Siim, B. G.; Johnson, R. S. Nat. Rev. Drug Discovery
2003, 2, 803.
(36) Carroll, V. A.; Ashcroft, M. Cancer Res. 2006, 66, 6264.
(37) Melillo, G. Mol. Cancer Res. 2006, 4, 601.
(38) Harada, H.; Inoue, M.; Itasaka, S.; Hirota, K.; Morinibu, A.;
Shinomiya, K.; Zeng, L.; Ou, G.; Zhu, Y.; Yoshimura, M.; McKenna,
W. G.; Muschel, R. J.; Hiraoka, M. Nat. Commun. 2012, 3, 783.
(39) Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001.
(40) Mullard, A. Nat. Rev. Drug Discovery 2012, 11, 173.
(6) Bardos, J. I.; Ashcroft, M. Bioessays 2004, 26, 262.
(7) Jaakkola, P.; Mole, D. R.; Tian, Y. M.; Wilson, M. I.; Gielbert, J.;
Gaskell, S. J.; Kriegsheim, A.; Hebestreit, H. F.; Mukherji, M.;
Schofield, C. J.; Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P. J. Science
2001, 292, 468.
10424
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Journal of the American Chemical Society
Article
(41) Birts, C. N.; Nijjar, S. K.; Mardle, C. A.; Hoakwie, F.; Duriez, P.
J.; Blaydes, J. P.; Tavassoli, A. Chem. Sci. 2013, 4, 3046.
(42) Scheuermann, T. H.; Li, Q.; Ma, H. W.; Key, J.; Zhang, L.;
Chen, R.; Garcia, J. A.; Naidoo, J.; Longgood, J.; Frantz, D. E.;
Tambar, U. K.; Gardner, K. H.; Bruick, R. K. Nat. Chem. Biol. 2013, 9,
271.
(43) Hubbi, M. E.; Kshitiz; Gilkes, D. M.; Rey, S.; Wong, C. C.; Luo,
W.; Kim, D. H.; Dang, C. V.; Levchenko, A.; Semenza, G. L. Sci.
Signaling 2013, 6, ra10.
(44) Huang, L. E. Science 2013, 339, 1285.
(45) Spurr, I. B.; Birts, C. N.; Cuda, F.; Benkovic, S. J.; Blaydes, J. P.;
Tavassoli, A. ChemBioChem 2012, 13, 1628.
(46) Wang, X. H.; Cavell, B. E.; Syed Alwi, S. S.; Packham, G.
Biochem. Pharmacol. 2009, 78, 261.
10425
dx.doi.org/10.1021/ja402993u | J. Am. Chem. Soc. 2013, 135, 10418−10425