The "gatekeeper" Residue Influences the Mode of Binding of Acetyl Indoles to Bromodomains

Article abstract of DOI:10.1021/acs.jmedchem.5b01757

Small-molecule hits for the bromodomains of CREBBP and BAZ2B have been identified by scaffold hopping followed by docking of a set of ～200 compounds containing the acetyl indole scaffold. Chemical synthesis of nearly 30 derivatives has resulted in ligands of representatives of three subfamilies of human bromodomains with favorable ligand efficiency. The X-ray crystal structures of three different bromodomains (CREBBP, BAZ2B, and BRPF1b) in complex with acetyl indole derivatives reveal the influence of the gatekeeper residue on the orientation of small-molecule ligands in the acetyl lysine binding site.

Full text of DOI:10.1021/acs.jmedchem.5b01757

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Article
The “gatekeeper” residue influences the binding
mode of acetyl indoles to bromodomains
Andrea Unzue, Hongtao Zhao, Graziano Lolli, Jing Dong, Jian Zhu,
Melanie Zechner, Aymeric Dolbois, Amedeo Caflisch, and Cristina Nevado
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01757 • Publication Date (Web): 16 Mar 2016
Downloaded from http://pubs.acs.org on March 17, 2016
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The “gatekeeper” residue influences the binding
mode of acetyl indoles to bromodomains
Andrea Unzue,[a] Hongtao Zhao,[b] Graziano Lolli,[b] Jing Dong,[b] Jian Zhu,[b] Melanie
Zechner,[a] Aymeric Dolbois,[a] Amedeo Caflisch,*[b] Cristina Nevado*[a]
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Department of Chemistry[a] and Department of Biochemistry,[b] University of Zürich
Winterthurerstrasse 190, CHꢀ8057 Zürich, Switzerland.
ABSTRACT Smallꢀmolecule hits for the bromodomains of CREBBP and BAZ2B have been
identified by scaffold hopping followed by docking of a set of about 200 compounds
containing the acetyl indole scaffold. Chemical synthesis of nearly 30 derivatives has resulted
in ligands of representatives of three subfamilies of human bromodomains with favorable
ligand efficiency. The Xꢀray crystal structures of three different bromodomains (CREBBP,
BAZ2B, and BRPF1b) in complex with acetyl indole derivatives reveal the influence of the
gatekeeper residue on the orientation of smallꢀmolecule ligands in the acetyl lysine binding
site.
Nomenclature
Capital letters are used to label generic chemical blueprints. 1a-i correspond to commercially
available compounds. Arabic numbers 1ꢀ50 are used for synthetic intermediates and selfꢀ
made products.
Introduction
Acetylation of lysine residues is an important postꢀtranslational modification of histone
proteins that contributes to the regulation of chromatin structure and transcription.1,
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Bromodomains are protein modules with fourꢀhelix bundle topology that specifically
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recognize (‘‘read’’) acetylated lysine residues, as well as butyryllysine and crotonyllysine,3
and are considered protein targets of interest for the development of chemical probes and
clinical tools to treat cancer, inflammation and other diseases.4ꢀ8
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The BET (bromodomain and extra terminal) subfamily (BRD2/3/4/T) has been widely
addressed, and as a consequence, several potent and selective inhibitors have been developed,
some of which are currently undergoing clinical trials for the treatment of NUT midline
carcinoma (NMC), solid tumors, leukaemia, lymphoma, haematological malignancies,
atherosclerosis and type II diabetes.7, 9, 10 In contrast, the specific function and potential
pharmacological relevance of other bromodomains, including CREB binding protein
(CREBBP), E1A binding protein p300 (EP300), BRD7/9, and bromodomain adjacent to zinc
finger domain (BAZ2B), is much less understood, and thus, small molecule inhibitors will be
valuable tools to unravel their biological roles.
The bromodomains of EP300 and CREBBP, which belong to the same subfamily and
share 96% sequence identity,11 play important roles in DNA replication and repair, cell
growth and cell cycle regulation and genomic stability.12, 13 As an example, EP300 and
CREBBP are able to acetylate p53 on its K382 residue through the histone acetyl transferase
(HAT) domain upon extracellular stress or DNA damage and they are also known to
specifically bind to acetylated p53 via their bromodomain module.14 As a consequence,
changes in the p53ꢀdependent activation of target genes result in cell cycle arrest, senescence
or apoptosis.15ꢀ17 On the one hand, chromosome translocations resulting in gene fusions
containing CREBBP or EP300 have been linked to leukemias and lymphomas.18, 19 On the
other hand, CREBBP and EP300 are mutated in solid tumors and Bꢀcell lymphoma,
suggesting they possess a tumor suppressing role.13, 20 Thus, because both oncogene or tumor
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suppressor roles have been reported for CREBBP and EP300,18, 21 the development of
chemical probes will be instrumental for the analysis of their biological function(s).
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The bromodomains of BRD7 and BRD9 belong to the same subfamily and share 72 %
identity.22 Both BRD7 and BRD9 are part of the SWI/SNF (SWItch/Sucrose Nonꢀ
Fermentable) chromatin remodelling complex, which plays a key role in the regulation of
gene expression.23ꢀ25 Recent reports have linked BRD9 to oncology, including nonꢀsmall cell
lung cancer26 and cervical cancer.27 Its paralogue, BRD7, is frequently downꢀregulated in
cancer28ꢀ30 and is able to regulate the tumor suppressor protein p53.31ꢀ33 The bromodomain of
BRPF1b (bromodomainꢀPHD finger protein 1b) belongs to the same subfamily as BRD7 and
BRD9. Despite the function of the BRPF1b bromodomain not being yet fully understood, the
availability of BRPF1b ligands might help elucidating its role.34ꢀ36
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BAZ2B is another bromodomainꢀcontaining protein whose role in physiology and
disease is not clear. Biophysical screening of a library of 1300 fragments resulted in the
identification of 10 small molecules that bind in the micromolar range to BAZ2B.37 Recently,
the first submicromolar selective CREBBP,11, 38, 39 BAZ2B40, 41 and BRD7/922, 42, 43 inhibitors
have been reported.
Here we present the result of a combined scaffold hopping and docking approach that
has enabled the discovery of acetyl indoles as ligands of the bromodomains of CREBBP,
BAZ2B, BRPF1b and/or BRD9, which belong to three different subfamilies that lie outside
of the BET bromodomain subfamily. A comparative analysis of four crystal structures of
bromodomain/acetyl indole complexes show the importance of the soꢀcalled gatekeeper
residue on the binding mode of the ligand.
Results and Discussion
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In silico screening by scaffold hopping and docking
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In the past few years, our groups have successfully identified several low µM to nM kinase44ꢀ
and bromodomain39, 48, 49 inhibitors by highꢀthroughput virtual screening campaigns. In
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this work, we decided to dock a small subset of compounds containing a moiety identified by
scaffold hopping (see Materials and Methods). First, the ZINC allꢀnow library was
decomposed into about 600,000 fragments retaining key functional groups.46 These fragments
were queried by the indolizine fragment A, which is present in the potent BAZ2B ligand
GSK2801 (1a)41 and more recently in BRD7 and BRD9 ligands.22 The acetyl indole B was
identified as the top ranking fragment with an activityꢀoriented fingerprint similarity of 0.975
with respect to A. The high similarity is due to almost identical geometry and connecting
vectors in the fragments A and B (Figure 1).
Figure 1. (Left) GSK2801 (1a), a nanomolar chemical probe for BAZ2A and BAZ2B
bromodomains.22, 41 (Right) Coreꢀfragment hopping by activityꢀoriented fingerprint using
fragment A as the query molecule. A and B respresent generic chemical blueprints.
In a second step, we retrieved about 200 commercially available compounds containing
48, 49
fragment B. As in our previous fragmentꢀbased virtual screening approach,46,
the
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retrieved compounds were docked into crystal structures of the targets, namely, the
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bromodomains of CREBBP (PDB code 4A9K) and BAZ2B (PDB code 3Q2F). An inꢀhouse
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developed program for automatic docking was used.48,
The docking poses were
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subsequently reꢀscored by a transferable scoring function (see Experimental Section).45, 50ꢀ54
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Finally, 14 molecules were selected for experimental validation by means of a competition
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binding assay.55,
At 50 µM concentration, seven compounds showed significant
competition (i.e., a percentage of residual binding of CREBBP to the acetylated histone
peptide below 70% with respect to the DMSO control, Figure 2), which corresponds to a hit
rate of 50% for the in silico screening approach based on scaffold hopping and docking. The
most active compound 1b exhibits an equilibrium dissociation constant (Kd) of 20 µM for
CREBBP. Using the same threshold of 70% as for CREBBP, the hit rate for BAZ2B was
29% (Figure 2). Interestingly, at 50 µM concentration, only compound 1g shows significantly
higher affinity for BAZ2B than CREBBP.
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Figure 2. Binding affinity and 2D structures of acetyl indole derivatives 1b-i identified by
coreꢀfragment hopping and docking. The values are percentage of residual binding of the
CREBBP or BAZ2B bromodomain to an acetylated histone peptide at 50 µM compound
concentration with respect to the DMSO control. Thus, lower percentages indicate higher
affinity of the compound. Values in brackets are equilibrium dissociation constants (Kd)
determined by two independent doseꢀresponse measurements of 11 doses each. Compounds
1c, 1d, and 1i were measured as racemic mixture. All values were determined by the
BROMOscan competition binding assay.55, 56
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Binding mode of compound 1b: Validation by X-ray protein crystallography
The opposite selectivity towards CREBBP and BAZ2B observed for compounds 1b and 1g
(Figure 2) prompted us to study their binding mode. While we could not solve the structure
with compound 1g, the crystal structure of CREBBP in complex with compound 1b was
solved at 2.0 Å resolution (Figure 3, in green, PDB code 4TS8), which revealed an overall
binding mode essentially identical to the docked pose of compound 1b (Figure 3, in blue).
The binding of compound 1b in CREBBP is characterized by a lipophilic sandwich of its
bicyclic core between residues Phe1111, Val1174 and Ala1164 on one side, and Val1115,
Leu1120 and Ile1122 on the other side of the binding pocket. The carbonyl oxygen of the
acetylꢀindole acts as the acetylated lysine mimic and is engaged in hydrogen bonding
interactions with the side chains of the conserved Asn1168 (BC loop) and Tyr1125 (ZA loop)
where the latter is bridged by a water molecule. Another four water molecules present at the
bottom of the pocket are conserved. In addition, there is a water molecule bridging the
dihydroꢀpyrazole ring and the guanidinium group of Arg1173 (Figure 3).
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Figure 3. Crystal structure (green) and docked pose (blue) of the CREBBP bromodomain
(shown in grey) in complex with compound 1b (PDB code 4TS8). The conserved Tyr1125
and Asn1168 residues, together with Arg1173 (a characteristic residue for the CREBBP
bromodomain)11, 38, 39, 49 are shown as sticks. Hydrogen bonds are shown as yellow dashed
lines and the crystallographic water molecules are represented by red spheres.
Assessing the affinity difference between indolizine and indole ligands
Compound 1a is reported to be a potent ligand for the BAZ2B bromodomain with a Kd of 136
nM.41 To study the influence on the binding affinity of the position of the nitrogen atom in
our hit compound 1b, we decided to synthesize the indole analogue of indolizine 1a.
Commercially available 1Hꢀindolꢀ5ꢀol (1) was transformed into compound 3 via alkylation of
the phenol moiety with 1ꢀiodopropane, followed by introduction of an Nꢀbenzenesulfonyl
group and bromination at C3 in presence of molecular bromine (Scheme 1). Bromo indole 3
was then coupled to (2ꢀ(methylsulfonyl)phenyl)boronic acid affording compound 4 in
moderate yield. Removal of the sulfonyl group under basic conditions preceded the
incorporation of the acetyl group to give the indole analogue of compound 1a (5).
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Scheme 1a
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aReagents and reaction conditions: a) (i) 1ꢀiodopropane, K2CO3, acetone, reflux, 72 h, 84%;
(ii) Benzenesulfonyl chloride, TBAB, 50% NaOH, H2O, toluene, 0−25 °C, 69 h, 97%; b) Br2,
DMF, 25 °C, 7 h, 47%; c) (2ꢀ(methylsulfonyl)phenyl)boronic acid, 1M NaHCO3, Pd(PPh3)4,
DME, 25−85 °C, 15 min, 33%; d) (i) 2M NaOH, MeOH, 85 °C, 5 h, 69%; (ii) AcCl, NaOH,
TBAHS, DCM, 25 °C, 13 h, 63%. TBAB: tetraꢀnꢀbutylammonium bromide; DMF: dimethyl
formamide; DME: 1,2ꢀdimethoxyethane; TBAHS: tetrabutylammonium hydrogen sulfate.
Interestingly, a 24 fold reduction of binding affinity towards BAZ2B was observed for acetyl
indole 5 with respect to 1a (IC50 values of 8.55 ꢁM and 0.36 ꢁM, respectively, determined by
AlphaScreen, see Supporting Information) which indicates that the position of the nitrogen
atom in the doubleꢀring system is crucial.
Synthesis
We decided to focus our derivatization campaign on compounds 1b and 1g as ligands.
Compound 1b was selected because of its ligand efficiency for CREBBP (0.34 kcal/mol per
heavy atom) and the availability of the crystal structure (Figure 3), while compound 1g was
chosen because of its selectivity towards BAZ2B (Figure 2).
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Synthesis of compound 1b and derivatives
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The synthesis of the CREBBP hit 1b is shown in Scheme 2. The carboxylic acid of
commercially available 2ꢀ(1 ꢀindolꢀ3ꢀyl)acetic acid (6) was transformed into the
H
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corresponding methyl ester. Acetylation of the N atom afforded indole 7. Deprotonation of 7
in the presence of in situ generated lithium diisopropylamide (LDA) followed by reaction
with acetic anhydride or 4ꢀmethoxyphenylacetic anhydride delivered intermediates 8 and 9
respectively. Cyclization in the presence of hydrazine hydrate afforded the hit compound 1b
and derivative 10 in moderate yields (Scheme 2).
Scheme 2a
aReagents and reaction conditions: a) (i) MeOH, H2SO4, 25 °C, 1 h, 94%; (ii) Ac2O,
DMAP, Et3N, 25 °C, 17 h, 90%; b) (iPr)2NH, nꢀBuLi, THF, −75 °C, 1 h, then, acetic
anhydride or 4ꢀmethoxyphenylacetic anhydride, −78 °C, 4 h; c) Hydrazine hydrate,
camphoric acid, EtOH, toluene, 93 °C, 30 min−1 h, 29−51 % over two steps. DMAP: 4ꢀ
dimethylaminopyridine.
Hit compound 1b was then reacted with a variety of acid and sulfonyl chlorides affording
diꢀsubstituted (11ꢀ18) as well as monoꢀsubstituted dihydroꢀpyrazole derivatives (19ꢀ22) as
indicated in Scheme 3. Two more derivatives were prepared: upon condensation of 1,2ꢀ
dibromoethane with compound 1b in the presence of K2CO3 derivative 23 was obtained. The
reaction of 1b with
pꢀmethoxybenzyl bromide in the presence of NaH afforded Oꢀalkylated
product 24 (Scheme 3).
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Scheme 3a
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aReagents and reaction conditions: a) RꢀCl, Et3N, DCM, 0−25 °C, 5−12 h, 24−89 %; b) 1,2ꢀ
dibromoethane, K2CO3, DMF, 80 °C, 7 h, 41 %; c) PMBꢀBr, NaH, TBAI, DMF, 0−25 °C,
1.5 h, 23 %. PMB: 4ꢀMethoxybenzyl ether; TBAI: tetrabutylammonium iodide.
Finally, we decided to incorporate the propoxy group at position 5 of the indole, for which a
similar protocol was applied to 1
(Scheme 4).
Hꢀindolꢀ5ꢀol (1) delivering the final products 27 and 28
Scheme 4a
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aReagents and reaction conditions: a) (i) 1ꢀiodopropane, K2CO3, acetone, reflux, 72 h, 84%;
(ii) (COCl)2, Et2O, 0 °C, then NaOMe in MeOH, −78 °C, 3 h; (iii) Pd/C, NaH2PO2, dioxane,
H2O, 25 °C, 12 h, 88 % over two steps; b) Ac2O, DMAP, Et3N, THF, 25 °C, 12 h, 42%; c)
(i) (iPr)2NH, nꢀBuLi, THF, −78 °C, 1 h, then, acetic anhydride, −78 °C, 4 h; (ii) Hydrazine
hydrate, camphoric acid, EtOH, toluene, reflux, 4 h, 18 %; d) 4ꢀMethoxybenzoyl chloride,
Et3N, DCM, 0−25 °C, 12 h, 19 %.
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Synthesis of derivatives of compound 1g
The common intermediate for the synthesis of derivatives of ligand 1g was obtained from
the commercially available nonꢀnatural amino acid 29 in four steps with a 20 % overall yield
(Scheme 5). Condensation of intermediate 32 with diverse commercially available anilines
afforded acetyl indole intermediates 33-38, which were then acylated in the presence of
diverse acid chlorides affording compounds 39-46. Four additional derivatives were prepared
by cleaving the methoxy subtituent in the presence of BBr3 to obtain phenol derivatives 47-50
(Scheme 5).
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Scheme 5a
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aReagents and reaction conditions: a) (i) Chloroacetic acid, K2CO3, H2O, 90 °C, 16 h; (ii)
Ac2O, 25 °C, 30 min, then 37% HCl for 12h, 33% over two steps; b) Ac2O, Et3N, reflux, 30
min, 89%; c) Na2SO3, reflux, 2 h, 83%; d) Method I: Aniline, AcOH, reflux, 1−8 h, 26−56%.
Method II: aniline, pTSA, toluene, reflux, 2.5−4 h, 50−52%; e) For MeCOCl, toluene, reflux,
6 h, 13−78%; if R4=aryl, R4COCl, toluene, Et3N, DMAP, 100 °C, 1 h, 13−21%; f) BBr3,
DCM, 0−25 °C, 1ꢀ12 h, 47−84%. pTSA: pꢀtoluenesulfonic acid.
Biophysical Characterization
Compound 1b and derivatives
Compound 1b was screened against a panel of ten different bromodomains using a thermal
shift assay (Table 1). This initial screening revealed BRD9 bromodomain as a potential off
target for compound 1b with a shift in the melting temperature of 1.3 °C, which translated
into a KD of 5.3 µM, as measured by a competition binding assay,55, 56 and a ligand efficiency
(LE) value of 0.38 kcal/mol per heavy atom. This result is in line with the recent work of
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Brennan and coꢀworkers, who developed BRD7 and BRD9 nanomolar potent indolizine
derivatives starting from the BAZ2B inhibitor 1a.22 Importantly, no activity of compound 1b
was observed for the tested BET family members, BRD4(1) and BRD4(2) (see Supporting
Information).
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46
47
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50
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Due to the involvement of the acetyl group of compound 1b in hydrogen bonds with the
conserved Asn1168 and Tyr1125 residues of CREBBP (Figure 3), we decided to maintain
this moiety and introduce the main modifications at the dihydroꢀpyrazole ring in order to gain
affinity towards CREBBP.
On the outset, we decided to modulate the interactions of the solvent exposed NH groups of
the dihydroꢀpyrazole ring. To do so, monoꢀ and diꢀsubstituted amide and sulfonamides that
could establish new interactions with the surrounding aminoacid residues including the
characteristic Leu1109 and Arg1173 residues in the CREBBP bromodomain and His42 and
Phe44 in BRD9 were incorporated (compounds 11ꢀ22, Table 1). The presence of methyl
substituted sulfonamides in compound 11 provided a KD of 6.6 µM in CREBBP, which is a
threeꢀfold binding affinity improvement with respect to the hit compound 1b and corresponds
to a LE value of 0.26 kcal/mol per heavy atom.
In an effort to form πꢀstacking interactions with Arg1173 in CREBBP,11, 38 and residues
Phe43 and Phe44 in BRD9, aromatic substituents, some of them electronꢀrich, were
incorporated (compounds 14ꢀ18 and 20ꢀ21). Compound 14 yielded the most active ligand
towards BRD9 with a KD of 3.5 µM. Interestingly, the presence of a pꢀmethoxybenzoate
substituent in compound 20 resulted in a 2ꢀfold improvement of binding affinity with a
thermal shift of 3.3 °C in CREBBP and a KD of 9.3 µM. At the same time, compound 20
retained the activity towards BRD9 with a thermal shift of 5.1 °C and a KD of 6.3 µM. The
presence of a morpholine ring at the same position (derivative 22) could only slightly
improve the binding affinity towards CREBBP with a KD of 12 µM.
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A dihydropyrazoloꢀoxazole ring was installed (compound 23), which allowed us to revert
the hydrogen bond donor capacity of the dihydroꢀpyrazole ring bearing two NH groups to a
hydrogen bond acceptor fused ring. Remarkably, compound 23 showed a KD of 6.2 µM in
CREBBP and retained the LE of the initial hit (0.34 kcal/mol per heavy atom), which makes
it an attractive lead for further optimization. On the other hand, compound 24 exhibited a
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23
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39
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50
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drop in the thermal shift, probably due to steric clashes of the pꢀmethoxybenzyl group.
We then aimed at establishing new interactions with the hydrophobic residues located on
top of the binding site, Ile1122 and Leu1120 in CREBBP and Ala54 and Ile53 in BRD9, by
substitution of the indole moiety at the 5 position with a propoxy group (Table 1, compounds
27 and 28), a modification proven to be successful in our previous work and in compound
1a.39, 41 Unfortunately, the presence of the propoxy substituent retained the activity of ca. 10
ꢁM.
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Table 1. Evaluation of compounds 10-24, 27, 28 derived from 1b.
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10
11
12
13
14
15
ꢂTm (°C)[a]
CBP EP300 BRD9 BRD4(1)
Kd (ꢁM) [b]
CBP BRD9
Cmpd
R1
H
R2
H
R3
H
H
H
H
H
R4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.3
0.4
1.3
‒0.1
(0.1)
‒0.9
(0.4)
0.6
(0.5)
‒0.4
(0.5)
0.0
Me
20.0
5.3
ꢀ
1b
10
11
12
13
(0.1) (0.1)
‒0.1 ‒0.6
(0.2) (0.2)
(0.1)
‒0.8
(0.7)
1.0
(0.8)
‒0.5
(0.3)
‒1.0
(0.7)
H
H
ꢀ
6.6
ꢀ
2.2
(0.2) (0.1)
1.4 ‒0.7
(0.5) (0.7)
1.5 0.0
(0.4) (0.2)
2.8 1.0
2.7
ꢀSO2Me
ꢀSO2Me
Me
Me
Me
ꢀ
ꢀ
ꢀ
(0.3)
2.8
1.2
(0.5)
H
Me
17
3.5
14
(0.7) (0.3)
(1.6)
‒0.6 ‒0.3
(0.1) (0.1)
0.2
(0.2)
‒0.3
(0.1)
H
H
H
Me
Me
Me
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
15
16
17
‒0.9 ‒0.3
(0.1) (0.1)
0.3
(0.3)
0.1
(0.2)
‒0.2
0.0
‒0.5
(0.7)
‒0.9
(0.3)
(0.7) (0.2)
‒0.5 0.0
(0.6) (0.1)
‒2.3
(0.5)
H
H
H
Me
Me
Me
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
18
19
20
‒0.4 ‒0.4
(0.2) (0.2)
0.8
(0.5)
‒1.6
(0.3)
H
H
3.3
5.3
5.1
0.2
(0.7)
9.3
6.3
(0.7) (0.9)
(1.5)
‒0.6 1.0
(0.8) (0.7)
‒0.5
(0.4)
H
H
H
H
Me
Me
ꢀ
ꢀ
ꢀ
ꢀ
21
22
1.4 2.5
(0.3) (0.1)
‒0.6
(0.7)
0.6
(0.3)
12
3.5
2.2
1.8
1.5
(0.4)
6.2
15
ꢀ
23
(0.2) (0.4)
(1.0)
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0.1
0.0
0.3
(0.3)
‒0.3
(0.2)
ꢀ
ꢀ
24
(0.1) (0.1)
7
8
9
0.4
0.4
0.7
(0.1)
0.5
(0.3)
H
H
H
OPr
OPr
Me
Me
ꢀ
ꢀ
27
28
(0.2) (0.1)
10
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2.7
(1.0)
‒2.7
(1.2)
ꢀ
ꢀ
9.8
>50
[a] Median value of the shift in the melting temperature. The total number of measurements
for each compound and bromodomain was between 7 and 24. Ligand and protein
concentrations were 100 µM and 2 µM, respectively. SEM values are given in parenthesis.
The similar thermal shift values measured with the CREBBP bromodomain and its paralogue,
EP300, are consistent with the fact that identical residues are present in the acetylꢀlysine
binding site of both proteins. [b] Kd values were determined by a competition binding assay55,
56
in duplicates. Doseꢀresponse data and fitting curves can be found in the Supporting
Information. Dashes indicate data not acquired.
Importantly, with the exception of compounds 14 and 23, all derivatives displayed ꢂTm
values of less than 0.6 °C for BRD4(1), one of the most promiscuous bromodomains.11, 38, 57
Compound 1g and derivatives
Intrigued by the selectivity difference observed in the hit compound 1g towards BAZ2B
(Figure 2), we decided to examine its binding mode more closely. As we could not obtain
crystals of the complex of compound 1g with BAZ2B, several analogues were synthesized
(Table 2) aiming not only to obtain a crystal structure of the complex but also to improve the
binding affinity towards BAZ2B. The chloroacetamide moiety was replaced by metabolically
more stable amides in all derivatives to avoid covalent binding to the protein.
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Table 2. Evaluation of compounds 39-50 derived from 1g.
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23
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Kd (ꢁM)[a]
%Ctrl BAZ2B[b]
91.3
Cmpd
R1
H
R2
H
R3
H
R4
BAZ2B
>50
1g
H
H
CN
OMe
H
H
H
F
Acetyl
Acetyl
Acetyl
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
99.7
73.5
91.4
71.9
86.3
89.6
39
40
41
42
43
44
H
Me
OMe
H
H
OMe Acetyl
Acetyl
H
H
H
OMe Acetyl
OMe
Me
Me
H
H
ꢀ
77.5
87.3
45
46
OMe
140
Me
OH
H
H
H
H
OH
H
Acetyl
Acetyl
Acetyl
23
ꢀ
54.2
88.4
86.3
47
48
49
OH
ꢀ
29.8
(IC50 = 27 µM)
Me
H
OH
39
50
Kd values were determined by a competition binding assay55, 56 in duplicates. Doseꢀ
[a]
[b]
response data and fitting curves are in the Suppl. Inform.
Percentage of the measured
signal (i.e., percentage binding of acetylated histone peptide to BAZ2B) relative to the
negative control at a compound concentration of 50 µM. Lower values indicate stronger
binding of the compounds. The AlphaScreen competition binding assay was performed at
Reaction Biology. Dashes indicate data not acquired.
The affinity of the synthesized derivatives 39-50 against BAZ2B was assessed by an
AlphaScreen competition binding assay at a compound concentration of 50 ꢁM. Compound
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47, bearing an
oꢀmethyl and mꢀhydroxy substituent at the benzene ring, showed 54%
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8
reduction of signal relative to the negative control DMSO in the AlphaScreen binding assay,
which translated into a KD of 23 ꢁM and a LE value of 0.27 kcal/mol per heavy atom. Upon
substitution of the acetyl substituent at R4 by a m-CF3ꢀphenyl group, an IC50 value of 27 ꢁM
in the AlphaScreen assay and a KD of 39 ꢁM (BROMOscan) were measured for compound
50.
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Crystal structures of the most potent derivatives, 47 and 50, in complex with BAZ2B were
solved at 1.71 Å and 1.78 Å resolution, respectively. Compounds 47 and 50 (Figure 5A, B)
have essentially identical binding modes in BAZ2B. Similarly to the binding mode of the hit
1b in CREBBP (Figure 3), the Nꢀacetyl substituent of the indole moiety of compounds 47 and
50 is engaged in hydrogen bonds with the side chain of the conserved Tyr1901 and Asn1944,
where the hydrogen bond with Tyr1901 is bridged by a water molecule. An additional
hydrogen bond is formed between the carbonyl group of the acetamide and the backbone NH
of Asn1894 in the ZA loop. The o-methyl substituent of the phenol ring provides sufficient
steric hindrance to block the conformation of compounds 47 and 50 with their hydroxyphenyl
ring pointing towards Trp1887, which is the first residue of the WPF segment in BAZ2B.
The additional trifuoromethylbenzoate of compound 50 points towards the solvent (Figure
5B) which explains the similar affinity for BAZ2B of compounds 47 and 50 (Table 2).
As mentioned above, the bromodomains of BRD9 and BRPF1/3 belong to the same
subfamily. Aiming to obtain further structural information, we took advantage of the
availability in our laboratory of crystals of the apo state of the bromodomain of BRPF1b
which were soaked in a solution of compound 1b. In this way, we solved the crystal structure
of the complex of compound 1b with BRPF1b at a resolution of 1.35 Å (PDB code 5D7X).
Interestingly, the size of the soꢀcalled gatekeeper residue, small (Val1174 in CREBBP) and
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large (Phe714 in BRPF1b), has a noticeable influence on the orientation of the indole moiety
(Figure 5C). Furthermore, the presence of the bulkier Phe714 residue in the BRPF1b
bromodomain has a strong effect on the orientation of the dihydroꢀpyrazole ring which is
rotated by about 180° in BRPF1b with respect to the complex with CREBBP. Despite these
structural differences compound 1b has very similar affinity for the bromodomains of
CREBBP and BRPF1b with Kd values of 20 µM and 15 µM, respectively (see Supporting
Information).
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The superposition of the Xꢀray structures of compound 1b in CREBBP and BRPF1b with
compound 47 in BAZ2B provides additional evidence on the influence of the gatekeeper
residue for ligand binding (Figure 5, D). Interestingly, the 6ꢀmemberedꢀring of the indole
moiety of compound 47 is located between the indole of compound 1b in BRPF1b and
CREBBP because the gatekeeper residue in BAZ2B (Ile1950) is smaller than the one in
BRPF1b (Phe714) and bigger than that in CREBBP (Val1174). It is important to note that in
these three crystal structures (viz., the complexes 1b/CREBBP, 1b/BRPF1b, and 47/BAZ2B)
there are no crystal contacts in the binding site that could affect the orientation of the acetyl
indole scaffold. Moreover, the structure of BAZ2B is essentially identical in the complex
with compounds 47 and 50, and the same is observed for the structures of CREBBP in the
complex with compound 1b and a previously reported acetyl benzene ligand (PDB code
4TQN) which differ only by a small rigidꢀbody displacement of the ZA loop. It is also
interesting to compare with the orientation of the scaffold of the BAZ2B inhibitor 1a. The
same progressive tilting of the indole emerges from the structural superposition of the
complex of BAZ2B and the nanomolar inhibitor 1a (PDB code 4RVR)41 with the crystal
structures of the hit compound 1b in CREBBP and BRPF1b (Figure 5, E). Overall, these
structural data suggest that acetyl indole mimics the acetylated lysine side chain in its rather
unselective binding to bromodomains. In addition, the precise orientation of the indole
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doubleꢀring system and the substituent at its position 3 are influenced by the size of the
gatekeeper residue which is different in different bromodomains.
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Figure 5. The crystal structures show the influence of the gatekeeper on the binding mode of
the ligand. A) Crystal structures of the BAZ2B bromodomain in complex with compound 47
(PDB code 5E73). B) Same as A) for the complex with compound 50 (PDB code 5E74). C)
Crystal structure of the complex of compound 1b (yellow) and the BRPF1b bromodomain
(blue) (PDB code 5D7X) structurally superposed to the crystal structure with CREBBP
(compound 1b shown in green and CREBBP side chains shown in grey). D) Superposition of
the crystal structures of compound 47 (brown) in BAZ2B (pink) and compound 1b (green
and yellow), in CREBBP (grey) and BRPF1b (blue), respectively. The side chain of the
gatekeeper is shown in cylinders with labels for the residue number. E) Same as D) for the
BAZ2B inhibitor 1a (brown) (PDB code 4RVR). In all panels, hydrogen bonds are shown as
yellow dashed lines and the crystallographic water molecules are represented by spheres.
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Conclusions
We have discovered in silico a series of small molecule antagonists of the bromodomains
of CREBBP and BAZ2B by docking about 200 compounds containing an acetyl indole
moiety, which we had identified by scaffold hopping from a potent BAZ2B ligand.
Considering that only 14 compounds were tested in vitro (by a competition binding assay),
the hit rate of the in silico screening based on scaffold hopping and docking is 50% and 29%
for CREBBP and BAZ2B, respectively. One of the original hits (compound 1b) has an
equilibrium dissociation constant of 20 µM, 15 µM, and 5.3 µM for CREBBP, BRPF1b, and
BRD9, respectively. With a relatively small derivatization campaign (i.e., ca. 30 derivatives)
we were able to improve the affinity for CREBBP and BAZ2B, and maintain the ligand
efficiency (0.34 kcal/mol per heavy atom for both hit 1b and derivative 23 in CREBBP) or
even slightly improve it (from <0.26 for hit 1g to 0.27 kcal/mol per heavy atom for
compound 47 in BAZ2B). The crystal structures of three acetyl indole derivatives in
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complex with three different bromodomains confirm the binding mode predicted by docking
with the acetyl oxygen of the ligand involved as hydrogen bond acceptor in two hydrogen
bonds with the conserved Asn side chain in the BC loop and a structural water that acts as
bridge to the conserved Tyr of the ZA loop. Moreover, the Xꢀray structures show that the
size of the gatekeeper side chain (Val1174, Ile1950, and Phe714 in the bromodomains of
CREBBP, BAZ2B, and BRPF1b, respectively) influences the orientation of the indole
moiety. This structural information can be used to further improve the selectivity for a single
bromodomain target or a small subset of bromodomains sharing the same gatekeeper residue.
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EXPERIMENTAL SECTION
1. Scaffold hopping. We have used an activityꢀoriented fingerprint that consists of 1)
chemical features such as hybridization states and different types of hydrogen bonding donors
and acceptors; 2) twoꢀdimensional topology index as a way to reflect spatial arrangement of
such features; and 3) threeꢀdimensional shape descriptors (Figure S1 in the supporting
information). A similarity coefficient is computed between two fingerprints ranging from 0 to
1, with 1 being the highest similarity, where the two molecules are not necessarily identical.
As such, scaffold hopping resembles pharmacophore mapping, but distinguishes itself by
using a molecular fingerprint.
2. Docking and scoring. The genetic algorithmꢀbased program for flexible ligand docking
50ꢀ52
has been described in previous applications.48,
About 20 docking poses for each
compound were first minimized by the CHARMM program58 and subsequently ranked by a
transferable scoring function.45, 50ꢀ54
49
3. Assays. Thermal shift measurements were carried out as previously described.39,
Thermal shift assays detect, by a fluorescent dye, the increase in thermal stability of a protein
in the presence of a ligand.59 BROMOscan technology is a competition experiment that uses
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an immobilized ligand and a DNA tagged bromodomain protein.55, 56 Compounds that bind to
the bromodomain of interest will prevent bromodomain binding to the immobilized ligand.
The amount of bromodomain captured on the solid support is then quantified by qPCR and
dissociation constants are calculated. AlphaScreen assays consist of a donor bead that is able
to transfer singlet oxygen to an acceptor bead that is in close proximity, and as a result, the
acceptor bead emits a lumininescent/fluorescent signal. In the presence of a bromodomain
ligand, the donorꢀacceptor complex is disrupted leading to a loss of singlet oxygen transfer
and loss of the fluorescent signal. Further details about the assays can be found in the
Supporting Information.
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4. X-ray crystallography. The Hisꢀtagged human bromodomains of CREBBP (residues
1081ꢀ1097), BRPF1b (residues 626ꢀ740), and BAZ2B (residues 1858ꢀ1972) were expressed
in E.coli. The purification procedures are reported in the Supporting Information. The
inhibitors were soaked into apo crystals of the bromodomains of BRPF1b and BAZ2B while
the structure of the complex of compound 1b and CREBBP was obtained by coꢀ
crystallization as described in the Supporting Information. Data collection and refinement
statistics are given in Table S1.
ANCILLARY INFORMATION
Supporting Information. General procedures for scaffold hopping, synthesis and
characterization, biophysical and biological evaluation of final compounds, and Xꢀray crystal
structure refinement data is available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The PDB accession codes for CREBBP and BRPF1b in complex with the hit compound 1b
are 4TS8 and 5D7X, respectively. Coordinates and structure factors for the BAZ2B
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bromodomain in complex with compounds 47 and 50 have been deposited in the PDB with
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accession codes 5E73 and 5E74, respectively.
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9
AUTHOR INFORMATION
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Corresponding Author
*Amedeo Caflisch. Eꢀmail: caflisch@bioc.uzh.ch; Phone number: +41 44 635 55 21
* Cristina Nevado. Eꢀmail: cristina.nevado@chem.uzh.ch; Phone number: +41 44 635 39 45
Funding Sources
This work was supported by the Swiss Cancer Society (Krebsliga, KFSꢀ3098) and the Swiss
National Science Foundation (315230_149897). We thank the Structural Genomics
Consortium at Oxford University for providing the plasmids of all the bromodomains but
EP300 (provided by AddGene).
ACKNOWLEDGMENT
We thank Dr. Emilie Frugier and Dr. Dimitrios Spiliotopous for interesting discussions and
help with the thermal shift assay. We thank Lisa Caflisch, Ursina Suter, and Cecilia Ferdenzi
for protein purification and Dr. Alvaro Salvador and Anna Coppola for their efforts towards
the synthesis of the hit compound 1b. Nicholas Deerain is acknowledged for performing part
of the thermal shift assays. We are grateful to the staff at PXI and PXIII beamlines, Swiss
Light Source, Paul Scherrer Institute (Villigen, Switzerland) and at XDR1 beamline,
ELETTRA Synchrotron Light Source (Trieste, Italy) for onꢀsite assistance.
ABREVIATIONS
BAZ2B, bromodomain adjacent to zinc finger domain, 2B ; BET, bromodomain and extra
terminal domain; BRD2, 3, 4, 7, 9 bromodomain containing 2, 3, 4, 7, 9; BRD4(1),
first/second bromodomain of BRD4; BRPF1b, bromodomain and PHD finger containing, 1;
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CBP, CREB binding protein; CREBBP, CREB binding protein; DMAP: 4ꢀ
dimethylaminopyridine; DME: 1,2ꢀdimethoxyethane; DMF: dimethyl formamide; DMSO,
dimethyl sulfoxide; EP300, E1A binding protein p300; HAT, histone acetyl transferase; LE,
ligand efficiency; NMC, NUT midline carcinoma; NUT, nuclear protein in testis; PHD, plant
homeodomain; PMB, 4ꢀmethoxybenzyl ether; SWI/SNF, SWItch/Sucrose nonꢀfermentable;
TBAB: tetraꢀnꢀbutylammonium bromide; TBAHS: tetrabutylammonium hydrogen sulfate;
TBAI, tetrabutylammonium iodide.
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