Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**

Article abstract of DOI:10.1002/anie.202008625

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9–33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.

Full text of DOI:10.1002/anie.202008625

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Selective N-terminal BRD4 bromodomain inhibitors by targeting
non-conserved residues and structured water displacement
Authors: William Charles Krause Pomerantz, Huarui Cui, Anand
Divakaran, Anil K. Pandey, Jorden A. Johnson, Huda Zahid,
Zachariah J. Hoell, Mikael O. Ellingson, Ke Shi, Hideki Aihara,
and Daniel A. Harki
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202008625
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10.1002/anie.202008625
Angewandte Chemie International Edition
RESEARCH ARTICLE
Selective N-terminal BET bromodomain inhibitors by targeting
non-conserved residues and structured water displacement
Huarui Cui, Anand Divakaran, Anil K. Pandey, Jorden A. Johnson, Huda Zahid, Zachariah J. Hoell,
Mikael O. Ellingson, Ke Shi, Hideki Aihara, Daniel A. Harki, and William C.K. Pomerantz*
[a]
H. Cui, A.K. Pandey, J.A. Johnson H. Zahid, Z. J. Hoell, M. O. Ellingson, Prof. W. C. K. Pomerantz
Department of Chemistry, University of Minnesota-Twin Cities
207 Pleasant St. SE, Minneapolis, MN 55455 (USA)
E-mail: wcp@umn.edu
[b]
[c]
A. Divakaran, Prof. D.A. Harki, Prof. W. C. K. Pomerantz
Department of Medicinal Chemistry, University of Minnesota-Twin Cities
2231 6th St. SE, Minneapolis, MN 55455 (USA)
Prof. Dr. H. Aihara, Dr. K.Shi
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota-Twin Cities
321 Church St. SE, Minneapolis, MN 55455 (USA)
epigenetic regulation of disease.[10,11]
Abstract: Bromodomain and extra-terminal (BET) family proteins,
BRD2-4 and T, are important drug targets; however, the biological
functions of each bromodomain remain ill-defined. Chemical probes
that selectively inhibit a single BET bromodomain are lacking,
although pan inhibitors of the first (D1), and second (D2),
bromodomain are known. Here, we develop selective BET D1
inhibitors with preferred binding to BRD4 D1. In competitive inhibition
assays we show that our lead compound is 9-33 fold selective for
BRD4 D1 over the other BET bromodomains. X-ray crystallography
supports a role for the selectivity based on reorganization of a non-
conserved lysine and displacement of an additional structured water
in the BRD4 D1 binding site relative to our prior lead. Whereas pan-
D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1
inhibition in MM.1S cells is insufficient for stopping Myc expression
and may lead to its upregulation. Future analysis of BRD4 D1 gene
regulation may shed light on differential BET bromodomain functions.
Selective inhibition of BET bromodomains is difficult due to the
high sequence similarity in the acetyl-lysine binding site between
each of the eight domains. Overall the D1 bromodomain of BET
protein family members are more similar than the individual
domains within the same protein (e.g. BRD4 D1:BRD4 D2 = 49%
similarity, BRD4 D1:BRD2 D1 = 80% similarity, Fig. 1B).[12]
Despite these differences, only three amino acids in the BRD4 D1
and D2 bromodomain binding sites are non-conserved.[13] Given
the high sequence similarity amongst BET bromodomains, small
molecule inhibitors for a single BET bromodomain are lacking;
however, potent pan-D2 inhibitors (e.g ABBV-744, Fig. 1A and
GSK046/GSK620) [13,14] and a pan-D1 inhibitor, GSK778[14] were
disclosed this year.
We were motivated to develop D1 selective inhibitors within
the BET family with a specific focus on BRD4, as BRD4 proteins
with impaired D1 function are sufficient for inhibiting chromatin
binding.[14,15] However, when pan-D1-selective inhibitors are used
over pan-BET inhibitors, divergent biological effects can be
Introduction
observed. In the case of
a pan-D1 inhibitor, Olinone,
Epigenetics involves the dynamic modification of DNA, RNA
and histone proteins resulting in heritable changes to gene
expression.[1] One emerging protein-drug class for epigenetic
therapy are bromodomain-containing proteins. Bromodomains
are ~110 amino acid structural motifs that function via binding to
distinct lysine acylation states, most commonly N-ε-acetylated-
lysine on histones at both enhancers and gene promoters.[2,3]
Bromodomain and extra-terminal (BET) family proteins, BRD2, 3,
4, and T each contain two N-terminal bromodomains (D1 and D2)
and are several of the most heavily studied bromodomain-
containing proteins.[4] In the context of disease, histone
hyperacetylation at super-enhancer regions and recruitment of
BET proteins promotes transcription of pro-survival and
proliferative genes such as MYC leading to their malignant role in
multiple cancers.[3,5,6] Despite the importance of BET proteins for
regulating transcription, the individual contributions of each of
their two bromodomains remains unclear. Several mechanisms
have been proposed including differential nucleosomal
interactions and simultaneous engagement of both acetylated
transcription factors and nucleosomes.[7–9] Small molecule
inhibitors of individual BET bromodomains will improve our
understanding of the molecular mechanisms underlying
oligodendrocyte progenitor cell differentiation is induced, whereas
pan-BET inhibitors inhibit this process.[16] We also reported a pan-
D1 inhibitor, 1,4,5-trisubstituted imidazole, 1 (Fig. 1A, formerly V),
possessing dual kinase/bromodomain activity.[17] 1 was reported
to bind to BET D1 bromodomains with a slight preference for
BRD4 D1 (16 and 6-fold selective over BRD2 and BRD3 D1
respectively). Similar to Olinone, its affinity was modest (1.2 μM
and 3.4 μM for 1 and Olinone, respectively), while also inhibiting
MAP kinase p38α (Kd = 0.47 nM). We attributed part of the BET
D1 selectivity to arise from displacement and reorganization of
structured water molecules in BRD4 D1 (Fig. 1C). Here, we
exploit our deeper access into the bromodomain binding site to
displace an additional water. Our new inhibitors have high affinity
and are 9-33 fold selective for BRD4 D1 over six additional BET
bromodomains, including > 25-fold selective over BET D2
domains. We further show a structure-based design for removing
binding to p38α, which was affecting interpretation of the
functional effects of BRD4 inhibition[18]. Finally, we conduct a
preliminary analysis in BRD4-dependent multiple myeloma cell
1
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10.1002/anie.202008625
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RESEARCH ARTICLE
Figure 1. (A) Representative BET bromodomain inhibitors and (B) the sequence similarity in each bromodomain relative to BRD4
D1. Percent similarities relative to BRD4 D1 calculated by PDB Sequence & Structure Alignment. (C) Co-crystal structures of BRD4
D1 with (+)-JQ1, 1, and 6. Structured waters are shown in red (PDB ID: 3MXF, 6MH1, 6WGX).
line MM.1S to test if BRD4 D1 inhibition versus pan-BET inhibition
was sufficient to block transcription of a key oncogene MYC. Our
work on domain-selective inhibition of BRD4 allow the study of
divergent BET bromodomain function and guide future
therapeutic designs.
conditions, (+)-JQ1 had a reproducible IC50 comparable to the
reported Kd of 50-75 nM (Table 1, Fig. S4).[21,22] Similar to FA, the
relative trend of affinity was observed for inhibitors 1-4, with 4
possessing the lowest IC50 value of 0.64 µM. In all cases, IC50
values from AlphaScreen were within 3-fold of the FA IC50 values
(Table 1, Fig. S3, S4).
To further improve the affinity and selectivity of our inhibitor,
we subsequently targeted one of the three non-conserved
residues between BRD4 D1 and D2, namely an Asp144 in BRD4
D1 (His433 in D2) used to develop prior pan-D1 inhibitors.[17] Our
co-crystal structure of 1 with BRD4 D1 showed the piperidyl group
to be solvent exposed providing a potential vector for attaching
polar groups to target the surface-exposed Asp144 (Fig. S2).
Several positively charged alkyl amino group were tested;
however, only the ethylamino group, 5, and N,N-dimethyl-
ethylamino group, 6, led to reasonable potency gains relative to 3
(2.4 and 2.1-fold respectively, Table 1) in the FA assays. Similar
effects were confirmed by AlphaScreen (Table 1). To test the
effect of a favorable electrostatic interaction, the ethylamino group
was acetylated to yield 7. In this case, the IC50 increased by 3.2-
fold. This result supports the need for a basic amine attached to
the piperidyl group.
Given the ~80-fold increase in potency from 1 to 5, we next
sought to evaluate if the selectivity for D1 exhibited by 1 was
maintained by 5 using our FA assay. To test the selectivity of
these molecules, BRD4 D2 and BRD2 D1 were selected as
representative BET D2 and D1 proteins. In both cases, 5 was
unable to fully displace Fl-JQ1 from the bromodomains (BRD2 D1,
61% inhibited at 100 µM and BRD4 D2 IC50 > 100 µM, Fig. 2),
whereas, pan-BET inhibitor, (+)-JQ1 displaced Fl-JQ1 from both
bromodomains. These results demonstrated that while inhibitor
potency could be enhanced from 1 to 5, selectivity against both a
BET D2 and a BET D1 domain was maintained.
Results and Discussion
We began our investigation through analysis of the co-crystal
structure of 1 with BRD4 D1 (PDB: 6MH1). In the case of
acetylated histones and acetylated lysine mimetic drugs (e.g. (+)-
JQ1), the methyl group of the acetyl groups, points directly into
the binding pocket which has up to 6 structured waters (Fig. 1C).
In the case of 1, the para-fluorophenyl group binds deeper into
the pocket, displacing two water molecules (Fig. 1C).[17] Having
access to this unexplored binding pocket, we evaluated the effect
of the size of the para substituent to fill the pocket and modulate
the structured water network (Table 1, Fig. S3). Three new
analogues were synthesized (2-4, Scheme S1). The relative
affinities of these inhibitors against BRD4 D1 were tested by
competitive inhibition fluorescence anisotropy (FA) assays in
which a fluorescein-labeled pan-BET inhibitor, (+)-JQ1 (Fl-JQ1)
was used as a tracer. In the retesting of 1, the inhibitory potency
was 6-fold weaker than our previous report that used a different
tracer.[17] Removing the F atom altogether had minimal effect on
potency (2, IC50 = 4.5 µM), whereas a larger methyl group, 3,
further improved activity (IC50 = 1.2 µM). However, the most
significant effect resulted when a trifluoromethyl group was
installed, (4, IC50 = 0.31 µM), leading to a 34-fold enhanced
potency relative to 1.
To verify the affinity in an orthogonal assay, we used an
AlphaScreen competitive inhibition for BRD4 D1 using
tetraacetylated H4 histone peptide as a more native-like ligand.
Due to the low concentration of both protein and histone used in
this assay, IC50 values can be used to approximate the Ki of the
inhibitor if conditions are appropriately controlled.[19,20] Under our
a
2
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10.1002/anie.202008625
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RESEARCH ARTICLE
Table 1. IC50 values of 1 and analogues against BRD4 D1 by fluorescence anisotropy and AlphaScreen. Kinase activity against
p38α.
N
N
R1
R1
N
N
N
N
N
N
R
R
NH
O
N
1 - 7
8
BRD4 D1 IC50 by
FAa(µM)
0.037 ± 0.008
11 ± 0.8
BRD4 D1 IC50 by
p38α
Kd (nM)
Cpd
R =
R1 =
c
AlphaScreenb(µM)
(+)-JQ1
-
F
H
CH3
CF3
CF3
CF3
CF3
CF3
-
0.051 ± 0.02†
-
1
2
3
4
5
6
7
8
H
H
H
H
3.8
2.6
1.1
0.64
0.29
0.20
1.5
0.47
-
-
260
1900
-
-
4.5 ± 0.2
1.2 ± 0.2
0.31 ± 0.06
0.13 ± 0.01
0.15 ± 0.03
0.42 ± 0.01
1.1 ± 0.2
(CH2)2NH2
(CH2)2N(CH3)2
(CH2)2NHCOCH3
(CH2)2NH2
1.3
>30,000
a Data represents the mean and standard deviation of three biological replicates.
b Data represents the mean of two technical replicates except (+)-JQ1. † Data represents the mean and standard deviation of six
biological replicates.
c Kd values were determined by KINOMEscan. Data represents the mean of two biological replicates.
absolute values with our AlphaScreen data, therefore we limited
our analysis to a relative comparison of the commercial data.
Consistent with our FP data, 5 was selective for BRD4 D1 over
BRD4 D2 (28-fold) and displayed similar selectivity against the D2
of BRD2, and 3 (25 and 33-fold respectively). 5 was less selective
for BRD4 D1 over other BET D1 domains (9-19 fold) (Fig. 2B, Fig.
S5). This D1 selectivity was similar to improved relative to 1. We
retested the selectivity in an orthogonal assay with a commercial
phage-display assay at DiscoverX and measured a similar
selectivity for the closest off-target BRD2 D1 of 8.5-fold (Table
S1). 4 was also tested against BRD4 D1 and BRD4 D2 to
evaluate if the ethylamino group of 5 improved the selectivity for
D1 over D2. In this case, 4 maintained selectivity for BRD4 D1
(Fig. S6). This result indicates that the ethylamino group can
improve affinity, but not selectivity.
To more broadly evaluate bromodomain selectivity, we
showed that 5 displayed no measurable affinity against six
additional bromodomains (p300, BRD1, SMARCA2, SMARCA4,
PBRM1(5), and PCAF) that 1 previously inhibited by > 35%
(Table S1). During the preparation of this manuscript, Liu et al.
reported two BRD4 D1 selective inhibitors with 9-10 fold
selectivity for BRD4 D1 over D2 and nanomolar potency. To the
best of our knowledge, 5 and these new inhibitors are the first
small molecule inhibitors with preferred binding to BRD4 D1 with
sub-micromolar potency.[23]
As a final design strategy, we sought to remove the potent
p38α binding affinity of our prior inhibitor 1.[17] The p-F phenyl
group was originally designed to target a secondary hydrophobic
Figure 2. Competitive binding experiments of 5 and (+)-JQ1
pocket in the kinase. Gallagher et al. showed that having a p-CF3
by fluorescence anisotropy and a commercial AlphaScreen
phenyl group in the kinase binding pocket based on similar
assay demonstrates selectivity for BRD4 D1. (A) Competitive
analogues led to a 25-fold decrease in IC50 compared with a F
binding curves of 5 (in red) and (+)-JQ1 (in black) by fluorescence
atom.[24] We therefore determined the p38α binding affinity for our
anisotropy against BRD2 D1, BRD4 D1, and BRD4 D2. (B) IC50
inhibitors 4 and 5 using a commercial kinase binding assay. In
values of 5 and (+)-JQ1 by fluorescence anisotropy and a
the case of 4, the binding affinity decreased by 550-fold compared
commercial AlphaScreen assay.
with 1. The affinity was further attenuated to micromolar levels
a Data represents the mean and SEM of biological replicates.
with 5. The role of the ethylamino group is unclear. Previously,
we mitigated p38α kinase activity using a related scaffold which
b
IC50 values were determined by a commercial AlphaScreen
assay. Data represents the mean of two biological replicates.
converted the pyrimidine to a 2,6-disubstituted pyridine and used
c 61% inhibition at 100 µM.
an exocyclic ether.[18] To further remove p38α kinase activity,
molecule 8 was synthesized. In this case, p38α binding could be
The observed BRD4 D1 selectivity led us to further assess
BET bromodomain selectivity via a commercial AlphaScreen
service. We could not control for identical conditions to compare
removed, but at the expense of BRD4 D1 affinity (Table 1). CK1
is a known off-target of p38α inhibitors, [25] for which 5 still inhibits
3
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10.1002/anie.202008625
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RESEARCH ARTICLE
(Fig. S10). Future analogs will be designed to remove affinity for
this protein.
and BRD4 D1 bound to 1 (PDB:6MH1) or 6 (PBD:6WGX, grey).
The Lys141 side chain was removed for clarity. (C) Possible water
network bridging 6 to D144. Three waters are indicated in red
sphere and B-factor are labeled in yellow, for which higher B-
factors indicate less stable waters.
We used crystallography to obtain higher-resolution structural
information to better understand the molecular basis for the
affinity and selectivity of our inhibitors. Although we were unable
to co-crystallize 5 with BRD4 D1, a co-crystal structure with 6 was
obtained at 1.53 Å resolution (PDB: 6WGX). Similar to 1, the
imidazole ring maintains a key hydrogen bond with the conserved
Asn140 (Fig. 3A). As designed, the p-CF3 phenyl group of 6 can
fill the binding pocket and displaces an additional structured water
molecule (Fig. 1C, Fig. 3A). This observation is consistent with
computational analysis of the structured water network of BRD4
D1, which indicates higher energy waters in the binding pocket
relative to BRD4 D2, and the bromodomains of BRD2 and 3.[26,27]
A second amino acid difference in the binding site between
BET D1 and D2 is the residue next to the conserved Asn; Lys vs
Pro, respectively, leading to a more flexible backbone for D1. We
analyzed a select set of pan-BET inhibitors, including a 1,4,5-
trisubstituted triazole analogue of 1 co-crystallized with BRD4 D1,
and compared the backbone Cα-Cα differences at Asn140 and
Lys141 relative to a H4 histone co-crystal structure for reference
(Table S3). The movement of the Cα of Asn140 ranged from
0.36-0.87 Å (mean = 0.69 Å), and the Cα movement for Lys141
ranged from 0.25-1.3 Å (mean 0.98 Å). Alternatively, for our three
D1 selective 1,4,5-trisubstituted imidazoles (1, IV, and 6, Fig.
S13), we found a larger movement for the Cα of Asn140 of 0.89-
0.94 Å (mean = 0.92 Å, Fig. 3B). The largest displacement was
observed for 6 at the Cα of Lys141, with a 1.7 Å displacement with
values ranging from 0.99-1.7 Å (mean = 1.5 Å, Fig. 3B, Table S3).
Gilan et al. attributed part of their pan-D1 selectivity through
reorganization of Lys141.[14]
A final analysis shows the non-conserved Asp144 residue
remained 10 Å away from the ethylamino group of 6. Additional
unfit electron density could be accounted for suggesting a flexible
ethylamino side-chain (Fig. S12).However, we also note a
structured water molecule that forms a bridging hydrogen-bond
between D144 and N140 (Fig. 3C). The ethylamino group of 6,
may engage this water molecule through a network of two
additional waters. Targeting this structured water has recently
been proposed by Wellaway et al. to help drive D1 selectivity.[28]
New inhibitor designs based on our inhibitor scaffold would need
to target this water directly to improve affinity and selectivity. From
this structural analysis, we attribute our inhibitor selectivity to at
least two key differences in structured water displacement and
backbone reorganization of the binding site and highlight a
potential third mechanism through engaging a new structured
water molecule bridging non-conserved D144 and N140.
Having developed a series of compounds with BRD4 D1
affinity and selectivity distinct from other pan-BET, pan-D1 or pan-
D2 inhibitors, we evaluated their cellular activity. MM.1S multiple
myeloma cells are highly BRD4-dependent due to super-
enhancer regulation of MYC.[3] As such, antiproliferation EC50
values correspond well to in vitro potency for pan-BET inhibitors
under our assay conditions (e.g. (+)-JQ1 = 0.12 µM, Fig. S14A).
We evaluated whether BRD4 D1 inhibitors have similar effects as
pan-BET inhibitors. While compounds 4, 5, and 6 reduced cellular
proliferation, the EC50 for growth inhibition was in the low micro-
molar range (2.2-4.6 µM, Fig. S14A); higher than the in vitro
potency. A comparison of salt and free-base forms of 5 gave
similar EC50 values, suggesting the observed anti-proliferative
activity cannot be solely attributed to a counterion effect (Fig.
S14B).
Figure 3. Structural analysis of 6 and BRD4 D1 interactions.
(A) Co-crystal structure of 6 with BRD4 D1. Three structured
waters are indicated in red sphere. The imidazole ring forms a
conserved hydrogen bond with Asn140. The distance between N,
N-dimethyl amino group and Asp144 is indicated. (B) Backbone
overlay of BRD4 D1 bound to H4 K5ac,K8ac (PDB: 3UVW, wheat)
Figure 4. Target engagement, cellular permeability, and
transcriptional effects of 4, 5, and 6 in MM.1S cells. (A)
4
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10.1002/anie.202008625
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RESEARCH ARTICLE
Cellular target engagement of BRD4 and cell permeability of 4, 5,
6 by isothermal dose-response stability profiling after 1 h.
treatment of MM.1S cells. Quantified densitometry relative to 5
µM (+)-JQ1 intensity levels shown in red. (B) Representative
western blot of expressed c-Myc levels after 8 h. treatment of
MM.1S cells. Quantified densitometry relative to untreated
(DMSO) intensity levels in red. Full images of representative blots
shown in Figure S15.
of selectivity to arise from at least two mechanisms: flexibility in a
D1-conserved YNKP motif and displacement of structured-waters
in the acetyl-lysine binding site of BRD4 D1, where an additional
water can be displaced relative to our previous reports.[17] This
binding mode is distinct from the binding mode of the BRD4 D1
inhibitors described by Liu et al.[23] With a useful set of tool
compounds in hand, we have conducted
a preliminary
investigation on how a BRD4 D1 selective inhibitor can regulate
oncogene expression in Myc-sensitive MM.1S cells. In contrast to
the current literature, showing in many cases, pan-BD1 inhibitors
phenocopy pan-BET inhibitors,[14] our results reveal an increase
in Myc expression at concentrations mirroring biochemical
potency, followed by downregulation at concentrations where
other BET bromodomains may be inhibited. This effect is similar
to data reported by Raux et al. using a different chemical scaffold.
[32] The origins of these effects are currently under investigation.
Cellular target engagement of BRD4 was tested to help
explain why our compounds had higher than expected MM.1S
EC50 values. We conducted thermal stability profiling in an
isothermal dose-response format using an established CETSA
assay.[29] After
a
1-hour incubation,
a
dose-dependent
stabilization of BRD4 was observed with compounds 4, 5, 6 and
control (+)-JQ1 (Fig. 4A). In contrast to our antiproliferation
experiments, BRD4 stabilization was observed at submicromolar
concentrations similar to our biochemical potency for BRD4 D1.
This result shows that target engagement and cell permeability
cannot explain the lack of cell activity for 4-6. An unexplained
finding for inhibitors 5 and 6 at concentrations exceeding 1 μM,
was an increased stabilization of BRD4 over a saturating
concentration of (+)-JQ1 at 5 μM.
Pan-BET bromodomain inhibitors have widely shown efficacy
downregulating MYC in hematological cancers, which is thought
to be a main mechanism for cellular growth inhibition.[6] Here, we
were surprised to find elevated c-Myc protein levels relative to
DMSO treated cells at submicromolar concentrations of
compounds 4-6. Elevated c-Myc protein levels were not observed
with control (+)-JQ1 (Fig. 4B). Downregulation of c-Myc was
observed when compound concentrations were >10-fold above
their respective biochemical IC50 values, at concentrations
comparable to the MM.1S antiproliferation EC50. Together these
results highlight the dependence of this cell line on c-Myc
expression.
While biochemical studies with protein mutants or pan-D1
inhibitors show BRD4 D1 inhibition is sufficient to displace BRD4
from chromatin,[14,15,30] our data suggest BRD4 D1 inhibition alone
may be insufficient for decreasing c-Myc protein levels in MM.1S
cells. Although a decrease in c-Myc is observed at high compound
concentrations, given our moderate 9-33 fold selectivity for BRD4
D1 and residual p38 activity, we cannot rule out effects from
inhibiting other BET bromodomains or kinases such as CK1, or
drug efflux, although we have reported on structurally related but
less selective inhibitors which do not show these effects.[17,18,31]
Reanalyzing the data of Raux et al., who reported a BRD4 D1
selective inhibitor with micromolar affinity (Kd = 1.4 μM),[32] we
note a similar trend of high antiproliferation EC50 values (27 μΜ),
and a Myc response to inhibitor that first increases and then finally
is suppressed by 50 μM inhibitor. Due to the established role of
D1 in maintaining chromatin occupancy of BRD4,[14] it would be of
interest to evaluate how chromatin occupancy for all BET proteins
correlates with c-Myc expression in the presence of a BRD4 D1
inhibitor and to compare how overall BET expression levels are
altered as a result.[33] The effects of our inhibitors on Myc
expression have only been studied in one cell line and merit
further assessment. While the inhibitors of Liu et al. were not
used to evaluate Myc expression, these inhibitors effectively
inhibited inflammatory gene expression in human small airway
epithelial cells, demonstrating a contextual dependence for
studying the effects of selective BET inhibitors.[23] These results
support a broader cellular investigation of the transcriptional
effects of selective BRD4 D1 inhibition.
Acknowledgements
We would like to dedicate this manuscript in honor of François
Diederich who inspired a generation of scientists for tackling
problems in molecular recognition. We gratefully acknowledge
support for this research from the Masonic Cancer Center at the
University of Minnesota, (W.C.K.P and D.A.H) the NIH (R35-
GM118047 to H.A. and R01-GM110129 to D.A.H), the American
Heart Association (15SDG25710427 to W.C.K.P.). J.A.J. was
supported by
a NIH Biotechnology training grant (T32-
GM008347-23) and A.D. was supported by the NIH chemistry-
biology interface training grant (T32-GM008700). This work is
based on research conducted at the NE-CAT beamlines at the
Advanced Photon Source, which are supported by the NIH (P30-
GM124165). The Pilatus 6M detector on 24-ID-C beamline is
funded by a NIH-ORIP HEI grant (S10-RR029205). We thank staff
at the NE-CAT beamlines for assistance in data collection, Dr.
Peter Ycas for help preparing the His9-BRD4 D1 plasmid, and Dr.
Brian Van Ness for providing MM.1S cells.
Keywords: BET bromodomains • Inhibitors • BRD4 D1
selectivity • Structured waters • Myc regulation
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The twin-bromodomain-containing BET proteins, BRD2-4 and T are important drug targets for inflammation, cancer, and heart
disease. However, potent and selective inhibitors for a single BET bromodomain are lacking. Here we describe the structure-based
design of inhibitors with preferred binding for the first bromodomain of BRD4, and provide design rules for future inhibitors. Cell-
based studies identify differential effects relative to pan-BET inhibitors.
Institute and/or researcher Twitter usernames: @UMNChemistry, @UMN_MedChem, @fewill26
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