Identification of a Chemical Probe for Family VIII Bromodomains through Optimization of a Fragment Hit

Article abstract of DOI:10.1021/acs.jmedchem.6b00012

The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.

Full text of DOI:10.1021/acs.jmedchem.6b00012

Article
pubs.acs.org/jmc
Identification of a Chemical Probe for Family VIII Bromodomains
through Optimization of a Fragment Hit
Brian S. Gerstenberger,† John D. Trzupek,† Cynthia Tallant,‡,§ Oleg Fedorov,‡,§
Panagis Filippakopoulos,‡,∥ Paul E. Brennan,‡,§ Vita Fedele,‡,§ Sarah Martin,‡,§ Sarah Picaud,‡,§
Catherine Rogers,‡,§ Mihir Parikh,⊥ Alexandria Taylor,⊥ Brian Samas,# Alison O’Mahony,∇ Ellen Berg,∇
Gabriel Pallares,○ Adam D. Torrey,○ Daniel K. Treiber,○ Ivan J. Samardjiev,□ Brian T. Nasipak,¶
Teresita Padilla-Benavides,¶ Qiong Wu,¶ Anthony N. Imbalzano,¶ Jeffrey A. Nickerson,¶
Mark E. Bunnage,† Susanne Muller,‡,§ Stefan Knapp,‡,§,∞ and Dafydd R. Owen*,†
̈
†Pfizer Worldwide Medicinal Chemistry, 610 Main Street, Cambridge, Massachusetts 02139, United States
‡Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom
§Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom
∥Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3
7DQ, United Kingdom
⊥Pfizer Pharmaceutical Sciences, Eastern Point Road, Groton, Connecticut 06340, United States
#Pfizer Worldwide Medicinal Chemistry, Eastern Point Road, Groton, Connecticut 06340, United States
∇Bioseek Inc., Division of DiscoveRx, 310 Utah Avenue, South San Francisco, California 94080, United States
○KinomeScan, Division of DiscoveRx, 11180 Roselle Street, Suite D, San Diego, California 92121, United States
□Eurofins Lancaster PPS, Eastern Point Road, Groton, Connecticut 06340, United States
¶Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655,
United States
∞Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences (BMLS), Johann Wolfgang Goethe University,
Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
S
* Supporting Information
ABSTRACT: The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an
acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-
competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader
bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding
mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other
bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening
hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported.
We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte
and myoblast cell differentiation assays.
proteins in humans.3 Bromodomains selectively recognize ε-N-
lysine acetylation motifs, a key event in the reading process of
INTRODUCTION
■
Bromodomains are essential interaction motifs for the recruit-
ment of transcriptional regulators to acetylated chromatin.1 They
these post-translational modifications that are important
were first identified as a conserved sequence motif present in the
Drosophila homologue of Brahma (BRM),2 and they constitute a
Received: February 2, 2016
family of 61 highly diverse interaction domains present in 46
© XXXX American Chemical Society
A
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components of the so-called “epigenetic code”. The recent
discovery of potent and highly specific inhibitors for the
bromodomain and extraterminal domain (BET) family of
bromodomains4 has stimulated intensive research activity
particularly in oncology, where BET proteins regulate the
expression of key oncogenes. The first wave of inhibitors from
this BET bromodomain class has entered clinical testing, and
their progress has garnered significant attention.5 As a family of
protein targets, bromodomains contain a central, deep, and
largely hydrophobic acetyl-lysine binding pocket that is an
attractive site for the development of selective and potent protein
interaction inhibitors that mimic the endogenous ligands. The
predicted favorable druggability of these acetyl-lysine interaction
domains6 suggests that other bromodomain members beyond
the BET family may also be selectively targeted. Mammalian
switch/sucrose nonfermentable (SWI/SNF) complexes play a
key role in cell differentiation and proliferation, and represent an
essential component of the embryonic stem cell (ESC) core
pluripotency transcriptional network.7 Brahma-related gene-1
(BRG1, SWI/SNF related, matrix associated, actin dependent
regulator of chromatin, subfamily a 4 (SMARCA4)) and the
related protein BRM (SMARCA2) are the central ATPase
components of these multisubunit complexes. In addition to an
essential role in pluripotency and development, genetic lesions of
SWI/SNF complexes have been strongly linked to cancer
development and a synthetic lethal relationship between
SMARCA4 and SMARCA2 has been reported.8 SMARCA2
and SMARCA4 are multidomain proteins that contain both
DNA and protein interaction modules within their structure.
Importantly, these domains include a C-terminal bromodomain
in each protein.9 The multidomain PB1 protein contains six
bromodomains classified to family VIII of bromodomain
containing proteins based on structural and sequence homol-
ogy.3 Protein polybromo-1 (PB1) appears in the polybromo,
Brg1-associated factors (PBAF) complex (human analogue of
SWI/SNF) alongside either SMARCA2 or SMARCA4. Inhib-
itors targeting the SMARCA2, SMARCA4, and PB1 bromodo-
mains would be useful tools to study the modulation of these
SWI/SNF mediated processes, and any chemical probes
identified could also offer starting points for druglike molecules
of potential therapeutic utility.
explained by the identification of a rare binding mode in the field
of bromodomain small molecule inhibitors where the phenolic
functionality displaced water molecules typically retained in
other bromodomain inhibitor complex binding modes.10 Very
few other examples of such deep pocket binding have been
reported in bromodomains.11 The crystal structure and
characterization of the salicylic acid fragment hit 1 have been
reported elsewhere.12 With a weak but ligand efficient fragment
hit successfully crystallized in PB1(5), further molecular
optimization was guided toward exploiting lipophilic interactions
for larger ligands derived from the salicylic acid lead 1. These
changes were designed to enhance potency levels closer to those
set as an expectation of a quality chemical probe, around 100 nM
in a biochemical or biophysical assay.13 The PB1(5) small
molecule X-ray structure points to asparagine N707 and tyrosine
Y664 as key hydrogen bond interactions for the phenolic and
carbonyl functional groups of 1 with the protein. The defined
orientation of the carbonyl group, in accepting a hydrogen bond
from N707, directs a potential substituent in more elaborated
ligands out of the protein, through and over a lipophilic surface of
amino acid side chains. Maintaining the phenolic and carbonyl
substituents off the phenyl group as substructural requirements,
structure−activity relationships (SAR) were initially explored
primarily through commercial purchase and file screening from
the Pfizer chemical compound library. Enamides proved to be
good surrogates for the previously stated key elements of the
salicylic acid pharmacophore. Commercially available 2 was
significantly more active than salicylic acid 1 in the primary DSF
assay that was used to assess SAR, with compounds being tested
at 10 μM (Table 1).
The DSF thermal shift of 4.9 °C for 2 in PB1(5) at 10 μM
indicated significantly enhanced binding to the protein compared
to salicylic acid 1 and that the acidic functional group was not
essential. The predicted binding mode for 2 was confirmed by a
crystal structure in PB1(5) showing the dimethylamino portion
of the enamide emerging toward the protein surface (Figure 2A).
A number of commercially available enamides showed similar
levels of activity. The selection of enamides available was further
enhanced by a simple one-step chemical synthesis of analogs in
which both primary and secondary amines could be function-
alized with chromone-3-carboxylic acid through a decarbox-
ylative ring opening (Scheme 1).14
The protocol was sufficiently robust to be run using parallel
synthetic chemistry methods with a high success rate for the
amine monomer set selected as the library variable. The first
library run yielded 130 compounds at a success rate of 73%, and a
number of pyrrolidine, piperidine, and piperazine based
compounds possessed significant activity against PB1(5),
SMARCA2, and SMARCA4 as judged by DSF (compounds
3−8, Table 1). For compounds such as 3, Tm shift values
increased by over 2 °C against PB1(5), SMARCA2 (both
isoforms), and SMARCA4 when compared to the simple
dimethylamine derivative 2. A crystal structure in PB1(5) was
secured for compound 3 which exemplifies the key features of the
interaction mode (Figure 2B−D). Compounds derived from
primary amine monomers showed poor binding. A small number
of functionalized chromone-3-carboxylic acid templates were run
in the library (as a negative SAR control), and as the crystal
structure of the initial fragment hit 1 suggested, room deep in the
bromodomain pocket was restricted and a specific substitution
pattern on the phenyl group was required for activity. As an
example, the closely related pyrrolidines 9 and 10 show that the
addition of a methoxy group ortho to the phenol eliminated
RESULTS AND DISCUSSION
■
In the search for chemical starting points for family VIII
bromodomains, a fragment screen against the PB1(5)
bromodomain using differential scanning fluorimetry (DSF)
successfully identified salicylic acid 1 as weak but highly ligand
efficient hit (Figure 1). This was confirmed as a direct interaction
with the protein by isothermal titration calorimetry (ITC). Of
particular note was the high selectivity for family VIII
bromodomains over other bromodomain family members for
what is a very small fragment molecule. This selectivity was
Figure 1. Fragment hit 1, a commercially sourced screening hit 2, and a
novel, chemically synthesized analogue 3 with representative DSF data
at 10 μM against the PB1(5) bromodomain.
B
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a
Table 1. DSF Thermal Shift (°C Stabilization, 10 μM Compound) SAR for Family VIII Bromodomains
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Table 1. continued
a
Values are derived from at least three determinations with standard deviations quoted.
family VIII activity completely. In general, the first round of
compound synthesis yielded analogs with a relatively limited
interaction with PB1(2) as determined by DSF, indicating the
potential for intrafamily VIII selectivity for compounds derived
from pyrrolidines, piperidines, and piperazines. This was not
easily explained by the crystal structures available or protein
sequence differences, but this contrasts with earlier examples
from the BET family where all chemotypes inhibit all members
of this bromodomain subfamily, and selectivity between BRD2,
-3, -4, and T has yet to be established through conventional
modes of inhibition.15
suppliers. 320 compounds were delivered at a 77% synthetic
success rate using the same parallel synthesis protocol.
Physicochemical properties were constrained such that they
would not compromise potential cell penetration for com-
pounds. Targets were designed within cLogP lower and upper
limits of 1 and 4. Once again, compounds with significant PB1(5)
activity were identified by DSF with Tm shifts of >8 °C
(compounds 11−15, Table 1). These fused bicyclic compounds
presented a different family VIII selectivity profile compared to
the first library, with PB1(2) activity now observed alongside
PB1(5), SMARCA2, and SMARCA4.
A second round of library chemistry looked at a greater
number of fused, bicyclic amines based on compound 3. This
compound had stood out in the first round as showing notable
levels of PB1(2) activity in the DSF assay compared to other
analogs. These bicyclic amines were primarily sourced, being
slightly more complex in structure, from the Pfizer compound
collection of secondary amines and not from commercial
Through use of the DSF assay, identification of both
moderately selective and “pan” profiles for family VIII
bromodomain binders from the same chemotype offered the
chance to design chemical tools that could be used in
conjunction with one another to establish the roles of these
bromodomain containing proteins in SWI/SNF complexes,
where both PB1(2) and PB1(5) bromodomain containing
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Figure 2. PB1 complexes with hydroxyphenylenamides. (A) Complex of PB1(5) with 2. The ligand occupies the acetyl-lysine cavity of the
bromodomain directly coordinating to the conserved asparagine (N707) and Y664. Key residues are annotated, and secondary structure elements are
displayed. The ligand’s 2FcFo map contoured at 2σ is displayed in the inset. (B) Complex of PB1(5) with 3. As in (A) the compound binds in an acetyl-
lysine mimetic fashion, directly engaging the conserved asparagine (N707) and Y664 as well as A703 via a backbone interaction. Key residues and
secondary structure elements are annotated. The ligand’s 2FcFo map contoured at 2σ is displayed in the inset. (C) Comparison of apo PB1(5) (PDB
code 3G0J) and PB1(5)-3 complex. The ligand inserts deep into the hydrophobic pocket of the protein, resulting in distortion of the plasticity and an
outward motion of the ZA-loop. Key residues that change orientation are highlighted in yellow. The four conserved water molecules found in most
bromodomain structures (w1−4) have been displaced in the complex. (D) Side view of 3 bound onto PB1(5) overlaid with the four conserved water
molecules found on the apo-PB1(5) structure. The ligand inserts deep into the cavity displacing the four water molecules (w1−4) and engaging the
protein via N707 and A703 while stacking over M699.
given the potential for chemical instability of the embedded
enamide within the structure. This functional group could be
hydrolytically unstable as a masked 1,3-dicarbonyl and
importantly render the compounds unsuitable for their primary
intended use as chemical probes for cell biology. Chemical
stability assessment of compound 11 confirmed the expected
potential for hydrolysis, and the rate of decomposition was
considered unsuitable for use in extended cellular assays. The
next round of compound design focused on combining strategies
for chemical stability with the potent binding affinity for family
VIII bromodomains. Factors influencing the mechanism for
hydrolysis such as leaving group ability of the amine, steric
hindrance of the hydrolyzed center, and likelihood for forming an
intermediate iminium ion were considered. Compound 15 had
indicated that substitution next to the hydrolytically displaced
nitrogen atom of the departing amine was tolerated. The bridged
piperazine proposed, in what was finally identified as the
nominated chemical probe 1612 (PFI-3, Scheme 2), conferred
significantly enhanced chemical stability over the earlier
prototype 11.
Scheme 1. General Method for Library Chemistry Amenable
Synthesis of Family VIII Bromodomain Binders
proteins can be present alongside either SMARCA2 or
SMARCA4. In total, some 400 compounds were synthesized in
these two rounds of library chemistry and the most potent
compounds (initially judged by Tm shift in DSF assays) were
progressed to isothermal titration calorimetry (ITC) for absolute
binding affinity determination. Compound 11 met the
pharmacological probe criteria with particularly potent binding,
as determined by ITC, to PB1(2), PB1(5), and SMARCA4 at
152 nM, 28 nM, and 49 nM, respectively.
Despite this promising profile and the relative ease with which
SAR could be explored through the one-step chemical synthesis
of analogs, the utility of the chemotype remained in question
Scheme 2. Synthetic Route to Compound 16 (PFI-3, PF-6687252)
E
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Figure 3. Compound 17 is a potent pan-inhibitor of SMARCA2/SMARCA4 and PB1 families. (A) Chemical structure of inhibitor 17. (B) Isothermal
titration data of the interaction of compound 17. Top panel shows the raw binding exotherms for each SMARCA2 injection into the cell containing the
inhibitor solution. Bottom panel displays normalized binding enthalpies and the corresponding nonlinear least-squares fits (insert) for the two families
SMARCA2/SMARCA4 and PB1 bromodomains. (C) 2Fo − Fc electron density map contoured at 1.5σ around the inhibitor at 1.7 Å resolution. (D)
Complex of SMARCA2 with compound 17. The hydroxyphenyl propenone core scaffold adopts the same conformation of previous crystallized
analogues in the acetyl-lysine binding site. The cyclopropanamine group is pointing toward the ZA-loop stabilized by hydrophobic interactions with the
lipophilic side chains of L1418, P1413, and E1417. (E) Surface representation of the SMARCA2 acetyl-lysine binding site in complex with compound
17.
The improved chemical stability over a range of conditions
relevant to cellular biology studies confirmed compound 16 as a
first in class chemical probe for PB1(5), SMARCA2, and
SMARCA4 while offering considerable selectivity over PB1(2).
Compound 16 had a half-life of >250 h in PBS, pH 7.4, at 20 °C.
In contrast, compound 11 had a half-life of 20 h under the same
conditions. Greater than 90% of the compound 16 was recovered
after 1 week in cell media at 37 °C (Supporting Information data,
Table T1). Strong Tm shift data for 16 (with the exception of
PB1(2) were confirmed by <100 nM ITC KD determinations for
PB1(5) and SMARCA4.12 Compound 16 showed complete
selectivity over all non family VIII bromodomains as judged by
both a Tm shift panel of 40 bromodomain proteins and the
BROMOscan screen from DiscoveRx.16,17
With just three bromodomains targeted by compound 16, the
attraction of a less selective family VIII profile, particularly a
compound with PB1(2) activity, was sought in order to offer
tools of orthogonal selectivity combinations for cellular biology
experiments. The chemical synthesis libraries initially performed
had identified compound 11 as a potent binder of PB1(2) in
addition to its SMARCA2, SMARCA4, and PB1(5) activity and,
but for its insufficient chemical stability, would have been
nominated as a chemical probe. Having established strategies for
conferring chemical stability in the more selective and syntheti-
cally expedient piperazine system that yielded compound 16, a
similar bicylization approach was applied to compound 11. This
required a considerably lengthier and complex synthetic route
that ultimately yielded compound 17 (Supporting Information
Scheme S1). Compound 17 (Figure 3A) is a potent binder to
most of the family VIII bromodomains as determined by ITC
(Figure 3B) and is not active against the remaining
bromodomain proteins as judged by a Tm shift panel of 40
bromodomains and also BROMOscan profiling from DiscoveRx
(Supporting Information Figure F3 and Figure F4). A protein
cocrystal structure for compound 17 was solved in SMARCA 2
(Figure 3C and Figure 3D), and this was also used to infer the
absolute stereochemistry of the compound that was prepared as a
single enantiomer, having used chiral supercritical fluid
chromatography (SFC) to separate a racemic amine inter-
mediate during the synthesis (Supporting Information Scheme
S1). A small molecule X-ray structure of a heavy atom containing
amide derivative of amine 17f (Supporting Information Scheme
S1) was also secured, and this provided definitive confirmation of
the absolute stereochemistry of compound 17 (Supporting
Information Figure F1)
The cellular activity of compound 17 for SMARCA2 was
assessed using a fluorescence recovery after photobleaching
(FRAP) assay (Figure 4)18 U2OS cells were treated with the
histone deacetylase (HDAC) inhibitor suberoylanilide hydroxa-
mic acid (SAHA) to induce hyperacetylation, resulting in a better
assay window. Cells were transfected with a green fluorescent
protein (GFP)-SMARCA2 construct or as control a GFP-
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Figure 4. SMARCA2 and PB1 FRAP assays. (A) Half-life of fluorescence recovery (t1/2) for transfected cells expressing GFP-tagged SMARCA2 (WT)
treated with compound 17 at different doses or mutant SMARCA2 (N1464F). Shown are bars representing the mean t1/2 calculated from individual
recovery curves of at least 20 cells per group, and error bars depict the standard error of the mean (SEM). (B) Raw data fluorescent recovery curves
corresponding to the fluorescence recovery shown in (A). (C) Half-recovery times of fluorescence recovery (t1/2) for transfected cells expressing GFP-
tagged PB1 (WT) treated with compound 17 at different doses or mutant PB1 (N263A or N707F). Shown are bars representing the mean t1/2 calculated
from individual recovery curves of at least 20 cells per group, and error bars depict the standard error of the mean (SEM). (D) Raw data fluorescent
recovery curves corresponding to the fluorescence recovery shown in panel (C). ∗ indicates a p-value compared to WT treated with 2.5 μM SAHA:
(∗∗∗∗) p < 0.0001; (∗∗∗) p < 0.001; (∗) p < 0.03.
SMARCA2 construct, in which the conserved asparagine
(N1464F) essential for recognizing the acetylated lysine had
been mutated. Compound 17 accelerated FRAP half-recovery
time (t1/2) significantly in a dose dependent manner to the level
of the N1464 mutant, indicating that compound 17 was able to
bind to the SMARCA2 bromodomain and displace full length
SMARCA2 from chromatin (Figure 4A and Figure 4B). We next
tested if compound 17 was able to also displace the GFP-linked
PB1 from chromatin. Again, SAHA was used to increase the assay
window. The assay did show a larger variability than the
SMARCA2 assay, possibly due to the presence of six
bromodomains acting as chromatin binding and scaffolding
domains.19 Mutation of the conserved asparagine in the second
(N263A) and fifth (N707F) bromodomain of PB1, respectively,
indicated that a significant difference in half-recovery time could
only be achieved by preventing the second bromodomain of PB1
from binding to chromatin. Compound 17 was able to displace
PB1 from chromatin at 1 μM concentration but not at a lower
concentration of 100 nM as observed in the SMARCA2 FRAP
assay. These data are in good agreement with the mutant data
indicating that the second bromodomain, for which 17 has a
lower potency, is the relevant domain for chromatin binding
(Figure 4C and Figure 4D). As expected, the inactive compound
18 had no effect on the half-recovery time of GFP-PB1.
With novel tools for family VIII bromodomains in hand,
known to be cell penetrant through target engagement assays,
phenotypes for the molecules were sought. The BioMAP primary
human cell platform from DiscoveRx has been shown to
successfully identify potential phenotypes for bromodomain
chemical probes.20 The early and strong phenotype derived from
binding BET bromodomains by small molecules is very clearly
identified by BioMAP systems with broad effects at multiple
concentrations detected across a panel of cell-based assays
modeling various aspects of human disease biology. With
nothing known about potential phenotypes derived from small
molecule binding to family VIII bromodomains, compound 16
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Figure 5. Contrast in BioMAP profile of the (A) BET bromodomain inhibitor PFI-1 and the (B) family VIII bromodomain inhibitor PFI-3 (16) in 12
human primary cell-based systems against a number of cellular end points. Compounds were tested over 5 concentrations as indicated, and effects were
normalized to vehicle and expressed as log ratio. The gray shading is the 95% vehicle control envelope for historical control data. Values that fall outside
this envelope at two or more consecutive concentrations and with an effect size of >20% ((|log10 ratio| > 0.1) are denoted as statistically significant and
are annotated. The protein-based parameters tested in each system are listed along the bottom of the graph.
(PFI-3) was profiled in BioMAP by DiscoveRx (Figure 5). Under
the standardized conditions run in the BioMAP assay platform
protocol, compound 16 had a largely “silent” profile based on
statistically significant effects outside the historical control
envelope (Figure 2B). The compound showed no inhibitory
effects against a large number (148) of cellular end points in 12
primary human cell-based systems. This is in stark contrast to our
previously disclosed BET inhibitor PFI-1 profiled in the same
platform which exhibited strong inhibition of inflammation,
immune activation, tissue/matrix remodeling, and hemostasis-
related biomarkers, illustrating that not all bromodomain
containing proteins will present obvious phenotypes when
bound to potent small molecule inhibitors.
With no obvious phenotype found in the BioMAP system, the
potential for PB1 mediated cellular efficacy of 16 and 17 was
tested using two clear cell renal cell carcinoma (ccRCC) cell lines
(786-O and Rcc4), the former having a wild type PB1 expression
and the latter having PB1 downregulation.21 On the basis of this
role for PB1, compound treatment of a cell line with a wild type
expression should result in an increase in proliferation.21
However, treatment with either compound 16 or 17 (that bind
to the PB1 bromodomains) did not show any effect on cell line
proliferation (Supporting Information Figure F5).
SMARCA4 proved to be the functionally important domain with
respect to cancer cell line efficacy.23 Cellular activity in FRAP
assay systems and a stem cell phenotype have been disclosed
elsewhere for compound 16,12 but additional non-oncology
phenotypes for compounds 16 and 17 are now reported here.
The SWI/SNF complex mediates a broad spectrum of
biological processes including cell proliferation, differentiation,
DNA replication and repair. Mouse knockout models demon-
strate that SMARCA4 is necessary for early embryogenesis,
whereas SMARCA2 knockout mice are viable without overt
visible defects.24 Previous studies have established the role of
SMARCA4 in regulating the transcriptional cascade of gene
expression during muscle cell and adipocyte differentiation.25
Furthermore, it has been shown that the C-terminal region of
SMARCA4, which includes the bromodomain, is subjected to
post-translational modification that regulates SMARCA4 activity
in myogenesis.26 These observations prompted us to dissect the
function of the SMARCA4 bromodomain in the cell differ-
entiation process using the small molecule tools 16 and 17
instead of gene mutagenesis.
Cell viability was measured by standard 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and
neither compound showed significant cytotoxic effect on cells up
to 50 μM dose (Figure 6A). SMARCA4 in cell target engagement
was confirmed for compounds 16 and 17 through a SMARCA4
FRAP assay (Supporting Information Figure F6) analogous to
the SMARCA2 method shown in Figure 4. Both compounds
blocked C2C12 myoblast differentiation in cells grown in the
presence of either compound 16 or compound 17. Defects in cell
fusion, evident through shortened myotubes containing fewer
The potential importance of family VIII bromodomain
containing proteins in the chromatin remodeling SWI/SNF
complexes and their role in cancer has however been highlighted
through many recent publications.22 In combination with knock-
down experiments, compound 16 showed no role for
bromodomain inhibition in the cancer phenotypes highlighted.
The adenosine triphosphate ATPase domain of SMARCA2 and
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Figure 6. (A) Cytotoxicity tests for the compounds were conducted in C2C12 and C3H10T1/2 cells which were treated with increasing doses of vehicle
(DMSO), compound 16, or compound 17 for 72 h. Cell viability was measured by standard MTT assay. The compounds did not have significant
cytotoxic effect on cells up to 50 μM dose. Cells treated with the highest dose of 100 μM caused 50% cell death. (B) Compounds 16 and 17 block
differentiation in C2C12 myoblasts normally seen within 48 h. This is assessed by monitoring the expression of the early marker of differentiation,
myogenin, and by monitoring cells undergoing fusion to become myotubes that express heavy chain (MyHC). Blockage of myogenin expression
required significantly higher concentrations of both compounds. (C) Compounds 16 and 17 attenuated adipocyte differentiation. On addition of an
adipogenic cocktail, C3H10T1/2 mesenchymal cells can be induced to differentiate to adipocytes that are manifested by the accumulation of lipid
droplets visualized by Oil Red O staining. In the presence of increasing doses of both compounds, there were fewer cells differentiated and lipid
accumulation was decreased dramatically. Compound 16 was more effective than compound 17 in blocking adipogenesis.
nuclei than controls, as well as failure to induce MyHC
expression were observed in cells treated with 1 μM compound
16 or 5 μM compound 17 (Figure 6B). Compounds 16 and 17
also attenuated adipocyte differentiation, and lipid accumulation
was decreased dramatically. Significant effects were seen at 1 μM
for compound 16 and 5 μM for compound 17. Near complete
inhibition of adipogenesis was achieved at 20 μM compound 16
(Figure 6C).
Compound 16 (PFI-3) is a first in class chemical probe for
family VIII bromodomains and was discovered through
optimization of the fragment hit salicylic acid 1. By use of two
rounds of library synthesis and optimization for chemical
stability, all guided by structure based design strategies,
I
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mixture was stirred for another 16 h at 110 °C. TLC (DCM/MeOH =
10:1) showed the reaction was complete. Water (3 mL) was added, and
the mixture was extracted with EA (10 × 3). The organic layer was dried
by anhydrous Na2SO4 and purified by column chromatography to give
16b as a yellow solid (620 mg, 89%). 1H NMR (400 MHz, DMSO-d6) δ
= 1.31−1.46 (m, 9 H), 1.90 (d, J = 11.32 Hz, 1 H), 3.18 (d, J = 9.76 Hz, 1
H), 3.25 (d, J = 9.37 Hz, 1 H), 3.29−3.40 (m, 1 H), 3.49 (t, J = 8.39 Hz, 1
H), 4.39−4.54 (m, 1 H), 4.78 (d, J = 6.63 Hz, 1 H), 6.49−6.63 (m, 1 H),
7.46−7.56 (m, 1 H), 8.07 (d, J = 5.07 Hz, 1 H).
compound 16 was nominated as a chemical probe and has been
used to identify novel examples of small molecule mediated
phenotypes in myoblasts and adipocytes. The identification of
compound 17 offers a cell permeable, family VIII bromodomain
tool compound with additional PB1(2) activity over and above
that of PFI-3 (16). Although the use of compounds 16 and 17
alongside one another did not offer any significant differentiation
in phenotype in the cellular systems studied in this work, they
stand as well characterized tool compounds to further study
hypotheses surrounding the role of certain bromodomain
containing proteins within the SWI/SNF complex, in cells.
(E)-1-(2-Hydroxyphenyl)-3-((1R,4R)-5-(pyridin-2-yl)-2,5-
diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one (16, PFI-3, PF-
06687252). To a solution of the starting material 16b (620 mg, 2.25
mmol, 1.0 equiv) in methanol (1 mL) was added 4 N HCl in dioxane (5
mL, 20 mmol, 9 equiv). The reaction was homogeneous and slightly
yellow in color. The reaction was stirred for 30 min, and LCMS/TLC
indicated the consumption of the starting material and a new peak with
the correct M + H (=176). The reaction was concentrated to a yellow
solid and used without further purification as the HCl salt. LCMS = 90%,
t = 0.16, m/z = 176.0. To a solution of the crude amine (477 mg, 2.25
mmol, 1.0 equiv) in ethanol (11.3 mL) was added diisopropylethyl-
amine (1.98 mL, 11.3 mmol, 5.0 equiv). The reaction was stirred and
was homogeneous. To the reaction was added chromone-3-carboxylic
acid (428 mg, 2.25, 1.0 equiv). The reaction was stirred at room
temperature and followed by LCMS and TLC. The reaction was judged
complete after 3 h, and the reaction was concentrated to an oil residue.
The residue was purified via silica column (80 g, 20% ethyl acetate in
heptane to 100% over 10 column volumes; desired material started
eluting at ∼80% ethyl acetate and peaked at ∼85% ethyl acetate) to
provide the desired material 16 as a yellow solid. 1H NMR (400 MHz,
DMSO-d6) δ = 2.02−2.12 (m, 2 H), 3.34−3.41 (m, 2 H), 3.53 (d, J =
11.32 Hz, 1 H), 3.62 (d, J = 8.98 Hz, 1 H), 4.80 (s, 1 H), 4.98 (s, 1 H),
5.65−5.90 (m, 2 H), 6.50−6.66 (m, 2 H), 6.76−6.83 (m, 2 H), 7.35 (t, J
= 7.61 Hz, 1 H), 7.49−7.59 (m, 1 H), 7.83−7.90 (m, 1 H), 8.10 (d, J =
SELECTED EXPERIMENTAL PROCEDURES
■
Chemistry Methods and Compound Characterization. Proton
(1H NMR), carbon (13C NMR), and fluorine (19F NMR) magnetic
resonance spectra where obtained in DMSO-d6 at 400, 100, 376 MHz,
respectively, unless otherwise noted. The following abbreviations were
utilized to describe peak patterns when appropriate: br = broad, s =
singlet, d = doublet, and m = multiplet. High-resolution mass
measurements were obtained on an Agilent ToF mass spectrometer.
All air and moisture sensitive reactions were carried out under an
atmosphere of dry nitrogen using heat-dried glassware and standard
syringe techniques. Tetrahydrofuran (THF) and acetonitrile were
purchased from EMD anhydrous and were used without further drying.
Flash chromatography was performed using an Analogix Intelliflash 280
or Biotage SP1 purification system with Sepra Si 50 silica gel using ethyl
acetate/heptane mixtures as solvent unless otherwise indicated. HPLC
was carried out on an Agella Venusil ASB C18 column (21.2 mm × 150
mm, 5 μm). A flow rate of 0.5−150 mL/min was used with mobile phase
A consisting of water + 0.1% modifier (v/v) and mobile phase B
consisting of acetonitrile + 0.1% modifier (v/v). The modifier was
formic acid, trifluoroacetate, ammonia acetate, or hydrochloric acid.
Quality control (QC) analysis was performed using a LCMS method.
Acidic runs were carried out on a Shimadzu XB-C18 (2.1 mm × 30 mm,
5 μm), X-Bridge (50 mm × 4.6 mm, 5 μm), Gemini NX C18 (50 mm ×
4.6, 3 μm), or Gemini NX C18 (50 mm × 4.6, 5 μm). A flow rate of 1.0−
1.2 mL/min was used with mobile phase A of water + 0.1% modifier (v/
v) and mobile phase B of acetonitrile + 0.1% modifier (v/v). For acidic
runs the modifier was trifluoroacetic acid. A Shimadzu 20AB pump ran a
gradient elution from 0% to 98% B over 2 min followed by a 1 min hold
at 95% B. Detection was achieved using a Shimadzu 10A detector set at
220 or 260 nm followed in series by a Shimadzu MS2010EV or Applied
Biosystem API 2000 mass spectrometer in parallel.
1
4.68 Hz, 1 H), 8.26 (d, J = 12.10 Hz, 1 H); H NMR (400 MHz,
chloroform-d) δ = 2.04−2.20 (m, 2 H), 3.35−3.55 (m, 3 H), 3.69 (d, J =
8.53 Hz, 1 H), 4.44 (br s, 1 H), 5.08 (br s, 1 H), 5.71 (d, J = 12.05 Hz, 1
H), 6.36 (d, J = 8.53 Hz, 1 H), 6.62−6.67 (m, 1 H), 6.79 (t, J = 7.78 Hz, 1
H), 6.92 (d, J = 7.53 Hz, 1 H), 7.34 (t, J = 7.03 Hz, 1 H), 7.48 (t, J = 8.28
Hz, 1 H), 7.61 (d, J = 9.03 Hz, 1 H), 8.09−8.20 (m, 2 H), 13.85 (s, 1 H);
13C NMR (DMSO-d6) δ = 36.9, 54.2, 55.6, 55.9, 63.9, 89.8, 107.2, 112,4,
117.4, 117.9, 119.9, 128.8, 133.8, 137.3, 147.9, 150.4, 156.9, 162.4,
189.9; LCMS = 100%, t = 0.60 m/z = 322.1; HRMS [M + H] for
C19H20N3O2, calcd, 322.1550, found, 322.1551; optical rotation, 4.4 mg
in 2 mL of MeOH, cell length of 1 dm; measured rotation (deg) =
+0.2652; [α] = 120.5.
(E)-3-(4,5-Dihydro-1H-pyrazolo[3,4-c]pyridin-6(7H)-yl)-1-(2-
hydroxyphenyl)prop-2-en-1-one (3). To a mixture of 4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-c]pyridine (203 mg, 1.65 mmol) in EtOH
(5 mL) and DMF (0.8 mL) was added DIPEA (303 mg, 3 mmol)
followed by chromone-3-carboxylic acid (285 mg, 1.5 mmol). The
mixture was stirred at 35 °C for 16 h. TLC (DCM/MeOH = 10/1)
showed the starting material was consumed. The volatiles were
evaporated off, and the residue was treated with DCM. Insoluble
material was removed via filtration. The filtrate was concentrated and
the residue was purified by flash column (EA in PE, 0−62%) to give 3
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00012.
Supplementary biological methods and chemistry exper-
imental procedures; cellular activity; X-ray statistics
(PDF)
1
(90 mg, 20%) as a yellow solid. H NMR (400 MHz, methanol-d4) δ
8.11 (d, J = 12.3 Hz, 1H), 7.90 (dd, J = 8.1, 1.6 Hz, 1H), 7.49 (s, 1H),
7.44−7.28 (m, 1H), 6.96−6.80 (m, 2H), 3.79 (t, J = 5.8 Hz, 2H), 3.33
(p, J = 1.6 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H); 13C NMR (100 MHz,
methanol-d4) δ 163.7, 135.2, 130.0, 121.7, 119.6, 118.8, 113.9, 21.9;
HRMS [M + H] for C15H15N3O2, calcd, 270.1237, found, 270.1232;
LCMS = 95.7%, m/z = 270.1 (M + H). HPLC = 97%, t = 3.02 min;
HRMS [M + H] for C15H16N3O2, calcd, 270.1237, found, 271.236.
(1R,4R)-tert-Butyl 5-(Pyridin-2-yl)-2,5-diazabicyclo[2.2.1]-
heptane-2-carboxylate (16b). To a stirred mixture of (1R,4R)-tert-
butyl 2,5-diazabicyclo[2.2.1]heptane-2-carboxylate 16a (500 mg, 2.52
mmol, [α]20 49.8° (c 0.79, EtOH)) in toluene (∼12.6 mL) were added
2-bromopyridine (0.481 mL,5.04 mmol), potassium tert-butoxide (1 M
in THF, 2.52 mL, 2.52 mmol), palladium acetate (28 mg, 0.126 mmol),
and BINAP (78 mg, 0.126 mmol) at room temperature, and then the
Molecular formula strings (CSV)
Accession Codes
Coordinates have been deposited with the Protein Data Bank:
compound 2, 5E7D; compound 3, 4Q0N; compound 17,
5KDH.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-617-674-7337. E-mail: dafydd.owen@pfizer.com.
Notes
The authors declare no competing financial interest.
J
DOI: 10.1021/acs.jmedchem.6b00012
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(10) Gallenkamp, D.; Gelato, K. A.; Haendler, B.; Weinmann, H.
Bromodomains and their pharmacological inhibitors. ChemMedChem
2014, 9, 438−464.
(11) Harner, M. J.; Chauder, B. A.; Phan, J.; Fesik, S. W. Fragment-
based screening of the bromodomain of ATAD2. J. Med. Chem. 2014,
57, 9687−9692.
(12) Fedorov, O.; Castex, J.; Tallant, C.; Owen, D. R.; Martin, S.;
Aldeghi, M.; Monteiro, O.; Filippakopoulos, P.; Picaud, S.; Trzupek, J.
D.; Gerstenberger, B. S.; Bountra, C.; Willmann, D.; Wells, C.; Philpott,
M.; Rogers, C.; Biggin, P. E.; Brennan, P.; Bunnage, M. E.; Schuele, R.;
ACKNOWLEDGMENTS
■
The Structural Genomics Consortium is a registered charity (No.
1097737) that receives funds from AbbVie, Bayer, Boehringer
Ingelheim, the Canadian Institutes for Health Research, the
Canada Foundation for Innovation, Genome Canada, Glax-
oSmithKline, Janssen, Lilly Canada, Merck, the Novartis
Research Foundation, the Ontario Ministry of Economic
Development and Innovation, Pfizer, Takeda, and the Wellcome
Trust. Aspects of this work were supported by NIH Grants
CA185926, CA82834, and GM56244.
Gunther, T.; Knapp, S.; Muller, S. Selective targeting of the BRG/PB1
̈
̈
bromodomains impairs embryonic and trophoblast stem cells
maintenance. Sci. Adv. 2015, 1, e1500723.
ABBREVIATIONS USED
■
(13) (a) Frye, S. V. The art of the chemical probe. Nat. Chem. Biol.
2010, 6, 159−161. (b) Bunnage, M. E.; Chekler, E. L.; Piatnitski; Jones,
L. H. Target validation using chemical probes. Nat. Chem. Biol. 2013, 9,
195−199. (c) Arrowsmith, C. H.; Audia, J. E.; Austin, C.; Baell, J.;
Bennett, J.; Blagg, J.; Bountra, C.; Brennan, P. E.; Brown, P. J.; Bunnage,
M. E.; Buser-Doepner, C.; Campbell, R. M.; Carter, A. J.; Cohen, P.;
Copeland, R. A.; Cravatt, B.; Dahlin, J. L.; Dhanak, D.; Edwards, A. M.;
Frye, S. V.; Gray, N.; Grimshaw, C. E.; Hepworth, D.; Howe, T.; Huber,
K. V. M.; Jin, J.; Knapp, S.; Kotz, J. D.; Kruger, R. G.; Lowe, D.; Mader,
BET, bromodomain and extra C-terminal domain; SWI/SNF,
switch/sucrose nonfermentable; BRG1, Brahma-related gene 1;
SMARCA, switch/sucrose nonfermentable related, matrix
associated, actin dependent regulator of chromatin, subfamily
a; PB1, protein polybromo; DSF, differential scanning
fluorimetry; ITC, isothermal titration calorimetry; SAR,
structure−activity relationship; NT, not tested; DIPEA,
diisopropylethylamine; EtOH, ethanol; RT, room temperature;
BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; OAc, ac-
etate; NaO-t-Bu, sodium tert-butoxide; MeOH, methanol;
FRAP, fluorescence recovery after photobleaching; HDAC,
histone deacetylase; SAHA, suberoylanilide hydroxamic acid;
GFP, green fluorescent protein; ccRCC, cell renal cell carcinoma;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; PFI-3, inhibitor that is commercially available and is also
known as PF-6687252
M. M.; Marsden, B.; Mueller-Fahrnow, A.; Muller, S.; O’Hagan, R. C.;
̈
Overington, J. P.; Owen, D. R.; Rosenberg, S. H.; Roth, B.; Ross, R.;
Schapira, M.; Schreiber, S. L.; Shoichet, B.; Sundstrom, M.; Superti-
Furga, G.; Taunton, J.; Toledo-Sherman, L.; Walpole, C.; Walters, M. A.;
Willson, T. M.; Workman, P.; Young, R. N.; Zuercher, W. J. The promise
and peril of chemical probes. Nat. Chem. Biol. 2015, 11, 536−541.
(14) Ghosh, C. K.; Khan, S. Heterocyclic systems; 10. Defunctionaliza-
tion of 4-oxo-4H-[1]-benzopyran-3-carboxylic acids and -3-carboxalde-
hydes. Synthesis 1981, 1981 (9), 719−721.
(15) Muller, S.; Knapp, S. Discovery of BET bromodomain inhibitors
̈
and their role in target validation. MedChemComm 2014, 5 (3), 288−
REFERENCES
■
296.
(1) Filippakopoulos, P.; Knapp, S. Targeting bromodomains:
epigenetic readers of lysine acetylation. Nat. Rev. Drug Discovery 2014,
13, 337−56.
(2) Tamkun, J. W.; Deuring, R.; Scott, M. P.; Kissinger, M.; Pattatucci,
A. M.; Kaufman, T. C.; Kennison, J. A. Brahma: A regulator of
Drosophila homeotic genes structurally related to the yeast transcrip-
tional activator SNF2/SWI2. Cell 1992, 68, 561−572.
(3) Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.
P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.;
(16) Philpott, M.; Yang, J.; Tumber, T.; Fedorov, O.; Uttarkar, S.;
Filippakopoulos, P.; Picaud, S.; Keates, T.; Felletar, I.; Ciulli, A.; Knapp,
S.; Heightman, T. D. Mol. BioSyst. 2011, 7, 2899−2908.
(17) Ciceri, P.; Muller, S.; O’Mahony, A.; Fedorov, O.;
̈
Filippakopoulos, P.; Hunt, J. P.; Lasater, E. A.; Pallares, G.; Picaud, S.;
Wells, C.; Martin, S.; Wodicka, L. M.; Shah, N. P.; Treiber, D. K.; Knapp,
S. Dual kinase-bromodomain inhibitors for rationally designed
polypharmacology. Nat. Chem. Biol. 2014, 10, 305−312.
̈
(18) Philpott, M.; Rogers, C. M.; Yapp, C.; Wells, C.; Lambert, J.-P.;
Strain-Damerell, C.; Burgess-Brown, N. A.; Gingras, A.-C.; Knapp, S.;
Gingras, A. C.; Arrowsmith, C. H.; Knapp, S. Histone recognition and
large-scale structural analysis of the human bromodomain family. Cell
2012, 149, 214−231.
Muller, S. Assessing cellular efficacy of bromodomain inhibitors using
̈
fluorescence recovery after photobleaching. Epigenet. Chromatin 2014,
7, 14.
(4) (a) Brand, M.; Measures, A. M.; Wilson, B. G.; Cortopassi, W. A.;
Alexander, R.; Hoss, M.; Hewings, D. S.; Rooney, T. P. C.; Paton, R. S.;
Conway, S. J. Small molecule inhibitors of bromodomain-acetyl-lysine
interactions. ACS Chem. Biol. 2015, 10, 22−39. (b) Garnier, J.-M.;
Sharp, P. P.; Burns, C. J. BET bromodomain inhibitors: a patent review.
Expert Opin. Ther. Pat. 2014, 24, 185−99. (c) Chung, C.-W. Small
molecule bromodomain inhibitors: extending the druggable genome.
Prog. Med. Chem. 2012, 51, 1−55.
(19) Thompson, M. Polybromo-1: the chromatin targeting subunit of
the PBAF complex. Biochimie 2009, 91 (3), 309−319.
(20) Hammitzsch, A.; Tallant, C.; Fedorov, O.; O’Mahony, A.;
Brennan, P. E.; Hay, D. A.; Martinez, F. O.; Al-Mossawi, M. H.; de Wit,
J.; Vecellio, M.; Wells, C.; Wordsworth, P.; Muller, S.; Knapp, S.;
̈
Bowness, P. CBP30, a selective CBP/p300 bromodomain inhibitor,
suppresses human Th17 responses. Proc. Natl. Acad. Sci. U. S. A. 2015,
112, 10768−10773.
(5) Jung, M.; Gelato, K. A.; Fernandez-Montalvan, A.; Siegel, S.;
Haendler, B. Targeting BET bromodomains for cancer treatment.
Epigenomics 2015, 7, 487−501.
(21) Pawłowski, R.; Muhl, S. M.; Sulser, T.; Krek, W.; Moch, H.;
̈
Schraml, P. Loss of PBRM1 expression is associated with renal cell
carcinoma progression. Int. J. Cancer 2013, 132, E11−E17.
(6) Vidler, L. R.; Brown, N.; Knapp, S.; Hoelder, S. Druggability
analysis and structural classification of bromodomain acetyl-lysine
binding sites. J. Med. Chem. 2012, 55 (17), 7346−7359.
(22) (a) Helming, K. C.; Wang, X.; Roberts, C. W. M. Cancer
vulnerabilities of mutant SWI/SNF complexes in cancer. Cancer Cell
2014, 26, 309−317. (b) Hoffman, G. R.; Rahal, R.; Buxton, F.; Xiang, K.;
McAllister, G.; Frias, E.; Bagdasarian, L.; Huber, J.; Lindeman, A.; Chen,
D.; Romero, R.; Ramadan, N.; Phadke, T.; Haas, K.; Jaskelioff, M.;
Wilson, B. G.; Meyer, M. J.; Saenz-Vash, V.; Zhai, H.; Myer, V. E.;
Porter, J. A.; Keen, N.; McLaughlin, M. E.; Mickanin, C.; Roberts, C. W.
M.; Stegmeier, F.; Jagani, Z. Functional epigenetics approach identifies
BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient
cancers. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 3128−3133.
(7) Ho, L.; Crabtree, G. R. Chromatin remodelling during develop-
ment. Nature 2010, 463, 474−484.
(8) Masliah-Planchon, J.; Bieche, I.; Guinebretiere, J.-M.; Bourdeaut,
F.; Delattre, O. SWI/SNF chromatin remodeling and human
malignancies. Annu. Rev. Pathol.: Mech. Dis. 2015, 10, 145−171.
(9) Singh, M.; Popowicz, G. M.; Krajewski, M.; Holak, T. A. Structural
ramification for acetyl-lysine recognition by the bromodomain of human
BRG1 protein, a central ATPase of the SWI/SNF remodeling complex.
ChemBioChem 2007, 8, 1308−1316.
K
DOI: 10.1021/acs.jmedchem.6b00012
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(23) Vangamudi, B.; Paul, T. A.; Shah, P. K.; Kost-Alimova, M.;
Nottebaum, L.; Shi, X.; Zhan, Y.; Leo, E.; Mahadeshwar, H. S.;
Protopopov, A.; Futreal, A.; Tieu, T. N.; Peoples, M.; Heffernan, T. P.;
Marszalek, J. R.; Toniatti, C.; Petrocchi, A.; Verhelle, D.; Owen, D. R.;
Draetta, G.; Jones, P.; Palmer, W. S.; Sharma, S.; Andersen, J. K. The
SMARCA2/4 ATPase domain surpasses the bromodomain as a drug
target in SWI/SNF-mutant cancers: Insights from cDNA rescue and
PFI-3 inhibitor studies. Cancer Res. 2015, 75, 3865−3878.
(24) (a) Bultman, S.; Gebuhr, T.; Yee, D.; La Mantia, C.; Nicholson, J.;
Gilliam, A.; Randazzo, F.; Metzger, D.; Chambon, P.; Crabtree, G.;
Magnuson, T. A. Brg1 null mutation in the mouse reveals functional
differences among mammalian SWI/SNF complexes. Mol. Cell 2000, 6,
1287−1295. (b) Reyes, J. C.; Barra, J.; Muchardt, C.; Camus, A.;
Babinet, C.; Yaniv, M. Altered control of cellular proliferation in the
absence of mammalian brahma (SNF2alpha). EMBO J. 1998, 17, 6979−
6991.
(25) (a) Ohkawa, Y.; Yoshimura, S.; Higashi, C.; Marfella, C. G.;
Dacwag, C. S.; Tachibana, T.; Imbalzano, A. N. Myogenin and the SWI/
SNF ATPase Brg1 maintain myogenic gene expression at different
stages of skeletal myogenesis. J. Biol. Chem. 2007, 282, 6564−6570.
(b) Ohkawa, Y.; Marfella, C. G.; Imbalzano, A. N. Skeletal muscle
specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1.
EMBO J. 2006, 25, 490−501. (c) de la Serna, I. L.; Carlson, K. A.;
Imbalzano, A. N. Mammalian SWI/SNF complexes promote MyoD-
mediated muscle differentiation. Nat. Genet. 2001, 27, 187−190.
(d) Salma, N.; Xiao, H.; Mueller, E.; Imbalzano, A. N. Temporal
recruitment of transcription factors and SWI/SNF chromatin-
remodeling enzymes during adipogenic induction of the peroxisome
proliferator-activated receptor gamma nuclear hormone receptor. Mol.
Cell. Biol. 2004, 24, 4651−4663.
(26) Nasipak, B. T.; Padilla-Benavides, T.; Green, K. M.; Leszyk, J. D.;
Mao, W.; Konda, S.; Sif, S.; Shaffer, S. A.; Ohkawa, Y.; Imbalzano, A. N.
Opposing calcium-dependent signalling pathways control skeletal
muscle differentiation by regulating a chromatin remodelling enzyme.
Nat. Commun. 2015, 6, 7441.
L
DOI: 10.1021/acs.jmedchem.6b00012
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