Binding model for the interaction of anticancer arylsulfonamides with the p300 transcription cofactor

Article abstract of DOI:10.1021/ml300042k

Hypoxia inducible factors (HIFs) are transcription factors that activate expression of multiple gene products and promote tumor adaptation to a hypoxic environment. To become transcriptionally active, HIFs associate with cofactors p300 or CBP. Previously, we found that arylsulfonamides can antagonize HIF transcription in a bioassay, block the p300/HIF-1α interaction, and exert potent anticancer activity in several animal models. In the present work, KCN1-bead affinity pull down, ¹⁴C-labeled KCN1 binding, and KCN1-surface plasmon resonance measurements provide initial support for a mechanism in which KCN1 can bind to the CH1 domain of p300 and likely prevent the p300/HIF-1α assembly. Using a previously reported NMR structure of the p300/HIF-1α complex, we have identified potential binding sites in the p300-CH1 domain. A two-site binding model coupled with IC₅₀ values has allowed establishment of a modest ROC-based enrichment and creation of a guide for future analogue synthesis.

Full text of DOI:10.1021/ml300042k

Letter
pubs.acs.org/acsmedchemlett
Binding Model for the Interaction of Anticancer Arylsulfonamides
with the p300 Transcription Cofactor
Qi Shi,† Shaoman Yin,§ Stefan Kaluz,§,⊥ Nanting Ni,# Narra Sarojini Devi,§ Jiyoung Mun,∇
Danzhu Wang,# Krishna Damera,# Weixuan Chen,# Sarah Burroughs,# Suazette Reid Mooring,#
Mark M. Goodman,*,∇,⊥,∥ Erwin G. Van Meir,*,§,∥,⊥ Binghe Wang,*,# and James P. Snyder*,†,‡
†Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States
‡Emory Institute for Drug Discovery, Emory University, Atlanta, Georgia 30322, United States
§Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, Emory University School of Medicine, Atlanta, Georgia
30322, United States
∥Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia 30322, United States
⊥Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, United States
#Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302-4098,
United States
∇Radiology and Imaging Sciences, Emory University, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: Hypoxia inducible factors (HIFs) are transcription factors that activate
expression of multiple gene products and promote tumor adaptation to a hypoxic
environment. To become transcriptionally active, HIFs associate with cofactors p300
or CBP. Previously, we found that arylsulfonamides can antagonize HIF transcription
in a bioassay, block the p300/HIF-1α interaction, and exert potent anticancer activity
in several animal models. In the present work, KCN1-bead affinity pull down, 14C-
labeled KCN1 binding, and KCN1-surface plasmon resonance measurements provide
initial support for a mechanism in which KCN1 can bind to the CH1 domain of p300
and likely prevent the p300/HIF-1α assembly. Using a previously reported NMR
structure of the p300/HIF-1α complex, we have identified potential binding sites in
the p300-CH1 domain. A two-site binding model coupled with IC50 values has allowed
establishment of a modest ROC-based enrichment and creation of a guide for future
analogue synthesis.
KEYWORDS: hypoxia, solid tumors, p300, HIF arylsulfonamide inhibitors, binding model, QSAR, KCN1
constitutively expressed β-subunits. The HIF heterodimer
translocates to the nucleus and associates through its C-
terminal activation domain (CAD) with the CH1 domains of
cofactors p300 or CREB binding protein (CBP) to form a
functional transcription factor. Activation of the HIF tran-
scription pathway induces the expression of over a hundred
genes, which encode proteins involved in the regulation of
glucose metabolism, cell migration, and neovascularization.5,6
HIF-1α overexpression is a common occurrence in most
human cancers and has been associated with drug resistance
mechanisms.2,7−10 Thus, because of the central role of HIF in
cancer, it has become an important target for cancer therapy
using different approaches.9,11−15 In previous screening studies
for the identification of small molecule inhibitors of the HIF
pathway, we identified arylsulfonamides as a new scaffold that
ypoxia is a condition in which insufficient oxygen is
Hsupplied to tissues. It is prevalent in fast-growing solid
tumor tissues, due to inadequate development of vascular
supply.1 The resulting perturbation in physiology renders the
tumor more resistant to chemo- and radiotherapies.2 Tumors
develop adaptive mechanisms to grow in a hypoxic micro-
environment, including a switch to glycolytic metabolism and
the activation of signals for the recruitment of new
vasculature.3−5 The hypoxia inducible factor (HIF) pathway
plays a crucial role for tumor cell growth under oxygen-
deprived conditions.5 HIFs are a family of heterodimeric
transcription factors composed of α- and β-subunits. The α-
subunits are regulated at the post-translational level by a family
of prolylhydroxylases (PHDs), which under normoxic con-
ditions tag them with a hydroxyl group causing recognition by
the Von Hippel Lindau protein, a component of an E3
ubiquitin ligase complex, and initiates their degradation by the
proteasome. The PHDs require oxygen to function; hence,
under hypoxia, HIFα subunits are stabilized and bind to the
Received: February 18, 2012
Accepted: June 21, 2012
Published: June 21, 2012
© 2012 American Chemical Society
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can interfere with HIF transcription using a hypoxia responsive
element (HRE)-driven gene luciferase-based reporter
assay.16−18 KCN1, one of the lead compounds (Figure 1),
interaction between KCN1 and HIF-1α was observed. This
experiment supports the hypothesis that KCN1 binds to p300,
but the exact binding site is not revealed.
Recombinant fusion peptides containing glutathione S-
transferase (GST) and the CH1 domain of p300 (GST-p300-
CH1) were produced in bacteria, purified, and then incubated
with 14C-KCN1 (see the Supporting Information for the
synthesis). After washing, the bound activity was counted in a
scintillation counter and shown to be significantly greater than
that obtained with GST-only peptides used as a control (Figure
3, left). This experiment supports the hypothesis that KCN1
can bind to the CH1 domain of p300.
Figure 1. Structure of KCN1 highlighted by three key substituent
groups.
was further investigated and demonstrated strong antitumor
activity against malignant gliomas, pancreatic cancer, and
metastatic uveal melanoma in animal models.19−21 These
studies further suggested that KCN1 might inactivate HIF
transcriptional activity by disrupting the interaction of the
p300/CBP cofactors with HIF-1α.21 In the present work, we
provide initial evidence for a mechanism of action where KCN1
can bind to the CH1 domain of p300. Finally, we established a
molecular model for the possible binding of KCN1 and its
analogues to p300 through computational modeling of putative
binding sites.
Figure 3. 14C-KCN1 binds to the p300 CH1 domain. The
recombinant proteins size, quantities, and integrity were verified by
Coomassie-stained gel electrophoresis (right).
Conceptually, KCN1 could inhibit HIF transcriptional
function by disrupting the p300/HIF-1α interaction by binding
separately to p300 or HIF-1α or by associating with the
complex such that function is attenuated. Our previous studies
have shown that KCN1 can disrupt the interaction between
p300 or CBP with HIF-1α using coimmunoprecipitation
studies, excluding the latter possibility.21 While p300 is able
to maintain its 3D architecture even in the absence of HIF-1α,
the C-terminal transactivation domain (CAD) of the latter is
disordered when uncomplexed with p300.22,27 Therefore, a
reasonable hypothesis is that KCN1 binds to p300 and thereby
blocks induced fit by HIF-1α. We have utilized affinity pull
down analysis with KCN1 coupled beads, 14C-KCN1 binding
to p300, and surface plasmon resonance (SPR) measurements
of the KCN1/p300-CH1 interaction to test this hypothesis.
Using nuclear extracts from hypoxic human glioma cells,
binding of p300 and HIF-1α was probed in affinity pull down
experiments with KCN1 immobilized on agarose beads through
chemical cross-linking with an aliphatic linker (see the
Supporting Information for the synthesis). Retained proteins
were subjected to Western blot analysis. As Figure 2 illustrates,
KCN1-coupled beads pulled down a fraction of the cellular
p300 (+ lane), while little nonspecific binding was observed
with beads alone used as a control (− lane). No detectable
SPR methodology has been widely used for defining the
kinetics and affinities for the interactions between a wide variety
of macromolecular entities and high- and low-affinity small
molecules.23−26 In the present instance, KCN1 was covalently
tethered to a gold surface as shown in Figure 4.
Figure 4. KCN1 attached to a gold surface.
Recombinant p300-CH1 peptides (after cleavage of the GST
moiety) were streamed over the KCN1-enriched surface in a
range of concentrations as illustrated by Figure 5. It shows that
SPR signals respond to the different protein concentrations,
which clearly indicates the existence of binding between KCN1
and p300-CH1. Analysis of the curve shapes and concentration
dependence permits an estimate that KCN1 binds to p300-
CH1 with a Kd value of ∼345 nM. Using a 1:1 kinetic binding
model (A + B = AB), the association and dissociation processes
were fitted to obtain the on (ka) and off (kd) rates separately.
Consequently, the Kd value was calculated as kd/ka ∼ 345 nM.
These results and the biochemical assays encouraged us to
build a model of the p300-CH1/KCN1 complex and employ it
for prospective generation of new analogs for bioactivity testing.
Neither the affinity pull down, radiolabeling binding assay,
nor the SPR experiment provides information on the precise
location of the presumed binding site for KCN1 on the CH1
Figure 2. Western blot analysis pull down of p300 (upper panel) and
HIF-1α (lower panel) proteins using KCN1-coupled agarose beads (+
lanes). A fraction (10%) of the cell extract before pull down was used
as a control to verify protein expression in the cell extract (Input).
Uncoupled beads were used as a control for nonspecific binding (−
lanes).
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Figure 7. Two favored docking sites for KCN1 resulting from Glide/
MM-GBSA scoring.
Figure 5. SPR sensorgrams after subtraction of control illustrate
KCN1 binding to p300; protein concentrations from 3 to 60 μg/mL.
domain of p300. In addition, no crystal structure of a small
molecule complexed with p300 is available. Therefore, we
sought to obtain binding site information from the structure of
p300, previously reported mutants, and molecular docking.
Figure 6 illustrates the structure of the p300(CH1 domain)/
Figure 8. Detailed protein−ligand contacts for site 1 illustrating a
substantial hydrophobic contact surface (cyan; Leu345 and Ile400 lie
beneath the KCN1 ligand) and supplemental polar anchors (dotted
lines; yellow).
Leu376, and Ile40031 complemented by π−π stacking with
His349. The dimethoxyphenyl ring is located above Leu346,
and one of the OMe units is stacked against Trp403, while the
dimethylbenzopyran faces the side chains of Ile400 and
Met379. Supplemental polar anchors are present for one of
the aromatic methoxy groups (Lys350), the SO2 moiety
(Lys404), and the benzopyran oxygen (Ser401 H-bond).
These suggested outcomes are supported by a random
mutation study for the p300/HIF-1α complex.32 Leu344,
Leu345, Cys388, and Cys393 on p300 were determined to be
crucial for interaction with HIF-1α (Figure 9). Cys388 and
Cys393 coordinate to a zinc ion, explaining that disruption of
the p300 zinc finger by random mutations at the two residues
has interrupted the p300 structure and consequently its ability
Figure 6. p300 (yellow)/HIF-1α (purple) complex; the insert reveals
four potential binding sites for KCN1.
HIF-1α(CAD) complex derived by NMR spectroscopy (PDB
code: 1L3E27). p300-CH1 consists of three major helices nearly
perpendicular to each other. HIF-1α -CAD primarily presents
loops that lay on the surface of p300, but two small helices (A
and B) bind to orthogonal intersections of the helices of p300-
CH1. The helix−helix interactions are characterized by a
significant degree of hydrophobicity, which undoubtedly
contributes to the mutual binding.
p300-CH1 was extracted from the complex of Figure 6 and
inspected graphically to reveal four possible clefts available for
ligand binding at the junctions of the three helices (Figure 6
insert). Each was subjected to KCN1 docking with Glide
followed by Prime MM-GBSA rescoring.28,29 Remarkably, the
top two best-scoring sites (sites 1 and 2, Figures 6 and 7) are
coincident with the binding locus of the two HIF-1α helices (A
and B). Similar results were obtained with the structure of the
CBP-CH1 cofactor determined by NMR.30 Examination of the
detailed ligand-p300 interactions at site 1 reveals substantial
hydrophobic contacts (Figure 8). The N-phenyl moiety is
nestled in a deep pocket surrounded by Leu345, Leu346,
Figure 9. p300-HIF-1α complex illustrating the spatial relationship of
residues critical for complex formation.
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to interact with HIF-1α. As described above, KCN1 docking
leads to intimate contacts between Leu344 and Leu345 and the
ligand fragments R1 and R2 (Figure 1). In a similar vein, two
residues critical for HIF-1α binding to p300, Leu818 and
Leu822,32 form a hydrophobic cluster with p300 Leu344 and
Leu345 side chains (Figure 9). Thus, it is reasonable to
hypothesize that one or both of the helix−helix interaction sites
in the p300/HIF-1α complex might serve as binding centers for
KCN1 and its analogues.
A series of recently reported KCN1 analogs were selected for
quantitative structure−activity relationship (QSAR) investiga-
tion (Figure 1 and Tables S1 and S2 in the Supporting
Information).16−18 The analogues were subjected to Glide
docking at each of the two favored binding sites (Figure 6)
followed by energy rescoring with Prime MM-GBSA to obtain
estimates of binding free energy.28,29 The IC50 values for
inhibition of HIF transcriptional activity in a glioblastoma cell
reporter assay were obtained from single triplicate runs as
previously described.16−18 To minimize possible biological
variability from measurements made over time, an IC50 value
for each analogue scaled to the corresponding KCN1 IC50
reference determined in the same experiment was employed for
the correlations. Thus, the ratio of the IC50 value of the
analogue and the corresponding IC50 value of KCN1 was used
as a measurement of activity (ΔIC50 ∼ K) and applied to the
QSAR analysis.
uncomplexed with p300, it is reasonable to assume that
occupancy of these two critical sites will prevent the induced fit
with HIF-1α. On the basis of the large protein−protein
interface and the energy needed to disrupt a pre-existing
complex, we believe that KCN1 action most likely occurs when
de novo-stabilized HIF-1α assembles into a 3D structure with
p300. This also partly explains why an affinity approach with
KCN1 beads on a cell extract with preformed HIF-1α-p300
complexes pulls down only a relatively small fraction of total
p300. Another reason is that only a fraction of total p300
associates with HIF, as it is bound to other cellular proteins.
The proposed two-site binding model explains many of the
experimental results, provides a useful preliminary ROC
analysis, and may prove applicable to the identification of
novel KCN1 analogues with improved potency. Further
validation of the model will be performed with an expanded
set of analogues.
EXPERIMENTAL PROCEDURES
■
KCN1 linked to agarose beads (see the Supporting Information) was
used to pull down KCN1-interacting proteins. Nuclear extracts
prepared from hypoxic (1% O2) LN229 human glioma cells using
the NE-PER kit (Pierce) were precleared with ethanolamine activated
agarose beads to eliminate nonspecific protein binding to the beads.
Bound proteins were separated by denaturing gel electrophoresis
(SDS-PAGE), and Western blots were performed using anti-p300 and
anti-HIF-1α antibodies as previously described.12,34
A variety of approaches employing full and split data sets,
explcit ligand docking, and ligand-only pharmacophores failed
to generate a quantitative SAR with r2 greater than 0.7. It would
appear that significant scatter in the present data precludes
development of a robjust QSAR. An alternative approach is the
ROC (receiver operating characteristic) methodology,33 which
uses a binary classifier to assess whether the predicted binding
energy values are useful for enrichment of the active
compounds. In the present context, we define true positives
as compounds with an average IC50 value <650 nM. Five of 30
analogues fall in this category based on the cell-based reporter
assay: 648, 306, 478, 378, and 280 nM activity (See Table 10,
ref 17).
Plotting selectivity versus sensitivity (i.e., the fraction of true
positives vs the fraction of false positives at a given threshold
setting), the AUCs (area under curve) for both sites 1 and 2 are
∼0.7, the ideal being 0.8−1. This indicates that the estimated
energy values (MM-GBSA) can, to some extent, differentiate
active from inactive analogues. Thus, we assume that
compounds demonstrating favorable energies at both sites are
likely to be active. In a test study carried out for the 30 available
KCN1 analogues, only three survived: KCN1, KCN2, and
KCN5, all of which are true positives. It is noteworthy that the
latter two analogues are the most potent agents among the test
molecules. This outcome indicates that the estimated energy
values at both sites can enrich the active compounds. In the
future, prior to a full-scale QSAR, structures with equal or
better estimated potency values will be considered high priority
for synthesis.
GST-only and GST-p300-CH1 proteins were expressed and
purified as described previously.35 For the SPR experiments, p300-
CH1 was cleaved from the purified GST fusion protein (10 mg) with
50 units of thrombin (GE Healthcare) in PBS at room temperature
overnight, and its concentration was evaluated by the BCA Protein
Assay (Thermo Scientific).
Binding and kinetics measurements were performed with a BIAcore
T200 system and carboxylic acid coated sensor chips (CM-5 chips
with five channels from BIAcore, two of which were used here). After
activation of the surface with EDC/NHS, KCN1-amine (500 nM; see
the Supporting Information for the synthesis of the free amine) in
HBS-EP+ buffer was immobilized on the flow cell gold surface by
covalent capture. One channel was used to immobilize KCN1-amine,
and the other was left blank as a control. Recombinant p300-CH1
peptides in PBS were injected at a flow rate of 50 μL/min. A 1 M
NaCl solution was used to dissociate the p300 from the KCN1
compound for surface regeneration. Injection of the protein
(association) was followed by injection of running buffer (dissocia-
tion). To reduce the probability of nonspecific binding to the chip
surface, 50 μL/L of surfactant P20 was added to the p300 buffers in
the binding experiments.
Two-dimensional structures of KCN1 and its analogues were
modeled by Chemdraw and then submitted to Ligprep in Maestro 9.0
to obtain 3D structures. The p300-CH1/HIF-1α CAD protein
complex (PDB code 1L3E) was processed by the protein preparation
wizard followed by removal of HIF-1α CAD. The p300 receptor was
refined with the OPLS2005 force field by Impref minimization. Four
docking sites, as shown in Figure 6, were selected for KCN1 Glide
docking to determine the most favorable interaction pharmacophores.
For other analogues, only sites 1 and 2 in Figure 6 were chosen for
QSAR development. The receptor grid was generated at both
presumed binding sites using the Glide receptor grid protocol without
applying constraints. Ligand docking was accomplished with Glide SP
precision (Maestro 9.0) for flexible docking of the ligands.28 The top
20 poses at each site were subsequently submitted to Prime MM-
GBSA (all atoms of the receptor are frozen) for rescoring. The two
best-scored KCN1 binding sites (1 and 2) were subsequently
employed for linear regression and ROC evaluation.
On the basis of affinity pull down analysis, 14C-KCN1
binding, KCN1/p300-CH1 SPR binding measurements, and
evaluation of the NMR structure of the p300/HIF-1α complex,
we provide a model illustrating how KCN1 and its analogues
might antagonize the p300/HIF-1α interaction by occupying
the two binding sites on p300-CH1 that are normally engaged
by the two inducible helices of HIF-1α CAD when p300 and
HIF-1α are associated. Because HIF-1α is disordered when
For each ligand, the rescored top pose was selected, and the
calculated MM-GBSA ΔG value was used as an estimate of binding
energy. On the experimental side, certain compound IC50 values were
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measured at different times with KCN1 as a standard in each case. The
ratio of the IC50 value of a given ligand to the corresponding IC50 value
of KCN1 was used as a measurement of activity (ΔIC50 ∼ K) and
applied to the attempted QSAR correlations. The relative binding
energies (ΔE) and activities (K) were fitted by the equation ΔE = −
RT ln K.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of KCN1-amine, immobilization of the latter on a
gold surface and agarose beads, synthesis of 14C-KCN1, and a
list of structures employed in the p300/KCN1 analogue
analyses. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: 404-727-9366. Fax: 404-712-5689. E-mail: mgoodma@
emory.edu (M.M.G.). Tel: 404-778-5563. Fax: 404-778-5550.
E-mail: evanmei@emory.edu(E.G.V.M.). Tel: 404-413-5544.
Fax: 404-413-5505. E-mail: wang@gsu.edu (B.W.). Tel: 404-
727-2415. Fax: 404-712-8679. E-mail: jsnyder@emory.edu
(J.P.S.).
Funding
This work was supported by EmTechBio, the Southeastern
Brain Tumor Foundation, the University Research Council, the
NIH [R01 CA86335, CA116804 (E.G.V.M.), P30 CA138292
(Winship), GM084933 and GM86925 (B.W.), and P50
CA128301 (M.M.G.)], the V Foundation, the Max Cure
Foundation, and the Samuel Waxman Cancer Research
Foundation (E.G.V.M.). J.P.S. and Q.S. are likewise grateful
to Prof. Dennis Liotta for generous support.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
HIF, hypoxia inducible factor; CBP, CREB binding protein;
SPR, surface plasmon resonance; PHDs, prolylhydroxylases;
QSAR, quantitative structure−activity relationship; GST,
glutathione S-transferase
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