Development of inhibitors of the PAS-B domain of the HIF-2α transcription factor

Article abstract of DOI:10.1021/jm301847z

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity relationship study of small molecules designed to inhibit HIF-2α-ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor-protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α-ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action.

Full text of DOI:10.1021/jm301847z

Article
pubs.acs.org/jmc
Development of Inhibitors of the PAS‑B Domain of the HIF-2α
Transcription Factor
Jamie L. Rogers,‡ Liela Bayeh,‡ Thomas H. Scheuermann,‡ Jamie Longgood, Jason Key, Jacinth Naidoo,
Lisa Melito, Cameron Shokri, Doug E. Frantz, Richard K. Bruick, Kevin H. Gardner,* John B. MacMillan,*
and Uttam K. Tambar*
Departments of Biochemistry and Biophysics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas,
Texas 75390-9038, United States
S
* Supporting Information
ABSTRACT: Hypoxia inducible factors (HIFs) are heterodi-
meric transcription factors induced in a variety of pathophysio-
logical settings, including cancer. We describe the first detailed
structure−activity relationship study of small molecules designed
to inhibit HIF-2α−ARNT heterodimerization by binding an
internal cavity of the HIF-2α PAS-B domain. Through a series of
biophysical characterizations of inhibitor−protein interactions
(NMR and X-ray crystallography), we have established the
structural requirements for artificial inhibitors of the HIF-2α−
ARNT PAS-B interaction. These results may serve as a
foundation for discovering therapeutic agents that function by
a novel mode of action.
potential consequences of global HIF attenuation on important
physiological pathways throughout a therapeutically relevant
window. Although a handful of high throughput screen (HTS)-
derived small molecules or natural products have been reported
to modulate the pathway, almost all of these directly interfere
with targets other than the HIF proteins themselves, giving rise
to pleiotropic effects and limiting their utility in testing HIF-
centric hypotheses.6 Indeed, HIFs themselves are an example of
a traditionally challenging target for pharmacological inter-
vention: it is a large, intracellular multiprotein complex without
any catalytic active sites that are typically used for small
molecule substrate binding. Moreover, much of the HIF
complexes exist in an extended conformation, reducing the
availability of potential ligand binding sites.
Human HIF transcription factors are heterodimers com-
posed of one of three regulated HIF-α (HIF-1α, HIF-2α/
EPAS-1, or HIF-3α) subunits and a constitutive ARNT (also
known as HIF-β) subunit, all members of the bHLH−PAS
(basic helix−loop−helix−period-ARNT-single minded) fam-
ily.8 HIF PAS domains stabilize the HIF heterodimers via
protein−protein interactions across subunits as mutations or
deletions in the PAS domains attenuate HIF heterodimer
formation and transcriptional activity.9 Though large, hydro-
phobic protein−protein interfaces are notoriously difficult to
disrupt directly with small molecules, PAS domains provide an
attractive opportunity. Notably, many PAS-mediated protein−
INTRODUCTION
■
Mammalian cells employ pathways by which they recognize and
respond to metabolic and environmental cues. Though
promoting normal developmental and physiological adaptions
in response to stress, such pathways are also frequently
misappropriated in the context of human disease. For example,
mammalian cells express a hypoxic response pathway by which
changes in cellular oxygen availability are sensed. When oxygen
availability is low, a family of hypoxia inducible factors (HIFs)
are upregulated.1 These transcription factors coordinate the
expression of hundreds of downstream target genes that
promote appropriate responses such as anaerobic metabolism
and increased oxygen delivery.2 However, HIFs also participate
in pathophysiological settings, including cancer.1,3 HIFs are
often induced within the hypoxic environment of tumors,
where they are conscripted to promote changes in tumor
metabolism, angiogenesis, metastasis, and many other pro-
tumorigenic responses.4 In fact, the impact of HIF on cancer
can be so great that there is often selective pressure for
mutations that uncouple HIF induction from oxygen
availability, resulting in constitutive HIF activity.5
While expression studies and genetic model systems provide
compelling evidence that HIF inhibition may impair tumor
progression in many settings, small molecule tools offer several
advantages for investigating HIF as a therapeutic target.5 Such
reagents offer the potential to assess the consequences of in
vivo HIF attenuation on therapeutically relevant time scales in a
range of model systems, many of which are not amenable to
genetic manipulation. Small molecules can also help address the
Received: December 14, 2012
Published: January 30, 2013
© 2013 American Chemical Society
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protein interactions are regulated by allosteric conformational
changes induced by cofactors that bind within the core of the
PAS domain itself.10 We hypothesized that HIF PAS domains
might likewise be amenable to binding small molecule
antagonists within their cores to induce conformational changes
that disrupt HIF dimerization (Figure 1).
a luminescent signal triggered by diffusion of singlet oxygen
from a donor bead and subsequent detection on an acceptor. In
the presence of a small molecule that disrupts the protein−
protein interaction, this luminescent signal is extinguished. A
screen of the 203520 compounds in this library provided us
with approximately 20 candidates suitable for further study by
analogue synthesis or purchase. Recently, our laboratories
characterized one such synthetic small molecule that binds the
HIF-2α PAS-B internal cavity and exhibits an AlphaScreen IC50
value of approximately 0.1 μM.7 Isothermal titration calorim-
etry measurements confirm in vitro binding in the same range
(KD = 80−90 nM). In cell culture, these compounds interfere
with HIF-2 driven transcription with low-μM potency.
Herein, we describe a full account of the synthesis and
evaluation of a series of compounds that culminated in the
identification of our most active small molecule binder of the
HIF-2α PAS-B domain. In the process, we elucidate the
structural features of the most active compound that lead to a
disruption of the heterodimeric structure of the HIF tran-
scription factor. We discuss a focused subset of the hundreds of
small molecule inhibitors of HIF-2α that were synthesized and
evaluated by our laboratories. This work represents the first
detailed SAR study of small molecules designed to inhibit HIF-
2α−ARNT heterodimerization, which may serve as a
foundation for discovering therapeutic agents that function by
a novel mode of action.
Figure 1. Basis of small molecule regulation of protein−protein
interactions in HIF-2. (A) Crystal structure of the HIF-2α−ARNT
PAS-B heterodimer9b (PDB code: 3F1P), highlighting the internal
cavity within HIF-2α PAS-B (gray surface, internal waters represented
as red spheres). Side chains lining the cavity are provided by a mix of
hydrophobic and polar residues as shown. (B) Schematic for small
molecule regulation of HIF-2, with ligand binding to the HIF-2α PAS-
B cavity, distorting the adjacent β-sheet that also provides the ARNT
PAS-B binding surface.7
RESULTS
■
Compound 1 was chosen as the starting point for our SAR
campaign because of its superior activity over other promising
scaffolds identified in our initial screen of >200000 structurally
diverse small molecules (Figure 2), exhibiting an IC50 of 0.4 μM
Notably, one of the two PAS domains in HIF-2α (PAS-B) is
particularly well-suited in this regard. Recently, our groups used
X-ray crystallography and NMR to identify a large (290 Å3)
water-filled cavity in the core of this domain (Figure 1).9b,11
Cavities of this size are rare and strongly suggestive of a missing
cofactor or ligand-binding site. An initial NMR-based small
molecule screen identified a number of artificial ligands for the
HIF-2α PAS-B domain, most of which were two substituted
aromatic rings connected by short (one- or two-atom) linkers.
These initial findings demonstrated that this cavity could
accommodate ligand binding to induce conformational changes
that weaken the protein−protein interaction between purified
PAS domains from the HIF-2α and ARNT subunits. However,
these initial lead molecules lacked the efficacy and pharmaco-
logical attributes required to modulate HIF-2 gene expression
in cells.
To identify superior HIF-2 antagonist candidates, we
screened a collection of >200000 structurally diverse small
molecules from an in-house compound library using a
commercially available luminescence proximity (AlphaScreen,
from Perkin-Elmer) assay format. AlphaScreen is a homoge-
neous, bead-based luminescence proximity assay,12 which
monitors the formation of a complex between two tagged
proteins (i.e., GST-HIF-2α PAS-B* and ARNT PAS-B*-FLAG
domains, where PAS-B* designates the HIF-2α E247R and
ARNT PAS-B R362E variants used to crystallize complexes
with small molecule ligands)9b to bring cognate donor and
acceptor beads into close proximity (Supporting Information
Figure S1). The beads constitute a pair that can be detected by
Figure 2. Compound identified from the initial high-throughput
screen and Strategic Molecular Subsections for SAR Studies.
in the AlphaScreen and a KD of 1.1 μM by ITC. This
compound contains three general structural units that can be
modified: the A-ring, the B-ring, and the one- or two-atom
linker.
We chose 4-nitrobenzoxadiazole as the initial A-ring for our
studies, and we systematically optimized the B-ring and the
linker to identify analogues with improved activity. Syntheti-
cally, analogues of 1 were accessed from the coupling of
commercially available 5-chloro-4-nitrobenzoxadiazole 2 and
heteroatom nucleophiles. This convergent strategy facilitated
the generation of a diverse array of structures that were
evaluated in the AlphaScreen.
Initially, benzoxadiazole 2 was coupled with a series of benzyl
amines in DMF at 100 °C to yield N-linked analogues 3−11
(Scheme 1). Thiophenols and phenol also reacted with
benzoxadiazole 2 in the presence of triethylamine in MeCN
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Scheme 1. Synthesis of Analogues of 1
at ambient temperature to furnish S-linked analogues 12−14
and O-linked analogue 15. The coupling of benzoxadiazole 2
with sterically and electronically diverse anilines at elevated
temperatures generated diarylamines 16−32. Compounds 3−
32 were evaluated for their ability to interrupt HIF-2α−ARNT
heterodimerization using the AlphaScreen described above
(Table 1). To better use this assay to identify allosteric
inhibitors that work by binding to the HIF-2α PAS-B cavity, we
sought to develop negative control proteins which would be
unable to bind ligands to the internal cavity while still folding
properly and retaining the ability to bind ARNT PAS-B. We
generated approximately 25 HIF-2α PAS-B variants for this
purpose, each of which contained point mutations in one or
more residues lining the internal cavity. Of this group, the
S304M mutation best met our design criteria, placing the newly
introduced long aliphatic methionine side chain into the
internal cavity (Figure 3A). This change does not grossly affect
the HIF-2α PAS-B protein fold, as judged by similarities in
backbone NMR chemical shifts (Supporting Information
Figure S2A) but very effectively interferes with binding of
small molecules into the hydrophobic core (Figure 3B,C).
Critically, HIF-2α PAS-B* (S304M) retains the ability to bind
ARNT PAS-B* (Supporting Information Figure S2B), essential
to its use as a control reagent with the AlphaScreen assay.
A number of molecules, designated as ND in Table 1,
typically inhibited heterodimerization of ARNT with the WT
and S304M mutant HIF-2α PAS-B, suggesting they are either
interfering with the assay conditions or cause disruption of
heterodimerization through an alternative pathway that may
not involve binding to the internal cavity. From the initial SAR
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a
The series of analogues most exhaustively explored was the
diarylamines, where a clear pattern of activity emerged.
Generally, monosubstitution of the B-ring resulted in inactive
compounds, with the exception of 19 (IC50 = 0.18 μM) and 21
(IC50 = 0.46 μM), which had m-Cl and m-CF3 substituents,
respectively. No analogues with monosubstitution in the para-
position showed inhibition of heterodimerization. The most
potent compounds to emerge from this series were the
disubstituted diarylamines, with analogues containing at least
one meta-substituent providing the most potent disruption in
the AlphaScreen (IC50 values ranging from 0.09 to 2.8 μM).
To further explore the most potent inhibitors, compounds
that had significant activity by AlphaScreen (i.e., IC50 < 2.0 μM)
were tested by ITC to obtain KD values for binding of small
molecules to the HIF-2α PAS-B domain (Table 2). Overall, we
Table 1. AlphaScreen IC50 for 3−32 (Values in μM)
compd
Alpha
compd
Alpha
3
>5
NA
ND
0.33
NA
2
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
NA
0.18
NA
0.46
2.8
4
5
6
7
8
2.1
9
>10
0.5
NA
ND
0.76
0.17
0.09
0.43
2
10
11
12
13
14
15
16
17
NA
ND
ND
ND
ND
NA
NA
0.12
0.1
Table 2. KD Values for Most Active Compounds (Values in
μM)
a
ND: compound disrupts the control. NA: no activity at the highest
compd
ITC KD
compd
ITC KD
concentration tested.
6
0.5
2
26
27
28
29
30
31
32
1.1
8
0.4
10
19
21
22
23
0.54
0.16
0.64
3
0.17
0.37
0.73
0.17
0.09
2.2
found good correlation between the IC50 values measured by
AlphaScreen and KD values measured by ITC. Ultimately,
compound 32 emerged as the most potent compound, both in
terms of the AlphaScreen assay and by ITC.
To explore the structural basis of the SAR trends we
observed in Tables 1 and 2, we performed NMR-based
structural analysis and X-ray crystallographic studies with the
HIF-2α PAS-B domain and our synthetic small molecules. For
the cocrystal structures, we utilized the engineered, high affinity
HIF2 PAS-B* heterodimer of HIF-2α E247R and ARNT PAS-
B R362E domains employed in the AlphaScreen assay.9b
A
Figure 3. A single internal mutation within the HIF-2α PAS-B cavity
attenuates ligand binding. (A) Two views of a model of the S304M
mutation (spheres) suggests that the new side chain will intrude upon
the apo-protein cavity (gray surface, from PDB code: 3F1P).9b (B,C)
ITC of wild-type (B) and S304M (C) complexes with compound 32,
demonstrating that the mutation reduces the affinity of the protein
over 50-fold, validating the biophysically characterized ligand binding
site.
comparison of the cocrystal structures of the mildly disrupting
mono-fluorinated analogue 23 (IC50 = 2.1 μM) and the
potently disrupting disubstituted analogue 32 reveals that both
inhibitors bind in the HIF-2α PAS-B internal cavity, fully
sequestered from bulk solvent (Figure 4A). The A-ring of both
23 and 32 is oriented to accommodate a weak electrostatic
interaction between the nitro-group and the H248 imidazole
side chain. The B-rings occupy similar binding sites, however,
they are oriented in slightly different conformations (Figure
4B). The m-F of the disubstituted B-ring of 32 packs against the
PAS-B β-sheet, and the m-Cl of 32 is positioned near the E
helix. In contrast, the m-F in compound 23 points away from
the β-sheet and occupies the same site near the E helix as the
m-Cl in compound 32 (Figure 4C).
The different orientations of the m-F in the B-ring of a series
of analogues may account for differences in activity, which is
supported by several observations. Notably, m-F of 32 packs
very tightly against the β-sheet surface of the binding site but
makes a modest contribution to the binding energy. A
comparison of the ITC data from 32 and 19, differing by a
m-F, demonstrates a 2-fold reduction in affinity (Table 2).
Extrapolating from the experimental structure of 32 bound
within HIF-2a, we believe the ortho, para substitution pattern of
the active benzylamine analogues 6 and 10 allow them to pack
studies, it was apparent that modifications of the linker had
deleterious effects on the activity. Of the nine benzylamine
derivatives, only 6 (IC50 = 0.33 μM) and 10 (IC50 = 0.5 μM)
demonstrated appreciable activity in the AlphaScreen. The
common feature of these two analogues is the ortho, para
substitution pattern with electron withdrawing substituents.
Further discussion of the activity of 6 and 10 will be included
below. Changing from N-linked to O- and S-linked analogues
diminished all activity, regardless of the substitution pattern
(compounds 12−15). On the basis of our initial success with
the aniline series 16−32 described below, we abandoned
further efforts with the O- and S-linked analogues. We believe
there may be a role for hydrogen bonding in the −NH of the
linker, which is not possible for the O- and S-linked analogues.
This is further demonstrated below, where N-methylation of
the linker reduces activity.
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Figure 4. Binding modes of HIF-2 antagonists. (A) The HIF-2 PAS-B*−23 ternary complex (PDB code: 4GS9) confirms that this inactive HTS-
lead analogue binds into the HIF-2α PAS-B internal cavity. To better view the internally bound ligand, secondary structural elements are indicated
along the ribbon diagram and the HIF-2α PAS-B surface (blue) has been cut away for residues that separate the binding site from bulk solvent (from
this perspective). Although present in these structures, the ARNT PAS-B domain has been omitted for clarity. (B) Comparison of the HIF-2 PAS-B
complexes with compounds 23 and 32 shows that the monosubstituted m-fluorinated B-ring of 23 flips roughly 180° relative to 32 (PDB code:
4GHI7). (C) Close protein−32 contacts at the m-fluorine site suggests that bound 23 adopts a lower energy bound conformation by placing its
single halogen where 32 positions its m-chlorine.
Figure 5. Solution NMR characterization of the 23 and 32 complexes with HIF-2α PAS-B. (A) 15N/1H HSQC spectra of apo HIF-2α PAS-B (black)
and its complexes with 23 (blue) and 32 (red) reveal sites differentially perturbed by the two analogues. (B) Chemical shift differences in HIF-2α
PAS-B observed between the complexes (with ligands 23 and 32) are plotted along the protein sequence. The red line at 0.033 ppm denotes the
average difference observed across all sites. (C) Chemical shift differences mapped onto the HIF-2α PAS-B−32 complex structure (PDB code:
4GHI) as a blue to red gradient, with spheres marking sites experiencing a greater than average difference.
their B-rings similarly to 32, placing one substituent against the
PAS-B β-sheet and one near the E helix.
collected from 15N-enriched HIF-2α PAS-B reveal a well-
ordered protein with significant spectral changes induced by
complexation with either 32 or 23 (Figure 5). At many sites, we
can assign the 23-bound state by inference from the
experimentally determined resonance assignments for both
ligand-free9a and 32-bound states.7 At several such sites, we
observe crosspeaks for 23-bound HIF-2α PAS-B located
between the corresponding peaks for apoprotein or the 32
complex, indicating that binding to 23 causes smaller protein
structural changes than binding to 32 (Figure 5A,B). Notably, a
number of the larger chemical shift differences localize to the
HIF-2α PAS-B β-sheet (Figure 5B,C), which constitutes the
ARNT PAS-B binding site.
Alternatively, the loss of the m-Cl (23) or the replacement of
the m-Cl with a m-F (30) weaken binding by 25- and 8-fold,
respectively. These observations suggest that the m-F binding
site of 32 is sterically strained in the HIF-2α PAS-B* ternary
complex and, in the absence of a m-Cl, adopts a lower energy
binding pose by flipping into the m-Cl binding site of 32.
Together, these observations suggest a mechanism where the
m-Cl of 32 pays the energetic cost of forcing the m-F into a
suboptimal pocket juxtaposed to the HIF-2α PAS-B β-sheet,
effecting conformational changes to the PAS-B heterodimeriza-
tion interface and weakening the important interaction between
the HIF-2α and ARNT PAS-B domains.
Once the 3-chloro-5-fluoro-aniline 32 emerged as the most
active compound and we could rationalize the necessity for the
B-ring substitutions, we examined modifications to the A-ring
to ascertain the role of the 4-nitrobenzoxadiazole functionality.
To further characterize the consequences of ligand binding in
solution, we examined the wild-type HIF-2α PAS-B domain by
solution NMR spectroscopy. 15N/1H HSQC NMR spectra
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The cocrystal structures of 32 and 23 indicated there was space
for additional steric bulk around the A-ring. In particular, the A-
ring of 32 was not filling the pocket para to the nitro group and
in the vicinity of the benzoxadiazole functionality. Moreover, in
all cocrystals with analogues that contain a nitro-group in the A-
ring (e.g., 32), the molecule is oriented to potentially
accommodate weak electrostatic interactions between its
nitro-group and the H248 imidazole side chain. We examined
a series of A-ring analogues of 32 that probed these
observations.
Since initial studies revealed the superiority of the NH-linked
analogues 16−32 over S-linked analogues 12−14 and O-linked
analogue 15, we synthesized N-methylated analogue 44 to
determine the importance of the bis-anilinic proton in
compound 32 (Scheme 3). Trifluoromethyl(3-chloro-5-
Scheme 3. Synthesis of N-Methylated Analogue 45
The first set of compounds we targeted involved replacement
of the benzoxadiazole with other aromatic and heteroaromatic
rings. For example, ortho-nitrophenyl analogue 35 was
generated by Buchwald−Hartwig coupling between ortho-
nitrochlorobenzene 33 and 3-chloro-5-fluoro-aniline 34
(Scheme 2).13 Nitrobenzotriazole 37 was accessed from
Scheme 2. A-Ring Modifications of Compound 32
fluorophenyl)carbamate 42 was converted to N-methyl-3-
chloro-5-fluoro-aniline 43 in a two-step sequence. This
intermediate was then coupled with benzoxadiazole 2 to
furnish N-methylated analogue 44. Compound 44 has an IC50
value >3 μM by AlphaScreen and was further shown by ITC to
have a KD > 10 μM.
We were also interested in studying the role of the nitro
functional group in compound 32. Because of the potentially
deleterious pharmacokinetic properties of a nitro group as well
as its weak interaction with the H248 imidazole side chain in
the HIF-2α cavity, we examined suitable replacements.
Omission of the nitro group in analogue 46 was achieved by
coupling 5-chloro-benzoxadiazole 45 and aniline 34 with a
slightly modified Buchwald−Hartwig amination protocol
(Scheme 4). The nitro functional group in 32 was also directly
Scheme 4. Conversion of 4-Nitro Functional Group
chloride precursor 36 via nucleophilic aromatic substitution.
Benzimidazole 39 and 2-aminobenzimidazole 41 were similarly
synthesized from 6-chloro-7-nitro-benzimidazoles 38 and 40,
respectively.
Evaluation of the four alternative A-ring heterocycles with
AlphaScreen revealed dramatically reduced activity for all of the
analogues, with IC50 values of ∼2 to 20 μM (Table 3). A few of
these analogues were evaluated by ITC, further supporting
attenuated binding to the HIF-2α PAS-B cavity. These results
suggest that the unique distribution of electron density in the
benzoxadiazole ring may play a key role in the optimal binding
and disrupting activity of analogue 32.
converted under reductive conditions into an amino group,
yielding 4-aminobenzoxadiazole 47. These changes led to
relatively insoluble compounds. Both 46 and 47 exhibited weak
activity in the AlphaScreen, although 46 was also active against
the S304M control. We further characterized these analogues
by ITC and NMR, with both methods demonstrating weak
binding (KD = 3 μM for 46, 6 μM for 47).
Table 3. AlphaScreen IC50 Values and KD Values for A-Ring
Analogues (Values in μM)
Primary aniline 47 was treated with a series of electrophiles,
including acetic anhydride, chloroacetylchloride, and maleic
anhydride (Scheme 5) to generate N-acyl analogues. These
transformations generated 4-amidobenzoxadiazoles 48−50.
Analogues 49 and 50 were especially compelling because of
their potential as irreversible disruptors of HIF-2α−ARNT
heterodimerzation through covalent interactions with the C339
side chain, which is proximal to the nitro group of compound
compd
Alpha
ITC KD
compd
Alpha
ITC KD
∼6
35
37
39
41
44
46
ND
>10
>5
4.3
47
48
49
50
54
55
> 30
NA
NA
NA
NA
NA
>20
>2
ND
NA
>10
5
5
∼3
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Scheme 5. Synthesis of 4-Amidobenzoxadiazoles
Scheme 7. Functionalization of the 7-Position of the
Benzoxadiazole
32 in the cocrystal structure. However, none of these analogues
exhibited activity.
To determine whether the 4-nitro group in compound 32
was beneficial because of its electron withdrawing properties,
we synthesized electronically analogous 4-sulfonamidobenzox-
adiazoles 54 and 55 (Scheme 6). These compounds were also
examining crystals of benzoxadiazole 57 that were suitable for
X-ray diffraction. 7-Bromobenzoxadiazole 57 was then
subjected to Suzuki−Miyaura cross-coupling conditions with
phenylboronic acid 58 to furnish 7-phenylbenzoxadiazole 59.15
Evaluation of 57 and 59 by AlphaScreen and ITC revealed
complete loss of activity by the addition of the −Br and −Ph
substituents. This result suggests the importance of the
electronic and steric environment of the A-ring for activity.
Scheme 6. Synthesis of 4-Sulfonamidobenzoxadiazoles
DISCUSSION
■
Taken together, our data provide a useful assessment of the
structural requirements for artificial inhibitors of the HIF-2α−
ARNT PAS-B interaction essential for assembling the HIF
transcription factor. Our compounds work by triggering
allosteric changes in the HIF-2α PAS-B β-sheet, initiated by
ligand binding to a completely buried internal cavity. This
binding site places certain structural restraints on the inhibitors,
as demonstrated by integrating these structure−activity
relationships with our prior structural work.7,9b,11 These
restraints can be satisfied by a variety of compounds with
two aromatic/heterocyclic rings coupled by short linkers,
including the nitrobenzoxadiazoles reported here, along with
modified trifluorotoluenes.9b,11 Both types of compounds can
utilize one- or two-atom linkers, with secondary amines being
most thoroughly explored.
Within the B-ring, we find that substitutions are most
tolerated at the positions where the apo- form of the cavity is
largest, corresponding to the meta-position of the ligand B-ring.
Crystal structures of the apo- and ligand-bound protein show
that the cavity approaches close to the para-position of the B-
ring, with a corresponding decrease in binding affinity and
disruption potency for para- vs meta-substituted compounds
(e.g., compare 17 vs 23, 18 vs 19). While explored in less detail
here, ortho-substitutions also appear to slightly decrease affinity
and potency.
examined because of the metabolic stability of sulfonamides as
electron withdrawing substituents. Synthetically, 5-chloroben-
zoxadiazole-4-sulfonyl chloride 51 was converted to 5-
chlorobenzoxadiazole-4-sulfonamides 52 and 53 in the
presence of methylamine and dimethylamine, respectively.
These intermediates were then coupled with aniline 34 to
furnish the desired 4-sulfonamidobenzoxadiazoles 54 and 55.
Replacement of the nitro group with the electronically similar
sulfonamides failed to result in compounds with equal activity.
Both 54 and 55 were inactive as disruptors, but had weak
affinity for the HIF-2α cavity, with KD values of 5 μM.
Finally, we functionalized the 7-position of the benzox-
adiazole ring system (Scheme 7) because X-ray cocrystal
structures of several small molecules and the HIF-2α PAS-B
domain indicated room for expansion in this chemical space of
our analogues. Dibromo-4-nitrobenzoxadiazole 56 was coupled
with aniline 34 under standard thermal conditions to yield 7-
bromo-4-nitrobenzoxadiazole 57. We anticipated the displace-
ment of the 5-bromo substituent in preference to displacement
of the 7-bromo substituent for two reasons: (1) the enhanced
stability of linear conjugation with the benzoxadiazole ring in
intermediate 60 over 61, and (2) the larger Coulombic term at
the 5-position.14 This regioselectivity was confirmed by
In contrast, the protein cavity near the A-ring appears to
accommodate a smaller range of groups, as exhibited by the
diversity of compounds reported here and previously.9b,11
These share a requirement for an electron-deficient aromatic
ring, here achieved by an NO2-substituted benzoxadiazole and
previously by a NO2, CF3-disubstituted phenyl ring. This ring
interacts with residues surrounding the cavity, including
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Y281.9b Notably, the nitro group that is common among these
compounds, often avoided in medicinal chemistry applica-
tions,16 appears to be essential for high affinity and potency.
Notably, its replacement by an NH2 worsens binding and
disruption by 30−60-fold (compare 32 vs 47), underscoring its
importance. This has been attributed to favorable interactions
with H2489b or involvement in an intramolecular H-bond,11 the
latter of which is also supported by the deleterious effects of N-
methylation (compare 32 vs 44).
AUTHOR INFORMATION
Corresponding Author
*For K.H.G.: phone, 214-645-6365; fax, 214-645-6353; e-mail,
kevin.gardner@utsouthwestern.edu. For J.B.M.: phone, 214-
648-8653; fax, 214-648-8856; e-mail, john.macmillan@
utsouthwestern.edu. For U.K.T.: phone, 214-648-0580; fax,
214-648-8856; e-mail, uttam.tambar@utsouthwestern.edu.
■
Author Contributions
‡These authors made equal contributions to the paper.
In addition to improving binding affinity and potency, these
diverse compounds enable studies that show the importance of
protein dynamics at all stages of ligand binding and action.
Entry of ligands from solvent into the pocket buried over 6 Å
within the protein requires substantial flexibility of several
helices.9a,b All of our compounds require some change in the
shape of the apo- cavity to accommodate chemical groups, as
perhaps best exemplified by the A-ring nitro group described
above, which displaces the M252 side chain. Indeed, ligand-
induced structural changes are essential for triggering the
allosteric changes required to displace ARNT PAS-B from the
other side of the HIF-2α PAS-B β-sheet. This is clearly
demonstrated with binding of compound 32 to the HIF-2α
PAS-B domain, which pushes on several β-sheet residues
(predominantly C339 and N341 in the Iβ strand and F244 in
Aβ) and bulges the sheet in such a way that distorts the ARNT
binding surface.7 SAR comparisons from compounds that bind
similarly but with lower potency (e.g., 32 vs 23; Figures 4 and
5) provide a critical (and practical) way to assess the relative
flexibility of the pocket for different ligands.
More broadly, it is becoming readily apparent that a wide
range of natural and artificial ligands are used by PAS domains
to disrupt protein−protein interactions via allosteric mecha-
nisms. Best studied among these are a group of photosensitive
flavin-binding PAS domains, known as LOV domains, which
harness the photochemically driven formation of a protein−
ligand bond to initiate similar conformational changes as we
observe with HIF-2α PAS-B.7,17 Studies of the aryl hydro-
carbon receptor (AhR), a transcription factor which responds
to a wide range of xenobiotic compounds and natural
metabolites,10a suggest that AhR may bind compounds
analogously to HIF-2α, folding the surrounding domain.18
On the basis of these data and the presence of the large,
hydrated cavity in the HIF-2α PAS-B domain, most typical of
apo- forms of ligand binding proteins, we postulate that there is
an endogenous ligand involved in HIF-2 regulation. If so, future
elucidation of such a natural ligand(s) may provide novel
insights into binding of small molecules in the HIF-2α PAS-B
pocket as well as insights into the metabolic regulation of HIF
in physiological settings.
Notes
The authors declare the following competing financial
interest(s): R.K.B., K.H.G., T.H.S., J.K., J.B.M., and U.K.T.
have received stock options and other financial compensation
from Peloton Therapeutics Inc.
ACKNOWLEDGMENTS
■
We acknowledge the following grants for funding this project:
NIH P01 CA095471, CPRIT RP100846, Cancer Center
support grant 5P30 CA142543, and the Welch Foundation I-
1689 (J.B.M.) and I-1568 (R.K.B.). J.B.M. is a Chilton/Bell
Foundation Endowed Scholar. U.K.T. and K.H.G. are W. W.
Caruth, Jr. Scholars in Biomedical Research. R.K.B. is the
Michael L. Rosenberg Scholar in Medical Research and was
supported by a Career Award in the Biomedical Sciences from
the Burroughs Wellcome Fund. This investigation was
conducted in a facility constructed with support from the
Research Facilities Improvement Program (grant no. C06 RR
15437-01) from the National Center for Research Resources,
National Institutes of Health. Results shown in this report are
derived from work performed at Argonne National Laboratory,
Structural Biology Center, at the Advanced Photon Source.
Argonne is operated by U. Chicago Argonne, LLC, for the U.S.
Department of Energy, Office of Biological and Environmental
Research, under contract DE-AC02-06CH11357
ABBREVIATIONS USED
■
HIFs, hypoxia inducible factors; ARNT, aryl hydrocarbon
receptor nuclear translocator; PAS, period-ARNT-single
minded; bHLH, basic helix−loop−helix; HTS, high-throughput
screen; ITC, isothermal calorimetry; AHR, aryl hydrocarbon
receptor
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ASSOCIATED CONTENT
■
S
* Supporting Information
General procedures, compound characterization, assay proce-
dures, data tables, and biophysical characterization. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
The atomic coordinates and structure factor amplitudes
associated with the crystal structure of HIF-2α PAS-B*−
ARNT PAS-B* bound to compound 23 (4GS9) have been
deposited in the PDB Macromolecular Database.
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