GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain

Article abstract of DOI:10.1021/acsmedchemlett.6b00092

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engag

Full text of DOI:10.1021/acsmedchemlett.6b00092

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Featured Letter
pubs.acs.org/acsmedchemlett
GSK6853, a Chemical Probe for Inhibition of the BRPF1
Bromodomain
Paul Bamborough,∥ Heather A. Barnett,§ Isabelle Becher,⊥ Mark J. Bird,§ Chun-wa Chung,∥
Peter D. Craggs,∥ Emmanuel H. Demont,*,† Hawa Diallo,† David J. Fallon,†,# Laurie J. Gordon,∥
Paola Grandi,⊥ Clare I. Hobbs,∥ Edward Hooper-Greenhill,‡ Emma J. Jones,∥ Robert P. Law,†,#
Armelle Le Gall,*,∥ David Lugo,‡ Anne-Marie Michon,⊥ Darren J. Mitchell,† Rab K. Prinjha,†
Robert J. Sheppard,*,† Allan J. B. Watson,# and Robert J. Watson†
‡
§
†Epinova Discovery Performance Unit, Quantitative Pharmacology, Experimental Medicine Unit, Flexible Discovery Unit, and
∥Platform Technology and Science, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.
⊥Cellzome GmbH, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany
#WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral
Street, Glasgow G1 1XL, U.K.
S
* Supporting Information
ABSTRACT: The BRPF (Bromodomain and PHD Finger-
containing) protein family are important scaffolding proteins
for assembly of MYST histone acetyltransferase complexes. A
selective benzimidazolone BRPF1 inhibitor showing micro-
molar activity in a cellular target engagement assay was recently
described. Herein, we report the optimization of this series
leading to the identification of a superior BRPF1 inhibitor
suitable for in vivo studies.
KEYWORDS: BRPF1, BRPF2, BRD1, BRPF3, BET, bromodomain, epigenetics, chemical probe, inhibitor
here are at least 56 bromodomain modules contained
assembly of MYST-family histone acetyltransferase com-
Twithin 42 human chromatin-regulator proteins. Of these, plexes.10,11 In addition to the bromodomain, which binds
1
acetylated histones,12 the proteins contain other histone-
the eight bromodomains of the BET (Bromodomain and Extra
binding domains (PZP and PWWP).13−15 The availability of
Terminal) subfamily (BRD2/3/4/T) have received most
potent and selective chemical probes for BRPF bromodomains
would significantly enhance our ability to dissect their biological
role and better understand the therapeutic opportunities.
We recently reported the discovery of the BRPF1
bromodomain inhibitor 1 (GSK5959) (Table 1).17 In the
BROMOscan panel of 34 bromodomain binding assays, it
showed 10 nM BRPF1 inhibition, 90-fold selectivity over
BRPF2, and over 500-fold selectivity over all members of the
BET family. Others have independently found related BRPF1
inhibitors with similar properties.18 Although the qualities of 1
made it an excellent probe to further elucidate the role of the
BRPF1 bromodomain in many situations, its physicochemical
properties and solubility are suboptimal. As part of our ongoing
efforts to develop bromodomain probe molecules we sought to
obtain an inhibitor with lower logD, solubility >100 μg/mL,
and cellular activity < 100 nM.
attention following the discovery that small-molecule inhibitors
found during cellular screening campaigns bind to these targets
and act by blocking their binding to Nε-acetyl-lysine (KAc)
modified histones.2−5 Inhibitors of BET proteins, such as I-
BET762, RVX-208, OTX-015, and CPI-0610, have progressed
into clinical evaluation for oncology and cardiovascular
disease.6,7
The phenotypic effects of inhibiting other bromodomains
remain to be discovered. The dearth of potent inhibitors of
these bromodomains is one factor, but the problem is
exacerbated by the significant and diverse phenotypic responses
associated with inhibition of the BET proteins.8 These can
complicate the interpretation of phenotypic readouts obtained
with poorly selective inhibitors. Biological evidence links several
non-BET bromodomain-containing proteins to disease,9 but
because of the multidomain architecture and scaffolding
function of these proteins the precise role of the bromodomain
is often uncertain. For example, the BRPF (Bromodomain and
PHD Finger-containing) family of BRPF1, BRPF2/BRD1, and
BRPF3 (Figure S1, Supporting Information) act to facilitate the
Received: March 2, 2016
Accepted: May 9, 2016
© XXXX American Chemical Society
A
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a
Table 1. Summary of the Properties of GSK5959
BRPF1
TR-FRET pIC50
BROMOscan pKd
NanoBRET pIC50
TR-FRET pIC50
TR-FRET pIC50
BROMOscan
7.1
8.0
6.0
BRPF 2/3
5.2/4.5
<4.3/<4.3
>90-fold
6.0
a
BRD4 BD1/2
Selectivity
16
Chrom logDpH 7.4
Figure 1. X-ray structure of BRPF1 with 1 (PDB 4UYE).
CLND solubility (μg/mL)
8
a
NH of 1. Compounds 6−12 were accessed via amide coupling
of the appropriate benzoic acids with the aromatic amine
(Scheme S1).
While introduction of these groups generally improved
solubility relative to 5, they all resulted in weaker inhibition of
BRPF1 (Table 3). This observation was particularly disappoint-
See also Table S1, Supporting Information.
Expansion of the SAR was carried out via analogue searches
within the GSK collection, and bespoke syntheses (Scheme
S1). The piperidine of 1 could be truncated to deliver the more
potent and ligand efficient inhibitor 2, albeit at the expense of
increased inhibition of BRD4 BD1 (Table 2). Further reduction
a
Table 3. Benzamide ortho-Position SAR
a
Table 2. SAR at the Benzimidazolone 6-Position
a
b
See also Table S1, Supporting Information. CLND (μg/mL).
a
b
See also Table S1, Supporting Information. CLND (μg/mL).
in the size of the 6-subsituent (3) was detrimental. However,
with the ether-linked analogues an increase in potency and
selectivity was observed on moving from 6-methoxy 4 to iso-
propoxy 5, while ligand efficiency (LE)19 was retained.
Although the discovery of 5 represented a step toward an
improved probe in terms of potency, LE, and logD, solubility
still remained to be addressed.
ing for side-chains containing hydrogen-bond donors (6 and 7),
which were no more potent than close analogues without this
functionality (10, 11), hydrogen bond acceptors (9), or basic
groups (8 and 12). These results suggest that introduction of
substituents larger than a methyl may give rise to steric clashes
with the residues of the ZA loop and suboptimal binding
conformations.
With the goals of expanding the diversity of substituents on
the 5-position of the benzimidazolone core, improving
solubility by lowering logD, and exploring alternatives to the
intramolecular methoxy/amide hydrogen-bond, further 5-
position analogues were synthesized (Schemes S1 and S2;
Table 4). All substituents showed improved solubility relative
to 5, but aliphatic groups, whether linear (14 and 15) or cyclic
The X-ray complex of BRPF1 with 1 (Figure 1)17 suggested
that the 3- and 4-positions on the benzamide provided vectors
suitable for projecting a solubilizing group directly out into
solvent, but presented no prospect of gaining interactions with
the protein. In contrast, elaboration of the phenyl 2-methoxy
also offered the potential to interact with residues of the ZA
loop, such as Glu655, while retaining the intramolecular
hydrogen-bond seen between the 2-methoxy and the amide
B
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a
The compounds in Table 4 all met the objective for
increased solubility relative to 5. However, it was disappointing
that the best balance between BRPF1 potency and selectivity
over BRD4 BD1 was achieved with the poorly soluble 2-
methoxyphenyl 5. Unable to improve upon the 2-methox-
ybenzaminde in the 5-position, we switched our efforts to the
benzimidazolone core itself. Inspection of the structure of 1
bound to BRPF1 (Figure 1) suggested it may be possible for a
chain possessing a solubilizing group to pass through the
channel between Phe714, Asn708, and Tyr707, although the
space for such a group was very limited. Extension of the 3-Me
to a hydroxyethyl (Scheme S3) (26) gave a substantial increase
in solubility, but with an unacceptable 40-fold drop in BRPF1
inhibition (Table 5).
Table 4. Modifications at the 5-Position
a
Table 5. Modifications at the N3-Position
a
See also Table S1, Supporting Information.
As the 3- and 5- positions seemed unproductive we returned
to the 6-position of the benzimidazolone. The crystal structure
of 1 in complex with BRPF1 (Figure 1) showed the proximity
of the backbone carbonyl of Asn651 to the C4 carbon of the
piperidine. It was postulated that introduction of a basic
nitrogen in this position could pick up an additional hydrogen
bond to this carbonyl, while also improving solubility.
a
b
See also Table S1, Supporting Information. CLND (μg/mL).
To test this hypothesis, compounds 28−31 were synthesized
via SNAr reaction or Suzuki chemistry on an appropriately
substituted core (Schemes S4 and S5). The piperazine 28 was
well tolerated, delivering the desired improvement in
physicochemical properties while maintaining potency at
BRPF1 (Table 6). Methylation of the piperazine to give 4-
methylpiperazine 29 was slightly detrimental. The C-linked
piperidine 30 and 1,2,3,6-tetrahydropyridine 31, however, were
significantly less potent, with the sp3 hybridized carbon being
more deleterious than the sp2. This is in accordance with our
previous observation that the 6-H and 6-Me (3) analogues were
∼10-fold less active than the 6-OMe (4). Collectively, the
results for compounds 1−5, 28, and 30−31 rule out steric
demands as the explanation for the reduced inhibition of
carbon-linked 6-substituents. Instead, they provide support for
the hypothesis put forward in our previous paper that linking
via a heteroatom plays an important role by electrostatically
influencing the conformation of the 5-benzamide.17 This is
further supported by the distribution of torsion angles and
distances in an analysis of small molecule X-ray structures
(Figure S3).
(16 and 17) and positively charged (16) or neutral (14−15,
17) had little effect on potency relative to the 5-H (13) and
were over 10-fold less potent than the 2-methoxybenzamide 5.
Consistent with our previous observations for a 6-
piperidine,17 the unsubstituted benzamide 18 is also weaker
than the 2-methoxy analogue 5. Presumably this is because the
unsubstituted phenyl of 18 is unable to form the intramolecular
hydrogen bond with the amide N−H. Anticipating that a 2-
pyridyl could mimic this aspect of the 2-methoxy substituent,
19 was synthesized and shown to reduce logD relative to 5
while maintaining potency. The other pyridyl isomers (20 and
21) had activity comparable to the phenyl 18. We interpret this
data as supporting the hypothesis that the intramolecular
hydrogen bond plays an important role in restricting the
degrees of freedom and increasing binding affinity (Figure S2).
Further evidence came from the observation that the activities
of compounds possessing a 2-heteroatom or 2-OMe group
(22−23) retain higher potency than those without. Incorpo-
ration of a 6-N into the phenyl ring of 5 is detrimental to
activity (compare 24 to 5), probably as a result of electrostatic
repulsion between the lone pair of the pyridyl nitrogen and the
amide carbonyl. This effect would be greater with the 6-OMe
(25), explaining the further drop in potency.
Despite the 40-fold variation in the BRPF1 activities of
analogues 1 and 27−31, there was comparatively little change
in potency versus the closely related BRPF2 bromodomain
C
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a
Table 6. Modifications at the 6-Position
Figure 2. (A) GRID map analysis using the BRPF1 structure of
compound 1 (PDB 4UYE). Methyl probe is shown in orange mesh.
(B). X-ray structure of BRPF1 (cyan, PDB 5G4R) with 34, overlaid
with BRPF2 apo (magenta, PDB 3RCW).
a
b
See also Table S1, Supporting Information. CLND (μg/mL).
between the isoforms, which the 2-methyl substituent is able
to exploit.
(Table S1). The largest selectivity window over BRPF2 is
therefore approximately 160-fold (27 and 28).
To facilitate interpretation of phenotypic assays we designed
a less active analogue of 34 in which the 5-amide was alkylated,
38 (Scheme S5, GSK9311). We reasoned that this would
prevent the 5-aryl group from adopting the conformation
observed in the structures of 1 and 34 because of the loss of all
internal hydrogen-bonding interactions, and steric clashes
between the amide N-alkyl group and both the piperazine
and the o-methoxy substituents. Usefully, this change resulted
in a 100-fold drop in BRPF1 inhibition. This result can be
rationalized by a crystal structure of 38 in BRPF1 in which the
benzamide group packs poorly against P658 and E661 (Figure
3).
Having achieved with 28 acceptable solubility, while
maintaining significant BRPF1 inhibition, we next sought to
investigate whether improved potency and selectivity was
readily achievable. Interrogating the crystal structure of 1 in
complex with BRPF1, it was intriguing to note that the 2-
position of the piperidine presented a vector toward Pro658
(Figure 2A). This residue is one of the few that are not
conserved across the BRPF subfamily, being substituted for
Ser592 in BRPF2 or Asn619 in BRPF3. Analysis of the BRPF1
site using GRID probes suggested the potential for favorable
interaction with a methyl group in the small indentation in the
surface near Pro658 (Figure 2A).20
This hypothesis was first explored with the 2-methyl
pyrrolidines 32 and 33. Indeed, a significant increase in
BRPF1 inhibition was observed, especially with the (R)-
enantiomer, with minimal effect on the inhibition of BRPF2
(Table S1, compare 32 with 27), BRPF3, and BRD4 (Table 6).
Transferring these findings to the more soluble piperazines
(34−35) again showed the (R)-enantiomer 34 to be the most
potent. The comparatively weaker inhibition with the 2-(S)-
enantiomer 35, and 3-methyl enantiomers (36−37), confirm
the specificity of this interaction.
Shortly afterward a 2.0 Å crystal structure of the BRPF1
bromodomain in complex with 34 was obtained (Figure 2B).
This showed that 34 binds to BRPF1 in a very similar way to 1.
As expected, the 2-methylpiperidine occupies the pocket close
to Pro658, and the piperazine 4-NH makes a direct hydrogen
bond to the backbone carbonyl of Asn651. Overlaying the apo
structure of BRPF2 highlights the structural differences
Figure 3. X-ray structures of BRPF1 with 34 (cyan, PDB 5G4R) and
38 (magenta, PDB 5G4S).
D
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a
Compound 34 was tested in the BROMOscan panel of
bromodomain binding assays (DiscoveRx). Consistent with the
data above, it showed excellent BRPF1 potency (pKd 9.5) and
greater than 1600-fold selectivity over all other bromodomains
tested (Table S2). To our knowledge this is the most potent
and selective inhibitor of any single bromodomain reported to
date.
All biochemical data presented thus far was determined using
recombinant, truncated bromodomains. Potent binding to full-
length endogenous BRPF1 (pIC50 8.6) was confirmed in a
chemoproteomic competition binding assay using pull-down of
lysate from HuT-78 cells followed by immuno readout (Figure
4). Selectivity over endogenous BRD3 was also shown.
Table 7. Summary of Properties of Compound 34
BRPF1
TR-FRET pIC50
BROMOscan pKd
NanoBRET pIC50
TR-FRET pIC50
TR-FRET pIC50
BROMOscan
8.1
9.5
b
7.7
BRPF 2/3
5.1/4.8
a
BRD4 BD1/2
4.7 /<4.3
c
selectivity
>1600-fold
2.0
16
Chrom logDpH 7.4
CLND solubility (μg/mL)
140
iv CLb (mL/min/kg)/t1/2 (h)
107/1.7
85/22
F% ip/po (3 mg/kg)
a
b
See also Table S1, Supporting Information. Promega Corp., Figure
c
S5. DiscoveRx Corp., Table S2.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00092.
■
S
Synthetic procedures, analytical data, methods, Figures
S1−S7, and Tables S1−S3 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: emmanuel.h.demont@gsk.com.
*E-mail: armelle.5.le-gall@gsk.com.
*E-mail: Robert.Sheppard@astrazeneca.com.
Figure 4. Potency for endogenous cellular BRPF1. pIC50 of 34 for
BRPF1 and BRD3 measured in a chemoproteomic competition
binding assay (Supplementary Methods, Supporting Information).
■
Present Addresses
Compounds 34 and 38 were assessed in a cellular protein
interaction assay measuring the displacement of NanoLuc-
tagged BRPF1 bromodomain from Halo-tagged histone H3.3
to demonstrate cell permeability and disruption of chromatin
binding (Supplementary Methods, Supporting Information).21
Compound 34 exhibited potent inhibition (Figure S7) with
pIC50 7.7, a 50-fold improvement over 1. The less active
analogue 38 gave a reduced effect (pIC50 5.4), in line with its
biochemical data.
Screening 34 against a panel of 48 unrelated assays revealed
only off-target activities that are relatively weak compared to
the BRPF1 potency (Table S3). However, to minimize the
chance of off-target effects we recommend that a concentration
of no higher than 1 μM should be used in cell-based assays. To
assess the suitability of compound 34 for in vivo studies, its
DMPK characteristics were determined in male CD1 mice via
iv, po, and ip administration. The results indicate that the ip
route of administration would be suitable for dosing this
molecule in potential PK/PD models. In order to be able to
compare biochemical potency measurements to free blood
concentrations, the fraction unbound (fub) in the CD1 mouse
was also determined with a resulting value of 7.9% (Table 7 and
Table S4).
(R.J.S.) Oncology Innovative Medicines and Early Develop-
ment, AstraZeneca, Cambridge Science Park, Milton Road,
Cambridge CB4 OWG, U.K.
(I.B.) Genome Biology Unit, European Molecular Biology
Laboratory, Meyerhofstrasse 1, Heidelberg, Germany.
Notes
The authors declare no competing financial interest.
Biographies
Armelle Le Gall gained her MSc in Chemoinformatics from the
University of Sheffield, U.K. After graduating, she joined GSK, where
she is currently an Investigator in the Molecular Design department.
She has been mainly involved in medicinal chemistry projects in the
Respiratory and Immuno-Inflammation Therapeutic areas. Most
recently, her research has focused on structure-based drug design, in
particular, including protein flexibility, and also high throughput
screening data analysis.
Robert J. Sheppard graduated with a degree in Chemistry from the
University of Oxford in 1997. After joining SmithKline Beecham he
worked on the design and synthesis of novel antibacterial agents
targeting tRNA synthetases, bacterial topoisomerase, and the
ribosome. In 2009 he moved to GSK’s Epinova Discovery Perform-
ance Unit as a team leader focussed on developing inhibitors of
epigenetic proteins including demethylases, deiminases, and bromo-
domains. Robert moved to AstraZeneca’s Oncology iMED in 2015
where he maintains a strong interest in the role of epigenetics in
cancer and immuno-oncology. He represents AstraZeneca in the
Kinetics for Drug Discovery Innovative Medicines Initiative.
To conclude, we have optimized compound 1 to produce 34
(GSK6853), a potent (300 pM), soluble, cell active (20 nM),
and highly selective inhibitor of the BRPF1 bromodomain, and
demonstrated properties suitable for cellular and in vivo studies.
We hope that disclosure of this chemical probe will stimulate
further investigation into the functions of the BRPF1
bromodomain.
ACKNOWLEDGMENTS
We thank Jacqui Men
Corporation for facilitating NanoBRET assays. Thanks to Tony
Cooper for chemistry and discussion, Eric Hortense and Steve
■
́
dez and Danette Daniels of Promega
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reviewed and carried out in accordance with Animals (Scientific
Procedures) Act 1986 and the GSK Policy on the Care, Welfare
and Treatment of Animals.
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ABBREVIATIONS
■
CLND, ChemiLuminescent Nitrogen Detection; LE, ligand
efficiency; KAc, acetyl-lysine; Sol, solubility
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