Structure-Guided Design and Development of Potent and Selective Dual Bromodomain 4 (BRD4)/Polo-like Kinase 1 (PLK1) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00765

The simultaneous inhibition of polo-like kinase 1 (PLK1) and BRD4 bromodomain by a single molecule could lead to the development of an effective therapeutic strategy for a variety of diseases in which PLK1 and BRD4 are implicated. Compound 23 has been found to be a potent dual kinase-bromodomain inhibitor (BRD4-BD1 IC₅₀ = 28 nM, PLK1 IC₅₀ = 40 nM). Compound 6 was found to be the most selective PLK1 inhibitor over BRD4 in our series (BRD4-BD1 IC₅₀ = 2579 nM, PLK1 IC₅₀ = 9.9 nM). Molecular docking studies with 23 and BRD4-BD1/PLK1 as well as with 6 corroborate the biochemical assay results.

Full text of DOI:10.1021/acs.jmedchem.8b00765

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Article
Structure-guided Design and Development of Potent and Selective
Dual Bromodomain 4 (BRD4)/Polo-like Kinase 1 (PLK1) Inhibitors
Shuai Liu, Hailemichael O Yosief, Lingling Dai, He Huang, Gagan Dhawan, Xiaofeng Zhang, Alex M. Muthengi,
Justin Roberts, Dennis L. Buckley, Jennifer A. Perry, Lei Wu, James E. Bradner, Jun Qi, and Wei Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00765 • Publication Date (Web): 20 Aug 2018
Downloaded from http://pubs.acs.org on August 20, 2018
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Structure-guided Design and Development of Potent and Selective Dual
Bromodomain 4 (BRD4)/Polo-like Kinase 1 (PLK1) Inhibitors
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Shuai Liu,a Hailemichael O. Yosief,a Lingling Dai,b,c He Huang,d Gagan Dhawan,a,e Xiaofeng Zhang,a
Alex M. Muthengi,a Justin Roberts,f Dennis L. Buckley,f Jennifer A. Perry,b Lei Wu,b James E.
Bradner,f,g* Jun Qi,b,h* Wei Zhanga,*
a
Department of Chemistry, University of MassachusettsꢀBoston, Boston, MA 02125, USA
b
Department of Cancer Biology, DanaꢀFarber Cancer Institute, Boston, MA 02215, USA
c
Phase I Clinical Trial Center & Department of Clinical Pharmacology, Xiangya Hospital, Central
South University, Changsha, Hunan, 410008, P.R. China
d
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794ꢀ3400
e Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhiꢀ
110019, India
f
Department of Medical Oncology, DanaꢀFarber Cancer Institute, Boston, MA 02215, USA
g
Novartis Institute of Biomedical Research Institute, Cambridge, MA 02139, USA
h
Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
Abstract: The simultaneous inhibition of poloꢀlike kinase 1 (PLK1) and BRD4 bromodomain by a
single molecule could lead to the development of an effective therapeutic strategy for a variety of
diseases in which PLK1 and BRD4 are implicated. Compound 23 has been found to be a potent dual
kinaseꢀbromodomain inhibitor (BRD4ꢀBD1 IC50 = 28 nM, PLK1 IC50 = 40 nM). Compound 6 was
found to be the most selective PLK1 inhibitor over BRD4 in our series (BRD4ꢀBD1 IC50 = 2,579 nM,
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PLK1 IC50 = 9.9 nM). Molecular docking studies with 23 and BRD4ꢀBD1/PLK1 as well as with 6
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corroborates the biochemical assay results.
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Keywords: Epigenetics, Bromodomains, BET, BRD4, PLK1 Kinases, BIꢀ2536, Antiꢀcancer, Structure
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INTRODUCTION
The bromodomain and extraꢀterminal domain (BET) family of proteins is essential for
recognizing acetylated lysines (KAc) on chromatin and has emerged as a potential therapeutic target
for the treatment of cancer, inflammation and viral infectious diseases.1ꢀ8 BET proteins, including
BRD2, BRD3, BRD4 and BRDT, are characterized by dual bromodomains (BD1 and BD2) that bind
to KAc on histones to facilitate the recruitment and stabilization of protein complexes, which plays an
active role in gene expression.9ꢀ13 With the development of the first small molecule inhibitor of BET
bromodomains, (+)ꢀJQ1, we were able to demonstrate that binding to BRD4 displaces BRD4 from
chromatin and interrupts the biological function of the protein.3,14 Since our initial reports, several
potent and selective BET inhibitors with different chemotypes have been reported to possess similar
binding affinities, selectivity profiles and cellular activity as (+)ꢀJQ1.15ꢀ19 More importantly, more than
twelve BET bromodomain inhibitors have progressed into clinical trials, including a derivative of
JQ1,20 for the treatment of hematologic malignancies, solid tumors and cardiovascular disease.21 With
multiple cancer studies demonstrating the development of resistance to single agents, including BET
inhibitors, strategies for combination therapies are being intently pursued.22ꢀ24
Recent reports highlight that inhibition of poloꢀlike kinase 1 (PLK1), a crucial cellꢀcycle
regulator, synergizes with BET inhibition in multiple cancer types, including prostate cancer and acute
myeloid leukemia (AML).25,26 While combinatorial inhibition of PLK1 and BET bromodomains with
two molecules is appears to be synergistic,26 achieving this with a single agent may increase potency
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for two diseaseꢀrelevant targets/pathways while decreasing toxicity. The concept has recently been
demonstrated with dual PI3KꢀBRD4 inhibitors have recently been reported to block expression of the
MYC oncogene, while markedly inhibiting cancer cell growth and metastasis in hepatocellular
carcinoma and neuroblastoma in vivo.27,28 The recent discovery that multiple kinase inhibitors,
including PLK1 inhibitor, BIꢀ2536, and the janus kinase 2 (JAK2) inhibitor, TG101209 (Fig. 1)
exhibit moderate to excellent inhibitory activity against BET family of proteins opens up the
possibility of creating polypharmacological compounds that target both kinases and BET proteins
equivalently.7,23,29ꢀ32
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In this study, we utilized the scaffold of BIꢀ2536 to develop inhibitors with varied inhibitory
activities against BRD4 and PLK1 in order to perform structureꢀactivity relationship (SAR) studies, as
well as understand the effect of inhibiting both proteins (PLK1 and BRD4) with a single agent in
cancer, as both the BET family of proteins and PLK1 are intently pursued targets for the treatment of
cancer.
Figure 1. Kinase inhibitors with nanomolar inhibitory activity against BRD4
First, we inspected the binding modes of BIꢀ2536 with BRD4ꢀBD1 and PLK1, respectively,
through coꢀcrystallization studies (Fig. 2). In the binding complex of BIꢀ2536 with BRD4ꢀBD1, the
methylated amide functions as an acetylated lysine mimic, where the carbonyl oxygen of the
dihydropteridinone ring forms a waterꢀmediated hydrogen bond with conserved acetyl lysine
recognition motif, asparagine (N140) in the binding pocket. The methyl group is orientated into a
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hydrophobic subꢀpocket formed by F83, I146, and C136. The NH group on the aniline and one of the
pyrimidine nitrogen atoms interact with the Q85 backbone amide through an intermediary water
molecule. The ethyl group that branches off the asymmetric dihydropteridinone carbon atom is
projected into a small hydrophobic subꢀpocket formed by V87, L92, L94, Y97, while the cyclopentyl
moiety and the Nꢀmethylpiperidine are both solvent exposed.29,30
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B
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Figure 2. Crystal structure of BIꢀ2536 (dark red) bound to A) BRD4ꢀBD1 (PDB ID: 4O74) and B)
PLK1 (PDB ID: 2RKU). The protein residues in contact with ligand showed in yellow sticks; hydrogen
bonding showed in red dashed lines; the rest residues near binding pocked showed in gray.
Based on structural information from the coꢀcrystals, we envisioned several strategies to improve
the BRD4ꢀBD1 binding affinity while reducing the PLK1 inhibition to achieve balanced dual
PLK1/BRD4ꢀBD1 inhibition (Fig. 3). First, we hypothesize that the replacement of the amide methyl
group at Site 1 with a slightly larger ethyl or isopropyl might enhance binding affinity to BRD4ꢀBD1.
Second, to decrease affinity for PLK1, the elaboration of the 3ꢀmethoxy group on the aromatic ring
(Site 2) with a bulkier group might modulate binding affinity. This methyl ether group is involved in
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critical hydrogen bonds with the PLK1 active site (Fig. 2B). The interruption of this interaction is
expected to reduce the PLK1 binding affinity of BIꢀ2536 but render its activity to BRD4ꢀBD1
unchanged. Third, modification on ethyl group might have more impact on PLK1 activity as the
pocket in PLK1 is narrower compared to the binding pocket in BRD4ꢀBD1. In the end, we will modify
the solvent exposed sites (Sites 4 and 5) to fully investigate the effect of each group on the binding
affinity of the parent compound toward BRD4ꢀBD1 and PLK1 based on the chemistry we established
previously developing fluorescentlyꢀtagged kinase inhibitors.31 Thus, we conducted a medicinal
chemistry campaign around the BIꢀ2536 core to develop small molecules with fineꢀtuned activities
against PLK1 and BET bromodomains.
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Figure. 3. Available modification sites on BIꢀ2536
RESULTS AND DISCUSSION
Utilizing the structural information from the coꢀcrystal structures of BIꢀ2535 with BRD4ꢀBD1 and
PLK1, a series of BIꢀ2536 analogs were synthesized using the general synthetic route shown in
Scheme 1. In brief, amino acids with varying alkyl side chains (different R1 group) were esterified
with thionyl chloride in MeOH followed by alkylation using reductive amination with cyclic or acyclic
ketones. The resulting secondary amine derivative E1 underwent a regioselective nucleophilic
aromatic substitution reaction with 2,6ꢀdichloroꢀ5ꢀnitropyrimidine to produce tertiary anilines. The
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nitro group was reduced using zinc dust in hot acetic acid generating the dihydropteridinone ring in
E2. Methylation of the amide produced the scaffold E3, which was used in a nucleophilic substitution
reaction with the aminobenzoic acid derivative and led to the formation of intermediate E4 with an
acid motif, which was coupled with different amines to produce the final BIꢀ2536 analogs. As planned
in Figure 3, the resulting analogs include derivatives with different substituent groups on the amide
bond (Site 1); different ethers at Site 2 with cyclic or acyclic motifs; varying substituent groups, such
as hydrogen, methyl, propyl and isobutyl on Site 3; and the solvent exposed Site 4 or 5. All
compounds in this focused library were then screened for their inhibitory activity against BRD4ꢀBD1
and BRDTꢀBD1 using AlphaScreen assay technology to obtain IC50 values (all IC50 values are for
BD1 of all of the bromodomains tested unless otherwise noted).32
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Scheme 1. Synthesis of BIꢀ2536 analogs 1-20
The modification of the cyclopentyl moiety at Site 4 with different cyclic and acyclic groups,
cyclohexyl (compound 1) and isopropyl (compound 2), created compounds with nanomolar inhibitory
activity against BRD4 (Table 1). These analogs, however, were less potent than the parent compound
BIꢀ2536, indicating that the cyclopentyl group is crucial for the potent inhibitory activity against
BRD4. These compounds, however, maintained low nanomolar inhibitory activity against PLK1
suggesting that modification on Site 4 is tolerated for PLK1 binding. On the other hand, unmethylated
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amide derivatives of compounds 1 and 2 (compound 3 and 4, respectively) resulted in more than 1000ꢀ
fold decrease in binding affinity for BRD4. Interestingly, the inhibitors with unmethylated amide,
compounds 3 and 4, displayed more than 250ꢀfold selectivity for PLK1 over BRD4, indicating the
importance of the methylated amide as an Nꢀacetylated lysine mimic for BRD4 binding. Thus, it is
critical to have a methylated amide at Site 1 to serve as Nꢀacetylated lysine mimic for bromodomain
inhibition. Taken together, the replacement of cyclopentyl group at site 4 together with an
unmethylated amide reduce the BRD4 activity of the inhibitors while maintaining PLK1 selectivity.
Next, we examined the modification of Site 3 with methyl (compound 5), propyl (compound 6),
hydrogen (compound 7) and isobutyl (compound 8) substituents. The analogue with methyl
substitution at the asymmetric dihydropteridinone carbon atom, compound 5, exhibited good
inhibitory activity against both BRD4 (IC50 = 84 nM) and PLK1 (IC50 = 11 nM). Selectivity profiling
against 32 bromodomains (BROMOscan®) and 468 kinases (KINOMEscan®)33 confirmed selective
activity for the BET subfamily and PLK kinases (PLK1ꢀ3) at 1 µM through profiling platform at
DiscoverX Specifically, 1 uM compound 5 exhibited selective activity against BRD4ꢀBD1 (12% of
control) and PLK kinases (0.3ꢀ0.5% of control) (Sup Inf Fig. 1). No significant difference in binding
activity was observed for the (S) and (R) isomers of the methyl substituent, which is similar to the
observation made on the (S) and (R) isomers of BIꢀ2536 by the Fletcher group. 32 However, replacing
the asymmetric ethyl group with bulky alkyl groups like propyl (compound 6) and isobutyl (compound
8) resulted in a significant decrease in binding affinity towards BRD4. While compound 6 displayed
excellent inhibitory activity against PLK1, with more than 250ꢀfold selectivity over BRD4, further
increasing the size of Site 3 to isobutyl, or deleting the methyl group resulted in a reduction of PLK1
inhibitory activity.
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Table 1. Structure Activity Relationships on BI-2536 Analogs
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IC50 (uM)
bPLK1
Compound
R4
R3
R1
Me
Me
Me
H
aBRD4ꢀBD1
0.205±0.008
0.65±0.024
0.186±0.013
>10
aBRDTꢀBD1
ND
Cp
(R)ꢀEt
(R)ꢀEt
(R)ꢀEt
(R)ꢀEt
(R)ꢀEt
(R)-Me
(R)-Pr
H
0.004
0.004
BI-2536
Cy
ND
1
iꢀPr
0.004
ND
2
iꢀPr
2.62
ND
3
Cy
H
>10
0.0372
0.0011
0.0099
0.0541
0.078
ND
4
5
Cp
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.084±0.006
2.579±0.180
6.633±0.530
18.8±3.4
0.342±0.015
8.468±0.076
25.99±9.70
ND
6
Cp
Cp
7
Cp
(R)ꢀiBu
(S)ꢀMe
(S)ꢀEt
(R)ꢀMe
(R)ꢀMe
(R)ꢀiBu
(R)ꢀiBu
8
9
Cp
0.107±0.002
0.489±0.017
0.31±0.01
0.421±0.016
>50
0.0087
0.0325
0.229±0.010
3.513±0.351
0.75±0.03
0.978±0.043
>50
Cp
10
Cy
0.0086
11
iꢀPr
tetrahydro(2H)pyran
iꢀPr
0.0252
0.322
0.218
ꢀ
12
13
41.9
ND
14
0.032±0.001
0.103±0.004
(S)-JQ1
aIC50 values were measured by AlphaScreenTM binding assay and reported as the average of 3 replicates ± SE.
bIC50 values were measured using Adapta assay format (ThermoFisher Scientific)
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With the improvement in BRD4 inhibitory activity with compound 5, we further examined this
compound through the replacement of the cyclopentyl group with cyclohexyl (compound 11),
tetrahydroꢀ2Hꢀpyran (compound 13) and isopropyl (compound 12) groups. None of these substitutions
improved potency towards BRD4 above that seen for compound 5, further indicating that substituting
the cyclopentyl moiety for the methyl group at the dihydropteridinone ring is optimal for BRD4
inhibition. On the other hand, the inhibitory activity of those analogs against PLK1 remained in the
low nanomolar range. Similarly replacing the cyclopentyl group of compound 8 with tetrahydroꢀ2Hꢀ
pyran (compound 13) and isopropyl (compound 14) groups did not improve potency towards BRD4
but showed modest inhibitory activity against PLK1 suggesting that PLK1 is tolerant of substitutions
at the asymmetric carbon and anilide nitrogen (Table 1). Taken together, the adjustment of substituent
groups at the Site 3 tunes down BRD4 activity with little impact on PLK1 activity, leading us to
discover the most PLK1 selective inhibitor in the series, compound 6 (> 260 fold).
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To increase the BRD4 selectivity of BIꢀ2536 analogs, we utilized compound 5 to further explore
the SAR around the terminal amine (Site 5) and the methoxy group at Site 2 to investigate their impact
on potency and selectivity (Table 2, synthetic route shown in Scheme 2). Replacement of the terminal
cyclic amine (aminoꢀpiperidine, Site 5) with different alkyl and aromatic amines (compounds 15, 16,
17, 18, 19 and 20) did not grossly impact the potency and selectivity for BRD4, implying that the
various amine replacements are not engaged in any significant interaction with the amino acid residues
in the binding
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Scheme 2. Synthesis of compounds 21 and 23-25
Table 2. Structure Activity
Relationship on Compound 5
Analogs
IC50 (uM)
Compound
R3
R2
R5
aBRD4ꢀBD1
0.084±0.006
0.107±0.003
0.095±0.002
0.183±0.009
0.104±0.050
0.109±0.003
0.094±0.002
0.059±0.001
0.253±0.007
0.028±0.001
0.596±0.014
0.69±0.018
bPLK1
0.0011
0.16
aBRDTꢀBD1
0.342±0.015
0.378±0.014
0.289±0.008
0.558±0.015
0.392±0.013
0.56±0.025
5
(R)-Me
(R)ꢀMe
(R)ꢀMe
(R)ꢀMe
(R)ꢀMe
(R)ꢀMe
(R)ꢀMe
(S)ꢀMe
(R)ꢀMe
(R)-Me
(R)ꢀPr
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OꢀCp
OꢀCp
O-Cp
OꢀCp
OꢀCp
4-amine, 1-methylpiperidine
hexamethylene amine
aminomethylpyridine
phenylethylamine
15
16
17
18
19
20
21
22
23
24
25
0.0084
0.027
0.021
0.009
0.095
0.127
0.457
0.04
N,Nꢀdimethylethylamine
aminopiperidine
piperidine
0.369±0.011
0.245±0.009
0.933±0.032
0.102±0.002
1.951±0.080
3.543±0.198
4ꢀamine, 1ꢀmethylpiperidine
OMe
4-amine, 1-methylpiperidine
4ꢀamine, 1ꢀmethylpiperidine
4ꢀamine, 1ꢀmethylpiperidine
0.047
ꢀ
aIC50 values were measured by AlphaScreenTM binding assay and reported as the average of 3 replicates ± SE.
bIC50 values were measured using Adapta assay format (ThermoFisher Scientific)
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pocket of BRD4. On the other hand, the modification of methoxy moiety (Site 2) with a cyclopentoxyl
group improved potency toward bromodomains while reducing the potency against PLK1. These
substitutions created a molecule that has relatively balanced potency toward both PLK1 and BRD4
with less than 2ꢀfold selectivity difference (Table 2). Notably, compound 23 exhibited improved
potency for BRD4 compared to our reference BET inhibitor, (+)ꢀJQ1, but reduced activity against
PLK1 compared to BIꢀ2536, making it one of the most potent BET bromodomain inhibitors with
reduced PLK1 inhibition in the series. This improvement can be overridden by introducing a larger
propyl group (compound 24) at the Site 1. Also, the (S)ꢀisomer of compound 23, compound 21,
retains good inhibitory activity against BRD4, but is less potent toward PLK1 suggesting that the
chirality change has more impact on PLK1 activity than BRD4 activity, which is consistent with the
SAR we established in the previous round. Finally, we examined the three compounds with the most
balanced BRD4 and PLK1 activity (compounds 5, 6, and 23) in a human acute myeloid leukemia
(AML) cell line, MOLM13. In general, BRD4 bromodomain inhibition causes a G1 arrest in the cell
cycle as demonstrated by JQ1, while dual PLK1/BRD4 inhibitors, such as BIꢀ2536, cause a G1/G2
arrest in the cell cycle. Twentyꢀfourꢀhour treatment with compound 23 caused cell cycle arrest in the
G1/G2 stage of the cell cycle, suggesting that it is functionally inhibiting both BRD4 and PLK1 (Sup
Inf Fig. 2).
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To gain a better understanding of the potency and predict the binding modes of our BIꢀ2536
analogs, we utilized molecular modeling with PLK1 and BRD4ꢀBD1 (Table 3). As described above,
compound 23 shows equivalent potency for both BRD4 and PLK1, while compound 6 demonstrates
the most selectivity for PLK1 over BRD4 in the series. To gain a better understanding of how
compound 23 interacts with BRD4, we further modeled the docking of 23 with BRD4ꢀBD1. As shown
in Fig. 4B, all of the interactions between residues on BRD4 and BIꢀ2536 are conserved when 23
resides in the active site of BRD4. The conserved phenyl ring in the center of compound 23 develops
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π−π stacking interactions with the aromatic rings in the WPF shelf (W81, P82, F83) of BRD4 as seen
with the parental compound BIꢀ2536. Furthermore, almost all of the original binding patterns
established in our coꢀcrystallography study of BIꢀ2536 and BRD4 (Figure 2) are strengthened as
indicated by the van der
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Docking score
IC50 (uM)
Ligandꢀreceptor interactions (vdw energy < ꢀ4 kcal/mol)
(Kcal/Mol)
Molecule
BRD4 PLK1
BRD4
PLK1
BRD4
PLK1
ꢀ7.34
ꢀ6.54
ꢀ6.63
ꢀ6.50
ꢀ7.02
ꢀ8.46
ꢀ8.68
ꢀ7.62
ꢀ8.23
ꢀ8.60
0.028
2.579
18.80
0.084
0.0400
0.0099
0.0780
0.0011
Pro82, Gln85, Val87, Ile146
Trp81, Pro82, Gln85, Val87, Leu92
Gln85, Val87, Asp88
Leu59, Cys133, Arg136, Phe183
Leu59, Arg136, Phe183
Gly63, Asp194
23
6
8
5
Val87, Asp88, Ile146
Leu59, Arg136, Phe183
Arg57, Leu59, Arg136, Phe183
BIꢀ2536
0.025 0.00083
Leu92, Ile146
Table 3. Docking results of some BI-2536 analogs on BRD4 and PLK1 (Details in SI)
Waals (vdw) interaction energy decomposition, where stronger interactions formed with P82, Q85,
V87, C136, N140, I146 and compound 23 (Fig. 4A). Although the L92, L94 and Y97 hydrophobic
subꢀpocket of BRD4 is moved further from the dihydropteridinone, the deep pocket formed by F83,
I146 and M149 made closer contact with the newly embedded cyclopentane to compensate or even
gain back the hydrophobic interaction loss (Fig. 4B). Overall, the docking analysis of compound 23
with BRD4 is consistent with the binding affinity defined in our biochemical assays.
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Figure 4. A. Comparison of the perꢀresidue decomposition of two BRD4ꢀBD1ꢀligand 23 in blue, BIꢀ2536 in red
intermolecular vdw interactions as a function of receptor sequence. B. Docking of 23 (cyan) with BRD4ꢀBD1
(PDBID: 4O74), protein residues in contact with ligand shown in yellow sticks, the rest in gray.
In modeling the docking between compound 23 and PLK1, we found that the C133 backbone
amide nitrogen of PLK1 conserves its hydrogen bonding to the nitrogen of dihydropteridinone in 23,
as indicated by unchanged dips at residue C133 from the vdw energy decomposition. The πꢀπ stacking
interaction between F83 and the dihydropteridinone in 23 is also conserved (Fig. 5A). The resulting
docking pose of 23 with PLK1 retains similar interactions as BIꢀ2536 with PLK1 (Fig. 5B). In
addition,
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Figure
5.
A.
Comparison of the
perꢀresidue decomposition of two PLK1ꢀligand 23 in blue, BIꢀ2536 in red intermolecular vdw interactions as a
function of receptor sequence. B. Docking of 23 (cyan) in PLK1 (PDBID: 2RKU), protein residues in contact
with ligand shown in yellow sticks, the rest in gray, hydrogen bonding in red dashed line.
the methyl group in 23, which replaced the original ethyl group in BIꢀ2536, does not reduce
interactions with A80 and K82 significantly. However, a notable difference upon the substitution of
cyclopentane for the methyl group in 23, is that the cyclopentane extends deeper into the hydrophobic
subꢀpocket between R57, F58 and L59 and creates additional interaction with R136, E140 that could
further stabilize the overall binding.
Molecular modeling of compound 6 with BRD4ꢀBD1 demonstrates that its docking pose
demonstrates drastic variation from the coꢀcrystal structure of BRD4ꢀBD1 bound to BIꢀ2536. When
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BIꢀ2536 binds to BRD4ꢀBD1, the dihydropteridinone and cyclopentane rings rest in between the WPF
shelf (W81, P82, F83) and the BC loop (particularly N140 through bridging water) of BRD4ꢀBD1,
with the ethyl group of BIꢀ2536 pointing away to make additional contacts with V87, L92, L94 and
Y97 (sharing the bridging water with N140) (Figs. 6A & 6B). With BIꢀ2536, Q85 interacts with the
inhibitor through waterꢀmediated hydrogen bonding effect. However, the longer alkyl chain of
compound 6 breaks this interaction due to steric clashes with L94 and Y97 (Fig. 6C). Therefore,
compound 6 has to adapt a novel orientation in the BRD4ꢀBD1 active site, which demonstrates much
weakened interactions by a striking positive vdw energy contribution from Y97. Although the phenyl
ring of compound 6 shifts into the pocket thereby enhancing binding with the WPF shelf, it still cannot
overcome the repulsive effect of the positive vdw effect. We believe that these adjustments contributed
significantly toward the reduced activity of 6 against BRD4ꢀBD1.
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C
Figure 6. A. Comparison of the perꢀresidue decomposition of two BRD4ꢀBD1ꢀligand 6 in gray, BIꢀ2536 in
dark red intermolecular vdw interactions as a function of receptor sequence. B. Docking of 6 (gray) in BRD4
(1) (PDBID: 4O74), protein residues in contact with ligand shown in yellow sticks, the rest in gray). C. Steric
clashes caused by the propyl group if 6 docked to BRD4(1) in the same pose as BIꢀ2536 does (bumps shown in
red dices).
Alternatively, the molecular docking of compound 6 with PLK1 shows great similarity with BIꢀ
2536 coꢀcrystalized with PLK1. The residue C133 conserves as hydrogen bonding receptor to the
nitrogen in the dihydropteridinone in 6, and the πꢀπ stacking interaction between F83 and the same
aromatic ring (Figs. 7A & 7B). Furthermore, the propyl group in 6, which replaced the original ethyl
group in BIꢀ2536 at Site 3, does not reduce interactions with A80 and K82 much. The docking pose
suggests that the carbonyl oxygen moved away from the active site, but may form hydrogen bonds
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with either R136, R57 or crystal water, as was observed in the BIꢀ2536ꢀPLK1 complex structure, to
reduce the entropy penalty when the new complex is forming. Again, the molecular docking analysis
is in agreement with the binding affinity assays that illustrate a potent interaction between PLK1 and
6. The detailed ligand interaction diagrams (LID) are listed in Supplement Figs 3 & 4.
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Figure 7. A. Comparison of the perꢀresidue decomposition of two PLK1ꢀligand 6 in gray, BIꢀ2536 in dark red
intermolecular vdw interactions as a function of receptor sequence. B. Docking of 6 (gray) in PLK1 (PDBID:
2RKU), protein residues in contact with ligand shown in yellow sticks, the rest in gray, hydrogen bonding in red
dashed line.
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CONCLUSION
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Our focused SAR study of the dual kinaseꢀbromodomain inhibitor BIꢀ2536 has led to the
identification of potent, balanced inhibitors of BRD4 and PLK1, as well as a more PLK1 selective
inhibitor. Through these studies, we determined the requirements for both PLK1 and BET protein
binding. We determined that the methylated amide and the cyclopentyl group of the BIꢀ2536 scaffold
is important for maintaining bromodomain binding activity but less important for PLK1 inhibitory
activity. Moreover, substituting the asymmetric ethyl moiety at the dihydropteridine ring with a methyl
group improved the inhibitory activity of resulting compounds against both BRD4 and PLK1, as seen
with compound 5. Installing bulky alkyl groups at the asymmetric carbon resulted in inhibitors with
potent and selective PLK1 activity but largely limited BRD4 activity, as seen with compound 6.
Further modification on the methoxy group of compound 5 led to the identification of compound 23, a
compound with increased potency for BRD4 with much reduced PLK1 activity. Our molecular
docking studies suggested possible binding orientations with BRD4ꢀBD1 and PLK1 and the binding
energies were in agreement with the observed SAR established through our medicinal chemistry
exercise.
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In summary, we have demonstrated that fineꢀtuning the selectivity for inhibitors demonstrating
polypharmacology can be achieved through structural guidance. We initiated an SAR campaign of BIꢀ
2536 in order to alter the PLK1 and BRD4 potency/selectivity of the compound and confirmed our
biochemical findings through molecular modeling. In this study, we discovered a set of compounds
with balanced selectivity against BRD4 and PLK1 that will entitle us to further explore the biological
outcome of dual BRD4/PLK1 inhibition. In particular, compound 23 is a wellꢀbalanced BRD4 and
PLK1 dual inhibitor when assayed biochemically and in cells. 23 demonstrates increased potency for
BRD4 and decreased potency for PLK1 as compared to the parent molecule BIꢀ2536. We envision
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that this set of molecules will generate sufficient PLK1 and BRD4 inhibition in order to study
simultaneous targeting of PLK1 and BRD4 in cancer.
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EXPERIMENTAL SECTION
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General Synthetic Information: All chemicals and solvents were purchased from commercial
suppliers and used as received. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on
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a Varian NMR spectrometer, and H NMR (400 MHz) and 13C NMR spectra (101 MHz) were
recorded on Agilent NMR spectrometers. LCꢀMS were performed on an Agilent 2100 LC with a 6130
quadrupole MS spectrometers. Accurate mass measurements were performed on a Fusion Lumos
Orbitrap mass spectrometer (Thermo Fisher Scientific) at a mass resolution setting of 500000
immediately after calibration or under lock mass calibration using solvent ions. A C18 column (5.0 ꢀm,
6.0 x 50 mm) was used for separation. The mobile phases were MeOH and H2O both containing
0.05% CF3CO2H. A linear gradient from 25:75 (v/v) MeOH/ H2O to 100% MeOH over 7.0 min at a
flow rate of 0.7 mL/min was used as a mobile phase. UV detections were conducted at 210 nm, 254
nm and 365 nm. Low resolution mass spectra were recorded by APCI (atmospheric pressure chemical
ionization) unless otherwise specified. Flash chromatography separation was performed on
YAMAZEN AIꢀ580 system with Agela silica gel (12 or 20 g, 230ꢀ400 mesh) cartridges. Final
products were purified on Angela HPꢀ100 preparativeꢀLC system with a Venusil PrepGꢀC18 column
(10 ꢀm, 120 Å, 21.2 mm x 250 mm). All biologically evaluated compounds were found to be >95%
pure as determined by NMR and LCMS.
Synthesis of BI-2536 analogs. The synthesis of compounds 1-20 shown in Tables 1 and 2 was
carried out following the reported procedures (Scheme 1).34 The synthesis of analogs of compound 5
including compounds 21, 23-25 listed in Table 2 was performed following the route shown in Scheme
2. Bromocyclopentane (1.3 equiv) was added slowly to a stirred suspension of potassium carbonate
(1.5 equiv) in DMF containing methylꢀ 3ꢀhydroxyꢀ4ꢀnitrobenzoate (1 equiv) at 65 oC for 4 h, then the
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product was treated by SnCl2·2H2O in EtOAc at 50 oC overnight. A mixture of ArꢀCl (1 equiv), Xꢀ
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4
Phos (28 mol %), Pd2(dba)3 (7 mol %), methyl 4ꢀaminoꢀ3ꢀ(cyclopentyloxy)benzoate (1.3 equiv), and
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o
K2CO3 (5 equiv) in tꢀBuOH was heated at 100 C in a sealedꢀtube for 8 h. The mixture was then
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diluted with EtOAc and washed three times with aqueous NaHCO3. The organic layer was dried over
anhydrous Na2SO4, filtered and condensed. The crude product was purified by preparativeꢀLC system
to get the final product.
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(R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxy-N-
1
(1-methylpiperidin-4-yl)benzamide (Compound 5). H NMR (300 MHz, CD3OD) δ 8.41 (1H, d, J
= 9.0Hz, 7.72 (1H, s), 7.41 ꢀ 7.31 (3H, m), 4.50 ꢀ 4.45 (1H, m), 4.31 ꢀ 4.24 (1H, q, J = 6.7 Hz), 4.02 ꢀ
3.82 (6H, s, NꢀCH3 and OꢀCH3), 3.27 ꢀ 3.26 (6H, m), 3.22 (3H, s), 2.57 ꢀ 2.47 (6H, m), 2.06 ꢀ 1.86
13
(4H, m), 1.25 (3H, d, J = 6.6 Hz); C NMR (75 MHz, CD3OD) δ 170.9, 166.6, 156.3, 153.0, 139.44,
138.7, 127.3, 121.2, 117.3, 117.3, 109.9, 59.5, 59.3, 56.4, 55.6, 55.02, 47.1, 44.9, 31.2, 30.5, 29.9,
28.5, 24.3, 23.8, 19.0; HRMS (ESI) m/z: (M+H)+ calcd for C27H37N7O3 536.3349, found 508.3031
(M+ +1).
(R)-4-((8-cyclopentyl-5-methyl-6-oxo-7-propyl-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-
methoxy-N-(1-methylpiperidin-4-yl)benzamide (Compound 6). 1H NMR (400 MHz, CDCl3) δ 8.54
(1H, d, J = 8.4 Hz), 7.68 (1H, s), 7.59 (1H, s), 7.51 (1H, s), 7.42 (1H, s), 7.42 (1H, d, J = 1.8 Hz), 4.56
– 4.46 (1H, m), 4.24 (1H, dd, J = 8.0, 3.7Hz), 3.97 (3H, s), 3.31 (3H, s), 2.86 – 2.84 (2H, m), 2.31 (3H,
s), 2.25 – 2.10 (4H, m), 2.07 – 2.04 (4H, m), 1.81 – 1.71 (14H, m), 1.37 – 1.21(4H, m), 0.87 (3H, t, J =
7.3 Hz); 13C NMR (101 MHz, CDCl3) δ 163.9, 155.0, 152.1, 147.2, 137.9, 133.2, 126.2, 126.0, 118.8,
116.4, 115.8, 109.0, 58.8, 58.1, 54.4, 35.8, 32.4, 29.6, 29.1, 28.2, 23.5, 23.0, 17.8, 13.9; HRMS (ESI)
m/z: (M+H)+ calcd for C29H41N7O3 536.3349, found 536.3342 (M+ +1).
(R)-4-((8-cyclopentyl-7-isobutyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-
1
methoxy-N-(1-methylpiperidin-4-yl)benzamide (Compound 8). H NMR (300 MHz, CD3OD) δ
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8.40 (1H, d, J = 9.0 Hz), 7.74 (1H, s), 7.42 – 7.38 (3H, m), 4.21 – 4.17 (1H, m), 3.91 (3H, s), 3.87 –
3.83 (1H, m), 3.23 (3H, s), 2.91 – 2.88 (1H, m), 2.27 (3H, s), 2.21 – 2.13 (2H, m), 2.02 – 1. 97 (2H,
m), 1.92 – 1.83 (2H, m), 1.74 – 1.54 (8H, m), 1.41 – 1.36 (1H, m), 0.92 (3H, d, J = 6.3 Hz), 0.78 (3H,
d, J = 6.6 Hz); 13C NMR (75 MHz, CD3OD) δ 169.1, 165.2, 156.2, 153.6, 148.4, 139.6, 134.0, 127.4,
121.2, 117.4, 117.3, 109.9, 59.6, 58.4, 56.4, 55.5, 45.8, 42.5, 32.0, 30.49, 30.4, 28.6, 25.7, 24.2, 23.9,
23.7, 21.7; HRMS (ESI) m/z: (M+H)+ calcd for C30H43N7O3 550.3506, found 550.3501 (M+ +1).
(R)-4-((8-isopropyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxy-N-(1-
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methylpiperidin-4-yl)benzamide (Compound 12). H NMR (300 MHz, CD3OD) δ 8.42 (1H, d, J =
9.3 Hz), 7.69 (1H, s), 7.38 (3H, m), 3.99 – 3.90 (5H, m), 3.90 (3H, s), 2.72 – 2.65 (4H, m), 2.55 (3H,
s), 1.86ꢀ1.76 (4H, m), 1.30 – 1.23 (9H, m); 13C NMR (75 MHz, CD3OD) δ 169.2, 166.4, 156.3, 152.4,
148.3, 139.5, 134.1, 127.1, 121.3, 117.2, 109.8, 56.4, 54.7, 53.6, 46.6, 44.4, 30.7, 28.5, 21.5, 19.9;
HRMS (ESI) m/z: (M+H)+ calcd for C25H35N7O3 482.2880, found 482.2884 (M+ +1).
(R)-4-((7-isobutyl-5-methyl-6-oxo-8-(tetrahydro-2H-pyran-4-yl)-5,6,7,8-tetrahydropteridin-2-
1
yl)amino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (Compound 13). H NMR (300 MHz,
CD3OD) δ 8.42 (1H, d, J = 9.0 Hz), 7.78 (1H, s), 7.45 – 7.42 (3H, m), 3.92 (3H, s), 3.56 – 3. 53 (2H,
m), 3.22 (3H, s), 3.03 – 2.99 (2H, m), 2.37 (3H, m), 1.96 – 1.92 (3H, m), 1.70 – 1.65 (4H, m), 1.55 –
1. 38 (5H, m), 0.94 (3H, d, J = 6.3 Hz), 0.79 (3H, d, J = 6.6 Hz); 13C NMR (75 MHz, CD3OD) δ 171.5,
171.4, 170.9, 169.1, 164.86, 153.0, 150.0, 140.1, 127.5, 121.2, 117.4, 110.0, 68.6, 68.5, 57.3, 56.4,
55.3, 55.2, 45.4, 43.4, 32.6, 31.6, 28.7, 25.8, 23.9, 21.7; HRMS (ESI) m/z: (M+H)+ calcd for
C30H43N7O4 566.3455, found 566.3448 (M+ +1).
(S)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxy-N-
1
(1-methylpiperidin-4-yl)benzamide (Compound 21). H NMR (300 MHz, CD3OD) δ 8.43 (1H, d, J
= 8.7 Hz), 7.75 (1H, s), 7.43 ꢀ 7.41 (3H, m), 4.31 ꢀ 4.29 (1H, q, J = 6.0 Hz), 3.94 (3H, s), 3.24 (3H, s),
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3.12ꢀ3.07 (3H, m), 2.90 ꢀ 2.87 (3H, m), 2.12 ꢀ 2.09 (3H, m), 1.91 ꢀ 1.89 (4H, m), 1.79 ꢀ 1.67 (6H, m),
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1.26 (3H, d, J = 6.9 Hz); C NMR (75 MHz, CD3OD) δ 171.1, 169.1, 166.6, 156.2, 153.0, 148.3,
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139.4, 134.0, 127.4, 121.2, 117.2, 109.9, 59.3, 56.4, 55.6, 55.4, 47.8, 45.7, 31.9, 30.5, 29.9, 28.5, 24.2,
23.8, 19.0; HRMS (ESI) m/z: (M+H)+ calcd for C31H43N7O3 562.3506, found 562.3501 (M+ +1).
(R)-4-((8-cyclopentyl-5,7-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-
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(cyclopentyloxy)-N-(1-methylpiperidin-4-yl)benzamide (Compound 23). H NMR (400 MHz,
CDCl3) δ 8.53 (1H, d, J = 8.0 Hz), 7.71 (1H, s), 7.60 (1H, s), 7.41 (1H, d, J = 1.9 Hz), 4.62 – 4.51
(1H, m), 4.32 – 4.30 (1H, m), 4.14 – 4.06 (1H, m), 3.97(3H, s), 3.31 (3H, s), 2.83 – 2.81 (3H, m), 2. 29
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( 3H, s), 2.20 – 2.10 (3H, m), 2.08 – 1.93 (4H, m), 1.32 (3H, d, J = 6.8 Hz); C NMR (101 MHz,
CDCl3) δ 166.6, 165.0, 155.2, 151.8, 147.2, 138.3, 133.1, 126.6, 118.8, 116.3, 116.0, 109.1, 57.6,
55.9, 54.4, 46.2, 32.5, 30.0, 29.5, 28.3, 23.7, 23.2, 19.0. HRMS (ESI) m/z: (M+H)+ calcd for
C31H43N7O3 562.3506, found 562.3508 (M+ +1).
(R)-4-((8-cyclopentyl-5-methyl-6-oxo-7-propyl-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-
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(cyclopentyloxy)-N-(1-methylpiperidin-4-yl)benzamide (Compound 24). H NMR (300 MHz,
CD3OD) δ 8.44 (1H, d, J= 8.1 Hz), 7.72 (1H, s), 7.42 ꢀ 7.39 (3H, m), 4.39 ꢀ 4.34 (1H, m), 4.27ꢀ 4.24
(1H, m), 3.85 – 3.80 (1H, m), 3.25 (3H, s), 2.86 ꢀ 2.60 (2H, m), 2.26 (3H, s), 2.16 – 2.09 (3H, m), 2.01
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– 1.80 (14H, m), 1.76 – 1.59 ( 8H, m), 1.26 – 1.17 (2H, m), 0.83 (3H, t, J = 7.2 Hz); C NMR (75
MHz, CD3OD) δ 169.12, 165.4, 156.0, 153.4, 146.5, 139.0, 134.8, 127.3, 120.8, 117.2, 112.4, 81.8,
60.67 60.5, 55.6, 49.7, 46.0, 36.8, 33.6, 32.2, 30.2, 29.9, 28.4, 24.8, 24.4, 24.2, 18.7, 14.0; HRMS
(ESI) m/z: (M+H)+ calcd for C33H47N7O3 590.3819, found 590.3815 (M+ +1).
4-((8-cyclopentyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-(cyclopentyloxy)-N-
(1-methylpiperidin-4-yl)benzamide (Compound 25).1H NMR (300 MHz, CD3OD) δ 8.43 (1H, d, J
= 8.4 Hz), 7.64 (1H, s), 7.42 –7.39 (3H, m), 4.06 (3H, s), 3.28 – 3.25 (4H, m), 3.23 (3H, s), 3.02 – 2.97
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(3H, m), 2.37 (3H, s), 2.30 – 2.26 (3H, m), 1.98 – 1.93 (7H, m), 1.74 – 1.68 (9H, m); C NMR (75
MHz, CD3OD) δ 169.1, 163.5, 156.1, 153.1, 146.4, 138.5, 134.8, 127.1, 120.8, 117.0, 116.8, 112.3,
81.8, 56.9, 55.37, 47.6, 45.9, 45.5, 33.6, 31.7, 28.0, 27.9, 24.8, 24.8; HRMS (ESI) m/z: (M+H)+ calcd
for C30H41N7O3 548.3350, found 548.3346 (M+ +1).
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Molecular Docking Software: For protein preparation: UCSF Chimera Dock Prep tool,35 UCSF
DOCK6.36 For compound structures and charge assignment method: ChemDraw 15.0, Chem3D 14.0,
GAFF, AM1ꢀBCC. For docking and energy decomposition analysis: UCSF DOCK6 suite. Dock poses,
visualization and illustration: UCSF Chimera ViewDock, PyMOL.
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Compounds and protein preparation: The synthesized compounds were built in ChemDraw,
and then their Cartesian coordinates’ construction and energy minimization using MM2 force field37,38
were generated in Chem3D. The crystal structures of BIꢀ2536 binding with BRD4ꢀBD1 (PDBID:
4O74) and PLK1 (PDBID: 2RKU) were downloaded from Protein Data Bank (PDB). Protein targets
were prepared using Chimera DockPrep tool, in which AMBER force field ff14SB39 was employed to
provide accurate descriptions for proteins. GAFF40and AM1ꢀBCC41 were used to generate parameters
and assign charges for all inhibitor molecules.
Docking and energy decomposition analysis: DOCK6 suite was used for docking compounds to
their protein targets. Flexible ligand docking was performed under the guidance of the anchorꢀandꢀ
grow algorithm. Dock Score was used as the criterion for ranking the binding poses. We visually
examined the top 10 ranked poses, and the highest ranked poses are reported in Table 3. In the case of
PLK1 and BRD4ꢀBD1, DOCK6 successfully sampled and topꢀscored the BIꢀ2536 docking poses to
reproduce the corresponding crystal binding poses with rmsd < 2 Å. DOCK Score is composed of Van
der Waals (vdw) and electrostatic interactions. The vdw composition of Dock score was further
decomposed onto every residue of its binder protein by using DOCK write_footprint function, which
generates both the vdw and electrostatic parts. Only the vdw composition was noticeably diversified
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and thus referred to represent the contribution of the protein target per residue in proteinꢀligand
interaction energy profiles as shown in Figures 4ꢀ7.
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Specific favorable and unfavorable interactions recognition: As listed in Table 3, the specific
residual information of the protein receptors interacting with BIꢀ2536 analogs was recognized by
referring to vdw energy and by visualization of binding poses. A threshold of < ꢀ4 kcal/mol in
decomposed vdw energy was used to include the first shell of residues around the molecules (see also
Sup Inf Fig Sꢀ3). The positive peak observed in perꢀresidue decomposition of BRD4ꢀDB1 compound
6, Figure 6A, was ascribed to the steric clashes of residue Y97, L94 with the propyl group on 6. The
disc was scripted in PyMOL software to show vdw overlaps or steric clashing (See also Sup Inf Fig Sꢀ
4).
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BRD4-BD1 and BRDT-BD1 activity assays: Compound potency was assessed by relative IC50
potency determined by an AlphaScreen biotinꢀJQ1 competition assay as reported previously by our
group.42 All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v
Tween20, pH 7.5 and allowed to equilibrate to room temperature prior to the addition to plates. After
addition of Alpha beads to master solutions, all subsequent steps were performed in low light
conditions. A 2x solution of components with final concentrations of BRD at 40 nM, Niꢀcoated
Acceptor Bead at 25 ꢁg/mL, and 20 nM biotinꢀJQ1 was added in 10 ꢁL to 384ꢀwell plates (AlphaPlate
ꢀ 384, PerkinElmer, USA). After a 1 min 1000 rpm spinꢀdown, 100 nL of compounds in DMSO from
stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The 2x, 10 ꢁL
streptavidinꢀcoated donor beads (25 ꢁg/ml) were added to the previous solution. Following this
addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates
were spun down again at 1000 rpm for 1 min. Next, the plates were incubated at room temperature
with the plate reader (for temperature equilibration) for 1 h prior to reading the assay. Signal is stable
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for up to 3 h after donor bead addition. AlphaScreen measurements were performed on an Envision
2104 (PerkinElmer, USA) utilizing the manufacturer’s protocol.
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PLK1 activity assay: The enzymatic activities against PLK1 were tested in ZꢀLyte assays with
ATP concentration of KM for each kinase per manufacturer’s protocol (Thermo Fisher).
All the compounds were tested in parallel with crossꢀlinker assay to rule out the assay
binder/interrupter.
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Selectivity profiling: Compound 5 was assayed at 1 uM in DiscoverX’s BROMOscan®
(bromoMAXSM) and KINOMEscan® (scanMAXSM) assay platforms. The results for single
concentration binding interactions for compound 5 are reported as % of control (DMSO).
Cell cycle study: MOLM13 cells were treated with 1 µM of JQ1, BI2536, TAE684, compound 5,
compound 6 or compound 23 for 24 h. Cells were subsequently harvested and stained with propidium
iodide (Calbiochem), followed by measurement of propidium iodideꢀmediated fluorescence with a
Guava H6T (Millipore Sigma).
ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge on the ACS Publications website at DOI: *****
BROMOscan and KINOMEscan data for compound 5 (CSV)
Molecularꢀformula strings (CSV)
Accession Codes
PDB codes are the following: 4O74 for BI2536: BRD4ꢀBD1; 2RKU for PLK1ꢀBI2536
AUTHOR INFORMATION
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Corresponding Authors
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*Eꢀmail:
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james.bradner@novartis.com. Tel.: +1 (617ꢀ871ꢀ8000. Fax: +1(617)ꢀ582ꢀ7370
jun_qi@dfci.harvard.edu. Tel.: +1 (617) 632ꢀ6629. Fax: +1(617) 582ꢀ7370
wei2.zhang@umb.edu. Tel.: +1 (617) 287ꢀ6147. Fax: +1 (617) 287ꢀ6030.
ORCID
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James E. Bradner: 0000ꢀ0002ꢀ2718ꢀ4415
Jun Qi: 0000ꢀ0002ꢀ1461ꢀ3356
Wei Zhang: 0000ꢀ0002ꢀ6097ꢀ2763
Author Contributions
All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was partially supported by National Institutes of Health U54 grant CA156732 (JEB and
WZ); U54 grant (HD093540ꢀ01 for JQ); P01 grant (CA066996 for JQ). We thank Jason Evans and
Dennis Zeh for performing HRMS analysis of some synthetic compounds.
ABBREVIATIONS USED
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BET, bromodomain and extraꢀterminal domain; BRD4, bromodomain 4; PLK1, poloꢀlike kinase 1;
AML, acute myeloid leukemia; JAK2, janus kinase 2; SAR, structureꢀactivity relationship; LID,
ligand interaction diagrams
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