Targeting low-druggability bromodomains: Fragment based screening and inhibitor design against the BAZ2B bromodomain

Article abstract of DOI:10.1021/jm401582c

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful tar

Full text of DOI:10.1021/jm401582c

Brief Article
pubs.acs.org/jmc
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Targeting Low-Druggability Bromodomains: Fragment Based
Screening and Inhibitor Design against the BAZ2B Bromodomain
Fleur M. Ferguson,† Oleg Fedorov,‡ Apirat Chaikuad,‡ Martin Philpott,‡ Joao R. C. Muniz,‡,⊥
Ildiko Felletar,‡,# Frank von Delft,‡ Tom Heightman,‡,∞ Stefan Knapp,‡,§ Chris Abell,†
and Alessio Ciulli*,†,∥
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K.
‡Nuffield Department of Clinical Medicine, SGC, University of Oxford, Oxford OX3 7DQ, U.K.
§Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, U.K.
S
* Supporting Information
ABSTRACT: Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET
bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower
pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and
structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results
provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.
approach would provide insights into the different function-
alities and the strategies required for ligand optimization in this
more challenging Kac-binding pocket.
INTRODUCTION
■
Bromodomains are epigenetic reader domains within proteins
that specifically recognize acetylated lysine (Kac) in histones
and other substrate proteins. There are 61 different
bromodomains spread across 46 proteins in the human
genome, many of which are medically relevant targets for
areas such as cancer, inflammation, and neurological disease.1,2
A subfamily of bromodomains of the bromo and extra terminal
(BET) proteins have been shown to have tractable Kac binding
pockets computationally3 and by the development of potent
inhibitors such as JQ14 and iBET.5 These compounds have
helped to unravel the biology and therapeutic potential of BET
proteins, leading to mounting interest in developing chemical
probes for other bromodomains in the human genome.
One bromodomain-containing protein whose biological role
is still elusive is the bromodomain adjacent to zinc finger
domain protein 2B (BAZ2B). A chemical probe would provide
a useful tool to help determine its function. The BAZ2B
bromodomain has an unusually small Kac-binding pocket
compared to the other 41 bromodomains for which structural
information is available (92−105 Å3 volume vs 131−221 Å3 in
BRD4(BD1)) which lacks many of the features of BET
bromodomains such as a ZA channel and a hydrophobic groove
adjacent to the WPF motif (Supporting Information Figure 1).3
Strategies that have been successfully employed in BET
bromodomain inhibitor optimization exploit the aforemen-
tioned structural features and are therefore not transferrable to
BAZ2B. Analysis by Vidler et al. predicts the BAZ2B
bromodomain to be one of the least druggable in the family.3
Consistent with this, reported inhibitors for other bromodo-
mains show no cross-reactivity with BAZ2B, even at relatively
early stages of the design process.6
RESULTS AND DISCUSSION
■
As a first step, an unbiased library of 1300 rule of three8
compliant commercially available fragments were screened.
Primary screening was performed using a competitive
AlphaScreen assay, which measures displacement of a histone
H3 peptide acetylated at K14 (H3Kac14).7 Initial hits were
defined as those that showed >50% inhibition at 1 mM
compound. For these fragments IC50 values were measured
using AlphaScreen, resulting in the identification of 10
fragments. All of these showed direct binding and displacement
when validated using orthogonal ligand-observed NMR
techniques STD,9 CPMG,10 and WaterLOGSY11 (Table 1), a
hit rate of 0.8%. The same fragment library was screened
against the bromodomains of BRD2-BD1 and CREBBP (see
Supporting Information Figure 2 for overlapping hits). The
initial hit rates for these proteins were much higher, 1.8% and
6.1% respectively, consistent with their predicted higher
ligandability.12
To elucidate the binding modes, the 10 fragment hits were
soaked into apo-crystals of BAZ2B. It was possible to solve
high-resolution crystal structures for fragments 1, 3, 6, and Kac
(Figure 1).
The ligand Kac makes hydrogen bonds to the side chain of
Asn1944 and to Tyr1901 via a highly conserved water, as has
been reported for other bromodomains.13 The alkyl chain has
an unusual kink, which orients toward the BC loop. The
carboxylic acid of Kac interacts with a water molecule that is
also bound to the side chain of Asn1944.
As the Kac binding site in all bromodomains is of a suitable
size and shape to bind to organic solvents and low MW
molecules (fragments),7 we reasoned that a fragment-based
Received: October 10, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
The acetyl group of the most potent fragment, tetrahydro-γ-
carboline (THγC) 6, bound to BAZ2B overlays with that in the
Kac structure (Figure 1d and Supporting Information Figure
4). The unsaturated ring allows the fragment to twist such that
the aromatic rings can make an edge-to-face π-stacking
interaction with the side chain of Trp1887. The poor solubility
of 6 meant direct ITC titration was not possible; therefore, a
KD of 65 μM was determined using competitive ITC
experiments with an H3Kac14 peptide (Table 2 and Supporting
Information Table 4).
Table 1. Structures, IC50 Values Determined by
AlphaScreen, and Ligand Efficiencies of Validated Fragment
a
Hits
Encouraged by these initial results, we sought to optimize
fragment 6 by varying the N1, N2, and aromatic substituents.
The 1-position of the THγC scaffold presented an attractive
vector for fragment growing to make interactions with the ZA
loop. In the crystal structure the N1 proton is not interacting
with the protein and the methylated compound (Table 2, 11)
retained potency, suggesting further modifications could be
exploited.
A set of compounds growing further from this position were
synthesized; however, all modifications resulted in a reduction
in potency (12−15, 28−32).
The distance between the backbone carbonyl oxygen of
Trp1887 and the chlorine of 6 is 3.5 Å (combined van der
Waals radii, 3.3 Å) and the angle between them is 109°,
indicating no halogen bonding is occurring.16 On the basis of
this observation, analogues were synthesized (16−21, 33−36)
with alternative aryl substituents that preliminary docking
studies17 suggested could hydrogen-bond to the protein and
also provide a vector for growing the molecule out toward the
ledge formed by residues 1893−1889, predicted to be a hotspot
by FTMap solvent mapping software (Supporting Information
Figure 5).18 Although some of these compounds showed a
desirable improvement in solubility, all lost some potency, the
best having an IC50 of 50 μM (19).
Another strategy implemented to optimize the aromatic
substituent of 6 was using it to tune the charge distribution of
the ring to strengthen the edge-to-face π−π interaction (22−
27, 37). As expected, molecules containing strongly electron-
withdrawing groups showed reduced potency, implying a
weakened π−π interaction (18, 22, 23, 37). However, those
with electron-donating groups (24, 25) did not result in the
expected improvement in affinity.
a
IC50 values are reported as the mean of three replicates ( standard
error of the mean).
Fragments 1 and 3 form hydrogen bonds to Asn1944 and the
conserved water via their carbonyl group in a similar manner to
Kac. The amide nitrogen forms a hydrogen bond to the
backbone carbonyl of Pro1888. A similar interaction is
observed by Magno et al. between the equivalent residue in
TAF1 and ε-NH of Kac in MD binding simulations.14 The
amide nitrogen of fragment 1 forms an intramolecular
hydrogen bond to the adjacent ether group, which restricts
rotational freedom and therefore may reduce the entropic
penalty of binding. The tetrahydropyran ring of 1 lies
orthogonal to its phenyl ring, providing an attractive trajectory
for growing the ligand toward Val1893 (Figure 1b and
Supplementary Figure 3).
Figure 1. Crystal Structures of the BAZ2B bromodomain in complex with (a) acetyllysine PDB 4NR9, (b) 1 PDB 4NRB, (c) 3 PDB 4NRC, and (d)
6 PDB 4NRA. The bridging water molecule essential for the acetyllysine interaction is shown black. Other binding site waters are not shown for
clarity. Hydrogen bonds are shown as red dashed lines. |2Fo| − |Fc| electron density maps contoured at 1σ for the bound ligands are shown in
subpanels.
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Journal of Medicinal Chemistry
Brief Article
Observation that the most ligand efficient fragments found in
the screen (7, 8, and 10) contain thioamides, conceivably a Kac
mimetic, led us to measure the KD of 7 using ITC, KD = 524
μM (Supporting Information Table 5). This encouraged us to
synthesize thioamide 38 and thiourea 39. Neither of these
bound to the protein. This indicates that the thioamide
functionality in fragment hits 7, 8, and 10 adopts an orientation
that cannot be achieved when attached to the larger THγC ring
and that the BAZ2B bromodomain does not recognize the
thiocarbonyl group.
Table 2. IC50 Values Determined by AlphaLISA for
Analogues of 6
a
Overlaying of the crystal structures of fragments 3 and 6
bound to BAZ2B (Figure 2) revealed that an additional H-bond
Figure 2. Overlay of the crystal structures of fragments 3 (shown
pink) and 6 (offset, shown cyan) illustrates the rationale for fragment
merging.
could potentially be gained by merging the Kac mimetic of 3
with the scaffold of 6 to generate compound 40. Urea-
containing 40 was synthesized and gratifyingly showed
improved solubility, an 8-fold reduction in KD (determined
by direct ITC titration), and a corresponding gain in ligand
efficiency (Table 3). The 8-bromo analogue 41 showed slightly
Table 3. IC50 Values Determined by AlphaLISA for
Analogues of 6 in Which the Acetyllysine Mimetic Group Is
a
Varied
Cpd
R1
R3
Me
X
IC50 (μM)
KD (μM)
38
39
40
41
Cl
Cl
Cl
Br
S
>100
NH2
S
>2000
NHMe
NHMe
O
O
9 ( 2)
14 ( 0.1)
8
17
a
IC50 values are reported as the mean of three replicates ( standard
error of the mean). KD determined by ITC (Supporting Information
Table 5).
weakened binding compared to 27. The results show that
optimization of the Kac bioisostere can play an important role
in bromodomain inhibitor design.
Fragments containing an N-methylamide, e.g., 1−3 and 5,
along with the THγC 6 were also hits against BRD2(1) and
CREBBP in our fragment screen, which led us to profile 27 and
40 against these proteins using differential scanning fluorimetry
(DSF), as previously described by Hewings et al.19 to allow for
comparison (Table 4).
a
IC50 values are reported as the mean of three replicates ( standard
error of the mean) and were not measured for molecules that resulted
in an inhibition below 70% at 200 μM compound (%I).15 KD values
determined by ITC (Supporting Information Tables 4 and 5).
Compound 27 proved the most potent of the tested
analogues, showing a 6-fold improvement in KD over 6. This
is possibly due to the weaker inductive electron-withdrawing
properties of bromine vs chlorine aromatic substituents and
increased hydrophobic contacts with the Trp1887 side chain.
Interestingly 27 causes a significant increase in thermal
stability against CREBBP and BRD2 (BD1), indicating that this
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Journal of Medicinal Chemistry
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Table 4. ΔTm in °C Measured by DSF against CREBBP and
AUTHOR INFORMATION
Corresponding Author
*Phone: +44-1382-386230. E-mail: a.ciulli@dundee.ac.uk.
■
a
BRD2 Bromodomains
bromodomain
Present Addresses
Cpd
CREBBP
BRD2 (BD1)
BRD2 (BD2)
∥A.C.: College of Life Sciences, University of Dundee, Dow
Street, Dundee, DD1 5EH, U.K.
27
40
8.1
2.1
7.3
3.5
−0.6
−0.2
⊥J.R.C.M.: Institute of Physics of Sao Carlos, University of Sao
Paulo, Avenida Trabalhador Saocarlense 400, Sao Carlos, SP
̃
̃
a
ΔTm values are reported as the mean of three replicates (standard
error of the mean all <0.1 °C). Compound concentration, 100 μM;
protein concentration, 2 μM.
̃
̃
13560-970, Brazil.
#I.F.: Genome Centre, University of Sussex, Sussex House,
Falmer, Brighton, BN1 9RH, U.K.
series may also provide useful leads for targeting these proteins.
Hewings et al. describe a ΔTm of 4 °C in CREBBP
corresponding to an IC50 of 28.1 μM;19 therefore, 27 is likely
to be a low micromolar lead for CREBBP. Compound 40
shows markedly lower thermal stabilization for the three other
bromodomains, implying that this compound may be more
selective.
∞T.H.: Astex Therapeutics, 436 Cambridge Science Park,
Cambridge, CB4 0QA, U.K.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the UK Biotechnology and
Biological Sciences Research Council (BBSRC, Grants BB/
J001201/1 and BB/G023123/1 to A.C.). F.M.F. is funded by a
BBSRC Ph.D studentship. The SGC is a registered charity (No.
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canada Foundation for Innovation,
Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly, the
Novartis Research Foundation, Takeda, the Ontario Ministry
of Research and Innovation, and the Wellcome Trust. We
thank Robert Pasteris and DuPont for the donation of 31−33
from their collection, Paul Brennan for useful discussions, and
Charlotte Sutherell for synthesis of 24−26, 35.
CONCLUSIONS
■
In summary, we describe biophysical screening, validation, and
structural characterization for a number of chemically diverse
fragment hits, which represent attractive starting points for
inhibitor design against the BAZ2B bromodomain. A novel
chemical series of BAZ2B bromodomain ligands based on a
THγC scaffold are disclosed and SARs discussed. Efforts
focused on optimization of interactions in and close to the Kac
binding pocket proved the most productive, compared to those
exploring its wider peripheries. These compounds make key
interactions with the WPF motif, which complements another
reported inhibitor, GSK2801,20 which makes key interactions
with residues on the opposite side of the pocket. We also show
that replacement of an acyl group with an N-methylamide as
the KAc bioisostere can lead to a gain in binding affinity.
Our successful targeting of one of the least druggable
bromodomains with small molecules highlights the possibility
that the family as a whole may be tractable to small molecule
modulation.
ABBREVIATIONS USED
■
Alpha, amplified luminescent proximity homogeneous assay;
BAZ2B, bromodomain adjacent to zinc-finger domain 2B; BD,
bromodomain, BET, bromodomain and extra terminal; BRD2,
bromodomain containing protein 2; CPMG, Carr−Purcell−
Meiboom−Gill; CREB, cyclic adenosine monophosphate
responsive element binding protein; CREBBP, CREB binding
protein; DSF, differential scanning fluorimetry, H3Kac14,
histone H3 acetylated at lysine 14; ITC, isothermal titration
calorimetry; Kac, Nε-acetyllysine; SAR, structure−activity
relationship; STD, saturation transfer difference; THγC,
tetrahydro-γ-carboline; WaterLOGSY, water ligand observed
via gradient spectroscopy; %I, percent inhibition of signal
relative to solvent-only control
EXPERIMENTAL SECTION
■
Chemistry. General directions are in the Supporting Information.
The syntheses of selected compounds are described below as
representative. A full description of the synthetic protocol and
spectroscopic analysis for each compound can be found in the
Supporting Information. The purity of all tested compounds was
analyzed by HPLC−MS (ESI) and is >95% unless otherwise stated.
General Procedure for Synthesis of THγCs 6, 18, 24−27.
Substituted phenylhydrazine hydrochloride (1 equiv) and N-acyl-4-
piperidone hydrochloride monohydrate (1 equiv) were refluxed in
absolute EtOH for 3 h. The mixture was cooled to room temperature
and the solvent concentrated in vacuo. The residue was purified by
automated flash chromatography on silica or recrystallized from
EtOH/H2O.
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ASSOCIATED CONTENT
* Supporting Information
Supplementary figures, supplementary tables, synthetic
schemes, experimental procedures, synthesis and character-
ization of organic molecules, biophysical assays, ITC data,
crystallographic refinement data. This material is available free
of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB accession codes of BAZ2B in complex with acetyllysine, 6,
1, and 3 are 4NR9, 4NRA, 4NRB, and 4NRC, respectively.
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