Selectivity, ligand deconstruction, and cellular activity analysis of a BPTF bromodomain inhibitor

Article abstract of DOI:10.1039/c8ob02599a

Bromodomain and PHD finger containing protein transcription factor (BPTF) is an epigenetic protein involved in chromatin remodelling and is a potential anticancer target. The BPTF bromodomain has one reported small molecule inhibitor (AU1, rac-1). Here, advances made on the structure-activity relationship of a BPTF bromodomain ligand are reported using a combination of experimental and molecular dynamics simulations leading to the active enatiomer (S)-1. Additionally, a ligand deconstruction analysis was conducted to characterize important pharmacophores for engaging the BPTF bromodomain. These studies have been enabled by a protein-based fluorine NMR approach, highlighting the versatility of the method for selectivity, ligand deconstruction, and ligand binding. To enable future analysis of biological activity, cell growth analyses in a panel of cancer cell lines were carried out using CRISPR-Cas9 and (S)-1 to identify cell-based model systems that are sensitive to BPTF inhibition.

Full text of DOI:10.1039/c8ob02599a

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Biomolecular Chemistry
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Selectivity, ligand deconstruction, and cellular
activity analysis of a BPTF bromodomain inhibitor†
Cite this: DOI: 10.1039/c8ob02599a
Steven E. Kirberger,‡a Peter D. Ycas,‡a Jorden A. Johnson,a Chen Chen,b
Michael F. Ciccone,b Rinette W. L. Woo,c Andrew K. Urick,a Huda Zahid,a Ke Shi,d
Hideki Aihara,d Sean D. McAllister,c Mohammed Kashani-Sabet,c Junwei Shi,e
a
Alex Dickson,f Camila O. dos Santos*b and William C. K. Pomerantz
*
Bromodomain and PHD finger containing protein transcription factor (BPTF) is an epigenetic protein
involved in chromatin remodelling and is a potential anticancer target. The BPTF bromodomain has one
reported small molecule inhibitor (AU1, rac-1). Here, advances made on the structure–activity relationship
of a BPTF bromodomain ligand are reported using a combination of experimental and molecular
dynamics simulations leading to the active enatiomer (S)-1. Additionally, a ligand deconstruction analysis
was conducted to characterize important pharmacophores for engaging the BPTF bromodomain. These
studies have been enabled by a protein-based fluorine NMR approach, highlighting the versatility of the
method for selectivity, ligand deconstruction, and ligand binding. To enable future analysis of biological
activity, cell growth analyses in a panel of cancer cell lines were carried out using CRISPR-Cas9 and (S)-1
to identify cell-based model systems that are sensitive to BPTF inhibition.
Received 20th October 2018,
Accepted 23rd January 2019
DOI: 10.1039/c8ob02599a
rsc.li/obc
finger and bromodomain via binding to trimethylated histone
Introduction
H3K4me3 and acetylated histone H4K16ac respectively.4
BPTF (bromodomain and PHD finger containing transcription
Motivating interests for therapeutic development, BPTF
factor) is the largest component of the NURF (nucleosome associates with the oncoprotein c-MYC and is required for
remodelling factor) complex and is essential for transcrip- tumorigenesis in high-expressing MYC cells.5 Dysregulation of
tional regulation of many processes in mammalian chromatin the protein has been implicated in a number of diseases
biology including early embryo development,1 stem cell pluri- including: pancreatic cancer,5 melanoma,6 colorectal cancer,7
potency,2 and T-cell immune function.1 The 3046 amino acid hepatocellular carcinoma,8 breast cancer,2 bladder cancer,9
protein was found to contain a bromodomain and two PHD
fingers, domains commonly found on histone and DNA
binding proteins (Fig. 1).3 To facilitate transcriptional regu-
lation, structural studies have shown that BPTF engages the
nucleosome in a bivalent manner with its C-terminal PHD
aDepartment of Chemistry, University of Minnesota, 207 Pleasant St. SE.,
Minneapolis, MN, 55455, USA. E-mail: wcp@umn.edu
bCold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724,
USA. E-mail: dossanto@cshl.edu
cCenter for Melanoma Research and Treatment, California Pacific Medical Center
Research Institute, San Francisco, CA 94107, USA
dDepartment of Biochemistry, Molecular Biology, and Biophysics, University of
Minnesota Twin Cities, Minneapolis, MN 55455, USA
ePerelman School of Medicine, University of Pennsylvania, 3400 Civic Center
Boulevard, Philadelphia, PA, 19104, USA
fDepartment of Biochemistry & Molecular Biology, Michigan State University,
East Lansing, MI, 48823, USA
Fig. 1 (Left) Structure of racemic AU1 (rac-1). (Right) Ribbon diagrams
from the crystal structures of the bromodomain of BPTF (gray) overlaid
with the bromodomain of 5FW BPTF (blue) with the fluorine of W2824
labeled in green. (Bottom) Domain scheme for BPTF with the plant
homeodomain (PHD) fingers and bromodomain (BRD) labeled.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02599a
‡These authors contributed equally to this manuscript.
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and lung cancer.10 Given the relevance of BPTF in disease and
its association with NURF, we sought to discover what role the
engagement of the bromodomain with acetylated histones on
chromatin plays in the context of disease states. Using a newly
developed protein-observed fluorine NMR (PrOF NMR) assay
for bromodomain ligand discovery,11 we reported the first
small molecule inhibitor of the BPTF bromodomain (AU1,
rac-1, Fig. 1), selective for BPTF over BRD4.12 Rac-1 was further
shown to be cell active, via reducing transcription in a BPTF-
specific reporter assay.12 Additionally, inhibitor treatment of
breast epithelial cells decreased c-MYC regulated genes, c-MYC
chromatin occupancy, and led to G1 cell cycle arrest.2
Despite the biological importance of BPTF, and the success
of chemical probe development for other bromodomains,13,14
outside of rac-1, ligands for the BPTF bromodomain are
lacking. Rac-1 has only a moderate potency (Kd = 2.8 μM), and
its selectivity profile has not been fully characterized. Here we
report our efforts to more thoroughly characterize rac-1 and
use PrOF NMR in structure–property studies for improving its
metabolic stability and selectivity. We further analyze the
mode and efficacy of binding through ligand deconstruction
in combination with weighted ensemble molecular dynamics
simulations. These studies highlight the versatility of PrOF
NMR for bromodomain ligand characterization, and identify
areas for improving future BPTF bromodomain inhibitors.
Fig. 2 (A) Ribbon structure of the acetylated lysine binding site of the
bromodomain of BPTF with 5FW (2824) displayed. The fluorine atom is
colored in cyan. N2881 is a key residue for binding of acetylated lysines
on histone proteins. (B) PrOF NMR using the BPTF bromodomain labeled
with 5FW at 2824 of rac-1 and both enantiomers against 5FW BPTF.
Each ligand is in a two-fold excess of the protein. Both experiments with
rac-1 and (S)-1 resulted in significant broadening of the fluorine
resonance.
the (S) enantiomer, more significant broadening of the 5FW
resonance into baseline than the racemate was observed
(Fig. 2B). Conversely, (R)-1 showed no broadening in the NMR
assay, indicating that only the (S) enantiomer significantly con-
tributed to the binding affinity originally reported, whereas the
(R) enantiomers of future analogs can serve as useful control
molecules.
Results and discussion
PrOF NMR identification of the active enantiomer of rac-1
For chemical probe development, having access to an inactive
stereoisomer with similar physicochemical properties can be a
useful control. In the original identification of AU1 (rac-1) as a
ligand for the BPTF bromodomain, the racemate was tested.
X-ray crystal structure of 5FW BPTF supports a lack of
structure perturbation from fluorine for PrOF NMR
experiments
Using our previously reported methods, we synthesized both To gain further insight into how (S)-1 interacts with the BPTF
the (R) and (S) enantiomers ((R)-1, and (S)-1) and tested them bromodomain, a co-crystal structure was highly desirable.
using PrOF NMR.2
Although to date we have been unsuccessful, during these
PrOF NMR is a structure-based method for characterizing studies we investigated the crystallization of the 5FW-BPTF
ligand interactions and protein binding sites. A comparative bromodomain. Previously, we characterized the stability of the
study has shown PrOF NMR to be comparable in speed and hit fluorine-labeled bromodomain by circular dichroism and
identification to ligand-observed NMR, however an advantage determined a negligible change in protein thermal stability
is the ability to rapidly quantify affinities of weak binding from fluorination relative to the unlabelled protein.12 To
ligands based on perturbation of fluorine resonances.15 The further verify that the native fold is intact, we have now solved
BPTF bromodomain contains a single tryptophan at position the crystal structures of the BPTF bromodomain in both the
2824 in the WPF shelf of the protein near the histone binding fluorinated and non-fluorinated forms. Alignment of the struc-
site (Fig. 2A). As described previously, for NMR analysis we tures gave a root-mean-square deviation (RMSD) of 0.1548 Å
labeled the BPTF bromodomain with 5-fluorotryptophan for the protein backbone and a RMSD of 0.3516 Å for W2824
(5FW).12 We monitored the 19F resonance upon addition of (see Fig. 1 and S2-1†). Proline 2836, far from the binding site,
both enantiomers. PrOF NMR binding isotherms can be used to had the largest RMSD at 0.8093 Å. The similarity in structure
quantify the dissociation constant based on measuring dose- of the 5FW and unlabeled BPTF further supports fluorine
dependent changes in chemical shifts of protein ligands that labeling and PrOF NMR as a viable binding assay. Given the
exhibit fast chemical exchange rates. In the case of moderate lack of a co-crystal structure of (S)-1, and further verification of
affinity ligands such as rac-1, significant resonance broaden- the non-perturbing nature of the fluorine in the bromodo-
ing is observed as the intermediate chemical exchange rate for main, we thus turned to indirect methods using PrOF NMR to
binding is longer, but not sufficiently long enough to resolve guide medicinal chemistry efforts using structure–activity
the bound and unbound state. In the PrOF NMR spectrum for relationships (SAR) and ligand deconstruction.
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(S)-1 Structure–activity relationships
order of magnitude loss in affinity relative to rac-1. Fast chemi-
cal exchange allowed for titration to derive a Kd = 30 μM
(Fig. 4). The ethyl amide (10) also showed fast exchange
binding behaviour consistent with a weak interaction, but the
affinity was unable to be quantified. A larger amide (11)
resulted in a complete loss of binding (Fig. S10 and 11†).
Unfortunately, upon changing the ester to an amide, we also
observed an attenuated selectivity with respect to BRD4(1) by
PrOF NMR (Fig. S34†). Future analogs containing the more
stable methyl amide will need to have improved binding
activity and selectivity through modifications on alternative
sites on the molecule.
In addition to a modest binding affinity of 2.8 μM, rac-1, and
by analogy (S)-1, possess several properties which we wished to
improve upon. One significant challenge was the low solubility
of rac-1, as observed by an increase in solution turbidity at
concentrations above ∼100 μM. Additionally, given its cellular
activity, for future use of (S)-1 analogs we were concerned
about the susceptibility to cellular esterase hydrolysis of the
methyl ester. Consistent with our concern regarding metabolic
liabilities, pharmacokinetics (PK) studies of (S)-1 in mice, pro-
duced a Cmax of 4540 ng mL−1 (10 μM) in the plasma, and a
rapid half-life at 0.68 h (Fig. S2†).
Due to the importance of the ester, we also explored
isomers of the compound, substituting the methyl ester
around the aromatic ring (2 and 3). In these cases, changing
the meta substitution of the methyl ester on the aryl urea
showed a complete loss of binding in both 2 and 3, indicating
meta substituted methyl esters were critical for binding
(Fig. S8†). Conversely, substitutions made on the aniline ring
consistently showed binding activity through our PrOF NMR
assay. Replacing the aniline nitrogen with an oxygen (5) or
methylating the nitrogen (6) maintained binding to BPTF but
resonance chemical shifts were in the fast-intermediate exchange
regime by PrOF NMR (Fig. S11 and S12†). Substituting the fluo-
rine for a benzyl amine (7) as well as a 3,5-dimethyl substitution
pattern (8) show similar characteristics as (S)-1 in an NMR titra-
Given the concerns above, we took advantage of our four-
step synthesis for SAR studies (see ESI† for synthetic schemes,
Fig. S5†). To test for ways to improve the stability of our com-
pound based on ester hydrolysis concerns, we synthesized new
analogs of (S)-1 (2–14, Fig. 3). We first tested the would-be
product from ester hydrolysis, 12, against 5FW BPTF by PrOF
NMR and determined that binding to the bromodomain was
eliminated. To improve the stability of the scaffold in the pres-
ence of cellular esterases for future cell-based studies, we
replaced the ester group of (S)-1 with a series of amides, 9–11.
For these analogs, only small amides were tolerated although
binding was weakened. The methyl amide (9) showed over an
Fig. 3 Structures of select rac-1 analogs. PrOF NMR titrations of all
compounds can be found in the ESI.†
Fig. 4 Sample titration data of 9, a broadening and shifting of the 5FW
BPTF resonance is observed. Full titration can be found in the ESI.†
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tion, indicating a reduced sensitivity to compound modification Fragment F1 which contains the amino-pyrimidine portion of
(Fig. S16†). The regioisomer of (S)-1, 13, also showed similar (S)-1 did not bind BPTF despite being a motif found in other
binding characteristics to the original scaffold by PrOF NMR bromodomain inhibitors.13,14 Alternatively, fragment F4 con-
(Fig. S21†). We conclude, from these PrOF NMR SAR studies, that taining the methyl ester displayed a Kd as determined by PrOF
the amino pyrimidine terminus of the molecule may be more NMR of 1.3 mM with the highest ligand efficiency of 0.28. This
solvent exposed when binding to the protein, whereas the methyl can be compared with rac-1 with a ligand efficiency of 0.22.
ester is bound more deeply in the bromodomain binding site.
We tested additional fragments, F3, F5, and F6 with results
(S)-1 has limited solubility in aqueous solution and the summarized in Fig. 5. In these cases, a similar ligand
broadening of its 19F resonance by NMR indicates a highly efficiency was maintained (0.22–0.23) of fragments tested.
associated state. We looked at the urea moiety within the Extending fragment F1 to F2 to include the amino-pyrrolidine
scaffold as a potential source of aggregation and sought to and the carbonyl group of the urea also bound with a Kd of
alter this functional group to improve the behaviour of (S)-1 in 1.3 mM, albeit with a reduced ligand efficiency of 0.17. We
aqueous solution. The urea was separated by inserting a note, that fragments can bind in multiple orientations if not
methylene as seen in compound 14, drastically improving the anchored by a strong non-covalent interaction.20 Despite this
water solubility to >1 mM at pH 7.4 as assessed by a lack of possibility, these data support that the binding of (S)-1 to the
solution turbidity. However, we observed a reduction in bromodomain of BPTF is the result of an additive effect of
binding affinity using PrOF NMR titrations when the urea was several weak contacts throughout the (S)-1 scaffold.
interrupted. We also attempted to reverse the substitution of
Molecular dynamic simulations of (S)-1 with BPTF
the pyrrolidine ring with the hopes of interrupting the aggre-
gation potential of the urea by reducing the hydrogen bond
capability of one of the nitrogens ((S)-4 and (R)-4). Upon this
change, we observed by PrOF NMR in (S)-4 a stoichiometric
binding at the concentration of protein that we used, indicat-
ing that its binding affinity was at least an order of magnitude
below our protein concentration, i.e., 5 μM or lower. We then
had this compound tested via AlphaScreen (Reaction Biology)
as a competition-based assay between the bromodomain and
an acylated histone; however, an IC50 of 70 μM was observed.
This difference in binding affinity observed via PrOF NMR and
AlphaScreen could potentially be due to the reduced DMSO con-
centration necessary for the AlphaScreen assay 0.25% vs. the
1% used in PrOF NMR, this reduction in co-solvent concen-
tration limits the solubility of this analog for the assay, and
points to further improvements that are needed for solubility
enhancement of our compounds.
We have not solved a co-crystal structure of (S)-1 to validate a
binding mode to support our SAR and ligand deconstruction
Ligand deconstruction of (S)-1 by PrOF NMR
Although PrOF NMR SAR experiments provided insights for
improving solubility and stability of the rac-1 scaffold and the
identification of the active enantiomer, (S)-1, none of the
changes led to a significant increase in affinity. These results
led us to explore a ligand deconstruction analysis of (S)-1 to
evaluate the contribution to binding of specific pharmaco-
phores of the parent scaffold.16,17 PrOF NMR has mainly been
used for hit identification, and only a preliminary ligand
deconstruction study was reported before.18 However, NMR is
well-suited for deconstruction analysis due to its ability to
quantify the affinities of weak binding fragments. We syn-
thesized fragments F1–F6 of the parent scaffold, which could
be used to test the relative contributions of each portion of the
molecule by PrOF NMR. In the original study which identified
rac-1 as a BPTF bromodomain ligand, apparent important
pharmacophores for binders of BPTF were the aryl urea and
the meta carbonyl group (ester or ketone).19 In light of this and
the SAR results described above, we predicted a stronger inter-
Fig. 5 Ligand deconstruction study of (S)-1. With the exception of F1,
all fragments bind 5FW BPTF with similar ligand efficiencies, indicating
binding contacts are dispersed throughout the molecule. Using PrOF
NMR, a binding isotherm can be generated to quantify weak binding
ligands. F6 is shown as a representative example. NB = No observable
action from this portion of the molecule when taken alone. binding.
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analysis, necessitating alternative methods to provide higher Bromodomain selectivity analysis of (S)-1
resolution information on binding. We therefore turned towards
As (S)-1 remained our best compound, we next conducted a
preliminary analysis of bromodomain selectivity for (S)-1.
Rac-1 was originally shown to be selective for BPTF over the
N-terminal BRD4 bromodomain, BRD4(1), which also con-
tains a WPF shelf and is a member of the bromodomain and
extra-terminal domain (BET) family of proteins. In addition
to 5FW-labeled BRD4(1), we tested three other WPF shelf-
containing proteins: a second BET bromodomain, 5FW
BRDT(1), PCAF, which is on the same branch of the phyloge-
netic tree as BPTF,23 and a highly homologous bromodo-
main from Plasmodium falciparum, PfGCN524 (Fig. 7).
Although PfGCN5 is not a human bromodomain, acetylated
peptides have been shown to bind to both the PfGCN5
bromodomain and BPTF with similar affinity.25 Additionally,
PCAF inhibitors also inhibit PfGCN5 suggesting high simi-
larity in binding sites of these structurally related bromo-
domains.26 In the PrOF NMR spectra, (S)-1 did not perturb
resonances for either BRD4(1) or BRDT(1). In the case of the
PCAF bromodomain, although PCAF and BPTF are very close
on the phylogenetic tree, only a small chemical shift was
observed with slight broadening, without a significant dose
dependence (see ESI†). These results indicate a moderate
level of selectivity is maintained for BPTF over the structu-
rally similar PCAF bromodomain. Finally, in the case of
PfGCN5, a significant broadening of the most downfield
resonance was observed indicating an interaction similar to
BPTF. We have tentatively assigned this resonance to the
tryptophan in the WPF shelf based on prior binding studies
with pan-inhibitor bromosporine.27 Interestingly, rac-1 has
shown antimalarial activity,28 consistent with bromodomain
targeting, although target identification for rac-1 has not
been carried out against the parasite. When potency is
increased, a more complete bromodomain profiling will be
conducted.
molecular dynamics (MD) simulations of (S)-1 binding to BPTF
using a recently reported weighted ensemble approach21 that
has been used to investigate bromodomain-ligand inter-
actions.22 The inclusion of full protein motion in explicit
solvent in our MD simulation versus our prior docking studies
was anticipated to improve our predicted binding pose.
Starting from multiple docked initial structures, a network
model of ligand poses was constructed, and the weights of
each state were used to predict an ensemble of probable poses
(Fig. 6A). These poses support that the methyl ester serves as
an acetylated lysine mimic as it can hydrogen bond to the con-
served N2881 of BPTF (Fig. 2, 6B, and S4†). The importance of
the methyl ester in the acetyl lysine binding site is consistent
with the high ligand efficiency of F4 and is consistent our SAR
study that alterations of the methyl ester such as substitutions
around the aromatic ring obliterate binding, as well as the
intolerance for large amide groups at this position. MD simu-
lations show the attached aryl ring also interacts with Phe2887
via a stacking interaction, while the urea is involved in
H-bonding interactions with D2834. Finally, the p-fluoroani-
line is exposed to solvent near W2824 in the WPF shelf, con-
sistent with our findings that other groups can be installed at
this position (e.g., 7, 8) and that replacement of the p-fluoroa-
niline with a chlorine maintains modest affinity (F3, Kd
=
35 μM). In addition to helping explain our SAR, this docking
pose will be used to guide future analog designs in the
absence of a co-crystal structure.
Fig. 6 (A) Conformation space network describing all of the binding
poses found and their interconnectivity. Each node represents a ligand
pose and nodes are colored according to the solvent accessible surface
area of the ligand (blue/black = unbound, green/yellow = fully bound).
The two starting poses, as well as the predicted most probable pose are
labeled. (B) Views of the most probable pose. A density isosurface was
computed using 10 random ligand structures taken from most probable
state (shown in transparent gray). Interacting residues are labeled.
Fig. 7 Selectivity of (S)-1 toward four different bromodomains, BRD4
(1), BRDT(1), PCAF, and PfGCN5. All experiments were carried out with
50 μM protein in the absence (black spectrum) or presence of 100 μM
(S)-1 (red spectrum).
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Kinase selectivity analysis of rac-1 stereoisomers
(S)-1 and (R)-1 treatment, to identify cell lines that were less
susceptible to off target effects shared by (S)-1 and (R)-1, and
were sensitive to BPTF inhibition. We first screened for cell
lines with increased BPTF gene expression levels, and identi-
fied HepG2 (Liver hepatocellular carcinoma cell line), K562
(Chronic myelogenous leukemia cell line), and MCF-7 cells
(Hormone positive breast cancer cell lines) amongst the cancer
cell lines with high levels of BPTF mRNA (Fig. S1A†). Next, we
utilized an all-in-one CRISPR-Cas9-GFP lentiviral vector and
single stranded guide RNA sequences (sgRNAs) specific to tar-
geting BPTF to define the effects of BPTF deletion on cellular
growth, as a readout for growth dependency (Fig. S1A–C†). The
percentage of GFP positive cells (GFP+), a mark for lentiviral
infection, was monitored for several weeks using flow cytome-
try, as a proxy for the effects of BPTF deletion of growth of
HepG2, K562 and MCF-7 cell lines. Control sgRNA for the
essential gene RNA Polymerase 3 (RPA3, cell depletion effect),
and vectors lacking specific sgRNAs (Empty vector) were used
for comparison. HepG2 liver cancer cells were the least sensi-
tive to BPTF depletion through the experimental time course
(Fig. 8A). Conversely, the percentage of GFP+ cells gradually
decreased over time in MCF-7 cells and K562 expressing BPTF
sgRNAs, similar to the RPA3 sgRNA depletion control experi-
ment. K562 cells were the most sensitive to BPTF depletion,
with the percentage of GFP+ cells dropping ∼2-fold over time.
Together, these results demonstrate that K562 and MCF-7 cells
are dependent on BPTF function for growth, providing useful
cell line models to define epigenetic mechanisms associated
with BPTF dependency, and for testing the efficacy of BPTF
bromodomain inhibitors.
Rac-1 was originally discovered using the published kinase
inhibitor set library.12 Therefore, we also tested for kinase
activity of (S)-1. In a recent report evaluating inhibition against
the kinome, rac-1 showed 82% inhibition of two kinases,
CDKL2 and TRKC, when tested at 1 μM.29 Four additional
kinases (CDK11, TRKB, HPK1, p38-δ) were inhibited between
51–68%. Based on this data, we utilized the KINOMEscan
service from DiscoverX to test (R)-1 and (S)-1 against CDKL2
and TRKC. (S)-1 Showed dissociation constants of 260 nM and
200 nM against CDKL2 and TRKC respectively, whereas (R)-1
had dissociation constants of 1200 nM and 400 nM against the
two kinases (Table 1). While there is a nearly five-fold differ-
ence in affinity toward CDKL2, there is only a two-fold differ-
ence toward TRKC, indicating that the stereocenter is not criti-
cal to the binding of these targets, in particular TRKC. These
kinases need to be considered in future cell-based studies.
We looked to make changes to limit the kinase binding of
the molecule, thereby reducing potential off-target effects
in vivo. The amino-pyrimidine pharmacophore found in (S)-1
is a known kinase-binding functionality which recognizes the
hinge region of the ATP binding pocket in kinases. Two of the
simplest changes that we envisioned were replacing the protic
nitrogen with an oxygen (5), and the alkylation of the protic
nitrogen (6). N-Methylation of the aniline nitrogen yielded stoi-
chiometric binding again in the PrOF NMR assay but a lack of
activity was observed in AlphaScreen, potentially for the same
solubility reason previously discussed. Upon exchanging the
nitrogen for an oxygen, binding was maintained, albeit wea-
kened relative to (S)-1, as the ligand approached fast-inter-
mediate exchange in the PrOF NMR assay. Encouragingly, after
testing these two compounds against CDKL2 and TRKC we
observed a drastic reduction in kinase binding in the N-methyl
case (6), and a complete loss of binding with compound 5
(Table 1). The phenoxypyrimidine motif will be a useful design
element for future ligands to remove kinase binding.
Given that additional loss of protein function beyond bro-
modomain effects can result from gene deletion studies, we
Cancer cell line sensitivity to BPTF gene deletion and rac-1
stereoisomers
Prior studies of rac-1, supported BPTF target engagement and
cellular activity.12 Here, our results led to the identification of
the active enantiomer, (S)-1. However, additional kinase off-
targets have now also been characterized. Prior to further
mechanistic studies, as a preliminary analysis of more global
cellular effects of (S)-1, we tested the sensitivity of cells to both
Table 1 Binding affinities of (S)-1 and analogs against known kinase
targets
Fig. 8 Sensitivity of cancer cell lines to (S)-1 and (R)-1. (A) CRISPR-Cas9
BPTF-targeting. HepG2, MCF-7 and K562 cell lines were infected with
all in one CRISPR-Cas9-GFP lentiviral particles, expressing sgRNAs tar-
geting RPA3 gene (depletion control) and the BPTF gene. Fold change
was calculated by comparing final measure of GFP to initial infection
GFP. n = 2 technical replicates. (B) Cell viability analysis in cancer cell
lines treated with 5 μM of either (S)-1 or (R)-1 for 72 h, following incu-
bation with AlamarBlue for 4 h. Error bars demonstrate standard devi-
ation across two biological replicates.
Compound number
TRKC Kd
CDKL2 Kd
(S)-1
(R)-1
5
200 nM
400 nM
NB
260 nM
1200 nM
NB
6
2600 nM
19 000 nM
NB = No significant binding.
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tested the effect of (S)-1 or (R)-1 on the growth of BPTF-depen- (C.O.D.S.) and Pershing Square Sohn Prize (C.O.D.S.) J.A.J. was
dent (K562 and MCF-7) and independent (HepG2) cancer cell supported by a National Institutes of Health Biotechnology
lines. In doing so, cancer cell lines were treated with either training grant 5T32GM008347-23 A.K.U. was supported by a
(S)-1, or (R)-1 at 5 μM for 72 hours, following quantification of UMN Doctoral Dissertation Fellowship. Cold Spring Harbor
cellular viability/growth using an AlamarBlue viability assay. Our Labs shared resources were supported through the Cancer
results show that HepG2 cells are not sensitive to treatment Center 5P30CA045508 and the cell lines were distributed
with either (S)-1 or (R)-1, consistent with BPTF deletion experi- through the Cold Spring Harbor Tissue Culture Facility. We
ments above (Fig. 8). Alternatively, K562 and MCF-7 cells were would like to thank Emily Sherman for early synthetic efforts
sensitive to treatment with (S)-1, demonstrated by a marked on rac-1 analogs.
decrease on cell viability. MCF-7 cells were also sensitive to the
inactive bromodomain inhibitor (R)-1, suggesting that for
MCF-7 cells, off-target effects in addition to those of BPTF
inhibition may be controlling cell growth in response to small
Notes and references
molecule treatment (Fig. 8). Alternatively, K562 cells were only
affected by (S)-1. These results support K562 cells as being a
useful model cell line for further evaluation of the effects of
(S)-1 and for defining the epigenetic mechanisms associated
with BPTF dependency with improved BPTF bromodomain
inhibitors.
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In conclusion, we have studied the SAR of rac-1, the first
reported inhibitor of the bromodomain of BPTF to show
selectivity over that of BRD4(1). We identified that the (S)-enan-
tiomer is the dominant stereoisomer for binding to the bromo-
domain. In addition, substitution of the amino-pyrimidine
moiety led to the diminished binding of the substrate to
CDKL2 and TRKC kinases. These results also showcase PrOF
NMR as a useful protein–ligand characterization tool for quan-
tifying weak binding ligands during ligand deconstruction,
SAR analysis, and testing selectivity against related bromo-
domains. Finally, cell-based experiments including BPTF
depletion, and chemical inhibition, support a BPTF depen-
dence in K562 cells, a chronic myelogenous leukemia cell line.
Richart and co-workers, identified cancer cell lines that could
be sensitive to BPTF inhibition with high c-Myc levels.5
Chronic myelogenous leukemia cells were identified to have
some of the highest c-Myc levels, supporting our BPTF sensi-
tivity studies. Together, these studies provide a framework for
the design of inhibitors for the BPTF bromodomain, an impor-
tant epigenetic effector domain lacking potent ligands.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes under the award numbers (R01GM121414, W.C.K.P,
R01GM130794, A.D., R35GM118047 H.A.) the National Cancer 15 A. K. Urick, L. P. Calle, J. F. Espinosa, H. Hu and
Institute (R01CA215755 M.K.) and Rita Allen Scholar grant
W. C. K. Pomerantz, ACS Chem. Biol., 2016, 11, 3154–3164.
This journal is © The Royal Society of Chemistry 2019
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