GNE-886: A Potent and Selective Inhibitor of the Cat Eye Syndrome Chromosome Region Candidate 2 Bromodomain (CECR2)

Article abstract of DOI:10.1021/acsmedchemlett.7b00132

The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.

Full text of DOI:10.1021/acsmedchemlett.7b00132

Letter
GNE-886: A Potent and Selective Inhibitor of the Cat Eye Syndrome
Chromosome Region Candidate 2 Bromodomain (CECR2)
Terry D. Crawford, James E. Audia, Steve Bellon, Daniel J. Burdick, Archana Bommi-Reddy, Alexandre
Côté, Richard T. Cummings, Martin Duplessis, Elizabeth Megan Flynn, Michael C. Hewitt, Hon-Ren
Huang, Hariharan Jayaram, Ying Jiang, Shivangi Joshi, James R. Kiefer, Jeremy M. Murray, Christopher
G. Nasveschuk, Adrianne Neiss, Eneida Pardo, F. Anthony Romero, Peter Sandy, Robert J. Sims,
Yong Tang, Alexander M. Taylor, Vickie Tsui, Jian Wang, Shumei Wang, Yongyun Wang, Zhaowu Xu,
Laura E. Zawadzke, Xiaoqin Zhu, Brian K. Albrecht, Steven R. Magnuson, and Andrea G. Cochran
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017
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GNE-886: A Potent and Selective Inhibitor of the Cat Eye Syndrome
Chromosome Region Candidate 2 Bromodomain (CECR2)
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Terry D. Crawford,*† James E. Audia,‡ Steve Bellon,‡ Daniel J. Burdick,† Archana Bommi-Reddy,‡ Al-
exandre Côté,‡ Richard T. Cummings,‡ Martin Duplessis,‡ E. Megan Flynn,† Michael Hewitt,‡ Hon-
Ren Huang,‡ Hariharan Jayaram,‡ Ying Jiang,§ Shivangi Joshi,‡ James R. Kiefer,† Jeremy Murray,†
Christopher G. Nasveschuk,‡ Arianne Neiss,‡ Eneida Pardo,‡ F. Anthony Romero,† Peter Sandy,‡ Rob-
ert J. Sims III,‡ Yong Tang,‡ Alexander M. Taylor,‡ Vickie Tsui,† Jian Wang,§ Shumei Wang,† Yongyun
Wang,§ Zhaowu Xu,§ Laura Zawadzke,‡ Xiaoqin Zhu,§ Brian K. Albrecht,‡ Steven R. Magnuson,† An-
drea G. Cochran†
†Genentech Inc., 1 DNA Way, South San Francisco, CA 94080
‡Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, MA 02142
§Wuxi Apptec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, People’s Republic of Chi-
na
KEYWORDS Epigenetics, bromodomain, CECR2, probe
ABSTRACT: The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely un-
known. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome
chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of
a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromo-
domain inhibitor (GNE-886) suitable for use as an in vitro tool compound.
Bromodomains are approximately 110 amino-acid residue
protein modules that function as epigenetic readers recog-
nizing acylated lysine residues on histone tails, as well as
other proteins.1-5 Bromodomains encompass 61 individual
modules located on 46 proteins,7-9 but the biological func-
tion of individual bromodomain family members remains
largely unclear. The most widely studied sub-class of bro-
modomains is the bromodomain and extra terminal domain
(BET) family, which is comprised of bromodomain-
containing protein 2 (BRD2), bromodomain-containing pro-
tein 3 (BRD3), bromodomain-containing protein 4 (BRD4),
and bromodomain testis-specific protein (BRDT), each of
which contains dual bromodomains.8,9 However, interest in
non-BET bromodomain inhibitors has increased significant-
ly, as demonstrated by the number of selective bromo-
domain inhibitor tool compounds that have been disclosed
recently.8-10
damage response (DDR) as CECR2 knockdown has been
shown to result in decreased -H2AX foci formation.13 Cecr2
deletion has also been associated with neural tube defect
(NTD) exencephaly.14 The detailed biological function of the
CECR2 bromodomain itself remains unknown, although
overexpression of the bromodomain can inhibit formation of
-H2AX and 53BP1 foci after irradiation of cells.13 This sug-
gests that the CECR2 bromodomain has a binding function
important in the response to DNA damage, and selective
small-molecule inhibitors will be critical in better defining
this function and any role it may have in disease states.
Recently, the Structural Genomics Consortium (SGC) dis-
closed a CECR2 inhibitor identified in collaboration with
Novartis.15 This compound, NVS-CECR2-1 has high-affinity
activity (CECR2 AlphaScreen IC50: 47 nM) with strong cellu-
lar activity in a Fluorescence recovery after photobleaching
(FRAP) assay at 0.1 M, however selectivity data across the
bromodomain family is not given. As part of our non-BET
bromodomain probe discovery platform16-18 we herein report
our efforts to identify a potent and selective CECR2 bromo-
domain inhibitor. To identify CECR2 lead chemical matter, a
collection of approximately 600 molecules from our internal
bromodomain medicinal chemistry collection was screened
Cat eye syndrome chromosome region candidate 2 (CECR2)
is a bromodomain containing transcription factor that, with
the ISWI-type catalytic subunit SMARCA1/SNF2L or
SMARCA5/SNF2H, forms the heterodimeric chromatin re-
modeling complex CERF (CECR2-containing remodeling
factor).11, 12 CECR2 has been found to be important in DNA
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mon “WPF” motif in CECR2)17 and Tyr467 from the Z helix.
in a single point CECR2 AlphaScreen (AS) assay. From this
screen we identified a number of hits, including 6-methyl-
N-(1-methylpiperidin-4-yl)-7-oxo-6,7-dihydro-1H-
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From the model, we envisioned that the introduction of a
tertiary amide would improve selectivity by creating unfa-
vorable interactions with Ile53 in BRD9 and the WPF shelf in
TAF1(2).
pyrrolo[2,3-c]pyridine-4-carboxamide 1. Subsequent titra-
tion in both AS and fluorescence resonance energy transfer
(FRET) assays revealed 1 to have pronounced selectivity
over the representative BET protein BRD4. Indeed, IC50s
were > 20 M for both BRD4 bromodomains. This was crit-
ically important, due to the strong phenotype that has been
reported for BET inhibitors.8,9 While compound 1 was found
to be more potent in TAF1(2) compared to CECR2, we ob-
served that compound 1 had slight selectivity over BRD9,
albeit only two-fold (Figure 1A). This was the only com-
pound from this screening set where we found a preference
for CECR2 over BRD9, and coupled with the BRD4 selectivi-
ty made 1 an attractive starting point for subsequent lead
optimization. We therefore endeavored to further improve
the CECR2 potency while increasing our selectivity window
against BRD4, BRD9, and TAF1(2) and maintaining high
selectivity over other non-BET family members.
Gratifyingly, we observed that the incorporation of the N-
methyl amide 2 produced a significant increase in selectivi-
ty over BRD9, as predicted by the model, as well as TAF1(2)
while maintaining CECR2 affinity (Table 1). Extending fur-
ther with an N-isopropyl piperidine 3 to the -methylbenzyl
piperidine 4 yielded a slight gain in potency although the
non-specific hydrophobic interactions yielded equivalent
potency improvements across our internal bromodomain
panel.
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We recently reported that small hydrophobic channels can
be induced in BRD9 and CECR2 bromodomains between
the tyrosine gatekeeper and the lipophilic shelf (GPP in
BRD9,WPF in CECR2) by replacing the pyrrolopyridone me-
thyl with small aliphatic unsaturated chains.17 In that study
we observed that in the case of CECR2, an allyl group ex-
tending from the pyridone nitrogen was the optimal substit-
uent, yielding CECR2 potency similar to the N-methyl ana-
logue and significantly enhanced selectivity over the majori-
ty of our internal bromodomain panel. Incorporation of the
N-allyl substituent onto our 4-carboxamide scaffold to make
compound 5 yielded a similar improvement in selectivity to
that we had previously reported. We were also pleased to
observe significant selectivity over BRD4(2), as well as CBP,
BRPF1, and TAF1(1) (TR-FRET IC50s >20 M, data not
shown), however only small improvements in selectivity
were observed over BRD9. This is not surprising as BRD9 is
able to form the same hydrophobic pocket as CECR2 to
accommodate the hydrophobic allyl chain. TAF1(2) potency
only decreased four-fold, which was expected as we have
found that TAF1(2) is able to tolerate such hydrophobic
substituents through rearrangement of the conserved bind-
ing-pocket water network. 17
Figure 1. A) Assay data for CECR2 screening hit 1. IC50 values
are the average from at least 2 independent experiments. B)
Model of 1 (cyan) in CECR2 structure (PDB code 3NXB): thick
sticks of 1 represents the conformation used to design the
tertiary amide, while wire frames of 1 represent other possible
conformations from the modeling. The CECR2 protein struc-
ture is shown as grey ribbon with grey sticks for sidechains
containing the WPF shelf (Trp457, Pro458 and Phe459), the
gatekeeper residue (Tyr520) and the conserved Asn514. Also
shown are the sidechain Tyr467 and its corresponding residue
Ile53 in BRD9 (orange).
To improve selectivity against TAF1(2), we next focused on
substitution at the pyrrolopyridone 2-position leading into
the BC loop region, which we hypothesized might not be
tolerated by TAF1(2) but might introduce further favorable
van der Waals interactions with CECR2. Introduction of a
methyl at the 2-position (Compounds 6, 7) did in fact result
in improved selectivity over TAF1(2) (Table 1). However, the
BRD9 affinity was increased at least five-fold. Interestingly,
we observed very little preference between enantiomers 6
and 7 in binding to any of the bromodomains assayed.
Routine crystallography proved to be challenging for our
CECR2 inhibitors, however we were confident in predicting
the binding mode of 1 in CECR2 based on previous work
with this class of chemical matter.17 Modeling of 1 bound to
the CECR2 structure suggests that the pyridone carbonyl
and N-methyl groups serve as the acetyl-lysine (KAc) mi-
metic, with the pyridone carbonyl and pyrrole forming a 2-
point hydrogen-bonding interaction with the highly con-
served Asn514 (Figure 1B). The gatekeeper Tyr520 forms a
-stacking interaction with the pyrrolopyridone. The N-
methylpiperidine amide at the 4-position appeared capable
of adopting multiple low-energy conformations, and three
of the most probable conformations are shown in Figure 1B.
In all three conformations, the piperidine extended into the
ZA channel forming hydrophobic interactions with Trp457
of the lipophilic shelf region (which conforms to the com-
A co-crystal structure of compound 6 bound to CECR2 (2.7
Å, PDB 5V84) confirmed the predicted overall binding mode
of the core (Figure 2). The allyl folds into the hydrophobic
channel with the backstop residue Phe459 from the WPF
shelf shifting slightly towards Met506 in order to accommo-
date binding. The allyl forms hydrophobic contacts with
Pro458 and Phe459 as well as with Tyr520, which also serve
to constrain the channel and prevent the deeper penetra-
tion that we have observed with crotonyl ligands binding to
BRD9. The amide was found to be nearly orthogonal to the
pyrrolopyridone core and occupied space that became
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Table 1. SAR for N-alkyl substitutions extending from the piperidine.a
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aAll bromodomain assays were run in TR-FRET format. IC50 data are an average of at least 2 independent experiments. Assay ta-
bles with standard deviations are located in the supporting information. A full table of data with standard deviations for these and
other N-alkyl substituents can be found in the Supporting Information.
the cavity where it makes hydrophobic interactions with the
WPF shelf and the gatekeeper. The piperidine makes addi-
tional hydrophobic contacts extending through the ZA
channel with a water mediated interaction between the
protonated piperidine and Asp464. The benzylic group was
not observed to make specific interactions and indeed had
significant rotational freedom as it exited the ZA channel
into solvent, which would explain that lack of potency dif-
ference between the separated enantiomers.
Additional substituted benzylic analogs yielded little to no
improvements in potency (data not shown). We therefore
examined more constrained substitutions on the piperidine
to reduce the entropic penalty resulting from the freely ro-
tating benzylic substituent, which led to the identification of
the 4-pyrimidine 8. Compound 8 has potency comparable
to 5, although selectivity versus BRD9 and TAF1(2) is de-
creased (Table 2). We hypothesized that the pyrimidine
heteroatoms may be able to form water-mediated interac-
tions to residues in the ZA channel, as docking exercises
did not indicate a potential for a direct hydrogen bonding
interaction. Additional substitution was found to be benefi-
cial at the 4-position of the pyrimidine ring. Starting from
Figure 2. Compound 6 bound to CECR2 [PDB 5V84]. B) Com-
parison of Compound 6 bound to CECR2 with Apo structure
residues Phe459, Tyr520, Met506 in red. Compound 6 is shown
as green sticks and H-bond interactions are represented as
dashes.
trifluoromethyl 9, we observed that increasing steric bulk to
the methoxy 10, cyclopropyl 11, and t-butyl 12 yielded
small potency increases for CECR2, with IC50s now below
100 nM.
accessible due to the induced fit. (Supporting Infor-
mation Figure S1). The carbonyl forms a direct hydrogen
bond with the ZA loop Asp464, while the N-methyl rests in
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Table 2. SAR for N-aryl substitutions on the piperidine core.a
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aAll bromodomain assays were run in TR-FRET format. IC50 data are an average of at least 2 independent experiments.
Introducing a methyl group at the pyrimidine 2-position to
make disubstituted analogs 13, 14, and 15 maintained the
improved CECR2 potency, although BRD9 potency was also
maintained. As the t-butyl group of 12 may serve as a spa-
tial isostere to a phenyl, we introduced increased steric bulk
with aromatic substituents. Five-membered aromatic rings
such as pyrazole 16 and furan 17 did not significantly im-
prove either selectivity or potency, nor did ortho-substituted
phenyl groups 18–20. Meta-substitution quickly proved
superior, possibly due to a decreased torsion-angle strain
relative to ortho-substituted analogs, as the 3-methoxy
phenyl 21 yielded significantly improved potency (16 nM).
Compound 21 had a wide selectivity over the 40 bromo-
domains tested, with only BRD9 (KD=2000 M), and its
homolog BRD7 (KD=1100 M), as well as TAF1(2) (KD=0.62
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M), and its homolog TAF1L(2) (KD=2400 M) showing
Terry D. Crawford: 0000-0003-4663-4116
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significant binding. While the selectivity window was re-
duced in comparison to the TR-FRET data, compound 21
still maintained a 2 log window over BET family members,
therefore minimizing the risk of observing BET inhibition
phenotypes. Compound 21 was also screened against a
panel of 35 diverse kinases, with no kinase inhibited over
20% at 1 M (Supporting Information). Finally, compound
21 (GNE-886) had a highly favorable measured kinetic sol-
ubility of 122 M and was therefore considered an appro-
priate in vitro tool compound for the CECR2 bromodomain
(Figure 3).
Funding:
All authors were employed by Constellation, Genentech, and
Wuxi, and experiments described were performed in Constel-
lation, Genentech, or Wuxi laboratories.
ACKNOWLEDGMENT
We thank the staff at beamline SER-CAT 22BM of the Ad-
vanced Photon Source. This research used resources of the
Advanced Photon Source, a U.S. Department of Energy (DOE)
Office of Science User Facility operated for the DOE Office of
Science by Argonne National Laboratory under Contract No.
DE-AC02-06CH11357. Phil Bergeron and Tom Pillow for assis-
tance during manuscript preparation. Mengling Wong, Michael
Hayes, and Amber Guillen for compound purification. Baiwei
Lin, Yanzhou Liu, Deven Wang, and Yutao Jiang are acknowl-
edged for analytical support. Grady Howes, Jan Seerveld, Hao
Zheng, Ted Peters, Gigi Yuen, and Jeff Blaney for help with
compound management and logistics are also recognized.
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ABBREVIATIONS
BET, bromodomain and extra terminal domain; BRD2, Bro-
modomain-containing protein 2; BRD3, Bromodomain-
containing protein 3; BRD4(1), The first bromodomain of
bromodomain-containing protein 4; BRD4(2), The second
bromodomain of bromodomain-containing protein 4; BRD9,
Bromodomain containing protein 9; BRDT, Bromodomain
testis-specific protein; BRPF1, Bromodomain and PHD fin-
ger containing, 1; CBP, CREB binding protein bromodomain;
CECR2, Cat eye syndrome critical region protein 2; TAF1(1),
The first bromodomain of human transcription initiation
factor TFIID subunit 1; TAF1(2), The second bromodomain
of human transcription initiation factor TFIID subunit 2.
Figure 3. Profile of GNE-886. All bromodomain assays were
run in FRET format. IC50 data are an average of at least 2 inde-
b
pendent experiments. LE = –RTln(IC50)/N heavy atoms. LLE =
pIC50–cLogP.
In summary, we identified pyrrolopyridone-4-carboxamide
compound 1 through screening of our internal medicinal
chemistry collection as a promising chemical lead for iden-
tification of a small molecule inhibitor of the CECR2 bromo-
domain. Structure-based lead optimization efforts involved
the inducement of a hydrophobic pocket with an allyl sub-
stituent, occupying a small cavity in the binding pocket with
an N-methyl amide, and introducing rigidified biaryl substit-
uents into the ZA channel towards solvent-exposed space.
This led to the identification of compound 21, suitable for
use in in vitro phenotypic screening.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedures and compound characterization for
compounds 1-21, protein and assay methods, CECR2 cellular
assay methods, dot images, curve, and replicate data for 21,
crystallography data and refinement for 6, modeling methods,
kinase and bromodomain selectivity data for 21.
AUTHOR INFORMATION
(7) Filippakopoulos, P.;Picaud, S.; Mangos, M.; Keates, T.; Lam-
bert, J.-P., Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Müller, S.;
Pawson, T.; Gingras, A.-C.; Arrowsmith, C. H.; Knapp, S. Histone
recognition and large-scale structural Analysis of the human bro-
modomain family. Cell 2012, 149, 214−231.
Corresponding Author
*E-mail: terrydc@gene.com
ORCID:
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Magnuson, S. R.; Grogan, J. L.; Bellon, S. F.; Albrecht, B. K.; Simms
III, R. J.; Lora, J. M. Regulatory T Cell modulation by CBP/EP300
bromodomain inhibition. J. Biol. Chem. 2016, 291, 13014–13027.
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GNE-886: A Potent and Selective Inhibitor of the Cat Eye
Syndrome Chromosome Region Candidate 2 Bromodomain
(CECR2)
Terry D. Crawford,*† James E. Audia,‡ Steve Bellon,‡ Daniel J. Burdick,† Archana Bommi-
Reddy,‡ Alexandre Côté,‡ Richard T. Cummings,‡ Martin Duplessis,‡ E. Megan Flynn,† Michael
Hewitt,‡ Hon-Ren Huang,‡ Hariharan Jayaram,‡ Ying Jiang,§ Shivangi Joshi,‡ James R. Kiefer,†
Jeremy Murray,† Christopher G. Nasveschuk,‡ Arianne Neiss,‡ Eneida Pardo,‡ F. Anthony
Romero,† Peter Sandy,‡ Robert J. Sims III,‡ Yong Tang,‡ Alexander M. Taylor,‡ Vickie Tsui,†
Jian Wang,§ Shumei Wang,† Yongyun Wang,§ Zhaowu Xu,§ Laura Zawadzke,‡ Xiaoqin Zhu,§
Brian K. Albrecht,‡ Steven R. Magnuson,† Andrea G. Cochran†
†Genentech Inc., 1 DNA Way, South San Francisco, CA 94080
‡Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, MA 02142
§Wuxi Apptec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, People’s Republic
of China
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