Structure-Based Identification of Inhibitory Fragments Targeting the p300/CBP-Associated Factor Bromodomain

Article abstract of DOI:10.1021/acs.jmedchem.5b01719

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b01719

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Brief Article
Structure-based identification of inhibitory fragments
targeting the p300/CBP-associated factor bromodomain
Apirat Chaikuad, Steffen Lang, Paul E Brennan, Claudia Temperini, Oleg Fedorov, Johan
Hollander, Ruta Nachane, Chris Abell, Susanne Müller, Gregg Siegal, and Stefan Knapp
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01719 • Publication Date (Web): 05 Jan 2016
Downloaded from http://pubs.acs.org on January 12, 2016
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Structure-based identification of inhibitory fragments targeting the
p300/CBP-associated factor bromodomain
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Apirat Chaikuad1, Steffen Lang5, Paul E.Brennan1, Claudia Temperini3, Oleg Fedorov1, Johan Hol-
lander4, Ruta Nachane4, Chris Abell5, Susanne Müller1, Gregg Siegal4, Stefan Knapp1,2.
1 Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, Uni-
versity of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK. 2 Institute for
Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, and Buchmann Institute for
Molecular Life Sciences, D-60438 Frankfurt am Main, Germany. 3 Leiden Institute of Chemistry, Leiden University,
Einsteinweg 55, Leiden, Netherlands. 4 ZoBio, Einsteinweg 55, Leiden, Netherlands, 5 Department of Chemistry,
Lensfield Road, Cambridge CB2 1EW, UK.
ABSTRACT: The P300/CBP-associated factor plays a central role in retroviral infection and cancer development and the
C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-
lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening ap-
proaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures
providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.
Bromodomains are epigenetic readers that specifically
recognize acetyl-lysine (Kac) marks on proteins. Target-
ing this protein interaction module with small molecules
has recently emerged as a potential therapeutic strategy
for the treatment of several diseases including cancer and
inflammation1, 2. To date most inhibitor development
efforts have been focused on the BET family of bromo-
domain proteins for which now several inhibitors entered
clinical testing. Bromodomains have good predicted
druggability3, and selective chemical tool compounds
have been developed even for less attractive binding sites
that possess open or highly charged acetyl-lysine binding
pockets such as BAZ24, 5 and ATAD26. In addition, potent
inhibitors have been developed for highly druggable bro-
modomains present in BRPF 7, 8, CBP9 and BRD910. Inter-
estingly, several recent inhibitor development projects
have highlighted the success of fragment-based ap-
proaches identifying inhibitors in particular for poorly
therapeutic potential as a targeting site for drug develop-
ment.
An important role of the PCAF acetyl-lysine recognition
module has already been demonstrated for the replication
of AIDS viruses. The PCAF bromodomain targets the HIV
TAT protein acetylated at K50, an essential association
that activates HIV-1 transcription and promotes the inte-
grated proviral replication19, 20. Development of PCAF
bromo-domain inhibitors has therefore been proposed as
a potential strategy for the treatment of AIDS19, 20 and this
strategy has been confirmed by early N1-aryl-propane-1,3-
diamine-based compounds that have been shown to pre-
vent PCAF/TAT association21.
This compound class has recently been followed up by
the development of a series of 2-(3-aminopropylamino)
pyridine 1-oxide derivatives that however did not lead to
improvement of the micromolar potency of current PCAF
bromodomain inhibitors22. This prompted us to identify
more diverse chemical starting points using fragment
screening by thermal shifts assays and NMR as well as
structure-based approaches.
druggable bromodomains5, 6, 8, 11, 12
.
P300/CBP-associated factor (PCAF; also known as His-
tone acetyltransferase KAT2B) is a multi-domain protein
that harbors an acetyltransferase (HAT) and E3 ubiquitin
ligase domains as well as a C-terminal bromodomain that
may associate with the HATs p300 and CBP13-15. While the
roles of the acetyltransferase and the E3 ubiquitin ligase
activities have been shown to be required for cell prolifer-
ation and apoptosis14-18, little is known about the regulato-
ry function of the PCAF bromodomain in cellular pro-
cesses. Selective inhibitors, so called chemical probes,
would therefore be interesting reagents to unravel the
functions of the PCAF bromodomain and to assess its
To extend structural knowledge from our previous apo
crystal structure23, we initially determined the crystal
structure of the PCAF-acetyl-lysine complex to provide
insights into acetyl-lysine recognition. The binding mode
of the acetyl-lysine in PCAF strongly resembled that ob-
served in other bromodomains with the Kac carbonyl
forming two canonical hydrogen bonds to a conserved
N803 and a conserved water molecule. This water is part
of a conserved intricate network of structural waters lo-
cated at the bottom of the pocket that bridge interaction
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with the ZA loop Y760 and the backbone carbonyl of E750 never more than 2%. Consistent with earlier predictions,
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(Figure 1a). Despite sharing the conserved interactions,
the Kac binding groove in PCAF was fenced on one side
by the bulky residue Y809, resulting in a tight and re-
stricted pocket (Figure 1b). Large aromatic residues in this
position are not common for human bromodomains,
present in less than a third of proteins in the family23.
we observed a TINS profile indicating that PCAF is highly
ligandable (Figure 2c). Based on the profile, 63 hits were
selected, a 6.6% hit rate. In contrast to TINS, binding of
Kac to immobilized PCAF in the presence of DMSO could
not be detected by SPR. Consequently, only those hits
that were readily soluble in an aqueous buffer were or-
thogonally validated by SPR. The binding of 17 hits could
be fully characterized by SPR and 3 were selected for
structural studies based on novel features (Supplementary
Figure S2 and Table S1). These fragments (9, 10 and 11)
also resulted in substantial Tm shifts (Figure 2).
Another interesting structural feature was the extension
of the binding pocket by a shelf created by the ZA loop
harboring a mixture of hydrophobic and polar surface
residues (Figure 1b). The wide ZA-loop pocket poses a
challenge for inhibitor development. However, recently
the development of Baz2 inhibitors demonstrated that
such large binding pockets can be efficiently occupied for
instance by aromatic ring systems of the BAZ2-ICR probe
that are aligned by intra-molecular aromatic π-stacking4.
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Figure 1. Structure of PCAF-Kac complex (pdb-code
5fe0). a) Detailed interactions between the bound acetyl-
lysine (Kac, shown in yellow stick) and the PCAF bromo-
domain. Water molecules are shown as pink spheres. b)
Electrostatic surface representation reveals a tight central
Kac pocket that is extended by wide cavities lined by the
ZA and BC loop regions. Inset shown │2Fo│-│Fc│ omit-
ted electron density map for the bound Kac.
The low affinities of the previously developed com-
pounds prompted us to search for other Kac mimetics
that would be compatible with the constraint nature of
the central PCAF pocket. We employed different tech-
niques to screen two fragment libraries. In one set of
experiments, we used a thermal stability shift assay to
screen a small fragment set at 1 mM and identified several
fragments that resulted in positive melting temperature
(Tm) shifts in the range of ~1-2 ˚C (Figure 2). These hits
were chemically diverse and contained different potential
Kac mimetic scaffolds. The highest Tm shifts were rec-
orded for 1-methylquinolin-2(1H)-one) (1, BR004), 1H-
indole-5-carboxamide (2, BR005), 2-methylisoindolin-1-
one (3, BR013), 4-methoxybenzo[d]isoxazol-3-amine (4,
MB360), 2,3-dihydrobenzo[b][1,4]dioxine-5-carboxamide
Figure 2. Fragments containing Kac mimetics identified as
potential binders for PCAF bromodomain. Chemical struc-
tures of the identified fragments are shown in a) together
with average Tm shifts (±SEM) using triplicate experimental
data listed in panel b). c) TINS profile. The T/R ratio of each
compound screened was calculated as a height weighted
average of the ratio of the peak amplitude of each NMR reso-
nance in the presence of PCAF over that in the presence of
the reference protein. The T/R ratios were then binned and
the frequency is plotted above. The asymmetry and tailing to
the left (i.e. a large number of compounds displaying prefer-
ential binding to PCAF) are indicative of high ligandability of
PCAF. The vertical red-dashed line indicates the cutoff for
definition of hits. Data of TINS hits are compiled in Supple-
mentary table 1.
(5,
MB364)
and
(4-(1,2,3-thiadiazol-4-
yl)phenyl)methanamine (7, MB093), most of which com-
prised known Kac mimetic scaffolds. In parallel, we used
Target Immobilized NMR Screening (TINS) to screen a
diverse collection of approximately 950 commercially
available fragments24. We first confirmed that binding of
Kac to immobilized PCAF could be observed in 2% DMSO
(Supplementary Figure S1). Subsequently mixtures of
fragments were screened with a maximum of 4 com-
pounds per mix such that the DMSO concentration was
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As expected from their chemical structure, all of these
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fragments occupied the Kac binding site through groups
that mimicked the hydrogen bond interaction of acetyl-
lysine. Since the co-crystalized compounds were small,
the contacts with the bromodomain were limited only to
the canonical hydrogen bond with N803 and the typical
water-mediated contact with Y760 (Figure 3a). However,
additional interactions were also observed for Fr11, of
which the 1-ethanol decoration was oriented towards the
open ZA cavity and formed both direct and water-
mediated hydrogen bonds to the backbones of the ZA
loop V752 and P751. Superimposition of all structures
revealed that the co-crystallized fragments fit tightly into
the narrow Kac pocket and most fragments formed aro-
matic interactions with Y809 that lines the central acetyl-
lysine binding groove of the PCAF bromomdomain (Fig-
ure 3b). No significant structural alterations were ob-
served when comparing all complexes suggesting that the
PCAF bromodomain contains a rigid acetyl-lysine binding
pocket. Some Kac mimetic groups of the identified frag-
ments were not specific for PCAF and have been previous-
ly shown to bind to other bromodomains, for example 1
also interacts with ATAD211 and the isoxazole 8 with
BRD4 and CREBBP25 with highly conserved binding
modes.
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With the success in identifying the Kac mimetic frag-
ments and obtaining the complex structures, we next
expanded the ligand series to more decorated compounds
choosing two diverse scaffolds (Figure 4a). Two com-
pounds,
CPD1
(12)
(N-(1,4-dimethyl-2-oxo-1,2-
dihydroquinolin-7-yl)acetamide) and CPD2 (13) (N-(1,4-
dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)methanesulfonam),
were derivatives based on fragment hit 1 containing an
additional 4-methyl group and two different decorations
at the 7th position, either with N-linked acetamide or
sulfonamide. A third inhibitor, CPD3 (14) (N-methyl-5-(2-
oxo-2-(phenylamino)ethoxy)-2-((tetrahydro-2H-pyran-4-
yl)oxy)benzamide), contained instead a benzene core har-
boring an acetamide extension, a Kac mimetic group
resembling fragment 1. In addition, this latter compound
was also further decorated by tetrahydropyran and N-
phenylacetamide linked to the benzene ring via an ethoxy
linkage at the ortho and meta positions relative to the
acetamide group, respectively.
Figure 3 Structures of PCAF in complexes with the identi-
fied Kac mimetic fragments. a) Detailed interactions between
the bound fragments (yellow stick) within the PCAF Kac
binding site. The conserved water molecules at the bottom of
the pocket are shown in pink spheres, and an additional
water molecule involving in additional water-mediated inter-
actions observed in the complex with 11 is highlighted by
magenta sphere. b) Superimposition of the bound fragments
and Kac revealed canonical acetyl-lysine mimetic binding
modes and no significant conformational changes upon
inhibitor binding within the pocket. Coordinates have been
deposited under accession codes: 5fe1, 5fe2, 5fe3, 5fe4, 5fe6,
5fe7.
The binding of these three compounds was initially con-
firmed by DSF with 12 and 13 showing a similar degree of
stabilization with ∆Tm shifts of ~2.6-2.9 ˚C at 1 mM con-
centration (Figure 4b). Due to signal interference, 14 was
used at half the concentration of the others, but nonethe-
less produced a similar ∆Tm shift of ~3.3 ˚C. As expected,
the increase of the ∆Tm shift values of these three ligands
suggested stronger binding to PCAF bromodomain. We
used isothermal titration calorimetry (ITC) to determine
binding affinities in solution.
We next attempted to verify the binding modes of the
identified fragments and successfully determined the
complex crystal structures for 7 Kac mimetic fragments.
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Figure 4. Compounds identified as PCAF binders. a) Chemical structures of three in-house available compounds that contain the
fragment-related Kac mimetic scaffolds. b) Average Tm shift values from triplicate experiments for the selected compounds. c)
ITC binding data for the interactions between 13 and 14 with PCAF with the isotherms of raw titration heat shown on the top and
the normalized binding heat with the single-site fitting (red line) shown at the bottom. The thermodynamic parameters includ-
ing equilibrium binding constants are the average from two repeats.
Figure 5 Crystal structures of PCAF in complexes with 12, 13 and 14. Detailed interactions between the bound compounds with
the PCAF bromodomain are shown. The conserved water at the bottom of the pocket is displayed in pink spheres, while an addi-
tional water molecule involving in contact between the 13 and protein is shown in magenta sphere. Insets show omitted electron
density map for the bound ligands. Coordinates have been deposited under accession codes: 5fe8, 5fe9, 5fdz.
In agreement with the DSF results, the measured Kd of 13
was observed to be ~21 µM, which was 3-fold higher than
that of the more decorated 14 (Kd of ~7 µM) (Figure 4c).
The increase in the affinity of the latter was due to signifi-
cantly larger negative binding enthalpy suggesting more
favorable polar interaction of this inhibitor. However, the
ligand efficiency (LE) decreased to 0.24 for 14 compared
to LE of 0.34 for 13. To gain insight into the selectivity of
the most potent fragment hits we performed a tempera-
ture shift screen using a panel of 48 recombinant bromo-
domains (Supplemental figure S3). This selectivity screen
revealed strong interaction of 14 to bromodomains pre-
sent in CECR2 (7.1 C), BRD7 (6.5 C) suggesting that this
fragment can also be optimized targeting other bromo-
domains. 12 and 13 were also screened using developed
Alphascreen assays. At 25 µM both inhibitors showed
strong inhibition for BRD9 (Supplementary Table S2).
This is not surprising since 1-methylquinoline-2-ones have
been developed into highly specific BRD9 inhibitors10.
decorations. All compounds could be unambiguously
positioned based on the well-defined electron density
map (Figure 5). As expected in complexes with 12 and 13,
the 1-methylquinoline-2-one group occupied the Kac
binding groove and formed similar canonical interactions
as observed in the PCAF complex with 1, while the deco-
ration of both compounds oriented towards the ZA pock-
ets. However, no contact between the 12 acetamide and
protein was observed. In contrast the 13 N-linked sulfon-
amide was observed to replace a water molecule that
bridged interactions between E756 and K753 in the Kac
complex (Figure 1a), enabling the formation of two direct
hydrogen bonds to these two residues as well as a water-
mediated contact to the carbonyl backbone of W746
(Figure 5). Interestingly, a sulfonamide at this analogous
position also engaged within the Kac site of BAZ2B in the
GSK2801 inhibitor complex5.
o
o
The binding mode of 14 was slightly different potentially
due to its larger decorations and its different acetyl-lysine
mimetic moiety (Figure 5). The acetamide interacted with
PCAF in a typical Kac mimetic manner with the benzene
The crystal structures of the ligand-PCAF complex al-
lowed us to evaluate the interactions of the introduced
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nary gradient pump, and micro plate sampler (Agilent
core stacking against the Y809 in a similar fashion ob-
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served also for the other fragments. The tetrahydropyran
expanded into the open ZA pocket, while the N-
phenylacetamide extension occupied the BC pocket with
the phenyl ring located above N803 and oriented perpen-
dicularly to Y802 and P804 within a distance for π-
stacking. However, no hydrogen bond contact was
formed between these substitutions and PCAF. Consider-
ing its affinity, such limited interactions were rather sur-
prising when compared to the binding mode of the weak-
er 13, of which the extended group engaged more hydro-
gen bond interactions. We hypothesize therefore that 14
may gain its increased binding potency through a larger
contact surface. Hence, an extension of the Kac-mimetic
fragments resulted in improved binding affinities despite
the lack of direct polar interaction with the protein. In
addition, the large surrounding space adjacent to the ZA
and BC loops provides few spatial constraints, enabling
the binding of diverse chemical moieties, albeit with little
shape complementarity. It is likely that elongated, flexible
and uncharged substituents that could span the interface
as well as engage a few interactions may be beneficial for
increasing ligand potency through targeting the ZA and
BC pockets in the PCAF bromodomain.
220). Separation was performed upon Centurysil C18-AQ
+ 5 μm, 50 mm × 4.6 mm (Johnson). The flow rate of the
mobile phase was kept at 3.5 mL/min. Mobile phases B
and C were acetonitrile with 0.35% CF3CO2H and water
with 0.35% CF3CO2H, respectively. The gradient condi-
tions were as follows: 0–0.5 min 1% B and 99% C, 3.7 min
90% B and 10% C, 5 min 99% B and 1% C. The injection
volume was 10 μL. 12 and 13 were ≥90% pure by HPLC at
254 nm and ≥99% by evaporative light scattering detec-
tion (ELSD). 14 was ≥99% pure by both methods.
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Thermal stability assays. The protein at 2 µM in 10
mM HEPES, pH 7.5 and 250 mM NaCl was mixed with the
fragments/compounds at 1 mM concentration (unless
stated). The assays, data evaluation and melting tempera-
ture (Tm) calculation were performed using a Real-Time
PCR Mx3005p machine (Stratagene) with the protocols
described previously26.
Isothermal Calorimetry. All isothermal calorimetry
(ITC) experiments were carried out on NanoITC (TA
instrument) at 15 ˚C in the buffer containing 25 mM
HEPES, pH 7.5, 150 mM NaCl and 0.5 mM TCEP. Titration
was performed by injecting PCAF (250 µM) into the reac-
tion cell containing the compounds (15-20 µM). The cor-
rected data of the integrated heat of titration were fitted
to an independent single binding site model based on the
manufactor protocol, from which the thermodynamics
parameters (ΔH and TΔS), equilibrium association and
dissociation constants (Ka and KD) and stoichiometry (n)
were calculated. All experiments were repeated twice.
Conclusion
The fragment-based approach coupled with structural
information presented here offers a platform expanding
the knowledge on PCAF Kac-mimetic scaffolds and bind-
ing modes to aid development of potent and selective
PCAF bromodomain inhibitors. The structural models
provide valuable data for the rational design of inhibitors
to the rigid and poorly enclosed PCAF Kac binding pock-
et. Comparison of inhibitor binding modes highlights the
importance of aromatic interactions with Y809 and pro-
vides several diverse acetyl-lysine mimetic chemotypes.
These chemotypes may serve as attractive starting points
for inhibitor development efforts targeting this interest-
ing bromodomain. All fragments that were co-crystallized
formed an acetyl-lysine mimetic hydrogen bond to N803.
Thus, for future development of these fragments into
more potent hits the fragment need to be grown rather
than linked in order to increase potency.
SPR. Recombinant PCAF was biotinylated on the N-
terminal AVI tag in vivo. Purified, biotinylated PCAF was
captured onto a NeutraAvidin-coated CM5 sensor chip
surface in 20 mM sodium phosphate pH7.5, 150 mM NaCl,
2 mM DTT and 0.05% P20 Tween. This buffer was subse-
quently used for all studies. The immobilization level was
10150 RU. All SPR experiments were performed using a
Biacore T200 instrument at 20°C. Fragment hits from
TINS were directly solubilized in the running buffer. Each
fragment was titrated at six different concentrations for
KD determination. An acetyl-Lys 16 peptide derived from
H4 was used to monitor the ligand binding capacity of the
immobilized PCAF bromodomain at regular intervals.
EXPERIMENTAL SECTION
Crystallization and structure determination. Apo
crystals of PCAF (~14-18 mg/mL) were obtained using the
vapor diffusion method at 4 ˚C and either single-PEG-
based (21-35% PEG 3350, 0.1 M Bis-Tris pH 5.5-7.0) or
PEG-smear-based (21-40% medium-molecular-weight
PEG smears buffered either with 0.1 M Bis-Tris pH 6.0-7.5
or 0.1 M Tris pH 7.5-8.8)27 reservoir solutions. To produce
the PCAF-ligand complex crystals, soaking were per-
formed using the mother liquor supplemented with the
fragments/compounds at ~10-20 mM concentration and
20% ethylene glycol. Diffraction data were collected ei-
ther in-house or at Diamond Light Source. Initial struc-
ture solution was achieved using molecular replacement
with Phaser28 from CCP4 suite29 and the published coor-
dinates of PCAF23. Manual model building alternated with
Recombinant protein production. The PCAF bromo-
domain (aa 715-831) containing an N-terminal His6-tag
was recombinantly expressed in E. coli Rosetta. The pro-
tein was initially purified by Ni2+-affinity chromatography,
and subsequently cleaved the tag with TEV protease. The
cleaved protein was further purified using size exclusion
chromatography, and stored in 25 mM HEPES, pH 7.5, 150
mM NaCl and 0.5 mM TCEP.
Identified fragments and compounds. Fragments
were purchased from chemical vendors and were at least
95% pure according vendor specifications. The purity and
correct structure of each compound was validated by
NMR. The purity of the synthesized compounds 12, 13 and
14 was assessed by analytical HPLC using an Agilent 1100
equipped with photodiode array detector (DAD), quater-
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refinement were performed in COOT30 and Refmac31,
respectively. Data collection and refinement statistics are
summarized in Supplementary Table S3.
Hoelder, S. Structure enabled design of BAZ2-ICR, a chemical
probe targeting the bromodomains of BAZ2A and BAZ2B. J Med
Chem 2015, 58, 2553-2559.
1
2
5.
Chen, P.; Chaikuad, A.; Bamborough, P.; Bantscheff,
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4
5
6
7
8
9
M.; Bountra, C.; Chung, C. W.; Fedorov, O.; Grandi, P.; Jung, D.;
Lesniak, R.; Lindon, M.; Muller, S.; Philpott, M.; Prinjha, R.;
Rogers, C.; Selenski, C.; Tallant, C.; Werner, T.; Willson, T. M.;
Knapp, S.; Drewry, D. H. Discovery and Characterization of
GSK2801, a Selective Chemical Probe for the Bromodomains
BAZ2A and BAZ2B. J Med Chem 2015. in press
Supporting Information. Crystallographic data collection
and refinement statistics, compound syntheses, selectivity
screening data. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
6.
Bamborough, P.; Chung, C. W.; Furze, R. C.; Grandi, P.;
Michon, A. M.; Sheppard, R. J.; Barnett, H.; Diallo, H.; Dixon, D.
P.; Douault, C.; Jones, E. J.; Karamshi, B.; Mitchell, D. J.; Prinjha,
R. K.; Rau, C.; Watson, R. J.; Werner, T.; Demont, E. H.
Structure-Based Optimization of Naphthyridones into Potent
ATAD2 Bromodomain Inhibitors. J Med Chem 2015. in press
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* Stefan Knapp, Goethe University Frankfurt, Department of
Pharmaceutical Chemistry and Buchmann Institute for Mo-
lecular Life Sciences, 60438 Frankfurt , Phone: +49 69 798
12799, e-mail: knapp@pharmchem.uni-frankfurt.de
7.
Bennett, J.; Fedorov, O.; Tallant, C.; Monteiro, O.;
Meier, J.; Gamble, V.; Savitsky, P.; Nunez-Alonso, G. A.;
Haendler, B.; Rogers, C.; Brennan, P. E.; Muller, S.; Knapp, S.
Notes
The coordinates and structure factors of all complexes have
been deposited to the protein data bank under accession
codes 5fdz, 5fe0, 5fe1, 5fe2, 5fe3, 5fe4, 5fe5, 5fe6, 5fe7, 5fe8
and 5fe9.
Discovery of
Bromodomains of TRIM24 and BRPF. J Med Chem 2015. in press.
8. Demont, E. H.; Chung, C. W.; Furze, R. C.; Grandi, P.;
a
Chemical Tool Inhibitor Targeting the
Michon, A. M.; Wellaway, C.; Barrett, N.; Bridges, A. M.; Craggs,
P. D.; Diallo, H.; Dixon, D. P.; Douault, C.; Emmons, A. J.; Jones,
E. J.; Karamshi, B. V.; Locke, K.; Mitchell, D. J.; Mouzon, B. H.;
Prinjha, R. K.; Roberts, A. D.; Sheppard, R. J.; Watson, R. J.;
Bamborough, P. Fragment-Based Discovery of Low-Micromolar
ATAD2 Bromodomain Inhibitors. J Med Chem 2015, 58, 5649-
5673.
All authors approved the final manuscript.
ACKNOWLEDGMENT
The authors thank the staff at Diamond Light Source for
assistance during data collection. The SGC is a registered
charity (number 1097737) that receives funds from AbbVie,
Bayer, Boehringer Ingelheim, the Canada Foundation for
Innovation, the Canadian Institutes for Health Research,
Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the
Novartis Research Foundation, the Ontario Ministry of Eco-
nomic Development and Innovation, Pfizer, Takeda, and the
Wellcome Trust [092809/Z/10/Z]. AC has been supported by
the European Union FP7 Grant No. 278568 “PRIMES”. SL was
supported by a German Academic Exchange Programme
DAAD Postdoctoral Fellowship.
9.
Hay, D. A.; Fedorov, O.; Martin, S.; Singleton, D. C.;
Tallant, C.; Wells, C.; Picaud, S.; Philpott, M.; Monteiro, O. P.;
Rogers, C. M.; Conway, S. J.; Rooney, T. P.; Tumber, A.; Yapp, C.;
Filippakopoulos, P.; Bunnage, M. E.; Muller, S.; Knapp, S.;
Schofield, C. J.; Brennan, P. E. Discovery and Optimization of
Small-Molecule Ligands for the CBP/p300 Bromodomains. J Am
Chem Soc 2014, 136, 9308-9319.
10.
Clark, P. G.; Vieira, L. C.; Tallant, C.; Fedorov, O.;
Singleton, D. C.; Rogers, C. M.; Monteiro, O. P.; Bennett, J. M.;
Baronio, R.; Muller, S.; Daniels, D. L.; Mendez, J.; Knapp, S.;
Brennan, P. E.; Dixon, D. J. LP99: Discovery and Synthesis of the
First Selective BRD7/9 Bromodomain Inhibitor. Angew Chem Int
Ed Engl 2015, 54, 6217-6221.
ABBREVIATIONS
11.
Chaikuad, A.; Petros, A. M.; Fedorov, O.; Xu, J.; Knapp,
PCAF, p300/CBP-associated factor; Kac, Nε-acetyllysine;
HIV, human immunodeficiency virus; AIDS, acquired im-
mune deficiency syndrome; ATAD2, ATPase family AAA
domain-containing protein 2; BAZ2B, Bromodomain adjacent
to zinc finger domain protein 2B; BET, Bromodomain and
Extra-Terminal; BRD4, Bromodomain-containing protein 4;
CREBBP, CREB-binding protein. SPR, surface plasmon reso-
nance. TINS, target immobilized NMR screening.
S. Structure-based approaches towards identification of
fragments for the low-druggability ATAD2 bromodomain
MedChemComm 2014, 5, 1843-1848.
12.
Ferguson, F. M.; Fedorov, O.; Chaikuad, A.; Philpott,
M.; Muniz, J. R.; Felletar, I.; von Delft, F.; Heightman, T.; Knapp,
S.; Abell, C.; Ciulli, A. Targeting low-druggability
bromodomains: fragment based screening and inhibitor design
against the BAZ2B bromodomain. J Med Chem 2013, 56, 10183-
10187.
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Table of Contents graphic
Diverse fragment screening approaches identified chemical starting points for the development of PCAF bromodomain
inhibitors.
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