Dual Inhibition of TAF1 and BET Bromodomains from the BI-2536 Kinase Inhibitor Scaffold

Article abstract of DOI:10.1021/acsmedchemlett.9b00243

Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.

Full text of DOI:10.1021/acsmedchemlett.9b00243

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Letter
Dual Inhibition of TAF1 and BET Bromodomains
from the BI-2536 Kinase Inhibitor Scaffold.
David Remillard, Dennis L. Buckley, Hyuk-Soo Seo, Fleur M. Ferguson,
Sirano Dhe-Paganon, James E. Bradner, and Nathanael S Gray
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00243 • Publication Date (Web): 13 Sep 2019
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Dual Inhibition of TAF1 and BET Bromodomains from the BI-2536
Kinase Inhibitor Scaffold.
‡
David Remillard1,†, Dennis L. Buckley2, , Hyuk-Soo Seo1, Fleur M. Ferguson1,3, Sirano Dhe-
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Paganon*1, James E. Bradner2* ‡, and Nathanael S. Gray1,3*.
1Department of Cancer Biology, Dana Farber Cancer Institute, 2Department of Medical Oncology, Dana Farber Cancer
Institute, 3 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston
MA, USA.
KEYWORDS: Bromodomain, Kinase Inhibitor, Epigenetics, BI-2536
ABSTRACT: Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in
cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships
within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to
TAF1(2), and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain
while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased
derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic datasets to examine the mechanisms of the ob-
served
TAF1
selectivity,
and
to
provide
a
resource
for
further
development
of
this
scaffold.
Featured in 46 human proteins, the bromodomain is
the largest class of binding module, or "reader domain",
for acetylated lysines (KAc), a pervasive modification in
epigenetic control.1 The development of JQ1 and related
probes for the Bromodomain and Extra Terminal Domain
(BET) subfamily first demonstrated the bromodomain’s
chemical tractability, competively occupying the hydro-
phobic KAc pocket, which is formed by a rim of loops be-
tween domain's common four-a-helical bundle structure.
These probes have provided widely used tools for the
study of BET function, in particular BRD4's role as a mas-
ter transcriptional coactivator, as well as broad therapeu-
tic investiations of BET inhibition in disease.2-4 Subsequent
clinical translations are currently underway.5-7 Broadly
ensuing efforts to extend bromodomain probe develop-
ment have now succeeded in providing selective tools for
nearly all existing subfamilies, enabling direct study of
domain specific contributions to protein function.8
gesting that as recently observed for other non-BET bro-
modomain targets, existing probes may not dramatically
impact protein function.25-27 However, recent studies have
suggested that TAF1 may collaborate with BRD4 to control
cancer cell proliferation in a bromodomain dependent
manner, potentially through cooperating transicriptional
effects.10 Indeed, two structurally dissimilar TAF1 inhibi-
tors have now been independently demonstrated to po-
tentiate the activity of BET inhibition in cultured cancer
13
models.10, These findings have engendered interest in
compounds that may offer dual BRD4/TAF1 inhibition.
Toward this objective, we herein report structure activity
relationships (SAR) toward dual TAF1/BRD4 inhibitors, as
well as TAF1-biased derivatives, from a single kinase in-
hibitor derived scaffold.
In the course of our own studies on bromodomain lig-
and development, we reported the first small molecule
ligand for TAF1, a mid-nanomolar binder accessed
through biased multicomponent library synthesis.9 How-
ever, further exploration of the compound's dimethylisox-
azole warhead revealed a challenging BRD4 bias. Among
other chemotypes previously described were several ki-
nase inhibitor scaffolds,28-29 examples of which have now
seen in-depth study and development as polypharmaco-
logical agents.30-31 In particular, previous work suggested
that the PLK1 kinase inhibitor BI-2536 harbored activity
on TAF1 through broad phage display (Ember, et al) and
melt temperature (TM) shifts panels (Ciceri, et al) .28-29, 32
Several recent studies have reported small molecule
ligands for the dual bromodomain containing TAF1.9-13 As
the largest of approximately 13 Tata-binding protein
(TBP) associated factors “TAFs” that assemble to comprise
the general transcription factor IID (TFIID),14-15 TAF1 scaf-
folds the pioneer factor for general eukaryotic transcrip-
tion.16-18 In-vitro transcription and temperature sensitive
mutant experiments have implied that TAF1 may be dis-
pensable for basal transcription,19 but directly mediates
transcription downstream of RB,20 B-Myb,21 and C-Jun,22
and is also critical to transcription downstream of key cell
cycle drivers.23-24 Despite these roles, no direct phenotypes
of TAF1 bromodomain inhibition have been reported, sug-
To assess the ability of BI-2536 to bind TAF1(2), we
first profiled the compound in dose via competitive phage
display (DiscoverX), confirming activity on the second
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Table 1. Substitutions to the BI-2536 KAc mimetic (R1),
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bromodomain of TAF1 (TAF1(2)) (170nM) in addition to
BRD4(1) (24 nM) (Table S1). To facilitate further explora-
tion of SAR within TAF1(2), we implemented an AlphaS-
creen assay leveraging a biotin functionalized derivative of
BI-2536 (SI, Synthetic Procedures A). Guided by previous-
ly published BRD4 cocrystal structures, we sought to de-
termine the influence of substituents buried within the
acetyl-lysine recognition pocket, as well as those posi-
tioned within the “ZA channel”, a known hotspot for bro-
modomain selectivity (Fig. 1).1, 8 Within both TAF1(2) and
BRD4(1), the imbedded methyl amide acetyl lysine mimet-
ic showed high sensitivity to modification (Table 1; 2,3).
Modifications of the ZA alkoxy substituent (4-7) were bet-
ter accommodated. Truncation of the buried asymmetric
ethyl group to a methyl substituent (8,9) improved poten-
cy, while substitution of the solvent projected 4-amino-
methylpipiridine solubilizing group was also well tolerat-
ed (Table S2).
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ZA-channel projecting alkoxy substituent (R2), and
asymmetric ethyl (R3).
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Table 2. Kinase inactivation by aminopyrimidine hinge-
binding motif replacement. Inset image: cartoon cutaway of
BI-2536 bound to PLK1 (2RKU) showing hinge binding H-
bonding (dashed) to key Cys residue backbone (sticks).
Figure 1. A) Chemical structure of BI-2536. B) Surface repre-
sentation of BI-2536-BRD4(1) cocrystal structure (4O74)29.
C) Cartoon cutaway of view as in (B) showing conserved wa-
ters (spheres), key residues (sticks) and H-bonding (dashed).
We next considered that the potent cytostatic activity
arising from the PLK1 kinase activity of this scaffold could
obscure biological studies of bromodomain inhibition. We
therefore sought to alter the H-bonding capabilities of the
imbedded aminopyrimidine hinge-binding motif, a well-
known approach for disrupting kinase binding (Table 2).33
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Replacement of the key hydrogen bond donating NH with
NMe as we previously reported,34 or with O,35 largely abol-
ished kinase activity, but resulted in both cases in dra-
matic and preferential loss of TAF1 bromodomain activity
(versus BRD4) (10,11). As this NH engages in water-
mediated H-bonding within BRD4, we considered that a
similar interaction may underlie an imporant contribution
to TAF1 affinity (Table 2, Fig. 1C). We therefore applied an
alternative strategy of removing hinge H-bond accepting
to the pyrimidine core through N to CH substitution,36 and
were pleased to find that this approach successfully dimin-
ished activity against the PLK1 kinase while better retain-
ing TAF1 affinity (Table 2; 12).
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Advancing this bromodomain selective scaffold, we
sought to assess further avenues toward potency on TAF1,
as well as to identify strategies to bias for TAF1 activity
relative to BRD4. Introducing the favored asymmetric
ethyl to methyl substitution into the kinase-selective pyri-
dopiperazone scaffold resulted in a potent dual inhibitor
of TAF1(2) (16 nM IC50) and BRD4(1) (37 nM IC50) (Ta-
ble 3; 13). Unexpectedly however, previously examined
substitutions of the methoxy ZA substituent, particularly H
replacement, were no longer favored, potentially reflecting
rearrangement of the water mediated interaction network
involving this substituent (14).29 To better understand the
binding mode of this series, we obtained an X-ray cocrystal
structure of 15 bound to TAF1(2). Interestingly, the re-
solved structure contains two protein molecules per unit
cell, with 15 showing a conserved core binding mode, but
exhibiting two dissimilar poses of the substituted 4-
aminobenzamide tail (Fig. 2, 6P38).
Table 3. Alkoxy substituent SAR for the kinase selective
dihydropyridopyrazinone scaffold.
Figure 2. A) Chemical structure of 15. B) Cocrystal structure
of 15 and TAF1(2) showing observed binding mode (I). C)
Observed binding mode (II) (6P38).
engage Asp1524, as well as direct aryl tail appendages
(Table S3). We observed broadly diminished affinity
across these analogs, suggesting that while the parent
methoxy-N-(1-methylpiperidin-4-yl)benzamide tail group
likely samples a variety of conformations, its poses are
nonetheless well positioned to contribute considerably to
affinity. Key interactions likely include offset pi-
stackingand backbone H-bon bonding to Phe1536 in Pose
(I), and T-stacking with Trp1526 in Pose (II). We note
however, the influence of crystal packing contacts within
the subcell featuring Pose (II) (Fig. S2).
We next turned our attention to examine SAR within
the dihydropyridopyrazone warhead, with a particular
interest in TAF1-biasing modifications. Here, a neutral
solubilizing tail was implemented to facilitate purifica-
tions; although we noted a cost in affinity against TAF1
(93nM for 17 vs 16nM for 13). We anticipated that TAF1’s
Val1547 might better accommodate bulky replacements
to the proximal cyclopentane moiety (Table 4; 17-22) rel-
ative to BRD4’s equivalently placed Leu94. Of this series,
expansion to a six membered ring best improved TAF1
bias, with tetrahydropyran (THP) substitution further
enhancing this effect (Table 4; 19,20). Further considering
To further probe the ZA SAR in light of the apparent
crystallographic flexibility, we undertook to study the role
of tail group positioning in TAF1(2) binding. We thus ex-
amined substituents that would preclude ligand planarity,
alternative tail compositions including addition of sulfon-
amide or methylene spacers which might allow improved
“WPF shelf” access as in related efforts,37 or which might
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Table 4. Cycloalkyl moiety expansions.
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Figure 3. A) TAF1(2)-15 cocrystal structure (6P38) showing
proximal Val1547 (gray spheres). B) Structural alignment
(Pymol) of BRD4 (4O74) showing proximal Leu94 (red
spheres). C) Schematic of tricyclization approach. Inset table:
aAlphaScreen IC50s from quadruplicate measurement, and
bIC50 Ratio: (BRD4(1)/TAF1(2)).
Table 5. Bicyclic core exploration.
that Leu94 of BRD4 packs between the cyclopentyl and
asymmetric ethyl substituents of BI-2536, we envisioned
that more severe steric discrimination of BRD4 might be
achieved by linking these positions through this space via
4-carbon bridgehead (Fig. 3). Gratifyingly the resulting
tricyclic warhead-containing compound 23 gave a 66-fold
improvement (vs. parent 17) in measured selectivity
(BRD4 IC50/TAF IC50).
Alongside cycloalkyl modifications, we concurrently
investigated substitutions to the warhead's pyridine ring
(24-30), as well as replacement of the embedded methyl
amide with extended acyl mimetics following the ap-
proach of Crawford et al (29, 30) (Table 5).11 Although we
found removal of the pyridyl nitrogen to be disfavored, we
noted that 7-methoxy analog 27 showed largely abrogated
BRD4 activity alongside only minor losses in TAF1 affinity
relative to its parent analog (24). This SAR is consistent
with the observations of Bennett et al,37 who found that
methoxy derivatization at an analogous position of a relat-
ed bicyclic TRIM24 inhibitor also diminished BRD4 bind-
ing. Alternatively, we found that the extended 1-butenyl
analog 30 also promoted TAF1 bias, as reported within an
alternative pyrrolopyridinone scaffold.11 As discussed pre-
viously, this permissiveness likely relates to the ability of
TAF1 to favorably rearrange the conserved water net-
work, in line with its ability to recognize natural lysine
butyrylation and crotonylation.11,38
Encouraged by their performance, we selected the tri-
cyclic core and N4-THP modifications for further examina-
tion. Combining these features with the preferred BI-2536
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tail group confirmed the respective contributions of the
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THP group to improved potency (31), and tricyclization to
TAF1 selectivity (32) (Table 6). As THP featuring com-
pound 31 offers attractive dual potency against both TAF1
and BRD4, it was prioritized for assessment of family-wide
selectivity. Profiling via the commercial phage-display
BROMOscan (DiscoveRx) assay confirmed potent dis-
placement to be largely restricted to TAF1(2) and the BET
family bromodomains, with additional activity observed
against the family IV bromodomain BRPF1 (Table S4).
9
To investigate whether structural features identified
in this study might be favorably combined, we prepared
analog 33 featuring both 1-butenyl and THP functionality.
We were pleased to observe that this analog showed
marked TAF1 bias, while still retaining considerable po-
tency, as well as PLK1 inactivity (Table 6, Table S5). To
pursue further structural insight into the selectivity pro-
moted by analogs 32 and 33, we resolved X-ray cocrystal
structures of binding modes within TAF1(2) (Fig. 4, 6P39,
6P3A) The obtained 32 cocrystal reveals the cycloalkyl
bridge to be asymmetrically projected as anticipated, ef-
fecting the intended steric contact with Val1574 (Fig. 4
A,B). Indeed, an accommodating rearrangement of this
side chain orientation is observed relative to the acyclic
analog shown in Figure 2, supporting that increased bur-
den of equivalent the Leu94 underpins diminished bind-
ing within BRD4(1). Contrastingly, within the 33 cocrystal,
we observe a distinct mode of bromodomain discrimina-
tion by projection of the 1-butenyl modification into the
floor of the KAc binding pocket (Fig. 4 C,D). Here, structur-
al comparisons reveal remarkable coherence to the 1-
butenyl pose adopted in previous work (Fig. S3).11 In ad-
dition to a nearly identical alkyl chain conformation, we
note also close coherence of proximal residues, including
shelf substituent Phe1528, backstop residue Ile1575, and
pocket residues Tyr1589(“gatekeeper”)/Tyr1541, while
in contrast distal ZA residues (Trp1526, Phe1536) show
significant discrepancy (Fig. S3). Although the current
structural resolution does not permit assignment of water
positions, these data strongly imply an equivalent
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Table 6. Lead Compounds.
Figure 4. Cocrystal structures of 32 (A,B), and 33 (C,D)
(6P39, 6P3A) showing binding modes to TAF1(2). A) Cutaway
of 32 binding within the KAc pocket, showing H-bonding
(dashes) to conserved Asn (sticks), and packing against WPF
shelf (spheres). B) Transparent surface view of 32 (spheres)
packing against Val1547 (spheres). C) Cutaway of 33 binding
within the KAc pocket, showing H-bonding (dashes) to con-
served Asn (sticks), packing against WPF shelf (spheres), and
position of “backstop” residue Ile1575 (sticks) D) Surface
representation of 33 showing extended conformation of 1-
butenyl group into the floor of the KAc pocket.
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The authors would like to thank Minoru Tanaka for technical
Page 6 of 9
rearrangement within the conserved water network as
observed previously.11 Together, these structural datasets
provide a resource for structure-based approaches toward
kinase inactive dual (BRD4(1)/TAF1(2)) and TAF1 selec-
tive compound development. Here, we highlight 31 as a
potent dual lead compound for TAF1/BRD4 inhibition
(Table 6). Toward TAF1 selectivity, we report three inde-
pendent approaches: 3 to 4-position cyclization (23, 32),
7-position ZA channel methoxy projection (27), and me-
thyl to 1-butenyl amide substitution as an extended acyl-
lysine mimetic strategy (30, 33). Conversely, we find the
NMe analog 10 to be over 100-fold selective for BRD4 over
TAF1 by IC50 ratio, offering a potential starting point to-
ward improving the selectivity of BET inhibitors based on
this scaffold.
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discussions.
ABBREVIATIONS
KAc, Acetyl-lysine; BRD, Bromodomain; BET, Bromodomain
and extra-terminal domain; TBP, Tata-Binding Protein; RB,
Retinoblastoma Protein; SAR, Structure-activity Relationships;
TM, Thermal-Melt; THP, tetrahydropyran.
REFERENCES
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Because of its role in transcription initiation, marked
effects on cell cycle, and potentially selective effects on
amplified gene expression, TAF1 is an attractive target for
both basic and translational research. Further, the multi-
ple purported enzymatic activities make TAF1 an excellent
candidate for targeted degradation approaches.39-43 To this
end, the compounds disclosed in this study offer new and
advanced starting points for TAF1 probe development. In
addition, previous studies of TAF1-BRD4 crosstalk have
also outlined functional synergy of BRD4 and TAF1.10 Ac-
cordingly, the potent dual BRD4 / TAF1 inhibitors report-
ed herein may offer single agent efficacy in these contexts,
and merit further biological investigation in this regard.
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ASSOCIATED CONTENT
Supporting Information
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Herait, P. E.; Berthon, C.; Thieblemont, C.; Raffoux, E.;
Magarotto, V.; Stathis, A.; Thomas, X.; Leleu, X.; Gomez-Roca,
C.; Odore, E.; Roumier, C.; Bourrdel, F.; Quesnel, B.; Zucca, E.;
Michallet, M.; Christian, R.; Cvitkovic, E.; Rezai, K.;
Preudhomme, C.; Facon, T.; Palumbo, A.; Dombret, H., Abstract
CT231: BET-bromodomain inhibitor OTX015 shows clinically
meaningful activity at nontoxic doses: interim results of an
ongoing phase I trial in hematologic malignancies. AACR: 2014.
The Supporting Information is available free of charge on the
ACS Publications website.
Supplemental Data (PDF)
Experimental Procedures (PDF)
Synthetic Procedures (PDF)
7.
Stathis, A.; Zucca, E.; Bekradda, M.; Gomez-Roca, C.;
AUTHOR INFORMATION
Corresponding Author
Delord, J.-P.; Rouge, T. d. L. M.; Uro-Coste, E.; de Braud, F.;
Pelosi, G.; French, C. A., Clinical response of carcinomas
harboring the BRD4–NUT oncoprotein to the targeted
bromodomain inhibitor OTX015/MK-8628. Cancer discovery
2016, 6 (5), 492-500.
*Nathanael_gray@ dfci.harvard.edu
*James.bradner@novartis.com
8.
Theodoulou, N. H.; Tomkinson, N. C. O.; Prinjha, R.
Crystallography Correspondence:
*dhepag@crystal.harvard.edu
K.; Humphreys, P. G., Progress in the Development of non-BET
Bromodomain Chemical Probes. ChemMedChem 2016, 11 (5),
477-487.
9.
McKeown, M. R.; Shaw, D. L.; Fu, H.; Liu, S.; Xu, X.;
Present Addresses
†Scripps Research, La Jolla, CA. ‡Novartis Institute for Biomedi-
cal Research, Cambridge, MA.
Marineau, J. J.; Huang, Y.; Zhang, X.; Buckley, D. L.; Kadam,
A.; Zhang, Z.; Blacklow, S. C.; Qi, J.; Zhang, W.; Bradner, J. E.,
Biased Multicomponent Reactions to Develop Novel
Bromodomain Inhibitors. Journal of Medicinal Chemistry 2014,
57 (21), 9019-9027.
Funding Sources
D.L.B. contributed to this work as a Merck Fellow of the Da-
mon Runyon Cancer Research Foundation (DRG-2196-14).
This work was supported by the Claudia Adams Barr Program
for Innovative Cancer Research (to D.L.B.).
10.
Sdelci, S.; Lardeau, C.-H.; Tallant, C.; Klepsch, F.;
Klaiber, B. o. r.; Bennett, J.; Rathert, P.; Schuster, M.; Penz, T.;
Fedorov, O.; Superti-Furga, G.; Bock, C.; Zuber, J.; Huber, K. V.
M.; Knapp, S.; ller, S. M. u.; Kubicek, S., Mapping the chemical
chromatin reactivation landscape identifies BRD4-TAF1 cross-
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Notes
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