Design, Synthesis, and Characterization of a Fluorescence Polarization Pan-BET Bromodomain Probe

Article abstract of DOI:10.1021/acsmedchemlett.8b00380

Several chemical probes have been developed for use in fluorescence polarization screening assays to aid in drug discovery for the bromodomain and extra-terminal domain (BET) proteins. However, few of those have been characterized in the literature. We ha

Full text of DOI:10.1021/acsmedchemlett.8b00380

Subscriber access provided by Kaohsiung Medical University
Letter
Design, Synthesis, and Characterization of a
Fluorescence Polarization Pan-BET Bromodomain Probe
Carolyn N. Paulson, Xianghong Guan, Alex M. Ayoub, Alice Chan, Rezaul M. Karim,
William C. K. Pomerantz, Ernst Schönbrunn, Gunda I. Georg, and Jon E. Hawkinson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00380 • Publication Date (Web): 31 Oct 2018
Downloaded from http://pubs.acs.org on November 1, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Design, Synthesis, and Characterization of a Fluorescence
Polarization Pan-BET Bromodomain Probe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Carolyn N. Paulson,†‡ Xianghong Guan,†‡ Alex M. Ayoub,# Alice Chan,§ Rezaul M. Karim,§ William C.
K. Pomerantz,# Ernst Schönbrunn,§ Gunda I. Georg,†* and Jon E. Hawkinson†*
†Department of Medicinal Chemistry and Institute for Therapeutics Discovery & Development, College of Pharmacy,
University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414
#Department of Chemistry, University of Minnesota, 207 Pleasant Street, SE, Minneapolis, MN 55455
§H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 and Department of Molecular
Medicine, University of South Florida, Tampa, Florida 33612
ABSTRACT: Several chemical probes have been developed for use in fluorescence polarization screening assays to aid in drug
discovery for the bromodomain and extra-terminal domain (BET) proteins. However, few of those have been characterized in the
literature. We have designed, synthesized, and thoroughly characterized a novel fluorescence polarization pan-BET chemical probe
suitable for high-throughput screening, structure-activity relationships, and hit-to-lead potency and selectivity assays to identify and
characterize BET bromodomain inhibitors.
KEYWORDS: bromodomain and extra-terminal domain, BET, pan-BET probe, fluorescence polarization
Bromodomain (BRD)-containing proteins are highly
conserved epigenetic regulators that recognize and bind to
acetylated lysine (KAc) residues of histones. BRDs recruit
proteins to macromolecular complexes essential for chromatin
remodeling and transcriptional control.1 The highly diverse
BRD family members share a conserved structural motif
comprised of a left-handed bundle of four alpha helices (αZ,
αA, αB, αC) that are linked by two variable loop regions (ZA
and BC loops) flanking a deep central hydrophobic cavity that
recognizes sequences containing ε-N-acetylated lysine
residues.2 Each of the BRD and extra-terminal domain (BET)
family members, BRD2, BRD3, BRD4, and BRDT, contains
two N- and C-terminal KAc binding domains (designated as
BD1 and BD2, respectively), which play important roles in
regulating the transcription of growth-promoting genes and
cell cycle regulators.3 While BRD2, 3, and 4 are ubiquitously
expressed, BRDT is exclusively found in the testis.4
Mutagenesis studies have shown that loss of BRDT-BD1
(BRDT-1) results in abnormal spermatids and complete
sterility in mice5 making BRDT-1 an attractive target for
developing non-hormonal male contraceptives.6 The greatest
challenge for BRD drug discovery efforts is identifying
compounds that are selective for individual BRD proteins due
to the high homology of KAc binding sites within BRD
families.7 In addition, the discovery of potent inhibitors with
high selectivity for BD1 over BD2 (or BD2 over BD1) has
been challenging because, although the BET BRD loop
regions are variable, the BD1 and BD2 substrate binding
pockets show high sequence similarity.7
BODIPY) is reported to have a KD of 50-110 nM for BRD4-
BD1 (BRD4-1)8-9 and only a 40 mA polarization signal
window while the Alexafluor488-IBET10 and FITC-JQ111-13
probes remain uncharacterized. This prompted us to develop a
novel FP small molecule chemical probe utilizing the high
affinity pan-BET inhibitor SG3-179 (Figures 1 and 3;
compound 5 in Ember et al.14). SG3-179-BODIPY probe 16
was selected for further characterization as described below.
Figure 1. Molecular docking predicts that compound 16 will
maintain high affinity for BRDT-1. Compound 16 orients
similarly to the parent compound SG3-179 and maintains the key
interaction with Asn109 of BRDT-1 (PDB: 4FLP). The
fluorophore is solvent exposed without the possibility of any
steric hindrance with the protein.
A general challenge for probe development is to avoid a
significant loss of binding affinity caused by steric hindrance
of the attached fluorescent label. To achieve this endpoint, we
docked SG3-179 to a representative BET-family protein
BRDT-1 (PDB: 4FLP) to reveal the binding mode of SG3-179
for BRDT-1 (Figure 1). The predicted binding mode is similar
Several chemical probes for use in fluorescence polarization
(FP) assays have been developed to aid in drug discovery
programs for BET proteins. Previously reported probes for
BET BRDs either have low binding affinities or have low
polarization signal windows, and most of them are not well
characterized. For example, BODIPY-conjugated BI6727 (BI-
*
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
to the later released crystal structure SG3-179 in complex with
Page 2 of 7
aldehyde 10,17 which was then oxidized to carboxylic acid 11.
Acrylic acid 12 was synthesized from aldehyde 10 by a
Knoevenagel condensation.18 A Heck-type reaction between
tetramethyl-BODIPY and methyl acrylate catalyzed by Pd(0)
yielded methyl acrylate 13.16 The double bond in 13 was
reduced with H2 to give methyl propionate 14, which was
hydrolyzed to yield acid 15. Fluorophores 11, 12 and 15 were
linked to ligand 9 (HOBt and EDC) to produce BODIPY
based FP probes 16-18.
BRD4-1 (PDB: 5F63).14 The diaminopyrimidine core of SG3-
179 forms a direct hydrogen bond with Asn 109 in the KAc
binding site of BRDT-1 and the water exposed piperidine
moiety is far away from this key interaction, which therefore
provided a reasonable position to connect to a fluorophore. We
chose BODIPY as the fluorophore due to its long fluorescence
lifetime (5-6 ns)15 and because tetra-methyl BODIPY is an
inexpensive fluorophore among BODIPY dyes and numerous
chemistries involving tetra-methyl BODIPY are available for
rapid linker incorporation.16-18 Although a longer linker length
may alleviate steric hindrance, local motion of fluorophores
attached by flexible linkers can decrease the polarization of the
bound probe (‘propeller effect’).19 In order to identify the
optimal balance between steric hindrance and unrestrained
fluorophore motion, linker length and flexibility were
explored.
1
2
3
4
5
6
7
8
Scheme 2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The synthetic route for SG3-179 based probes 16-18 is
shown in Schemes 1 and 2. The synthesis of the KAc binding
site ligand employed procedures similar to the methods
reported for the preparation of SG3-179 (Scheme 1).20-21
Protection of commercially available 4-chloro-3-nitroaniline
with Boc anhydride gave 1, followed by reduction of the nitro
group with hydrazine to provide aniline 2. Reaction of 2 with
tert-butylsulfinyl chloride furnished tert-butylsulfinyl 3 that
was further oxidized with mCPBA to sulfonamide 4.
Deprotection of 4 with TFA produced aniline 5, which was
reacted with 2,4-dichloro-5-methylpyrimidine to afford
chloropyrimidine 6. Intermediate 7 was obtained by coupling
Reagents: (a) DMF, POCl3, 0 °C, 2~3 h, then 50 °C, 3 h, 89%; (b)
NaClO2, H2O, NaH2PO4, rt, 15 h, 30%; (c) malonic acid, pyridine,
DMF, 90 °C, 5 h, 28%; (d) methyl acrylate, 20 mol % of
Pd(OAc)2, tert-BuOOBz, DMSO, 35 °C, 5 d, 57%; (e) Pd/C, H2,
MeCN, MeOH, rt, 12 h, 80%; (f) NaOH, DCM:MeOH:H2O, rt, 10
h, then neutralize, 98%; (g) intermediate 11, 12 or 15, HOBt,
EDC, DIPEA, DCM, rt, 6 h, 72%-93%.
4-amino-2-fluorobenzoic
aminopiperidine-1-carboxylate,
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
acid
and
tert-butyl
using
4-
1-
b]pyridinium 3-oxid hexafluorophosphate (HATU) in the
presence of N,N-diisopropylethylamine (DIEPA) in dry DMF.
Connection of fragment 6 and 7 by a Buchwald-Hartwig
coupling reaction yielded 8 which was then Boc deprotected
with TFA to give the KAc binding site ligand 9.
In comparison to compound 16, which has sharp UV
absorption and fluorescence excitation and emission peaks
similar to BODIPY, the peaks for compounds 17 and
particularly 18 are broadened and red-shifted (Figure S1).
Although it is not clear why compound 17 has a red-shifted
emission spectrum, the extended -conjugate system present
in compound 18 is expected to produce a higher LUMO level
and narrowed HOMO-LUMO gap,22 which likely explains the
red-shifted emission spectrum.
Scheme 1
The three probes (16-18) were then tested against BRDT-1
in a FP assay to assess probe affinity and signal window
(Figure S2). Whereas probes 16 and 18 have similar affinities,
probe 16 had the highest FP signal window (Bmax >100 mP) at
the standard BODIPY excitation and emission wavelengths,
and was therefore selected for further characterization. KD
values for all the BET family proteins (BRDT-1, BRDT-2,
BRDT-T, BRD2-1, BRD2-2, BRD3-1, BRD3-2, BRD4-1,
BRD4-2, and BRD4-T, where –T designates the BD1 and
BD2 tandem BRD protein), and two representative non-BET
family proteins (BRD7 and BRD9) were determined with FP
probe 16 (Table 1, Figures 2 and S3).
Reagents: (a) (Boc)2O, THF, reflux, 28 h, 58 %; (b) Pd/C,
hydrazine monohydrate, MeOH, reflux, 10 h, 64%; (c) 2-
methylpropane-2-sulfinic chloride, pyridine, DCM, rt, 20 h, 77%;
(d) mCPBA, DCM, rt, overnight, 84%; (e) TFA, DCM, rt, 2 h,
78%; (f) 2,4-dichloro-5-methylpyrimidine, DIPEA, isopropanol,
120 °C, 2 d, 36%; (g) tert-butyl 4-aminopiperidine-1-carboxylate,
HATU, DIPEA, DMF, rt, 12 h, 95%; (h) Compound 6, Pd2(dba)3,
FP probe 16 exhibited low nanomolar affinities for all of the
BET family proteins (6-56 nM), but had KD values in the low
micromolar range for the two non-BET family proteins (7-11
M) (Figure 2). The lower affinity of probe 16 for BRD7 and
BRD9 was expected based upon previously reported SG3-179
BROMOScan data.14 Therefore, compound 16 is a pan-BET
probe, but is unlikely to be a useful tool for non-BET BRDs.
Probe 16 has a slight preference for BD2 over BD1 BRDs,
(±)-2,2’-Bis(diphenylphosphino)-1,1’-binaphthalene
(BINAP),
NaOtBu, 1,4-dioxane, 110 °C, 5 h, 90%; (i) TFA, DCM, 12 h,
91%.
The synthesis of the fluorophore moiety commenced with
commercially available tetramethyl-BODIPY (Scheme 2) by
reaction with DMF in the presence of POCl3 to produce
*
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
ranging from 1.7- to 4.7-fold higher affinity for BD2
Parent compound SG3-179 has been reported to have an
IC50 ~20 nM for both BRDT-1 and BRD4-1 using an
AlphaScreen assay.14 SG3-179 FP Ki values are ~15 nM for
these BRDs. SG3-179 FP Ki values for BD1 domains of the
four BET BRDs correlate well with the KD values obtained for
the SG3-179-BODIPY probe 16. However, SG3-179 has 3- to
5-fold lower affinity for the BD2 domains of BRDT, BRD2,
and BRD4 based on SG3-179 Ki values relative to probe 16
KD values, perhaps due to a contribution of the fluorophore to
the binding of probe 16 to these proteins. ABBV-075, also
known as mivebresib, is a potent small molecule BET
inhibitor presently in a Phase I clinical trial for patients with
advanced hematologic malignancies and solid tumors.25
ABBV-075 has previously been reported to be selective for
tandem BRDT, BRD2, and BRD4 proteins (Ki = 1-2 nM) over
the tandem BRD3 protein (Ki = 12.2 nM) in TR-FRET
assays.25 When tested in the FP assay reported here, the IC50
values ranged from 9-32 nM for all of the BRD proteins.
These IC50 values likely underestimate the true affinity of
ABBV-075 and Ki values could not be calculated due to the
‘floor effect’ described above, consistent with the high affinity
values reported using the TR-FRET assay.25
1
2
3
4
5
6
7
8
depending on BET family member. In contrast to the BRD4-T
protein, the pan-BET probe 16 bound BRDT-T with 2- to 3-
fold higher affinity than the individual BRDT BRDs.
Although it has been argued that the two BRDs of BRDT
function independently due to the long flexible linker
connecting them,23 the higher affinity of the probe for BRDT-
T suggests that there may be cooperativity between the tandem
BRDs or that the 123 aa linker may stabilize one or both
BRDs.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Pan-BET probe 16 binds with high affinity to BET
BRDs, but only weakly to other BRD proteins. Error bars
represent ± SEM where n ≥ 3.
Table 1. Affinity of pan-BET FP probe 16 for BRD proteins.
Values are mean + SEM in nM, where n ≥ 3 independent
experiments.
KD, nM
d
BRDT
BRD4
BRD3
BRD2
BRD7
BRD9
,
Ten representative BET BRD inhibitors with varying
scaffolds and selectivities were chosen for competition studies
to establish that pan-BET probe 16 provides the expected
affinity and selectivity values for the BET BRDs. The
structures of the reference compounds SG3-179, ABBV-075,
Figure 3. Structures of the 10 reference BET inhibitors with
diverse chemical scaffolds selected to validate the FP assay. CAS
2096312-12-8 refers to compound 3s from Ayoub et al.9
The prototypic BET inhibitor, (+)-JQ1, is a thieno-triazolo-
1,4-diazepine with reported AlphaScreen and TR-FRET IC50
values ranging from 18-91 nM for BRD4-1,14, 26, 29, 30 14-33
nM for BRD4-2 and BRD3-1,29, 30 and 100-150 nM for BRDT-
1 and BRD2-1.14, 26 The FP assay provides similar IC50 values,
particularly for BRDT-1, although it indicates a higher
potency of (+)-JQ1 for BRD2-1, but a lower potency at
BRD3-1 and BRD4-1. (-)-JQ1, the inactive enantiomer of (+)-
JQ1, is considerably less potent with reported IC50 values of
8354 and >10000 nM for BRD4-1 and 52120 nM for BRD4-
2.29, 30 We report similar findings in our FP assay in which (-)-
JQ1 had IC50 values >10000 nM for all BET BRDs.
TG101209,
(+)-JQ1,
(-)-JQ1,
CAS
2098312-12-8,
bromosporine, BI2536, MS436, and RVX-208 are illustrated
in Figure 3. IC50 values were generated for all ten compounds
against the BET-family proteins and Ki values were calculated
using the equations of Nikolovska-Coleska et al.24 (Figures 4
and S4, Table 2). In some cases, Ki values could not be
determined because IC50 values were too low relative to the
protein concentration (30-130 nM) used in the assay. In these
cases, the apparent IC50 values likely underestimate the true
potency of the inhibitors. As protein concentrations could not
be lowered in order to maintain a sufficient signal window, an
alternate method must be employed to obtain accurate affinity
estimates for these very potent inhibitors due to this ‘floor
effect’. Additionally, Ki values are not reported for BRDT-T
and BRD4-T as the Ki calculation equation cannot be used for
proteins with > 1 ligand binding site.24
The dihydropyridopyrimidine compound (CAS registry
number: 2098312-12-8) is a potent BET inhibitor that has
been shown to have submicromolar affinity (BRDT-1, Ki =
200 nM; BRD4-1, Ki = 110 nM) in a FP assay using BI-
BODIPY.9 Three- to six-fold higher affinity values were
obtained using the SG3-179-BODIPY probe 16 for BRDT-1
*
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
and BRD4-1, respectively. The prior report by Ayoub et al.9
carried out the experiment at a 5-fold higher DMSO
concentration (0.5% vs. 0.1%), which is known to affect BRD
binding interactions.28 Additionally, 2098312-12-8 had higher
affinity for BRD2 and BRD3 BD1 and BD2 that exceeded the
sensitivity of the assay.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Competitive displacement of pan-BET probe 16 by 10 reference BET inhibitors at (a) BRDT-BD1 and (b) BRDT-BD2. Error
bars represent ± SEM where n ≥ 3. Z’ value for BRDT-1 = 0.78 ± 0.04 (mean ± SEM).
Table 2. Inhibitory potency (IC50) and affinity (Ki) of representative inhibitors for BET family BRDs determined using pan-BET
FP probe 16. Values are mean + SEM in nM, where n ≥ 3. Ki values are included for BRDs containing a single binding domain
(BD1 or BD2) if potency values are greater than the lower sensitivity limit of the assay (IC50 ≥ 1.5X BRD concentration)
Compound
BRDT-1
BRD2-1
BRD3-1
BRD4-1
BRDT-2
BRD2-2
BRD3-2
BRD4-2
BRDT-T
BRD4-T
SG3-179
ABBV-075
(+)-JQ1
CAS 2098312-
12-8
bromosporine
BI2536
TG101209
MS436
RVX-208
(-)-JQ1
Bromosporine is a promiscuous BRD inhibitor that was
developed to target the conserved KAc recognition site of
BRD-containing proteins. While IC50 and Ki information could
not be found in the literature, KD values for bromosporine
against all of the BET proteins using ITC have been reported
to be in the 40- 100 nM range for BRD2, BRD3, BRD4, and
BRDT BD1 and BD2, excluding BRDT-2 which had a
significantly higher KD value (KD = 172 nM).27 Similarly, Ki
values obtained in the FP assay for BRD4-1, BRD4-2, and
BRDT-1 ranged from 35 – 90 nM, and bromosporine had
lower affinity for BRDT-2 (Ki = 310 nM). In contrast,
bromosporine had higher affinities for BRD2 and BRD3 BD1
and BD2 in the FP assay that exceeded the sensitivity of the
assay.
was shown to be selective for BRD4-1 over BRDT-1 with IC50
values of 25 and 260 nM, respectively, in an AlphaScreen
assay.26 Similarly, in the FP assay, BI2536 had higher affinity
for BRD4-1 (Ki = 15 nM) compared to BRDT-1 (Ki = 71 nM).
We also found that BI2536 had high affinity for BRD2-2 (Ki =
9.3 nM).
TG101209, a dual BET BRD and JAK2 kinase inhibitor,
has been reported in the literature to have lower affinity for
BRD2-1 (IC50 = 680 nM) relative to BRD3-1, BRD4-1, and
BRDT-1 (IC50 = 130-290 nM).26 The Ki values determined in
the FP assay for these BRDs revealed a similar profile in
which TG101209 was 2-5 times less active against BRD2-1
(Ki = 700 nM) than the other BRD BD1s (Ki = 120-320 nM).
The BD1-selective diazobenzene-based inhibitor MS436
has been reported to have a Ki < 85 nM for BRD4-1 and had
>4-fold lower affinity for BD2.31 In our assay, MS436 showed
similar activity with a BRD4-1 Ki of 130 nM and 4-fold lower
affinity for BRD4-2. MS436 had ~2- to 4-fold lower affinity
for the other BET BRDs and showed lower selectivity for their
BD2 domains.
BI2536 is a potent and selective inhibitor of PLKI (IC50
=
0.83 nM) and has antitumor activity against relapsed or
refractory acute myeloid lymphoma (AML) and non-small cell
lung cancer in phase I/II clinical trials. It binds to the KAc
recognition site of BRD4 through an elaborate network of
hydrogen bonding and Van der Waals interactions.26 B12536
*
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
‡These authors contributed equally. All authors have given
approval to the final version of the manuscript.
RVX-208 has been evaluated in several clinical trials for the
treatment of atherosclerosis and associated cardiovascular
disease.32 RVX-208 has been reported to be a BD2-selective
inhibitor of BET proteins with KD values measured by ITC for
BD1 that are 8.5- to 23-fold higher than those determined for
BD2, with highest selectivity for BRD2 and BRD3.32 In the FP
assay, a similar trend was observed where RVX-208 is 3-17
times selective for BD2 over BD1 depending on the BRD
protein, with highest BD2 selectivity in BRD3.
1
2
3
4
5
6
7
Funding
Financial support for this project is gratefully acknowledged from
the NICHD: HHSN27520130001 and 1 U54 HD09354, and NSF-
CAREER Award CHE-1352091.
ACKNOWLEDGMENT
8
9
We thank Drs. Shameem Syeda and Sudhakar Jakkaraj for
providing (-)-JQ1 and (+)-JQ1 and Jiewei Jiang for providing
compound 2098312-12-8 for this study. We thank Brian Smith for
providing the BRD4-T plasmid construct. We also thank Dr.
Matthew Cuellar for performing LCMS purity analysis on both
synthesized and commercially obtained compounds.
Several compounds, including SG3-179, ABBV-075, (+)-
JQ1, TG101209, MS436, and RVX-208 inhibited the binding
of the pan-BET probe 16 to the BRDT-T and BRD4-T
proteins with IC50 values close to their respective BD1 and
BD2 domains, as expected assuming no effect of the 123 aa
linker on BRD affinity. In contrast, CAS 2098312-12-8,
bromosporine, and BI2536 had considerably lower IC50 values
for the tandem BRDT and BRD4 BRDs compared to the
individual BD1 and BD2 domains, up to > 80-fold higher
potency for BRD4-T over BRD4-2 in the case of
bromosporine. These latter inhibitors apparently bind to BRDs
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ABBREVIATIONS
BD1, binding domain 1; BD2, binding domain 2; BET,
bromodomain and extra-terminal domain; BRD, bromdomain;
BRD2, bromodomain containing protein 2; BRD3, bromodomain
containing protein 3; BRD4, bromodomain containing protein 4;
BRDT, testes specific bromodomain containing protein; FP,
fluorescence polarization; KAc, acetylated lysine.
in the tandem protein in
a cooperative manner or,
alternatively, the amino acid linker in the tandem proteins
induces conformational changes in the BD1 and/or BD2
domains that increase the affinity of these inhibitors, as
observed for probe 16 at BRDT-T.
REFERENCES
1. Kouzarides, T. Chromatin modifications and their function.
Cell. 2007, 128, 693-705.
2. Zeng, L. and Zhou, M.-M. Bromdomain: an acetyl-lysine
binding domain. FEBS Letters. 2002, 513, 124-128.
In conclusion, the novel pan-BET probe 16 has high affinity
for BD1, BD2 and tandem constructs of BET family members
and provides a high FP signal window sufficiently robust for
HTS (Z’= 0.78, Figure 4). It can be used to establish the SAR
for inhibitor series, determine the selectivity of inhibitors for
BET family members and their respective BD1 and BD2 KAc
recognition sites, and for HTS to identify novel BET BRD
inhibitor scaffolds.
3. Muller, S.; Filippakopoulos, P.; Knapp, S. Bromodomains as
therapeutic targets. Expert Rev. Mol. Med. 2011, 13, e29.
4. Jones, M. H.; Numata, M.; Shimane, M. Identification and
characterization of BRDT: a testis-specific gene related to the
bromodomain genes RING3 and Drosophila fsh. Genomics.
1997, 45, 529-534.
5. Shang, E.; Nickerson, H. D.; Duancheng, W.; Wang, X.;
Wolgemuth, D. J. The first bromodomain of Brdt, a testis-
specific member of the BET sub-family of double-
bromodomain-containing proteins, is essential for male germ
cell differentiation. Development 2007, 134, 3507-3515.
6. Miao, Z.; Guan, X.; Jiang, J.; Georg, G. I. BRDT inhibitors
for male contraceptive drug discovery: current status. In
Targeting Protein-Protein Interactions by Small Molecules,
Chunquan Sheng and Gunda I. Georg, Eds.; Springer Nature
Singapore Pte ltd.: Singapore, 2018. pp 287-315.
7. Shadrick, W. R.; Slavish, P. J.,; Chai, S. C.; Waddell, B.;
Connelly, M.; Low, J. A.; Tallant, C.; Young, B. M.;
Bharatham, N.; Knapp, S.; Boyd, V. A.; Morfouace, M.;
Roussel, M. F.; Chen, T.; Lee, R. E.; Guy, R. K.; Shelat, A.
A.; Potter, P. M. Exploiting a water network to achieve-
enthalpy-driven, bromodomain-selective BET inhibitors.
Bioorg. Med. Chem. 2018, 26, 25-36.
8. Urick, A. K.; Hawk, L. M. L.; Cassel, M. K.; Mishra, N. K.;
Liu, S.; Adhikari, N.; Zhang, W.; dos Santos, C. O.; Hall, J.
L.; Pomerantz, W. C. K. Dual screening of BPTF and Brd4
using protein-observed fluorine NMR uncovers new
bromodomain probe molecules. ACS Chem. Biol. 2015, 10,
2246-2256.
ASSOCIATED CONTENT
Supporting Information
Materials and methods; UV absorbance and fluorescence spectra
of probes 16-18; saturation binding curves and a table of KD
values of probes 16-18 for BRDT-1; saturation binding curves
with probe 16 for BRDT-2, BRDT-T, BRD2-1, BRD2-2, BRD3-
1, BRD3-2, BRD4-2, and BRD4-T; competitive displacement of
probe 16 by 10 representative BET inhibitors against BRDT-T,
BRD4-1, BRD4-2, BRD4-T, BRD3-1, BRD3-2, BRD2-1, and
BRD2-2; references; NMR spectra. The Supporting Information is
available free of charge on the ACS Publications website at DOI:
XXX.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: hawkinso@umn.edu
*E-mail: georg@umn.edu
ORCID
Carolyn N. Paulson: 0000-0001-8564-7345
Xianghong Guan: 0000-0002-7874-8065
Alex M. Ayoub: 0000-0003-0341-8747
Alice Chan: 0000-0001-6913-6764
Rezual M. Karim: 0000-0002-0424-127X
William C. K. Pomerantz: 0000-0002-0163-4078
Ernst Schönbrunn: 0000-0002-3589-3510
Gunda l. Georg: 0000-0002-8900-9460
Jon E. Hawkinson: 0000-0001-5461-7071
9. Ayoub, A. M.; Hawk, L. M. L.; Herzig, R. J.; Jiang, J.;
Wisniewski, A.J.; Gee, C. T.; Zhao, P.; Zhu, J.-Y.; Berndt, N.;
Offei-Addo, N. K.; Scott, T. G.; Qi, J.; Bradner, J. E.; Ward,
T. R.; Schönbrunn, E.; Georg, G. I.; Pomerantz, W. C. K.
BET Bromodomain inhibitors with one-step synthesis
discovered from virtual screen. J. Med. Chem. 2017, 60,
4805-4817.
10. Chung, C.; Coste, H.; White, J. H.; Mirguet, O.; Wilde, J.;
Gosmini, R. L.; Delves, C.; Magny, S. M.; Woodward, R.;
Hughes, S. A.; Boursier, E. V.; Flynn, H.; Bouillot, A. M.;
Bamborough, P.; Brusq, J.-M. G.; Gellibert, F. J.; Jones, E. J.;
Author Contributions
*
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
Riou, A. M.; Homes, P.; Martin, S. L.; Uings, I. J.; Toum, J.;
23. Miller, T. C.; Simon, B.; Rybin, V.; Grötsch, H.; Crtet, S.;
Khochbin, S.; Carlomagno, T.; Müller C. W.
A bromodomain-DNA interaction facilitates acetylation-
dependent bivalent nucleosome recognition by the BET
protein BRDT. Nat. Comm. 2016, 7, 13855.
24. Nikolovska-Coleska, Z.; Wang, R.; Pan, H.; Tomita, Y.; Li,
P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.;
Wang, S. Development and optimization of a binding assay
for the XIAP BIR3 domain using fluorescence polarization.
Anal. Biochem. 2004, 332, 261-273.
25. Bui, M. H.; Lin, X.; Albert, D. H.; Li, L.; Lam, L. T.; Faivre,
E. J.; Warder, S. E.; Huang, X.; Wilcox, D.; Donawho, C.
K.; Sheppard, G. S.; Wang, L.; Fidanze, S.; Pratt, J.K.; Liu,
D.; Hasvold, L.; Uziel, T.; Lu, X.; Kohlhapp, F.; Fang,
G.; Elmore, S. W.; Rosenberg, S. H.; McDaniel, K. F.; Kati,
W. M.; Shen, Y. Preclinical characterization of BET family
bromodomain inhibitor ABBV-075 suggests combination
therapeutic strategies. Cancer Res. 2017, 77, 2976-2989.
26. Ember, S. W. J.; Zhu, J.-Y.; Olesen, S. H.; Martin, M. P.;
Becker, A.; Berndt, N.; Georg, G. I.; Schönbrunn, E. Acetyl-
lysine binding site of bromodomain-containing protein 4
(BRD4) interacts with diverse kinase inhibitors. ACS Chem.
Biol. 2014, 9, 1160-1171.
27. Picaud, S.; Leonards, K.; Lambert, J.-P.; Dovey, O.; Wells,
C.; Fedorov, O.; Monteiro, O.; Fujisawa, T.; Wang, C.-Y.;
Lingard, H.; Tallant, C.; Nikbin, N.; Guetzoyan, L.; Ingham,
R.; Ley, S. V.; Brennan, P.; Muller, S.; Samsonova, A.;
Gingras, A.-C.; Schwaller, J.; Vassiliou, G.; Knapp, S.;
Filippakopoulos, P. Promiscuous targeting of bromodomains
by bromosporine identifies BET proteins as master regulators
of primary transcription response in leukemia. Sci. Adv. 2016,
2, e1600760.
Clément, C. A.; Boullay, A.-B.; Grimley, R. L.; Blandel, F.
M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.; Nicodeme, E.
Discovery and characterization of small molecule inhibitors
of the BET family bromodomains. J. Med. Chem. 2011, 54,
3827-3838.
1
2
3
4
5
6
7
8
11. Zhao, O.; Cao, D.; Chen, T.; Wang, Y.; Miao, Z.; Xu, Y.;
Chen, W.; Wang, X.; Li, Y.; Du, Z.; Xiong, B.; Li, J.; Xu, C.;
Zhang, N.; He, J.; Shen, J. Fragment-based drug discovery of
2-thiazolidinones as inhibitors of the histone reader BRD4
bromodomain. J. Med. Chem. 2013, 56, 3833-3851.
12. Zhao, L.; Wang, Y.; Cao, D.; Chen, T.; Wang, Q.; Li, Y.; Xu,
Y.; Zhang, N.; Wang, X.; Chen, D.; Chen, L.; Chen, Y. L.;
Xia, G.; Shi, Z.; Liu, Y. C.; Lin, Y.; Miao, Z.; Shen, J.;
Xiong, B. Fragment-based drug discovery of 2-
thiazolidinones as BRD4 inhibitors: 2. Structure-based
optimization. J. Med. Chem. 2015, 58, 1281-1297.
13. Yu, J.-I.: Chen, T.-T.; Zhou, C.; Lian, F.-L.; Tang, X.-L.;
Wen, Y.; Shen, J.-K.; Xu, Y.-C.; Xiong, B.; Zhang, N.-X.
NMR-base platform for fragment-based lead discovery used
in screening BRD4-targeted compounds. Acta Pharmacol.
Sin. 2016, 37, 984-993.
14. Ember, S. W.; Lambert, Q. T.; Berndt, N.; Gunawan, S.;
Ayaz, M.; Tauro, M.; Zhu, J. Y.; Cranfill, P. J.; Greninger, P.;
Lynch, C. C.; Benes, C. H.; Lawrence, H. R.; Reuther, G. W.,
Lawrence, N. J.; Schönbrunn, E. Potent dual BET bromodo
main-kinase inhibitors as value-added multitargeted chemical
probes and cancer therapeutics. Mol. Cancer Ther. 2017, 16,
1054-1067.
15. de Rezende, L. C. D.; Vaidergorn, M. M.; Moraes, J. C. B.;
Emery, F. D. Synthesis, photophysical properties and
solvatochromism of meso-substituted tetramethyl BODIPY
dyes. J. Fluoresc. 2014, 24, 257-266.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28. Philpott, M.; Yang, J.; Tumber, T.; Fedorov, O.; Uttarkar, S.;
Filippakopoulos, P.; Picaud, S.; Keates, T.; Felletar, I.; Ciulli,
A.; Knapp, S.; Heightman, T. D. Bromodomain-peptide
displacement assays for interactome mapping and inhibitor
discovery. Mol. BioSyst. 2011, 7, 2899–2908.
16. Thivierge, C.; Bandichhor, R.; Burgess, K. Spectral
dispersion and water solubilization of BODIPY dyes via
palladium-catalyzed C-H functionalization. Org. Lett. 2007,
9, 2135-2138.
17. Wu, W. H.; Cui, X. N.; Zhao, J. Z., Hetero Bodipy-dimers as
heavy atom-free triplet photosensitizers showing a long-lived
triplet excited state for triplet-triplet annihilation
upconversion. Sci. Rev. Chem. Commun. 2013, 49, 9009-
9011.
18. Zhu, S. L.; Bi, J. H.; Vegesna, G.; Zhang, J. T.; Luo, F. T.;
Valenzano, L.; Liu, H. Y., Functionalization of BODIPY dyes
at 2,6-positions through formyl groups. RSC Advances. 2013,
3, 4793-4800.
29. Li, J.; Wang, P.; Zhou, B.; Shi, J.; Liu, J.; Li, X.; Fan, L.;
Zheng, Y.; Ouyang, L. Development of 4,5-dihydro-
benzodiazepinone derivatives as a new chemical series of
BRD4 inhibitors. Eur. J. Med. Chem. 2016, 121, 294-299.
30. Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W.
B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.;
Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang,
Y.; Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.;
Heightman, T. D.; Thangue, N. L.; French, C. A.; Wiest, O.;
Kung, A. L.; Knapp, S.; Bradner, J. E. Selective inhibition of
BET bromodomains. Nature 2010, 468, 1067-1073.
19. Jameson,
D.
M.;
Ross,
J.
A.,
Fluorescence
polarization/anisotropy in diagnostics and imaging. Chem.
Rev. 2010, 110, 2685-2708.
20. Schönbrunn, E.; Lawrence, N. J.; Lawrence, H. R.
Preparation of substituted pyrimidinediamines as potent dual
BRD4-kinase inhibitors useful as cancer therapeutics. PCT
Int. Appl. 2016, WO 2016022460 A1 20160211.
31. Zhang,
G.; Plotnikov,
A.
N.; Rusinova,
E.; Shen,
T.; Morohashi, K.; Joshua, J.; Zeng, L.; Mujtaba, S.;
Ohlmeyer, M.; Zhou, M.-M. Structure-guided design of
potent diazobenzene inhibitors for the BET bromodomains. J.
Med. Chem. 2013, 56, 9251-9264.
21. Schönbrunn, E.; Lawrence, N. J.; Lawrence, H. R. 2,4-
Diaminopyrimidines as BRD4 and kinase inhibitors as cancer
32. Picaud, S.; Wells, C.; Felletar, I.; Brotherton, D.; Martin,
S.; Savitsky, P.; Diez-Dacal, B.; Philpott, M.; Bountra,
therapeutics
and
their
preparation.
PCT
Int.
C.; Lingard,
H.; Fedorov,
O.; Müller,
S.; Brennan,
Appl. 2017, WO 2017066428 A1 20170420
P.E.; Knapp, S.; Filippakopoulos, P. RVX-208, an inhibitor of
BET transcriptional regulators with selectivity for the second
bromodomain. Proc. Nat. Acad. Sci. U. S. A. 2013, 110,
19754-19759.
22. Rousseau, T.; Cravino, A.; Bura, T.; Ulrich, G.; Ziessel, R.;
Roncali, J. BODIPY derivatives as donor materials for bulk
heterojunction solar cells. Chem. Commun. 2009, 1673-1675.
*
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
For Table of Contents Use Only
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*
ACS Paragon Plus Environment