Discovery of Benzo[cd]indol-2(1H)-ones as Potent and Specific BET Bromodomain Inhibitors: Structure-Based Virtual Screening, Optimization, and Biological Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.5b01511

The discovery of inhibitors of bromodomain and extra terminal domain (BET) has achieved great progress, and at least seven inhibitors have progressed into clinical trials for the treatment of cancer or inflammatory diseases. Here, we describe the identification, optimization, and evaluation of benzo[cd]indol-2(1H)-one containing compounds as a new class of BET bromodomain inhibitors, starting from structure-based virtual screening (SBVS). Through structure-based optimization, potent compounds were obtained with significantly improved activity. The two most potent compounds bind to the BRD4 bromodomain, with K_d values of 124 and 137 nM. Selected compounds exhibited high selectivity over other non-BET subfamily members. Notably, compound 85 demonstrated a reasonable antiproliferation effect on MV4;11 leukemia cells and exhibited a good pharmacokinetic profile with high oral bioavailability (75.8%) and moderate half-life (T_1/2 = 3.95 h). The resulting lead molecule 85 represents a new, potent, and selective class of BET bromodomain inhibitors for the development of therapeutics to treat cancer and inflammatory diseases.

Full text of DOI:10.1021/acs.jmedchem.5b01511

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Article
Discovery of Benzo[cd]indol-2(1H)-ones as Potent and
Specific BET Bromodomain Inhibitors: Structure-Based
Virtual Screening, Optimization and Biological Evaluation
Xiaoqian Xue, Yan Zhang, Zhaoxuan Liu, Ming Song, Yanli Xing, Qiuping
Xiang, Zhen Wang, Zheng-Chao Tu, Yulai Zhou, Ke Ding, and Yong Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01511 • Publication Date (Web): 05 Jan 2016
Downloaded from http://pubs.acs.org on January 9, 2016
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Discovery of Benzo[cd]indol-2(1H)-ones as Potent and Specific BET Bromodomain
Inhibitors: Structure-Based Virtual Screening, Optimization and Biological
Evaluation
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Xiaoqian Xue,†,‡,# Yan Zhang,†, # Zhaoxuan Liu,†,§,# Ming Song,† Yanli Xing,†,§
Qiuping Xiang,†,‡ Zhen Wang,† Zhengchao Tu,† Yulai Zhou,§ Ke Ding,† and Yong
Xu†,*
†Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, No.190 Kaiyuan Avenue, Guangzhou Science Park,
Guangzhou, Guangdong 510530, China
‡University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049,
China.
§Department of Bioengineering School of Pharmaceutical Sciences, Jilin University,
No.1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
*Corresponding Author: Yong Xu, PhD, E-mail: xu_yong@gibh.ac.cn
#These authors contributed equally to this work.
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ABSTRACT
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The discovery of inhibitors of bromodomain and extra terminal domain (BET) has
achieved great progress, and at least seven inhibitors have progressed into clinical
trials for the treatment of cancer or inflammatory diseases. Here, we describe the
identification, optimization and evaluation of benzo[cd]indol-2(1H)-one containing
compounds as a new class of BET bromodomain inhibitors, starting from
structure-based virtual screening (SBVS). Through structure-based optimization,
potent compounds were obtained with significantly improved activity. The two most
potent compounds bind to the BRD4 bromodomain with Kd values of 124 and 137 nM.
Selected compounds exhibited high selectivity over other non-BET subfamily
members. Notably, compound 85 demonstrated a reasonable anti-proliferation effect
on MV4;11 leukemia cells and exhibited a good pharmacokinetic profile with high
oral bioavailability (75.8%) and moderate half-life (T1/2 = 3.95 h). The resulting lead
molecule 85 represents a new, potent, and selective class of BET bromodomain
inhibitors for the development of therapeutics to treat cancer and inflammatory
diseases.
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1. INTRODUCTION
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Acetylation of lysine residues is a post-translational modification that plays a key role
in the regulation of the chromatin structure and transcription.1,2 These changes in
histone and their effect on gene expression are regulated by three categories of
regulatory proteins, “writers”, “erasers”, and “readers”. Histone acetyltransferases
(HATs) act as a writer to add an acetyl group to form an acetylated lysine, whereas
histone deacetylases (HDACs) act as an eraser to remove the acetyl group from
acetylated lysine. The bromodomain (BRD) family of proteins recognizes and binds
to the acetylated lysine acting as a reader of lysine acetylation state.3,4
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The bromodomain was named after it was first identified in the Brahma gene from
Drosophila Melanogaster as a protein domain containing approximately 110 amino
acids.5 There are 61 human bromodomains that are found within 46 proteins in the
human genome. These bromodomain-containing proteins can be divided into 8
diverse bromodomain groups.2,4 Structural studies have revealed that bromodomains
have a conserved structural fold which consists of a left-handed four-helix bundle and
two interspersed ZA and BC loops which constitute the active acetyl lysine-binding
pocket (Figure S1).2,6,7 The site contains an evolutionarily conserved asparagine side
chain that acts as a hydrogen bond donor to the acetylated lysine side chain. A second
interaction occurs between the acetyl carbonyl oxygen atom and the phenol of a
conserved tyrosine via a structured water molecule. Despite this conserved overall
structure, different bromodomains recognize distinct acetylated lysines in different
proteins because the specific amino acid residues within the loops of each
bromodomain are critical for determining the acetyl lysine-binding specificity. The
architecture of their acetylated lysine binding pocket makes them attractive targets for
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the development of potent and specific inhibitors. Bromodomain-containing proteins
have been implicated in the development of diverse diseases such as cancer and
inflammation.8-15
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Recently, a number of small-molecule compounds with potent inhibitory activity
against bromodomain family proteins were reported in the literature.16-21 Known
bromodomain inhibitors mainly target the BET subfamily, including BRD2, BRD3,
BRD4 and BRDT. The first potent BET inhibitors reported were the diazepines 1
((+)-JQ1) and 2 (I-BET762) (Figure 1).8,22 1 was obtained after the simple
modification of the BRD4 inhibitors patented by Mitsubishi Pharmaceuticals, and 2 as
reported by Nicodeme et al., was derived from medicinal chemistry optimization of a
hit derived from a phenotypic screen designed to identify small molecules able to
enhance ApoA1 expression. 2 has entered clinical trials for NUT midline carcinoma.
The quinolinone-base compound 3 (RVX-208, Figure 1), which was initially
described as a compound that upregulated ApoA1 expression and increased the
high-density lipoprotein mass in vitro and in vivo, has recently been reported to be a
BET inhibitor.15 GSK has also reported the isoxazoloquinoline derivative 4
(I-BET151, Figure 1) as a BET inhibitor and has demonstrated efficacy in studies of
MLL-fusion leukemia.23,24 The dihydroquinazolinone 5 (PFI-1, Figure 1) was recently
reported to be a BET chemical probe derived from the optimization of a
fragment-screening hit.13,25 The compound exhibits modest activity against cell lines
carrying oncogenic rearrangements in the MLL locus, although its solubility and
pharmacokinetics are suboptimal.
Fragment-based screening methods have been extensively used to identify BET
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inhibitors for different chemotypes. Most of the compounds reported in these studies
are generally weak inhibitors but represent promising starting points for further
medicinal chemistry optimization. Chung et al. identified a weak fragment inhibitor
and obtained a potent and selective sulfonamide inhibitor 6 (Figure 1) with an IC50 of
500 nM through extensive medicinal chemistry optimization.26,27 Zhao et al. found
that thiazolidinone could mimic an acetylated lysine moiety and bind to BRD4 protein.
Further optimization led to a potent BRD4 inhibitor 7 (Figure 1) with an IC50 of 140
nM and favorable metabolic stability.28,29 Gehling et al. at Constellation
Pharmaceuticals reported isoxazole-based BET bromodomain inhibitor developed
using hits from a fragment screening. Further optimization led to its carboxamide
derivative with an IC50 value of 26 nM against BRD4(1) in the AlphaScreen assay.30
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Rapid progress in the development of bromodomain ligands has stimulated extensive
interest and has led to at least seven BET bromodomain inhibitors reaching clinical
trials.31,32 However, the chemotypes are still limited. The discovery of new potent and
specific BET inhibitors with improved pharmacokinetics and physicochemical
properties will greatly enhance our understanding of the therapeutic potential of
bromodomain inhibition for various human diseases.
In the present work, we report a structure-based virtual screening combined with free
energy evaluation, medicinal chemistry optimization, and biological evaluation of a
new class of potent, specific and bioavailable small-molecular BET inhibitors, the
benzo[cd]indol-2(1H)-ones.
2. RESULTS AND DISCUSSION
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2.1. Identification and Validation of Benzo[cd]indol-2(1H)-one Scaffold by
Structure-Based Virtual Screening.
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Virtual screening is a powerful tool for novel scaffold discovery. In this study, a
common virtual screening strategy was used as shown in Figure 2. Our in-house
library with approximately 10,000 compounds was screened through molecular
docking and binding free energy evaluation. After cluster analysis and visual
inspection, 15 representative compounds were selected for biological assays (Figure
S2). Using a thermal shift assay, 7 of the 15 compounds demonstrated a stabilized
effect for BRD4(1) protein with a temperature shift of more than 1.0 °C (Table S1).
The AlphaScreen assay further demonstrated that 2 of the compounds (8 (S32),
N-(4-(1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)phenyl)acetamide
(S35)) showed low micromolar level activity with IC50 values of 6.93 μM and 4.70
μM, respectively. The corresponding ligand efficiencies were approximately 0.25.
These two compounds bearing a benzo[cd]indol-2(1H)-one scaffold were previously
used as nuclear receptor inhibitors and plant hormone receptor agonists in our
previous studies.33,34
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To design more potent derivatives, we compared the binding mode of 1 and 8 bound
to BRD4(1) (Figure 1, Figure 3A and 3B). The binding mode predicted by molecular
docking study demonstrated that compound 8 binds snugly in the acetyl lysine
binding site. The carbonyl oxygen atom of the amide group interacts with the
conserved residues Asn140 and Tyr97 through direct and indirect hydrogen bonds via
a water molecule, respectively. The ethyl group attached to the amide group mimicked
the terminal methyl group of the acetyl lysine and occupied the small pocket
surrounded by residues Pro82, Phe83, Val87 and Ile146. The planar
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benzo[cd]indol-2(1H)-one scaffold binds to and forms extensive hydrophobic
interactions with the deep and narrow acetyl lysine binding pocket. N-substitution of
the sulfonamide group occupied the hydrophobic WPF shelf through the sulfonamide
bend angle. The sulfonamide group at the 3-position of N-benzyl sulfonamide forms
one more hydrogen bond with residue Asp145. To investigate the complex stability,
molecular dynamic simulations were performed. The results demonstrated that the
8-BRD4(1) complex is very stable with a RMSD values of approximately 2 Å for all
protein atoms and approximately 1 Å for the ligand during a 20 ns simulation time
(Figure S3). These important interactions mentioned above were maintained during
the simulation, which validated that the predicted binding mode is rational and can be
used for hit optimization. Therefore, we are confident that we can design and optimize
the ligand based on the predicted mode.
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To develop compounds with improved affinity for BRD4(1), we focused on two
regions to perform an extensive SAR study to enhance the protein-ligand interactions.
The first region is the hydrophobic WPF shelf, and the second region is the small
binding portion of acetyl lysine tail.
2.2 Chemistry.
The sulfonamide derivatives of benzo[cd]indol-2(1H)-one were designed and
synthesized as shown in Scheme 1. The commercially available
benzo[de]isochromene-1,3-dione (87) reacted with hydroxylamine hydrochloride and
PTSA in the presence of pyridine to produce compound 88, which was then reacted
with NaOH followed by HCl treatment to produce benzo[cd]indol-2(1H)-one (89).
Alkyl substitution products (90a-d) were obtained by addition of iodoalkane or
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bromoalkane. The sulfonyl chloride substitution products (91a-e) were obtained
through chlorosulfonic acid. Treatment of different amine compounds in the presence
of organic bases gave the desired sulfonamide products 9-13, 17-30, 32-35, and
37-55.
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The reversed sulfonamide derivatives of benzo[cd]indol-2(1H)-one were designed and
synthesized as shown in Scheme 2. 1-ethylbenzo[cd]indol-2(1H)-one (90a) was
reacted with nitric acid and acetic acid to produce nitro-substitution product (92).
Nitro reduction was achieved through iron and ammonium chloride. The reversed
sulfonamide products 56-80, 82, and 84-86 were prepared from
6-amino-1-ethylbenzo[cd] indol-2(1H)-one and commercially available sulfonyl
chlorides.
Compounds 14, 36, 81, 83 were synthesized by simple hydrolysis reaction (Scheme
S1). Compound 31 was synthesized by addition of trifluoroacetic acid (TFA) to
remove the t-butyloxycarbonyl group of compound 39 (Scheme S2) (for details see
supporting information).
Scheme 1. Synthesis of benzo[cd]indol-2(1H)-one derivatives with a sulfonamide
linker.
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Reagents and conditions: (1a) NH4OH·HCl, pyridine, PTSA, 95 °C, 1 h; (1b) (i) 2.7
mol/L NaOH, EtOH, H2O, reflux, 3 h; (ii) HCl, rt, 30 min; (1c) K2CO3, iodoalkane or
bromoalkane, DMF, 50°C, overnight; (1d) HSO3Cl, CHCl3, 0 °C to 50 °C, 6.0 h; (1e)
R2-NH2, pyridine, 80 °C, 1 h or R2-NH2, DIPEA, DCM, rt, 3 h.
Scheme 2. Synthesis of benzo[cd]indol-2(1H)-one derivatives with a reversed
sulfonamide linker.
Reagents and conditions: (2a) HNO3, acetic acid, 0 °C to 50 °C, 1 h; (2b) Fe, NH4Cl,
acetic acid, H2O, 50 °C, 20 min; (2c) R1SO2Cl, CH2Cl2, pyridine, rt, 2h.
2.3. Structure Activity Relationships of N-Substituent Sulfonamide Derivatives
of Benzo[cd]indol-2(1H)-one.
To find more potent analogues based on the experimentally determined and modeled
complex structures, we designed various substituents on the phenyl group to explore
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the chemical space for affinity improvement (Table 1). We preferentially evaluated
the phenyl group and 4-chlorophenyl group, which are used in many potent molecules,
such as 1. As shown in Figure 3B, the N-substituents sulfonamide of
benzo[cd]indol-2(1H)-one could reach the WPF shelf and form favorable van der
Waals interaction. Compound 9, with a phenyl group, is the most potent one (IC50 =
1.0 μM and ΔTm = 7.5 °C) compared with other compounds that have a substituted
phenyl group. Chloro-substituents at any position (10, 11, or 12) were tolerable and
the corresponding compounds showed weaker activity than 9. For the polar
substituent carboxy group, only the ortho-position compound 14 maintained the
activity with an IC50 value of 1.04 μM and a temperature shift of 7.2 °C in the thermal
shift assay. Compound 15, which bears a meta-position carboxy group showed
moderate activity, whereas compound 16, bears a para-position carboxy group, had no
activity. The biochemical activity results showed that ortho-substituents on the ring
were most favorable, whereas 13, which has an acetyl group at this position, was less
potent, with a 3-fold decrease of activity. For para-position substituents, replacement
of chlorine (10) with fluorine (17) resulted in a 2-fold increase of inhibitory activity,
which suggests that the size of the substituent at the 4-position is important for
compound activity. From the crystal structure of 17-BRD4(1) (Figure 4A, Figure S4),
we can see that the fluorine atom fit very well in the small pocket, whereas 16, with
the larger carboxy substituent, interfered with the protein.
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To further explore SAR, hydrophobic and flexible groups were incorporated into the
molecule (Table 2). These substituents made hydrophobic interactions with residues
Ile146, Trp81, Pro82, Phe83 and Met149 in the WPF pocket entrance. When the
substituents were alkyls, changing the chain from methyl to propyl, led to a
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significant potency improvement. However, further extension of the carbon chain
gradually decreased the activity.
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To understand the structural basis for the SAR, we determined the cocrystal structure
of 19 bound to BRD4(1). As shown in Figure 4B, the amino of sulfonamide has
H-bond interactions with a water molecule. This water molecule, along with other
water molecules, mediated an H-bond network and formed H-bond interactions with
Asp144 and Asp145. The crystal structure also revealed that the ethyl group well
occupied the hydrophobic cavity, and the addition of an extra carbon atom in 20
resulted in greater potency. For comparison, we synthesized compounds containing
polar group in the alkyl (26), which showed a significant loss of inhibition. However,
the incorporation of a boc amino group in the butane chain (25) showed moderate
activity.
When cycloalkyl groups were used to occupy the hydrophobic pocket, a large
increase in inhibition was found. Among them, analogue 28 was one of the best
compounds with strong biochemical activity (ΔTm = 10.5 °C; IC50 = 0.13 μM).
Expanding the size to cycloheptane (29, Figure 4D) resulted in 3.5 fold decreased
activity, which suggests that the optimal length of the substitution is approximately
2-4 heavy atoms. The crystal structure of 28 (Figure 4C) showed its binding mode
similar to 19 (Figure 4B). To confirm the importance of hydrophobic interactions, we
synthesized compounds with polar atoms in the cycloalkyl group (30, 31 and 32). The
results demonstrated that including polar atoms reduced the inhibitory activity. For
example, if the substituent atom is N (31), a loss of inhibition was observed, and the
O analogue (30) was better than the N analogue. Compounds 35 and 36 showed
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moderate activity, which is consistent with analogue 14 and can be explained by the
crystal structure of 36 (Figure 4E). When the carboxy group exposed to solvent was
present, the water-mediated H-bond network was formed and provided less optimal
arrangement (comparing with 28). The water molecules form several H-bond
interactions with the carbonyl group of Asp145 and amino group of Ile146 on the
main chain at the pocket entrance.
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As mentioned above, 25 showed moderate activity. Considering the flexibility of 25,
we further synthesized conformational constraint analogues 37, 38, 39 and 40.
Compounds 37 and 38 exhibited similar potency as 25, whereas 39 and 40 showed a
little decreased activity with IC50 values of 4.10 and 4.84 μM. Overall, the
modification with substituents attached to cycloalkyls near the hydrophobic pocket
did not improve the potency. Thereafter, flexible linkers with 1 to 3 methylenes were
used to attach polar substituents for improved activity (41-47). However, these
compounds exhibited moderate or weak activity. From the above SAR, we can see
that the most potent compounds in this series are 20 and 28.
As shown Figure 4C, in the co-crystal structure of 28 bound to BRD4(1) protein, a
small sub-pocket exists near the ethyl portion of the pyrrolidin-2-one ring. There are
two conserved water molecules in this sub-pocket. Oleg Fedorov et al. attempted to
displace the water molecules with polar group but failed to do so.35 To investigate a
suitable substitution, the preferred butyl and cyclohexyl group was maintained in the
R1 position, and R2 was replaced with hydrogen atoms or short-chain alkyl group
(48-55). The activity demonstrated that ethyl is the best substituent. To rationalize this
result, we superimposed two complexes of BRD4(1) with analogue 28 and acetylated
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lysine (Figure 5A). The ethyl group at the R2 position is the optimal size and fit very
well with the acetyl group of the acetylated lysine. A larger alkyl group may interfere
with residues around this pocket.
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2.4. Structure Activity Relationships of Reversed Sulfonamide Derivatives of
Benzo[cd]indol-2(1H)-one.
The sulfonamide linker provides a suitable angle turn for N-substitution to reach into
the hydrophobic pocket. To explore the chemical space, we also synthesized
compounds with a reversed sulfonamide linker. We combined the reversed
sulfonamide linker with the optimal alkyl and cycloalkyl motifs obtained above,
leading to compounds 56-60. These compounds displayed comparable inhibitory
activity to BRD4(1). We solved the complex structure of 58 bound with BRD4(1).
The structure (Figure 5B) showed that the NH of reversed sulfonamide linker formed
an H-bond interaction with the solvent water. The existing water presented suitable
geometry to enable good contact with two other waters. By comparing the crystal
structures of 58 and 19 bound to BRD4(1) (Figure 5B), we found that the
benzo[cd]indol-2(1H)-one scaffolds were overlapped in the KAc binding pocket. The
turn angle of the reversed sulfonamide linker was larger than that of the sulfonamide
linker, which resulted in the butyl motif of 58 being situated at the same position as
the ethyl motif of 19. The butyl analogue 58 (IC50 = 0.73 μM) showed a slight
increase in potency compared with its ethyl analogue 19 (IC50 = 1.09 μM). By
comparing the trends of activities for alkyl substituent compounds (20 vs 57, 21 vs
58), we concluded that the optimal substituent was approximately 3 heavy atoms for
sulfonamide analogues but 5 heavy atoms for reverse sulfonamide analogues.
Similarly, the cycloalkyl analogue 60 (IC50 = 0.21 μM) exhibited a slight potency
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As shown in Figure 5B, the larger rotation angles of the reversed sulfonamide resulted
in a shift of the substituent (58 vs 19). Based on the structure basis from this analysis,
we next explored the attachment of benzyl groups with various substituents. When the
substituent on the phenyl group was halogen, the compounds displayed moderate
potency. The 4-chloro analogue 64 showed similar activity as that of 10. The SAR of
compounds 61, 62 and 63 were consistent with their analogues 12, 11 and 10.
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Various types of 4-substituent analogues were tested, and decreased BRD4(1) activity
was observed compared with the 2-substituent compounds (63-65). Although no
structural information is available for this series of compounds, we can conclude that
the binding modes of these compounds are similar to that of 17 from molecular
docking study.
As shown in Table 3, when using a benzyl group as the substituent, the flexibility of
the molecule may be detrimental to its potency. We then investigated the effect of
various substitutions directly on the aromatic ring. The results for representative
examples of R3 modifications are summarized in Table 4. The thienyl derivative 66
and phenyl derivative 67 showed high binding affinity compared with the benzyl
derivatives listed in Table 4. A mono-substituent on the phenyl group with halogen
atoms (68, 69, and 70) led to a slight loss of activity compared with 67. This is
consistent with those compounds with a sulfonamide linker listed in Table 1.
Similar to the above results, the unsubstituted phenyl ring (67) maintained the optimal
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activity compared to mono-substitution. However the mono-substituted analogues
with reverse sulfonamide linker were more tolerated than sulfonamide compounds.
When two halogen atoms were added to the phenyl ring at different positions,
improved potency was obtained. When fluorine atoms were presented as
2,4-substitutions, the activities were better than that of other di-halogen substituents.
Among them, 73, which has a 2,4-difluoro-phenyl group exhibited significantly
improved activity, with an IC50 of 0.12 μM. The TSA assay also demonstrated its
effect of stabilizing the BRD4(1) protein with a temperature shift of 9.5 °C. From the
data listed in Table 4, we can see that the steric and electronic properties were both
important for improved activity. When 4-position substitutions were varied from
fluorine (68) to methyl (77), the compound potency was maintained.
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Different sized electron-withdrawing or electron-donating substitutions at 4-position
including methyl, cyano, and nitro (77-79), exhibited only moderate activity with IC50
values approximately 1 to 5 μM. The crystal structure of 71 (Figure 5D) bound to
BRD4(1) indicated that a size restriction exists in this area. Single atom substitution
was most favorable in this direction.
For the sulfonamide derivatives described above, we investigated the effect for
compounds with ortho-position substituents, which may form favorable interaction
with surrounding water molecules. We use a similar strategy and synthesized
compounds 80, 81, 82 and 83. No significant potency improvement was obtained for
these compounds, although a water-bridged H-bond network was formed, as shown in
the crystal structure of 80 bound to BRD4(1) protein (Figure 5E).
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We superimposed the structure of compound 67 with that of the literature-reported
active molecules. Surprisingly, the phenyl motif overlapped very well with that of 5.
This discovery encouraged us to design 2-methoxy compounds. The 2,5 di-substituent
derivative 85 exhibited a similar inhibitory activity as the unsubstituted 67, suggesting
that H-bond interaction with the water is beneficial to maintaining activity.
Replacement of 5-chloro (84) with 5-bromo (85) led to a slightly increased activity,
whereas it reduced the activity compared with that of 5-methoxy substituted 86.
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2.5. Evaluation of Binding Affinity and Cell Growth Inhibition Activity.
To further confirm the binding abilities to BRD4(1), representative compounds were
selected based on TSA and AlphaScreen assays. Isothermal titration calorimetry (ITC)
experiments were performed and the Kd values were determined and are listed in
Table 5. Most of the tested compounds exhibited Kd values in the nanomolar range.
The results suggested that these compounds had good binding activities for BRD4(1),
which were consistent with the data from the AlphaScreen and TSA assays.
Compounds 28 and 85 were identified as the most active compounds with Kd values
of 124 nM and 137 nM, respectively.
These potent BRD4(1) inhibitors were also evaluated for their cellular
anti-proliferation activities (Table 5). We selected sensitive cell lines such as leukemia
MV4;11, HL-60, and human colon cancer HT-29 cells to test the cellular proliferation
inhibition effects. Our compounds exhibited reasonable potency against MV4;11 and
HL-60 cell lines but were not well correlated with the other cell lines, which indicated
that these analogues were more active against leukemia cancers. Most of the tested
compounds revealed more than 60% inhibition at 10 μM in the MV4;11 cell line;
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however, 73 showed only 25% inhibition, and its good protein potency did not
translate into the cellular assay. Overall, considering the data from the above assays,
28 and 85 have good profiles for further evaluation.
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2.6. Evaluation of Bromodomains Selectivity.
To investigate the selectivity profile, representative compounds were tested against 12
bromodomain-containing proteins from different subgroups of the human
bromodomain family by thermal stability shift assay (Figure 6). Most of the tested
compounds showed excellent selectivity for BET bromodomains over other non-BET
bromodomain-containing proteins. The heat map showed that all of the inhibitors
tested showed better binding affinity to BRD4(1) than to the other non-BET
bromodomain-containing proteins. Some compounds displayed different
bromodomain activity profiles. Compounds 14, 60, 68, and 73 displayed moderate
activities for EP300. Compound 68 also exhibited weak potency for BRD9.
Compounds 17 and 67 showed appreciable inhibitory activity against PCAF, with a
temperature shifts of 6.4, and 4.7 °C, respectively, which indicates that these
compounds are promising starting points for developing PCAF inhibitors.
2.7. Assessment of Pharmacokinetic (PK) Properties and Metabolic Stability for
BET Bromodomain Inhibitors 28 and 85.
To assess the potential of this series of BRD4(1) inhibitors in vivo, we conducted
preliminary pharmacokinetic analysis for representative sulfonamide and reversed
sulfonamide derivatives 28 and 85. As listed in Table 6, the results showed that 85 had
favorable pharmacokinetic properties, whereas 28 displayed unfavorable
pharmacokinetic properties in rats. Compound 28 in the oral administration mode
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demonstrated poor drug exposure with an AUC value of 85.6 ± 39.9 μg/L*h and
resulted in lower oral bioavailability (1.7%) In contrast to 28, the reversed
sulfonamide derivative 85 exhibited excellent oral bioavailability (76.8%) and good
oral half-life (3.95 h). The results suggest that 85 may have good in vivo efficacy
when it is orally administered. These two leads were further evaluated for their in
vitro metabolic stability with rat liver microsome (Table 7). The results showed that
28 was not very stable in the rat liver microsome, as shown by T1/2 of 4.3 min and
34.3 min at 1 μM and 10 μM, respectively. However, 85 exhibited encouraging level
of microsomal stability with a T1/2 of 32 min and more than 40 min at 1 μM and 10
μM, respectively (Table 7). These two compounds were also tested in a Caco-2
monolayer assay to evaluate their cellular permeability (Table 8). Both 28 and 85
showed good cellular permeability with apparent permeability coefficients (Papp) over
10x10-6 cm s-1 and high recovery values from both directions. The efflux ratio of 28
and 85 were 1.3 and 2.4, respectively, which indicates that these two compounds are
not a favorable substrate for P-glycoprotein or any active transport system that might
transport it out of the cells. From above data, we conclude that poor oral
bioavailability of compound 28 may come from its poor metabolic stability but not
cell permeability. Overall, compound 85 exhibited favorable PK properties, cell
permeability and metabolic stability.
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3. CONCLUSIONS
In this study, we report the discovery of the benzo[cd]indol-2(1H)-one scaffold as a
new class of BET bromodomain inhibitors starting from structure-based virtual
screening approach, in conjunction with medicinal chemistry optimization and
biological evaluation. Determination of the high-resolution crystal structures provided
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guidance for extensive structure optimization and resulted in high-potency BET
inhibitors. The most promising compound 85 had an IC50 of 410 nM in the
AlphaScreen assay and a Kd value of 137 nM in the ITC assay. The compound also
exhibited reasonable inhibitory activity in the MV4;11 acute leukemia cell line. The
excellent bioavailability, cellular permeability, and half-life time make compound 85 a
good starting point for further optimization. In summary, these data indicate that the
promising compound 85 represents a new class of BET bromodomain inhibitors.
Optimization of this series may ultimately lead to a new class of BET inhibitors for
treating diseases such as cancers.
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4. EXPERIMENTAL SECTION
4.1 Computational Methods.
In the molecular docking study, the crystal structure of BRD4(1) in complex with the
inhibitor 1 (PDB ID: 3MXF.pdb) was used as the reference structure. Protein structure
preparation for docking studies includes water deletion, hydrogen atom addition and
protonation state adjustment. All of the ligand and protein preparation were performed
in Maestro (version 9.4, Schrödinger, LLC, New York, NY, 2013) implemented in the
Schrödinger program (http://www.schrödinger.com). Our in house chemical library
with approximately 10,000 compounds was selected for structure-based virtual
screening. This collection includes compounds with diverse structures from SPECS,
ChemDiv, Lifechamicals and ChemBridge. In this study, molecular docking study
was performed with the Glide program (version 6.1, Schrödinger, LLC, New York,
NY, 2013) using the SP score mode. Using hydrogen bond constraints with Asn140,
220 structures were selected based on the Glide SP docking score. These 220
structures were further assessed with MMGBSA evaluation, cluster analysis and
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visual inspection. At last, 15 representative compounds were kept according to their
predicted binding mode, docking score (lower than -6.5 kcal/mol), and binding free
energy score (lower than -60 kcal/mol). Finally, selected compounds were submitted
for subsequent biological evaluation.
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Molecular dynamics (MD) simulations were conducted by using AMBER 14 program.
The starting coordinates were obtained from docking results. For ligand coordinates,
predicted binding mode of 8-BRD4(1) was used. For protein coordinates, the protein
preparation panel in Schrodinger 2014 Suite was applied to assign the protonation
states and orientations of residues, which was then further processed by using LEaP
module in Amber program. Parameters of compounds were prepared by AM1-bcc
model and the other parameters were assigned from the AMBER GAFF force field
using ANTECHAMBER. Topology and parameter files for the protein, ligand and
complex were generated using the LEaP module in AMBER 14. TIP3PBOX water
molecules were added in cube periodic boxes, which were 10 Å × 10 Å × 10 Å. To
ensure overall neutrality of the system, appropriate Na+ and Cl− were added at
physiological concentration in the box. For each system, energy minimization and
MD simulation were carried out using the GPU version of the PMEMD program in
AMBER 14 program. The MD simulations were performed for up to 20 ns for each
complex system. The coordinates of the complexes were saved every 2 ps, those
snapshots were taken in production run for detailed analysis. Trajectories were
analyzed using the PTRAJ module in Amber 14.
4.2 General Chemistry.
Synthesis of the desired reagents and solvents were obtained from commercial
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suppliers and used without further purification. Flash chromatography was performed
using silica gel (300-400 mesh). All reactions were monitored by TLC, using silica
gel plates with fluorescence F254 and UV light visualization.1H-NMR spectra were
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recorded on a Bruker AV-400 spectrometer at 400 MHz and C-NMR spectra were
recorded on a Bruker AV-500 spectrometer at 500 MHz. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million
(ppm) units relative to internal control (TMS). The low-resolution of ESI-MS was
recorded on an Agilent 1200 HPLC-MSD mass spectrometer. The purity of
compounds was determined to be over 95% by reverse-phase high performance liquid
chromatography (HPLC) analysis. HPLC instrument: Dionex Summit HPLC (column:
Inertsil ODS-SP, 5.0 μm, 4.6 × 250 mm (GL Sciences Inc); detector: UVD170U;
injector: manual injector; pump: P680; detection wavelength: 254 nm; flow rate: 1.0
mL/min.
Procedure 1a of Scheme 1. Benzo[de]isochromene-1,3-dione (87) (6 g, 30 mmol)
and hydroxylamine hydrochloride (2 g, 30 mmol) were combined as a solution in
pyridine (40 mL).The reaction was conducted under reflux for 1 h followed by
cooling to 80 ˚C. To the reaction system mixture was added powdered
p-toluenesulfonyl chloride (11.5 g, 60 mmol). After the addition, the reaction was
performed under reflux for 1 h. After cooling to room temperature, the reaction
mixture was poured into ice water (300 mL) and stirred to precipitate crystals. The
precipitate was filtered and rinsed with additional cool water (100 mL) and saturated
NaHCO3 (100 mL) to give 1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl 4-methyl
benzenesulfonate (88) (8.6 g, 78%) as a yellow solid.
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Procedure 1b of Scheme 1. To a solution of 88 (8.6 g, 23.5 mmol) in ethanol (50 mL)
and water (40 mL) was added an aqueous solution of sodium hydroxide (2.7 mol/L,
30 mL) at room temperature. The mixture was heated to reflux temperature for 3 h
while distilling the ethanol. After the reaction was completed, the reaction mixture
was cooled to 75˚C, concentrated hydrochloric acid was added dropwise, and a yellow
precipitate was formed. Then, the mixture was further cooled. The precipitate was
collected by filtration and washed with water (100 mL×2). The resulting crude
product was purified by silica gel chromatography with dichloromethane to give
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Benzo[cd]indol-2(1H)-one (89) (3.3 g, 83%) as a yellow solid. H NMR (400 MHz,
DMSO) δ 10.75 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 6.8 Hz, 1H), 7.78 (t, J =
7.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 7.2 Hz,
1H).
Procedure 1c of Scheme 1. The product 89 (3.3 g, 20 mmol) and potassium
carbonate (5.4 g, 39 mmol) were dissolved in DMF (50 mL). Ethyl iodide (3.65 g,
23.5 mmol) was added dropwise. The reaction mixture was stirred at 50˚C overnight.
The reaction mixture was extracted with ethyl acetate (150 mL×2). The organic layer
was washed with brine and dried over Na2SO4. The solid was filtered off, and the
filtrate was concentrated under reduced pressure. The resulting crude product was
purified by silica gel chromatography with petroleum ether/ethyl acetate (10/1, v/v) to
yield 1-ethylbenzo[cd]indol-2(1H)-one (90a) (3.1 g, 78%) as a yellow oil. MS (APCI),
m/z for C13H11NO ([M + H]+): Calcd 197.24, found 198.0.
Procedure 1d of Scheme 1. To a solution of 90a (3.23 g, 16.5 mmol) in chloroform
(100 mL) was added batches of chlorosulfonic (5.8 g, 50 mmol) at 0 ˚C for 10 min.
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The reaction mixture was heated at 50 ˚C for 6 h. The mixture was then poured into
ice water and extracted with DCM (150 mL×2). The organic layer was washed with
brine and dried over Na2SO4. The solid was filtered off, and the filtrate was
concentrated under reduced pressure. The resulting crude product was purified by
silica gel chromatography with petroleum ether/ethyl acetate (5/1, v/v) to yield
1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl chloride (91a) (2.9 g, 59%) as a
yellow solid. 1H NMR (400 MHz, DMSO) δ 8.72 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 6.8
Hz, 1H), 7.84 (t, J = 7.2 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 3.89 (q, J = 6.8 Hz, 2H),
1.24 (t, J = 7.2 Hz, 3H).
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Procedure 1e of Scheme 1. A reaction mixture of compound 91a (70 mg, 0.237
mmol) and propan-1-amine (17 mg, 0.284 mmol) in pyridine (4 mL) was stirred at
80˚C for 1 h. Dilute HCl was added, the aqueous layer was extracted with ethyl
acetate (50 mL×3), and the organic layer was washed with water and brine, dried with
Na2SO4 and evaporated. The residue was purified by silica gel chromatography with
petroleum and ether/ethyl acetate (4/1, v/v) to afford 1-ethyl-2-oxo-N-propyl-1,2-
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dihydrobenzo[cd]indole-6-sulfonamide (20) (54 mg, 71%). H NMR (400 MHz,
CDCl3) δ 8.65 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 7.2 Hz, 1H),
7.92 – 7.68 (m, 1H), 6.92 (d, J = 7.6 Hz, 1H), 4.66 (t, J = 6.0 Hz, 1H), 3.98 (q, J = 7.2
Hz, 2H), 2.91 (dd, J = 13.2, 6.8 Hz, 2H), 1.46 (dt, J = 14.4, 7.2 Hz, 2H), 1.39 (t, J =
7.2 Hz, 3H), 0.80 (t, J = 7.2 Hz, 3H). 13C NMR (500 MHz, CDCl3) δ 167.7, 143.9,
132.9, 130.6, 129.5, 128.7, 126.9, 126.1, 125.4, 124.9, 103.0, 45.0, 35.2, 23.0, 14.0,
11.1. MS (APCI), m/z for C16H18N2O3S ([M - H]-): Calcd 318.39, found 317.0. HPLC
analysis: MeOH − H2O (80:20), 4.52 min, 99.78% purity.
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The synthesis of 9-13, 17-19, 21-26, 33, 37-47 can refer to 20. (Details see supporting
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Another procedure 1e of Scheme 1. A reaction mixture of compound 91a (70 mg,
0.237 mmol) and cyclohexanamine (28 mg, 0.284 mmol) in DCM (10 mL) was added
DIPEA (0.5 mL), the reaction mixture stirred at room temperature for 3 h. Added
water, extracted with DCM (50 mL×3), and the organic layer was washed with water
and brine, dried with Na2SO4 and evaporated. The residue was purified by silica gel
chromatography with petroleum and ether/ethyl acetate (3/1, v/v) to afford
N-cyclohexyl-1-ethyl-2-oxo-1, 2-dihydrobenzo [cd]indole-6-sulfonamide (28) (76 mg,
89%) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 8.4 Hz, 1H), 8.20
(d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.84 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 7.6
Hz, 1H), 4.52 (d, J = 7.6 Hz, 1H), 3.99 (q, J = 7.2 Hz, 2H), 3.29 – 2.92 (m, 1H), 1.75
– 1.63 (m, 2H), 1.58 – 1.50 (m, 1H), 1.49 – 1.46 (m, 1H), 1.40 (t, J = 7.2 Hz, 3H),
1.25 – 1.05 (m, 6H).13C NMR (500 MHz, CDCl3) δ 168.0, 144.0, 132.8, 130.7, 130.2
129.7, 127.1, 126.3, 125.5, 125.0, 103.2, 125.9, 53.0, 35.4, 34.3, 25.4, 24.9, 14.2. MS
(APCI), m/z for C19H22N2O3S ([M + H]+): Calcd 358.46, found 359.1. HPLC analysis:
MeOH − H2O (80:20), 5.67 min, 99.20% purity.
The synthesis of 27, 29-30, 32, 34-35, 48-55 can refer to 28. (Details see supporting
information)
Procedure 2a of Scheme 2. To a solution of 1-ethylbenzo[cd]indol-2(1H)-one (90a)
(2 g, 10.1 mmol) in AcOH (20 mL) was added HNO3 (0.64g, 10.1 mmol) at 0 °C then
the reaction mixture was stirred at 50 °C for 1 h. After the reaction was completed, the
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reaction mixture was cooled to rt. The reaction mixture was extracted with ethyl
acetate (150 mL×2). The organic layer was washed with brine and dried over Na2SO4.
The solid was filtered off, and the filtrate was concentrated under reduced pressure.
The resulting crude product was purified by silica gel chromatography with petroleum
ether/ethyl acetate (6/1, v/v) to yield 1-ethyl-6-nitrobenzo[cd]indol-2(1H)-one (92)
(1.8 g, 73%) as a yellow solid. MS (APCI), m/z for C13H10N2O3 ([M + H]+): Calcd
242.23, found 243.1.
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Procedure 2b of Scheme 2. A reaction mixture of Fe power (2.1 g, 37 mmol) and
NH4Cl (0.8 g, 14.9 mmol) in AcOH (20 mL) and water (80 mL) was heated at 50℃
for 5 min. 1-ethyl-6-nitrobenzo[cd]indol-2(1H)-one (1.8 g, 7.43 mmol) was dissolved
in DMF (15 mL) and added to the reaction mixture. After the reaction was completed,
the reaction mixture was cooled to rt. The reaction mixture was extracted with ethyl
acetate (150 mL×2). The organic layer was washed with brine and dried over Na2SO4.
The solid was filtered off, and the filtrate was concentrated under reduced pressure.
The resulting crude product was purified by silica gel chromatography with petroleum
ether/ethyl acetate (3/1, v/v) to yield 6-amino-1-ethylbenzo[cd]indol-2(1H)-one (93)
1
(1.38 g, 87.5%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 7.2 Hz,
1H), 8.00 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 6.73 (d, J = 7.6 Hz, 1H), 6.63
(d, J = 7.6 Hz, 1H), 4.10 (s, 2H), 3.95 (q, J = 7.2 Hz, 2H), 3.95 (t, J = 7.2 Hz, 3H).
Procedure 2c of Scheme 2. To a solution of compound 93 (70 mg, 0.33 mmol) and
cyclohexanesulfonyl chloride (66 mg, 0.36 mmol) in DCM (4 mL) was added
pyridine (0.5 mL).The mixture was stirred at rt for 2 h. The reaction mixture was
extracted with DCM (50 mL×2). The organic layer was washed with brine and dried
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over Na2SO4. The solid was filtered off, and the filtrate was concentrated under
reduced pressure. The resulting crude product was purified by silica gel
chromatography with petroleum ether/ethyl acetate (2/1, v/v) to yield
N-(1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)cyclohexanesulfonamide (60) (80
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mg, 67 %) as a yellow solid. H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.4 Hz, 1H),
8.09 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.00 – 6.72
(m, 2H), 3.96 (q, J = 7.2 Hz, 2H), 3.21 – 2.96 (m, 1H), 2.23 (d, J = 11.2 Hz, 2H), 1.87
(d, J = 7.6 Hz, 2H), 1.65 – 1.61 (m, 4H), 1.37 (t, J = 7.2 Hz, 3H), 1.19 (d, J = 9.2 Hz,
2H).13C NMR (500 MHz, CDCl3) δ 167.8, 138.2, 129.6, 127.5, 127.4, 126.7, 126.1,
125.2, 124.3, 124.2, 105.2, 61.0, 35.3, 26.9, 25.4, 25.3, 14.3. MS (ESI), m/z for
C19H22N2O3S ([M + H]+): Calcd 358.46, found 359.0. HPLC analysis: MeOH − H2O
(80:20), 5.17 min, 99.02% purity.
The synthesis of 56-59, 61-80, 82, 84-86 can refer to 60. (Details see supporting
information)
4.3 Biological Evaluation.
4.3.1 Protein Expression and Purification.
The bromodomains were expressed as a His6-fusion protein with a TEV cleavage site
between His6 and bromodomain using the pET24a expression vector (Novagen).
cDNA encoding bromodomain of human BRD2(1) (residues K77-N194), BRD3(1)
(residues P24-E144), BRD4(1) (residues N44-E168), BRDT(1) (residues N21-E137),
EP300 (residues A1040-G1161), CREBBP (residues R1081-G1197), BRD1 (residues
E556-A688), BRD9 (residues L14-Q134), PCAF (residues G715-D831), ASH1L
(residues E2433-E2564), BAZ2B (residues S1858-S1972) and TAF1(1) (residues
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R1377-D1503) were synthesized by Genscript. BL21 (DE3) cells transformed with
these expression plasmids were grown in LB broth at 25 ºC to an OD600 of
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isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 16 ºC for 16 h. Cells were harvested
by centrifugation (6000g for 15 min at 4 ºC, JLA 81,000 rotor, on a Beckman Coulter
Avanti J-20 XP centrifuge) and were frozen at -80 ºC as pellets for storage. Cells were
resuspended in extract buffer (50 mM HEPES, pH 7.5 at 25 ºC, 500 mM NaCl, 5 mM
imidazole, 5% glycerol and 0.5 mM TCEP (Tris(2-carboxyethyl)phosphine
hydrochloride)) and high-pressure homogenized using an JN3000 PLUS high pressure
homogenizer (JNBIO, Guangzhou, China) at 4 ºC. The lysate was collected on ice and
centrifuged at 12,000g for 40 min. The supernatant was loaded onto a 5-mL
NiSO4-loaded HisTrap HP column (Ni-NTA, GE Healthcare, NJ). The column was
washed with 20 mL extract buffer (50 mM HEPES, pH 7.5 at 25 ºC, 500 mM NaCl,
50 mM imidazole). The protein was eluted with a 50-500 mM imidazole gradient in
elute buffer with (50 mM HEPES, pH 7.5 at 25 ºC, 500 mM NaCl, 500 mM
imidazole). The protein was concentrated and further purified by a gel filtration
column (HiLoad, Superdex 75, 16/60, GE Healthcare). The sample purity of each
fraction was examined by SDS-PAGE and the sample concentration was determined
by Bradford assay. Purified proteins were concentrated and stored in the gel filtration
buffer (10 mM Hepes pH 7.5 at 25 ºC, 150 mM NaCl, 0.5 mM TCEP) and were used
for crystallization or stored at -80 ºC for AlphaScreen, TSA, or ITC assay.
4.3.2 AlphaScreen Assay.
Interactions between bromodomain-containing proteins (BCP) and ligands were
assessed by luminescence-based AlphaScreen technology (Perkin Elmer) as
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previously described in using a histidine detection kit from PerkinElmer (Norwalk,
CT). All of the reactions contained bromodomain-containing protein bound to nickel
acceptor beads (5 μg/mL) and biotinylated acetylated histone H4 peptide bound to
streptavidin donor beads (5 μg/mL) in the presence or absence of the indicated
amounts of control compounds 1, or candidate compounds. The C-terminal
biotinylated tetra-acetylated histone peptide H4 (bH4KAc4) sequence was
H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-Biotin-OH (synthesized by
Genscript). The experiments were conducted with various protein/peptide ratio as
follows for sensitive signal: BRD4(1) : bH4KAc4 = 50 nM : 50 nM; CREBBP :
bH4KAc4 = 200 nM : 50 nM. BRD1 : H4KAc4 = 100 nM : 50 nM; BRD9 : H4KAc4
= 150 nM : 50 nM; BRD2(2) : H4KAc4 = 150 nM : 100 nM; BRD3(2) : H4KAc4 =
100 nM : 50 nM; BRD4(2) : H4KAc4 = 100 nM : 50 nM; ATAD2 : H4KAc4 = 100
nM : 100 nM; BAZ2B : H3K14Ac = 150 nM : 100 nM.
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All reagents were diluted in the buffer (50 mM MOPS, pH 7.4, 50 mM NaF, 50 μM
CHAPS, and 0.1 mg/mL bovine serum albumin) and allowed to equilibrate at room
temperature prior to addition to low-volume 384-well plates (ProxiPlate-384 Plus,
PerkinElmer, USA). Plates were foil sealed to protect from light, incubated at room
temperature for 2 h and read on an EnSpire plate reader (PerkinElmer, USA). When
excited by a laser beam of 680 nm, the donor beam emits singlet oxygen that activates
thioxene derivatives in the acceptor beads, which releases photons of 520–620nm as
the binding signal. All experiments were carried out in triplicate on the same plate.
The results were based on an average of three experiments with standard errors
typically less than 10% of the measurements.
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4.3.3 Thermal Stability Shift Assay (TSA).
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Thermal stability shift assays were carried out using the Bio-Rad CFX96 Real-Time
PCR system. All reactions were buffered in 10 mM HEPES, pH 7.5, 150 mM NaCl at
a final concentration of 10 μM proteins and 200 μM compounds. The 20 μl reaction
mix was added to the wells of 96-well PCR plate. SYPRO Orange (ABI, Sigma) was
added as a fluorescence probe at a dilution of 1:1000 and incubated with compounds
on ice for 30 mins. Total DMSO concentration was restricted to 1% or less. Excitation
and emission filters for the SYPRO Orange dye were set to 465 nm and 590 nm,
respectively. The temperature was raised with a step of 0.3 °C per minute from 30 °C
to 75 °C, and fluorescence readings were taken at each (0.3 °C) interval. All
experiments were performed in triplicates. Melting temperatures (Tm) were calculated
by fitting the sigmoidal melt curve to the Boltzmann equation using GraphPad Prism.
ΔTm is the difference in Tm values calculated for reactions with and without
compounds.
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4.3.4 Isothermal Titration Calorimetry (ITC).
The ITC measurements were carried out using an ITC200 instrument (Microcal, GE
Healthcare). All experiments were performed at 25 °C while stirring at 1,000 rpm in
an ITC buffer (50 mM HEPES, 150 mM NaCl, 0.5 mM TCEP and pH 7.5). All
titrations of bromodomain-containing proteins into ligands were performed using an
initial injection of 0.5 µL followed by 20 identical injections of 2 µL with a duration
of 4 seconds per injection and a spacing of 180 seconds between injections. The stock
solutions of ligands and the bromodomain-containing proteins were diluted with the
ITC buffer to a compound concentration of 40-60 µM and protein concentration of
500-600 µM before titrations. The final concentration of DMSO in the reaction buffer
is less than 0.25% of the total volume. In order to estimate the background of the heat
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