Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains

Article abstract of DOI:10.1021/ja412434f

Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (K _d = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.

Full text of DOI:10.1021/ja412434f

Article
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Discovery and Optimization of Small-Molecule Ligands for the CBP/
p300 Bromodomains
Duncan A. Hay,†,‡ Oleg Fedorov,‡,§ Sarah Martin,‡,§ Dean C. Singleton,‡,§ Cynthia Tallant,‡,§
Christopher Wells,‡,§ Sarah Picaud,‡ Martin Philpott,‡,§ Octovi‡a,§P. Monteiro,‡,§ Catherine M. Rogers,‡,§
Stuart J. Conway,† Timothy P. C. Rooney,† Anthony Tumber, Clarence Yapp,‡,§
Panagis Filippakopoulos,‡ Mark E. Bunnage,⊥ Susanne Muller,‡,§ Stefan Knapp,‡,§
̈
Christopher J. Schofield,† and Paul E. Brennan*,‡,§
†Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3TA, U.K.
‡Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ,
U.K.
§Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford, OX3 7LD, U.K.
⊥Worldwide Medicinal Chemistry, Pfizer, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Small-molecule inhibitors that target bromodomains
outside of the bromodomain and extra-terminal (BET) sub-family
are lacking. Here, we describe highly potent and selective ligands for
the bromodomain module of the human lysine acetyl transferase
CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles.
Our starting point was a fragment hit, which was optimized into a
more potent and selective lead using parallel synthesis employing
Suzuki couplings, benzimidazole-forming reactions, and reductive
aminations. The selectivity of the lead compound against other
bromodomain family members was investigated using a thermal
stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity
issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of
BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two
distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues,
selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective,
displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching
(FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300;
therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300
bromodomains as therapeutic targets.
unmodified lysine side chains. These processes enable CBP/
p300 to exert context-dependent regulation of transcriptional
control. The sequence similarity between CBP and p300 is high
in the conserved functional domains, with the BRD having 96%
similarity (Figure 1A).
CBP and p53. Mutations of the p53 gene are common, with
around 50% of human cancers encoding such mutations.21−23
In response to cellular stress, p53 undergoes PTMs of the C-
and N-terminal regions, including acetylation at the C-terminal
region, which results in changes in the p53-dependent
activation of target genes leading to cell cycle arrest,
senescence, or apoptosis.23−25 Lysine acetylation at lysine 382
(K382) of p53 is responsible for recruitment of CBP via its
INTRODUCTION
■
CBP and p300. The CREB (cyclic-AMP response element
binding protein) binding protein (CBP) and E1A binding
protein (p300) are ubiquitously expressed pleiotropic lysine
acetyl transferases that play a key role as transcriptional co-
activators in human cells.1−5 CBP and p300 possess nine
conserved functional domains which bind to general and gene-
specific transcription factors such as the hypoxia-inducible
transcription factor (HIF) and the human tumor suppressor
protein p53 (Figure 1A).6−15 Both CBP and p300 possess a
single bromodomain (BRD) and a lysine acetyltransferase
(KAT) domain, which are involved in the post-translational
modification (PTM) and recruitment of histones and non-
histone proteins.16−20 The BRD is an acetyl-lysine (Kac)-
selective recognition module, whereas the KAT domain
transfers an acetyl group from acetyl co-enzyme A to
Received: December 12, 2013
Published: June 19, 2014
© 2014 American Chemical Society
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Figure 1. (A) Percent conservation and domain organization of human CBP (accession no. Q92793) and p300 (accession no. Q09472).
Abbreviations: ZF TAZ 1 and 2, zinc finger transcription adaptor putative zinc finger-type; KIX, kinase-inducible domain interacting domain; BRD,
bromodomain; RING, really interesting new gene; PHD, plant homeodomain; KAT, lysine acetyltransferase domain; ZF ZZ, zinc finger, ZZ-type;
NRC, nuclear receptor co-activator interlocking domain; CH1−3, cysteine−histidine-rich regions 1−3. (B) CBP BRD-fold depicting the four α-
helices (αZ, αA, αB, αC) and the ZA and BC loops that form the Kac binding pocket (derived from PDB 3P1C).27 (C) Structure of an acetyl lysine
(yellow, only partial structure shown) and CBP BRD complex. Key interactions are shown, including the hydrogen bond (dotted lines) from the Kac
carbonyl to N1168 and a water-mediated hydrogen bond from the Kac carbonyl to Y1125 (PDB 3P1C).27 (D) CBP BRD ligands with reported
affinities.28−31
BRD, as shown by NMR titration of the CBP BRD with
acetylated p53 peptides, and transfection of p53−/− cells with
mutated p53.12 Additionally, in a p21 luciferase assay, the CBP-
Kac interaction was shown to be crucial for p53-induced p21-
mediated G1 cell cycle arrest.
Additionally, chemo- and radio-therapy can cause p53-
mediated tissue damage of non-cancerous tissue, implying
that p53 inhibition could be used to protect healthy tissue
during these therapies.26 Thus, inhibition of the CBP BRD, and
therefore p53-mediated p21 activation, has potential clinical
applications.
myocardial ischemia.32−37 Thus, inhibitors of p53 activity
represent potential points of intervention in multiple diseases.
The CBP/p300 Bromodomain. Bromodomains are made
up of ∼110 residues arranged in a characteristic structure made
up of four α-helices (αZ, αA, αB, αC) connected by interhelical
loops, termed the BRD fold (Figure 1B). Two loop regions
(ZA and BC) form the largely hydrophobic Kac side-chain
binding pocket, which in most BRDs binds the Kac N-acetyl
group via a hydrogen bond between the acetyl carbonyl and the
NH2 of a well-conserved asparagine residue (N1168 in CBP)
and a water-mediated hydrogen bond from the acetyl carbonyl
to the phenolic hydroxyl group of a conserved tyrosine (Y1125
in CBP) (Figure 1C).27,38−42
In addition to its important tumor suppressor role,
hyperactive p53 is implicated in Alzheimer’s disease,
Parkinson’s disease, spinal cord diseases, multiple sclerosis,
ischemic brain injury, infectious and auto-immune diseases, and
The 61 known BRDs encoded by the human genome can be
clustered into sub-families on the basis of sequence and
structural similarity.27 Selective inhibitors of these sub-families
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could serve as tools to elucidate the function of these proteins,
and these have started to emerge in recent years. In particular,
potent and selective inhibitors of the BRD and extra-terminal
(BET) sub-family are now available from several different
structural classes.43−49
RESULTS AND DISCUSSION
■
An X-ray crystal structure of the reported dimethylisoxazole
compound 7 in complex with the CBP BRD illustrated two
potential regions which substituted analogues of 5 could
interact with, potentially leading to improvements in potency
and selectivity (Figure 3).43 Figure 3B shows how the
dimethylisoxazole of compound 7 mimics the key Kac binding
interactions of the CBP BRD with an H-bond to N1168 and a
water-mediated H-bond to Y1125. Figure 3C highlights the two
regions targeted for analogues of 5. Region 1 is comprised of
part of the “ZA-channel” and is largely hydrophobic, except for
Pioneering work on the inhibition of the CBP BRD emerged
from the Zhou group, which reported several compounds with
micromolar affinities (Figure 1D).28−30 The N-acetylindole
MS7972 (compound 1, Kd = 19.6 μM) was shown to block the
p53−CBP interaction at 50 μM in a competition assay.28
Ischemin (compound 2, Kd = 19 μM) inhibited p53-induced
p21 activation in a luciferase reporter-gene assay (IC50 = 5 μM)
and down-regulated p53 target gene expression under oxidative
stress conditions.29 The cyclic peptide 3 has also been shown to
bind the CBP BRD (Kd = 8 μM) and to inhibit p53 in the
reporter assay.30 These compounds demonstrated the potential
of CBP BRD inhibitors to modulate p53-mediated expression;
however, they are not very potent or well characterized in terms
of selectivity against other BRD-containing proteins. Recent
reports also describe potent but non-selective or moderately
selective CBP BRD inhibitors, including the sub-micromolar
dihydroquinoxalinone inhibitor 4.31,50 We now report on the
discovery of a series of potent and selective CBP/p300 BRD
inhibitors, and we demonstrate their inhibition of the CBP−
p53 interaction in cells. In addition, we demonstrate how
compounds from the series bind in the Kac binding pocket
using X-ray crystallography.
Our starting point for developing selective CBP/p300
inhibitors was the reported non-selective 3,5-dimethylisoxazole
BRD inhibitor, 5 (Figure 2).43 Compound 5 was considered to
Figure 2. Starting point for this project. Compound 5 is a non-
selective CBP and BRD4(1) inhibitor.43
be an attractive fragment to develop CBP BRD-selective
inhibitors because it has a low molecular weight (213 Da) and
reasonable LipE51 (3.5) and ligand efficiency (0.45)52,53 for
CBP, and because it has various points useful for the
introduction of diversity to the core scaffold. Our aim was to
develop the scaffold of compound 5 with the goal of achieving
potent compounds (Kd ≤ 0.1 μM) with selectivity over other
BRD sub-families (≥30-fold) in vitro and displaying target-
based cellular activity (IC50 ≤ 1 μM) to enable functional
studies in cellular systems.47,54
Several reports describe 1,3-dimethylisoxazoles as potent
inhibitors of the BET BRDs, including the benzimidazole
compound 6.43,45,46,55 Therefore, it was recognized that
obtaining selectivity for CBP/p300 over the BET BRDs
could represent a substantial challenge. Compounds would
therefore initially be screened against both CBP and BRD4(1)
BRDs as representative examples of their respective sub-
families. All in vitro screening and X-ray crystallography was
carried out using recombinant BRDs as surrogates of full-length
protein.
Figure 3. (A) Compound 7.43 (B) View from an X-ray crystal
structure of compound 7 (carbon = yellow) bound to CBP BRD
showing key H-bond interactions (PDB 4NR4). Kac (carbon =
magenta) from PDB 3P1C is overlaid for reference. The dimethyl-
isoxazole group is positioned to form an H-bond (gray dotted lines) to
N1168 and a water-mediated H-bond to Y1125. The N-3 of the
benzimidazole is positioned to form a water-mediated H-bond to the
carbonyl of P1114. (C) Surface representation of CBP complexed with
compound 7 with two shaded regions marked as potential areas to
introduce functionality onto the benzimidazole scaffold in order to
enhance potency and selectivity. Region 1 (blue) comprises the ZA
channel which has points for interaction with polar functionality, such
as the carbonyl on P1114, or the carboxamide of Q1113. Region 2
(green) is analogous to the “WPF shelf” in BRD4(1). R1173 was
identified as a potential residue to interact with in an otherwise
hydrophobic region.
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backbone carbonyls and the carboxamide of Q1113. Region 2 is
analogous to the WPF shelf of BRD4(1).56 The surface is
mostly hydrophobic but also has the side-chain of R1173 as a
potential site for ligand interactions that could give selectivity
for CBP over BRD4(1). On the basis of this analysis, it was
anticipated that analogues of compound 5 which possessed
substitution on the N-1 and C-2 may be able to interact with
regions 1 and 2, with the aim of improving potency and
selectivity. However, in initially library chemistry, other
substitution patterns were also included in anticipation that
unexpected interactions may occur.
using parallel synthesis for the coupled reduction/cyclization
step (Scheme 2). Dithionite-mediated reduction of the aryl
a
Scheme 2. Synthesis of Trisubstituted Benzimidazoles
Library Chemistry. In order to identify more potent and
selective leads for the CBP/p300 BRD, a set of Suzuki
couplings was carried out in parallel with a commercially
acquired 3,5-dimethylisoxazole-4-boronic acid pinacol ester and
a set of heteroaryl bromides (Scheme 1). The heteroaryl
a
Scheme 1. Parallel Suzuki Couplings
a
Reagents and conditions: 3,5-dimethylisoxazole-4-boronic acid
pinacol ester, Pd(dppf)Cl2 [dppf = 1,1′-bis(diphenylphosphino)-
ferrocene], NaHCO3, 1,2-dimethoxyethane (DME)/H2O, 100 °C,
5−93%. See Supporting Information (SI) for specific structures of
products s1−s82.
a
Reagents and conditions: (a) 3,5-dimethylisoxazole-4-boronic acid
pinacol ester, Pd(dppf)Cl2, NaHCO3, DME/H2O, 73%; (b) N,N-
dimethylethylenediamine, N,N-diisopropylethylamine (DIPEA), tetra-
hydrofuran (THF), 86%; (c) RCH2CH2CHO, Na2S2O4, H2O, EtOH,
dimethyl sulfoxide (DMSO), 80 °C, 8−54% (see SI for specific
structures of products s83−s100); (d) NH2CH2CH(OMe)2, DMSO;
(e) PhCH2CH2CHO, Na2S2O4, H2O, MeOH, 65% (two steps); (f)
CF3CO2H, H2O, CH2Cl2, microwave 150 °C, 99%; (g) amine (RH),
NaBH(OAc)3, THF, 35−86%; (h) Amine (RH), NaCNBH3, AcOH,
MeOH, 42%; (i) 4-(2-aminoethyl)morpholine, DIPEA, THF, 94%; (j)
Na2S2O4, H2O, EtOH, 80 °C, 87%; (k) RCH2CH2CO2H, T3P,
DIEPA, EtOAc, microwave, 150 °C, 31−62% (l) RCH2CH2CO2H,
T3P, DIPEA, EtOAc, reflux, 15−63%; (m) RCH2CH2CO2H, 6 M aq.
HCl, microwave 210 °C, 16−55% (see SI for specific structures of
s101−s116).
bromides were chosen from the Pfizer compound collection
and were selected so that products obtained would have
Lipinski rule of 5 compliant properties (e.g., clog P < 5 and
MW < 500), with the majority of the compounds targeted at
around clog P = 2−4 and MW = 300−400.57 The set chosen
consisted of 101 heteroaryl bromides, which had a variety of
substituents on the heterocyclic core in order to maximize
diversity. Reactions and workups were carried out in parallel,
and products were purified by automated preparative HPLC.
Product purity was assessed by UV, evaporative light scattering
detection, and MS. The 101 reactions delivered 83 target
compounds in sufficient yield and purity for biochemical testing
using differential scanning fluorimetry (DSF, ΔTm), a high-
throughput assay used as a surrogate for displacement assays.47
From this initial library, compound 8 emerged as a promising
hit, with ΔTm = 4.5 °C against CBP and 3.2 °C against
BRD4(1) at a compound concentration of 10 μM (Figure 4).
To improve potency and selectivity for CBP, analogues of
compound 8 varied in the C-2 position were then prepared
nitro group of compound 11 in the presence of R-aldehydes
efficiently afforded the desired benzimidazole targets which
were purified by automated HPLC. The most promising
compound from this set was compound 12, with an increased
ΔTm = 6.3 °C (4.5 °C for compound 8) against CBP while
maintaining BRD4(1) potency at ΔTm = 2.9 °C (3.2 °C for
compound 8) (Figure 4). A pIC50 = 6.3 was measured for
compounds 8 and 12, using a peptide displacement
AlphaScreen (amplified luminescent proximity homogeneous
assay screen).58 The CBP potency represents a significant
improvement on compound 5 (pIC50 = 5.4). These results
implied that differential 1,2,-disusbtitution of benzimidazoles
could lead to compounds with improved potency and
selectivity. It was therefore decided to optimize the substituents
on this template via focused synthesis to further improve
potency and selectivity.
N-1 Amine Optimization. Late-stage variation of the
amino moiety was achieved by reductive amination of an
aldehyde, obtained from the dimethyl acetal 13 (Scheme 2).
The compounds obtained were screened by DSF and
AlphaScreen (Table 1). Comparison of the screening
Figure 4. CBP-selective hits from initial parallel chemistry. Mean
values SEM (number of measurements).
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showed that further work needed to be done in order to obtain
sufficient selectivity for CBP/p300.
Table 1. Structure−Activity Relationships for CBP and
BRD4(1) Binding As Determined by DSF and AlphaScreen
a
Selectivity Optimization. In order to identify potential
avenues for improving the selectivity of the series, high-
resolution crystal structures were determined for compound 17
in complex with CBP and BRD4(1) (Figure 5). The results
suggested that differences in the observed binding modes could
be exploited to improve selectivity. In the CBP complex, the
morpholine moiety of compound 17 occupies an area in the ZA
channel, between the targeted regions 1 and 2. The phenethyl
group of compound 17 appears to be in a hydrophobic region
on the edge of the pocket. In BRD4(1), compound 17 adopts a
flipped binding mode with respect to side-chain orientation;
notably, a water-mediated hydrogen bond from the NH2 of the
Q85 carboxamide to the benzimidazole N-3 nitrogen is
apparent. The morpholine moiety points out toward solvent,
whereas the phenethyl group fits into a hydrophobic region
comprising the WPF shelf and ZA channel of BRD4(1).
C-2 Aryl Optimization. As the phenylethyl moiety of
compound 17 was observed to occupy different regions in the
CBP and BRD4(1) structures, it was decided to explore the
synthesis of a diverse set of compounds with substituted aryl
rings to test effects on selectivity. Additionally, it was
considered that the C-2 linked aryl group would be amenable
to late-stage variation, allowing for an efficient synthesis of
analogues. The targeted compounds were synthesized accord-
ing to Scheme 2. Thus, the phenylenediamine compound 22
was formed by an SNAr reaction of compound 10 followed by
dithionite-mediated nitro reduction. Precursor 22 was then
reacted with substituted carboxylic acids under dehydrating
conditions (propylphosphonic anhydride (T3P) or 6 M
aqueous HCl) to yield the target compounds 23−36 (Table
3) and s101−s116 (see SI).
for N-1 Analogues
a
Values are given as mean SEM (number of measurements).
The DSF results for compounds 23−25 (Table 3) suggest
that an electron-donating methyl group on the aryl ring is
tolerated, with the para-methyl analogue 25 showing an
increased ΔTm for CBP of 7.4 °C. The more strongly
electron-donating para-methoxy group in compound 26 gave
a further increase with ΔTm = 8.1 °C, and showed a larger
window over BRD4(1). Conversely, the strongly electron-
withdrawing nitro groups in compounds 27 and 28 were
detrimental to CBP binding. The results for compounds 29−31
indicated that halogens are tolerated on the phenyl ring, with
the meta-substituted fluorine analogue 30 appearing optimal,
with ΔTm = 7.1 °C against CBP. Compounds 32 and 33
combined the para-methoxy and meta-halogen moieties,
resulting in a further increase in CBP ΔTm to 9.0 and 9.6
°C, respectively. Analogues 34−36 demonstrated that the
phenyl group can be substituted for a heteroaryl group, with the
electron-rich indole-containing compound 36 representing the
optimal compound from this set with ΔTm = 8.9 °C against
CBP. It was encouraging that increases in CBP potency for the
best compounds in Table 3 were not accompanied by increases
in BRD4(1) potency.
Since the four aryl analogues 26, 32, 33, and 36 all indicated
improved potency in the ΔTm assay, the pIC50 against CBP was
determined using AlphaScreen (Table 4). The results were in
the range of 7.0−7.3, indicating that these were all promising
analogues warranting more detailed biophysical investigations
on their selectivity for CBP over BRD4(1). Thermodynamic
parameters for binding to CBP and BRD4(1) were therefore
determined by ITC for this set of compounds. The results are
summarized in Table 4.
techniques confirmed that DSF was an effective tool for ranking
the potency of compounds.59 The correlation was high enough
that the general and operationally simple DSF assay was felt to
be a useful surrogate for the more complex AlphaScreen in
further efforts to increase the potency of the compounds. The
results suggest that simple cyclic amino analogues 14−16 (CBP
ΔTm = 0.27−5.4 °C, pIC50 = 3.3−6.2) are not as potent as the
dimethylamino variant 12 (CBP ΔTm = 6.3 °C, pIC50 = 6.3).
However, the morpholine-containing analogue 17 gave a slight
improvement with ΔTm values of 6.5 and 2.7 °C for CBP and
BRD4(1) respectively and was potent in CBP AlphaScreen
(pIC50 = 6.8). Piperazines 18 and 19, thiomorpholine 20, and
substituted morpholine derivative 21 were less potent and/or
less selective for CBP over BRD4(1). With the best balance of
CBP potency and BRD4(1) selectivity in the primary assays,
the binding constants (Kd) of compound 17 were then
determined using isothermal titration calorimetry (ITC)
(Table 2). The Kd value of 17 was measured at 0.32 μM
against CBP, 0.35 μM against p300, and 0.94 μM against
BRD4(1). These results indicate that the ITC measured
selectivities of compound 17 with respect to BRD4(1) were
only 3.0-fold and 2.7-fold for CBP and p300, respectively. This
Table 2. ITC Results for Compound 17
CBP
p300
BRD4(1)
Kd (μM)
0.32 0.094
0.35 0.082
0.95 0.044
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Table 3. Structure−Activity Relationships for CBP and
BRD4(1) Binding As Determined by DSF Assay for a
a
Selection of the C-2 Analogues
Figure 5. Views from X-ray crystal structures of compound 17
complexed to CBP (PDB 4NR5) and BRD4(1) (PDB 4NR8) BRDs.
For CBP: (A) view showing the H-bond interactions between the
oxygen of the isoxazole of 17 and N1168 (3.02 Å), and between the
nitrogen of the isoxazole and a water (2.75 Å) (water = red spheres),
and (B) surface view with shaded regions indicating regions targeted
by N-1 and C-2 benzimidazole substitution according to Figure 3. For
BRD4(1): (C) view showing the H-bond interactions between the
oxygen of the isoxazole of 17 and N140 (3.08 Å), and between the
nitrogen of the isoxazole and a water (2.91 Å); the H-bond between
the benzimidazole N-3 of 17 and a water molecule is also shown (2.71
Å), and W81 from other ligand-bound structures (carbon = magenta)
is overlaid to illustrate the shift in W81 side-chain position (PDB
3MXF, 4E96, 4C67),47,49,60 and (D) surface view.
a
Values are given as mean ΔTm SEM (number of measurements).
Gratifyingly, the substituted aryl analogues are more potent
against CBP than compound 17, with Kd in the range of 0.022−
0.050 μM. Additionally, the selectivity for CBP over BRD4(1),
as determined by ITC Kd values, had improved to 11−22-fold
for these analogues, with the best results being those of
compound 36, which had a CBP Kd = 30 nM and was 22-fold
selective over BRD4(1). Although the selectivity had improved,
efforts continued in order to achieve a greater window over
BRD4(1). To achieve this objective, the obtained crystal
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Table 4. Determination of pIC50 and Kd of Selected C-2 Analogues by AlphaScreen and ITC
ITC Kd (μM)
a
Cmpd
CBP AlphaScreen pIC50
CBP
BRD4(1)
Selectivity
26
32
33
36
7.0 0.17 (4)
7.2 0.0080 (2)
7.0 0.69 (2)
7.3 0.18 (2)
0.050 0.0039
0.028 0.0024
0.022 0.0017
0.030 0.0021
0.55 0.0033
0.48 0.038
0.44 0.025
0.66 0.055
11-fold
17-fold
20-fold
22-fold
a
Values are given as mean pIC50 SEM (number of measurements).
structures (Figure 5) were employed in order to further guide
design.
Table 5. Structure−Activity Relationships for CBP and
BRD4(1) Binding As Determined by ΔTm Assay for the
Indole Target and Conformationally Constrained/Polar C-2
Analogues
Indole Analogue. The structure of compound 17
complexed to BRD4(1) reveals a water-mediated hydrogen
bond from the benzimidazole N-3 to the protein backbone
(P82) and to the carboxamide side chain of Q85; analogous
interactions are absent in the equivalent CBP structure (Figure
5A,C). These observations imply that, in the absence of other
effects, replacing the benzimidazole ring with an indole that
does not contain the nitrogen in compound 17 should
negatively impact BRD4(1) binding, but not CBP.
The synthesis of the targeted indole analogue 40 is shown in
Scheme 3. Aminophosphonium salt 38 was synthesized
a
Scheme 3
a
Reagents and conditions: (a) HNO3 conc., H2SO4 conc., 0 °C, 39%;
(b) PPh3, CHCl3, quant.; (c) (i) Zn/AcOH; (ii) HCl, quant. (2
steps); (d) 3-(3-chloro-4-methoxyphenyl)propanoyl chloride, N,N-
dimethylformamide (DMF)/pyridine, 56%; (e) KOt-Bu, toluene,
microwave 130 °C, 56%; (f) 4-(2-chloroethyl)morpholine hydro-
chloride, NaH, KI, DMF, 80 °C, 24%, (g) 3,5-dimethylisoxazole-4-
boronic acid pinacol ester, Pd(dppf)Cl2, NaHCO3, DME/H2O, 43%.
according to known procedures61 and then acylated. Base-
promoted cyclization yielded the indole intermediate 39, which
was alkylated and cross-coupled to give the target molecule 40.
As hoped, indole 40 was completely inactive against
BRD4(1) with a ΔTm < 1 °C (Table 5). Disappointingly, it
also gave a significantly lower ΔTm for CBP (2.0 °C) than the
equivalent benzimidazole analogue 32. This implies that
although the crystal structure of compound 17 bound to
CBP shows no H-bond to the protein backbone, interaction of
the electron-poor benzimidazole with the CBP protein is
favored over that of the electron-rich indole.
Conformationally Constrained Analogues. The ethyl-
ene moiety of compound 17, which links the benzimidazole C-2
and phenyl groups, sits in a hydrophobic region in BRD4(1)
and partly occupies the space termed the WPF shelf, which
contains a tryptophan residue protruding out of the pocket
(Figure 5C,D). The orientation of W81 in BRD4(1) in the
complex with compound 17 is unusual when compared to
other BRD4(1) ligand-bound structures (Figure 5C) and shows
a
Values are given as mean ΔTm SEM (number of measurements).
how BRD4(1) can accommodate hydrophobic groups in
orientations other than those that occupy the typical WPF
shelf.47,48,55 It was envisaged that polar and/or constrained
linkers could reduce the affinity for BRD4(1). An increase in
polarity may disfavor binding in the hydrophobic WPF region,
while BRD4(1) may be less able to accommodate conforma-
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tionally constrained linkers with reduced degrees of freedom, as
they would be less able to avoid a steric clash with the WPF
shelf by bond rotation.
Synthesis of the oxygen-linked targets is shown in Scheme 4.
1,1′-Carbonyldiimidazole (CDI)-mediated cyclization of
modification of the N-1 ethylene linker between the morpho-
line moiety and the N-1 position of the benzimidazole ring. It
was again proposed that by constraining the conformation of
the linker it would force unfavorable interactions in BRD4(1)
due to steric interactions with the WPF shelf or by changing the
orientation of the phenethyl group (Figure 5).
Racemic analogues containing methyl groups on the N-1
ethylene linker (compounds 52−55) were prepared according
to the methodology in Scheme 2. Screening results for these
compounds are shown in Table 6. While methyl groups were
not well tolerated next to the benzimidazole ring (compounds
52 and 54), they were tolerated next to the morpholine ring
(compounds 53 and 55). The ΔTm and AlphaScreen values for
the racemic compounds were encouraging enough to prompt
synthesis of the single enantiomers, according to the route
shown in Scheme 5. Commercially acquired chiral (R)- and
a
Scheme 4. Synthesis of O-Linked Targets
a
a
Scheme 5. Route to Single Enantiomers
Reagents and conditions: (a) CDI, THF, reflux, 78%; (b) BnBr,
Ag2CO3, toluene, 80 °C (18%); (c) 2-hydroxyacetic acid, 6 M aq. HCl,
microwave 180 °C, 62%; (d) 3-fluoro-4-methoxyphenol, 1,1′-
(azodicarbonyl)dipiperidine, P(n-Bu)3, CH2Cl2, 67%.
phenyleneiamine 22 gave a 2-oxo precursor. Alkylation tended
to favor N-substitution, but use of Ag2CO3 as base gave a
mixture of N- and O-alkylated isomers, which could be
separated to yield the desired target 41. Compound 22 was
also used to prepare a hydroxymethyl precursor, which was
reacted with a phenol using Tsunodu’s Mitsunobu conditions
to yield target compound 42.62 Analogues 43−51, which
possess additional substitution or conformational constraints
on the ethylene moiety linking the benzimidazole and aryl
groups, were synthesized by methodology analogous to that
described in Scheme 2 (see SI).
a
Reagents and conditions: (a) (R)- or (S)-1,2-diaminopropane
dihydrochloride, K2CO3, DMF, 80 °C, 38−41%; (b) 2-bromoethyl
ether, K2CO3, DMF, 70 °C, 27−37%; (c) Na2S2O4, H2O, EtOH, 80
°C; (d) R2CH2CH2CO2H, T3P, EtOAc, reflux, 38−52%.
Disappointingly, screening using DSF showed no improve-
ment in potency and selectivity for the O-linked analogues (41,
42) or conformational constrained compounds (43−51) over
the analogous ethylene linked compounds (Table 5). With no
indication of an improvement in selectivity, attention shifted to
(S)-1,2-diaminopropane reacted via SNAr with 10, predom-
inantly at the less sterically hindered 1-amino group. This
reaction gave an inseparable 4:1 mixture of isomers in favor of
the desired compound. The morpholine ring was formed by
Table 6. Structure−Activity Relationships for CBP and BRD4(1) Binding As Determined by DSF and AlphaScreen for
Conformationally Constrained N-1 Linkers
a
R
2
ΔTm (°C)
a
Cmpd
1
3
CBP
BRD4(1)
CBP AlphaScreen pIC50
ND
52
53
54
55
58
59
60
61
62
63
A
A
B
B
A
A
B
B
C
C
CH3
H
H
5.8 0.092 (3)
9.4 0.58 (5)
5.6 0.29 (3)
9.1 0.44 (5)
7.5 0.10 (3)
9.7 0.31 (4)
7.2 0.21 (3)
11 0.17 (3)
7.3 0.058 (3)
10 0.11 (3)
1.9 0.27 (2)
2.0 0.33 (3)
1.2 0.070 (2)
2.4 0.25 (3)
2.0 0.040 (2)
1.8 0.71 (4)
2.3 0.25 (2)
3.3 0.57 (2)
3.4 0.54 (2)
2.3 0.25 (2)
CH3
7.6 0.046 (2)
ND
CH3
H
H
CH3
7.0 0.15 (2)
6.3 0.10 (2)
7.1 0.049 (2)
6.3 0.054 (2)
7.3 0.050 (2)
6.7 0.065 (2)
7.2 0.033 (2)
H
(R)-CH3
(S)-CH3
(R)-CH3
(S)-CH3
(R)-CH3
(S)-CH3
H
H
H
H
H
a
Compounds are racemic except where indicated. Values are given as mean SEM (number of measurements).
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alkylation with 2-bromoethyl ether. At this stage, the isomeric
compounds could be separated by chromatography. Nitro-
reduction and benzimidazole formation yielded the target
compounds 58−63 in >99% ee, as determined by chiral HPLC.
Gratifyingly, the (R)- and (S)-enantiomers displayed clearly
different affinities for CBP, with the (S)-form giving a large
ΔTm of around 10 °C and high potency (pIC50 > 7.0) in
AlphaScreen for the three aryl variants tested (compounds 59,
61, and 63). The (S)-enantiomers were therefore analyzed by
ITC, and the results are shown in Table 7. The Kd for these
Table 7. ITC Determination of Kd for the Binding of (S)-
Methyl Analogues to CBP and BRD4(1)
ITC Kd (μM)
Cmpd
CBP
BRD4
CBP selectivity
a
59
0.021 0.0022
0.85 0.096
40-fold
250-fold
20-fold
16-fold
b
5.2 0.14
a
a
61
63
0.026 0.0026
0.039 0.0029
0.53 0.074
0.61 0.054
a
b
BRD4(1). BRD4(2).
analogues indicated high potency (0.021−0.039 μM). The
most selective compound, 59, was shown to be 40-fold selective
for CBP over BRD4(1) and 250-fold over BRD4(2).
Compound 59 was also potent against p300, with Kd = 0.032
μM (see SI for details). Introducing a chiral methyl onto the
morpholino-ethylene moiety had the desired effect of
improving selectivity over BRD4(1) while maintaining CBP/
p300 potency, and validated the design strategy of introducing
conformational restraints into the ethylene linker of the target
compounds.
Figure 6. (A) View from X-ray crystal structure of compound 59
(carbon = yellow) complexed to CBP, showing the H-bond interaction
between the oxygen of the isoxazole of 59 and N1168 (3.08 Å), and
the nitrogen of the isoxazole and a water molecule (2.83 Å) (water
molecules are red spheres) (PDB 4NR7). (B) Surface view from same
with shaded regions indicating regions which were targeted by N-1 and
C-2 benzimidazole substitution according to the strategy described in
Figure 3. Overlaid with crystal structure of compound 17 (carbon =
magenta) complexed to CBP. The aryl group of compound 59 and
R1173 form an apparent cation−π interaction.
Compound 59 was crystallized with CBP (Figures 6).
Although the binding mode is similar to that observed for
compound 17 (Figure 5A,B), the orientation of the ethylene-
linked aryl group is different. In the complex with CBP and 59,
there is an apparent cation−π interaction between the
guanidino group of R1173 and the aryl ring. This is made
possible because the R1173 side chain moves with respect to
the conformation observed in the complex with compound 17,
forming an induced binding pocket for the aryl ring (Figure
6B). This is consistent with the observation that electron-
donating groups on the aryl ring were preferred for CBP
binding, as these should have a stronger interaction with the
positively charged R1173. The chlorine atom on the aryl ring of
59 sits in a hydrophobic section of the induced pocket between
V1174 and F1177. The chiral methyl group sits below the aryl
ring, possibly helping to lock the ring in position. The induced
pocket has been reported for another series of CBP inhibitors
which also form cation−π interactions between the inhibitors
and the R1173 side chain. The combined findings perhaps
suggest that the interaction of aromatic groups with R1173
represents an important feature of potent CBP inhibitors.31
In order to investigate the wider selectivity of inhibitors in
the series, compounds 17 and 59, were screened against
representative members of the other BRD sub-families using
DSF (Figure 7). The values obtained correlated well with
available AlphaScreen IC50 values (Spearman rank correlation,
ρ 0.94, see SI). In the DSF panel, both compounds were
selective for CBP/p300 BRDs. Compound 59 was particularly
selective, with no significant ΔTm (>2 °C) against any other
BRDs apart from the BETs: BRD2(1), BRD3(1), and
BRD4(1) with ΔTm between 1 and 2 °C.
Cellular Assays. On-target cellular efficacy for the CBP
BRD was investigated using a fluorescence recovery after
photo-bleaching (FRAP) assay (Figure 8A).63 HeLa cells
transfected with a construct encoding a green fluorescent
protein (GFP)-tagged multimerized (3×) CBP BRD showed a
rapid recovery time (t1/2 = 0.59 s) upon photobleaching of a
small area of the nucleus. The broad-spectrum histone
deacetylase inhibitor, SAHA, was used to increase the extent
of global lysine acetylation, so increasing recovery time (t1/2
=
0.79 s) and expanding the assay window. An equivalent increase
in the recovery time was not observed in cells transfected with a
CBP BRD construct carrying the N1168F mutation, (see SI,
Figure S1), consistent with the critical role of N1168 in Kac
binding, and supporting the proposal that the increase in assay
window due to SAHA addition is due to BRD binding.
Treatment of SAHA-treated cells with compound 59 at 0.1 μM
was sufficient to reduce FRAP recovery times back to
unstimulated levels (t1/2 = 0.60 s), equivalent to the N1168F
mutant. The effect is indicative of displacement of the CBP
BRD from acetylated chromatin. The weaker and less selective
CBP inhibitor, compound 17 and the BRD4(1)-selective
inhibitor 6 were unable to significantly alter the FRAP at this
concentration (t1/2 = 0.66 and 0.74 s, respectively). Conversely,
in an equivalent assay using GFP-tagged BRD4, only the
BRD4-selective inhibitor 6 significantly affected FRAP recovery
times, with CBP inhibitors 17 and 59 showing no significant
effect at 0.1 μM (Figure 8B) demonstrating the specificity of
the compounds on their respective targets.
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Figure 7. Selectivity assessment of compounds 17 and 59 against human BRD families using DSF (ΔTm) binding assays. The 10 screened targets are
labeled in black on the phylogenetic tree of the human BRD family; BRDs that were not part of the screening panel are in gray.
Figure 8. Time dependence of fluorescent recovery in the bleached area in Fluorescence Recovery After Photobleaching (FRAP) assays with GFP-
tagged 3 × CBP BRD construct (A) and full-length BRD4 (B). Half-times of fluorescence recovery (t1/2) are shown as bars, which are color-coded:
blue, DMSO control; green, DMSO + SAHA; yellow, 0.1 μM BRD4(1)-selective inhibitor 6; orange, 0.1 μM compound 17; and red, 0.1 μM
†
compound 59. N1168F mutant (red hashed). Bars represent the mean
SEM t1/2 calculated from two or three independent experiments.
Significance of groups compared with the control was determined by t-tests: *p < 0.05, **p < 0.01, ****p < 0.0001. (C) Inhibition of p53-driven
luciferase activity by compound 59. RKO cells were transfected with p53 reporter plasmid. Cells were treated with compound 59 at the indicated
concentrations for 24 h and subsequently with doxorubicin at 0.3 μM for 16 h. Each value is the mean SEM of a representative experiment done in
eight replicates.
To investigate the effect of compound 59 on the CBP−p53
association in a cellular context, a luciferase reporter assay for
p53 induction was used. Doxorubicin induced p53 activity was
effectively inhibited by compound 59 in a dose-dependent
manner (IC50 = 1.5 μM) (Figure 8C). These results suggest
that the CBP BRD inhibitor 59 inhibits the CBP co-activation
of p53 target genes in cells and demonstrate the utility of 59 in
a cellular context. The effect on p53 regulation by compound
59 is most likely due to its CBP inhibition, not its weaker
BRD4 inhibition, as the much more potent BRD4 inhibitor,
JQ1, shows p53-mediated effects at similar concentrations.64,65
However, it was not possible to analyze the effects of less
selective CBP inhibitors in the p53 reporter gene assay due to
the confounding effects of BRD4 inhibition on p53.64,66
To test if CBP BRD inhibition was cytotoxic at the
concentrations where on-target efficacy was observed, U2OS
osteosarcoma cells were treated with compound 59 for 24 h,
and cell viability was determined using a standard 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
turnover assay (see SI, Figure S2). This showed that compound
59 had modest cytotoxicity (CC50 = 80 μM), well above the
levels where on-target efficacy was observed in the FRAP and
p53 reporter gene assays. After 72 h of treatment with
compound 59, cytotoxicity in U2OS cells increased (CC50 = 8.1
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μM), consistent with the effect of BRD4 inhibition at higher
concentration as previously reported.67
beamlines I02 and I24 for assistance with crystal testing and
data collection; Cerep for ligand profiling and ADME assays;
Prof. Darren Dixon and Peter Clark for help with ee
determinations; and Yue Zhu at Changchun Discovery Sciences
Ltd for synthesis of selected monomers.
In order to investigate if compound 59 could also serve as a
probe in animals, it was tested in in vitro ADME (absorption,
distribution, metabolism, and excretion) assays (see SI, Table
S6). In a human liver microsome (HLM) stability assay, no
compound was detected after 60 min, implying that the
metabolism of compound 59 may be too rapid for it to be
useful as an oral in vivo probe.
The selectivity of compound 59 against other target classes
was assessed using wide ligand profiling (see SI, Table S7).68
When tested against 136 GPCR, ion channel, enzyme, and
kinase targets, compound 59 showed IC50 < 1 μM only for the
adrenergic receptors α2C (0.11 μM) and α2A (0.57 μM),
phosphodiesterase-5 (PDE5) (0.15 μM), and platelet-activating
factor (PAF) (0.54 μM).
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In summary, potent, selective, and cell-active inhibitors of the
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paul.brennan@sgc.ox.ac.uk
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ACKNOWLEDGMENTS
■
Lambert, J.-P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller,
̈
The SGC is a registered charity (number 1097737) that
receives funds from AbbVie, Bayer, Boehringer Ingelheim, the
Canada Foundation for Innovation, the Canadian Institutes for
Health Research, Genome Canada, GlaxoSmithKline, Janssen,
Lilly Canada, the Novartis Research Foundation, the Ontario
Ministry of Economic Development and Innovation, Pfizer,
Takeda, and the Wellcome Trust [092809/Z/10/Z]. P.F. and
S.P. are supported by a Wellcome Trust Career-Development
Fellowship (095751/Z/11/Z). The authors thank the Euro-
pean Union, the Biotechnology and Biological Sciences
Research Council (BBSRC), and the British Heart Foundation
for funding. We also thank Diamond Light Source for
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