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Identification and Further Development of Potent TBK1 Inhibitors

André Richters,† Debjit Basu,† Julian Engel,† Meryem S. Ercanoglu,‡ Hyatt Balke-Want,‡ Roberta Tesch,†,§
Roman K. Thomas,‡ and Daniel Rauh*,†
†
Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Straße 6, 44227 Dortmund,
Germany
‡
Department of Translational Genomics, University of Cologne, Weyertal 115b, 50931 Cologne, Germany
§
Programa de Pós-Graduaçaõ em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio
de Janeiro, Rio de Janeiro, 21941-901 Brazil
*
S Supporting Information
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ABSTRACT: The cytosolic Ser/Thr kinase TBK1 was

discovered to be an essential element in the mediation of
signals that lead to tumor migration and progression. These
Downloaded via 112.211.6.52 on January 5, 2022 at 10:36:58 (UTC).

findings meet the need for the identification of novel tool

compounds and potential therapeutics to gain deeper insights
into TBK1 related signaling and its relevance in tumor
progression. Herein, we undertake the activity-based screening
for unique inhibitors of TBK1 and their subsequent
optimization. Initial screening approaches identified a selection
of TBK1 inhibitors that were optimized using methods of
medicinal chemistry. Variations of the structural characteristics
of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or
basic contributions to drug design approaches are anticipated for our improved small molecules.

T ANK-binding kinase 1 (TBK1) is a critical regulator of the

antiviral response and initiates type I interferon (IFN)
transcription through an endosomal toll-like receptor 3 (TLR3)
pathway. Subsequently, the interferon regulatory factors 3 and
7 (IRF3/7) are activated and then translocated to the nucleus
to initiate IFN gene transcription.1−4 Alternatively, cytosolic
activation occurs by the recognition of infiltrated viral RNA
through sensor proteins such as melanoma differentiation-
associated protein 5 (MDA5) or the retinoic acid-inducible
gene 1 protein (RIG-I). Consecutive TBK1 activation is
induced by adapter protein mediated phosphorylation of TBK1,
thereby initiating the same downstream cascades of IFN
production as in the endosomal pathway. TBK1 is involved in
the formation of a higher ordered multi enzyme complex and
interacts with several adapter proteins such as nuclear factor κB
(NFκB)-activating kinase-associated protein 1 (NAP1), TBK1-
binding protein 1 (SINTBAD), and TRAF family member-
associated NFκB activator (TANK) in an orchestrated
trafficking network.1,5 Besides its implication in the mediation
of antiviral responses, TBK1 has been proposed as a target for
cancer therapy since TBK1 knockout studies led to
antiproliferative effects in KRAS-mutated human alveolar
adenocarcinoma cells (A549).6 Based on this analysis, TBK1
has become a focus of medicinal chemistry approaches to
decipher the complex regulation and activation mechanism and
its associated signaling pathway.1,7 Constitutive explorations
investigating the role of TBK1 in promoting KRAS-driven
tumorigenicity and survival by activating autocrine C−C motif
chemokine 5 (CCL5) and intereukin 6 (IL-6) further
substantiate a potential role in cancer therapy.8 However,
TBK1 was also demonstrated to participate in tumor metastasis
and epithelial−mesenchymal transition (EMT), and therefore,
modulating TBK1 activity may curtail tumor growth and
propagation. Prevailing findings support this assumption, since
small molecule inhibitors have been discovered that block
TBK1 transferase activity and thereby hinder the ability of
tumor cells to further metastasize, proliferate, or undergo EMT
processes.9 Recently, TBK1 has been shown to activate
estrogen receptor α (ERα) by phosphorylation, thereby
stabilizing ERα and inducing transactivational activity and
modulating breast cancer growth. Ectopic expression of TBK1
in this context renders breast cancer cells resistant to tamoxifen
while suppression of TBK1 activity by treatment with BX795
(Figure 1),10,11 a known 3-phosphoinositide-dependent protein
kinase 1 (PDK1) and TBK1 inhibitor, in combination with
tamoxifen leads to synergistic antiproliferative effects in breast
cancer cells. Therefore, TBK1 may serve as a predictive marker
for tamoxifen resistance and as a potential therapeutic target in
breast cancer.12 Additionally, TBK1 is involved in mouse
double minute 2 (MDM2) mediated repression of estrogen
Special Issue: New Frontiers in Kinases
Received: November 7, 2014
Accepted: December 26, 2014
Published: December 26, 2014

© 2014 American Chemical Society 289 DOI: 10.1021/cb500908d

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potential target in cancer therapy.8,14 Since TBK1 may serve as

Figure 1. Representative scaffolds (6-aminopyrazolopyrimidine (1), 2-
aryl-azabenzimidazole (2), and pyrimidines (BX795, CYT387)) of
currently known TBK1 inhibitors revealing broad target specificity.
induced protein kinase B (Akt) activation by facilitating
phosphorylation induced degradation of hematopoietic PBX-
interaction protein (HPIP).13 Broader studies indicate potential
therapeutic use of TBK1 inhibitors, since they attenuate
radiation-induced EMT of human lung cancer cells (A549)
via activation of glycogen synthase kinase-3 β (GSK3β) and
simultaneous repression of zinc finger E-box-binding homeobox
1 (ZEB1).9 Similarly, studies on the impact of TBK1 as a
therapeutic target in HER2 positive breast cancer and KRAS-
driven tumorigenicity further emphasized that TBK1 is a
a potential therapeutic target, numerous studies have been
conducted to identify and develop new TBK1 inhibitors such as
(2- and 6-aryl-)azabenzimidazoles, 6-aminopyrazolopyrimi-
dines, and pyrimidines, respectively (Figure 1).15−18
The recently reported crystal structure of the full-length
TBK1 kinase in complex with the type I inhibitor BX795 has
provided the structural basis for medicinal chemistry
approaches.19 TBK1 consists of three superordinate domains:
a catalytic domain facilitating Ser/Thr kinase activity, a
ubiquitin-like domain (ULD) for substrate specificity, and a
scaffolding and dimerization domain (SDD) for structural
integrity and mediating adapter protein interactions to form a
multi-enzyme complex.19 Moreover, the presence of SDD and
ULD provided evidence for TBK1 existing as a homo-
dimer.1,20,21 Although TBK1 was proposed as a target in
various cancer types and particularly in facilitating EMT
processes, its associated tumor biology to large extent remains
unclear.9,22 Therefore, tool compounds for deciphering TBK1
biology as well as specific inhibitors are needed to more deeply
investigate its function with respect to the orchestrated network
of cellular signaling of the antiviral response, as well as its role
in cancer progression and metastasis.
Herein, we report the identification of a new class of
pyrimidine-based inhibitors of TBK1 by activity-based screen-
ing approaches. Subsequent ligand optimization by using

Figure 2. (A) Activity-based screening results as cluster analysis according to inhibitory effect and structural similarity. Colors encode for the
remaining TBK1 activity elicited at 13.2 μM compound concentrations. Ring sizes represent the discontinuity of binding: Marginal differences in the
compound’s structural composition result in dramatic differences of the inhibitory effect. The clustering was performed using SARANEA.36 (B)
Simplified screening workflow for Collections I and II as well as illustration of redundant scaffolds revealing divergent substitution patterns for
subsequent SAR studies. (C) Validated hit compounds derived from Collection I (% remaining activity in gray).

290 DOI: 10.1021/cb500908d

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Table 1. Collection of Tozasertib Derivatives and Screening Hits and Related IC50 Valuesa

compd R1 R2 R3 X IC50 [μM]

13 H H NO2 O 2.35 ± 0.28
14 H NO2 H O 1.41 ± 0.46
15a NO2 H H O 0.06 ± 0.03
15b t
Bu H H O 2.66 ± 0.11
15c i
Pr H H O 0.34 ± 0.07
15d Me H H O 0.38 ± 0.10
15e OMe H H O 0.65 ± 0.21
15f F H H O 0.84 ± 0.36
15g Cl H H O 1.19 ± 0.12
15h CF3 H H O 0.29 ± 0.11
15i Cl F H O 0.72 ± 0.05
15j Cl CF3 H O 3.00 ± 1.22
15k NH2 H H O 0.73 ± 0.30
22 H H NHCOCH2CH3 O nib
23 H NHCOCH2CH3 H O >10
24 NHCOCH2CH3 H H O 2.50 ± 0.39
tozasertib NHCO(cyclopropane) H H S 6.22 ± 2.86
axitinib 0.47 ± 0.04
bosutinib 0.67 ± 0.03
dovitinib 0.06 ± 0.01
erlotinib 2.20 ± 0.48
K252a <0.001
sunitinib 0.22 ± 0.01
tofacitinib 5.76 ± 0.36
a
IC50 values were determined in three independent duplicate experiments. bni = no inhibitory effect at 50 μM compound concentration

methods of medicinal chemistry and drug design led to
promising TBK1 inhibitors with activities in the nanomolar
range. In addition, we screened focused compound collections
to identify and validate further TBK1 inhibitors that were
provided by GSK and ROCHE in the course of promoting the
idea of public private partnerships among academic and
industrial research facilities.23
Activity-Based Screening for TBK1 Inhibitors and Hit
Validation. We initially screened two compound libraries,
comprised of 839 (Collection I) and 968 (Collection II)
molecules, for inhibitors of TBK1 using an activity-based assay
that quantified TBK1-dependent substrate phosphorylation in
presence and absence of test compound (see Methods). With
respect to Collection I, each small molecule that reduced TBK1

■
RESULTS AND DISCUSSION
Current studies in TBK1 biology have provided evidence for
this enzyme’s involvement in the progression and metastasis of
breast cancer cells as well as an implication in various lung and
colon cancers.9,12,24 Since TBK1 may represent a future drug
target in cancer therapy, the identification and development of
novel inhibitors to modulate TBK1 kinase activity is an
important area of research. Currently known inhibitors such as
BX79510,11 or CYT38725 (Figure 1) were potent TBK1
inhibitors but lacked specificity, which rendered them
unfeasible for application as molecular probes to decipher
TBK1 biology. We therefore sought to screen a wide array of
compounds, seeking to identify promising enzyme binders by
utilizing activity-based assay systems. Our ultimate goal was to
improve the inhibitory effects of the identified hits against
TBK1 for potential application to investigate TBK1 signaling
and for the development of more potent inhibitors or starting
compounds for constitutive drug design approaches.
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activity to 40% at 13.2 μM test concentration was considered as
a hit and subjected to further validation studies (Figure 2A,B).
A Z′-factor26 of 0.79, based on the consideration of at least
eight positive (BX795, 13.2 μM) and negative (DMSO)
controls, was calculated for the entire screen. In Collection I,
we identified eight compounds of commercial origin as
promising TBK1 inhibitors (Figure 2C). Validation studies
confirmed their inhibitory impact and the staurosporine
analogue K252a, exhibiting an IC50 value <0.001 μM, was
determined to be the most potent compound followed by
dovitinib (0.06 μM) and sunitinib (0.2 μM, Table 1).
Collection II originated 52 hits, which significantly reduced
TBK1 activity to less than 30% (Z′-factor: 0.68). This
compilation contained oxindoles (17), pyrimidines (12),
pyridopyrimidinones (5), commercial inhibitors (4), benzo-
thiophenes (3), quinolines (2), flavones (2), and other
(unique) scaffolds (7). Of these, seven selected scaffolds and
derivatives thereof were subjected to further validation studies
and confirmed as hits in activity-based assays with IC50 values
of 0.14 to 15 μM (Supporting Information Figure S1). Of note,
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Figure 3. SARANEA-supported cluster analysis of activity-based screening results according to inhibitory effect and structural similarity of the hit
compounds of the GSK- (A) and ROCHE-library (B), respectively. Representative derivatives of selected scaffolds are illustrated and assigned to the
respective cluster centers (roman numerals). The color code illustrates remaining TBK1 activity elicited at 10 μM compound concentrations.
we removed flavone scaffolds from the panel as they are
frequent hitters in HTS campaigns and recently were in-depth
described in the literature as pan assay interference compounds
(PAINS).27−29 However, we identified a pyrimidine scaffold
(Scaffold I, Figure 2B, IC50 = 8.6 μM), which illustrated a
similar substitution pattern as well as analog inhibitory potency
relative to tozasertib30 (IC50 = 6.2 μM). Comparative analysis
of the crystal structures of tozasertib in complex with Aurora A
(PDB-code: 3E5A) and TBK1 in complex with BX795 (PDB-
code: 4EUU) revealed that TBK1 and Aurora A share common
structural features with respect to the hinge region (Supporting
Information Figure S2). Therefore, we focused our optimiza-
tion efforts on the design of further analogues of Scaffold I.
In a second screening approach, we utilized compound
collections provided by GSK (Boston, MA; 360 compounds)
and ROCHE (Basel; 235 compounds) that were comprised of
inhibitors and intermediates that were already part of
pharmaceutical medicinal chemistry endeavors in the past.
Since these libraries contain high affinity inhibitors, we set the
cutoff to pick a hit compound to 10% remaining kinase activity
at 10 μM compound concentration (Supporting Information
Figure S3). We determined several core-structures (Figure 3)
revealing fairly divergent chemotypes that were shown to
significantly modulate TBK1 in activity-based assays. Since the
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appropriate cores were redundant throughout the libraries and
therefore were represented by sundry derivatives, we
immediately analyzed the respective hits with respect to the
corresponding structure−activity relationships (SARs). Con-
cerning the GSK library, we identified 14 compounds (Figure
4) represented by substituted imidazopyridines (25−29) and
oxindoles (30−34) as well as maleimide(-like) structures (35,
36) and pyrimidines (37, 38). Of note, we generated IC50
values within the range 0.14−1.6 μM for the imidazopyridines
25−29, which are structurally related to the known Akt
inhibitor GSK 690693.31 In detail, molecule 28 leads to the
most potent inhibition of TBK1 (IC50 = 0.14 μM) due to the
substitutions in 2- and 6-position, which seem to be superior to
the coincidental decoration of 2- and 5-position (25−27, IC50 =
0.72−0.92 μM) as well as solely 5-position decorated
compounds as 29 (IC50 = 1.6 μM). However, the 2-
methylbut-3-yn-2-ol decoration in 2-position of the imidazo-
pyridines appears to be crucial for gaining inhibitory activity,
since 29 remarkably drops in its effect toward TBK1. With
respect to the identified oxindoles, we likewise observed SARs.
Among these, the entity of hits was decorated with divergent
motifs on both 3- and 5-position and except 30 evoked IC50
values in the range 0.07 μM (33) to 0.28 μM (31). Thereby, 33
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Figure 4. GSK screening hits and corresponding IC50 values (determined in at least two independent duplicate experiments).
represented the most potent TBK1 inhibitor among the series
of identified GSK screening hits.
Based on the ROCHE library, we were able to originate 39
hits (Supporting Information Figure S4) that were significantly
characterized by a plethora of substituted bis-aryl maleimides
(39−63). Additionally, we detected several quinolines (64−70,
75), substituted oxindoles (71−74, 77), and an aminothiazole
(76). Interestingly, the vast majority of substitutions that was
found on the maleimide core translated into moderate to strong
TBK1 inhibition; thus, differences in potency could be linked to
the divergent substitution patterns (2- and 3-position). While a
1-methyl-1H-indole was found to be present among the entity
of maleimides, the majority of compounds were additionally
decorated with a further 1H-indole (linked through indole 3-
position), which was variably substituted at its amine function
thereby accounting for discriminative inhibitory effects.
Essentially, we observed a broad range of IC50 values starting
from 0.03 μM and ending up into 5.6 μM for 59 and 61
respectively. Among these structures with linear extensions on
the indole’s amine functionality, we illustrated that 59, 48, 58,
55, and 53 were most potent in our activity-based studies (IC50
= 0.03 μM, 0.27 μM, 0.38 μM, 0.46 μM, and 0.47 μM,
respectively). Notably, the most potent maleimide 59 features a
nitroguanidine moiety, which was already described to
introduce a large dipole moment to the compound, and this
property was thereby speculated to account for its remarkable
potency against protein kinase C (PKC).32
However, compound 39 decorated with a small aniline
substituent instead of a second 1H-indole moiety on the
maleimide’s 4-position represents an exception from the rule
and elicited an IC50 value of 0.23 μM. On the contrary,
substituents with larger spatial arrangement as shown for 60
(naphthyl) and 63 (tetrahydropyrazinoindole) were, in general,
found to be less potent on TBK1 (IC50 ≥ 20 μM). Besides
these, we found several quinolines (64, 65, 67-70, 75) with
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moderate inhibitory impact (IC50 = 0.40−6.1 μM). Further-
more, we identified substituted oxindoles (71−74, 77) as
effective modulators of TBK1 at which 77 (IC50 = 0.003 μM)
was shown to be the most potent inhibitor among the
collectivity of hits derived from the ROCHE screening library.
Design of Derivatives with Improved Inhibitory
Efficiency. Based on our screening results, we identified the
2,4,6-substituted pyrimidine tozasertib30 and the structurally
related Scaffold I derived from our in-house library as
candidates for subsequent medicinal chemistry approaches
involving inhibitor design and synthesis to aim for compounds
with enhanced inhibitory properties. Moderate inhibition of
TBK1 activity was shown for tozasertib and Scaffold I resulting
in IC50 values of 6.2 μM and ∼8.6 μM, respectively. Since these
molecules differ solely in the substituents attached to the 2-
position of the pyrimidine while retaining the same inhibitory
effect, we anticipated that this position would be productive for
derivatization. We chemically optimized this scaffold, paying
particular attention to the alteration of the para-linked
cyclopropylamide of tozasertib for SAR studies and to improve
the inhibitory effect. A crystal structure of tozasertib in complex
with Aurora A (PDB code: 3E5A) was used for in silico
superposition with TBK1 (PDB code: 4EUT) revealing that a
similar binding mode may be adopted (Supporting Information
Figure S2). As a first design approach, we replaced the
arylthioether by an arylether and converted the cyclo-
propylamide moiety into the corresponding propylamide,
which was attached to the accordant ortho-, meta-, and para-
positions. We then focused on the derivatization of the para-
position, since biochemical characterizations (see SAR Studies)
exhibited loss of inhibitory activity in decreasing order for the
meta- and ortho- substitutions. Various alternative substituents
along with electron withdrawing as well as donating properties
were introduced at the para-position including −NO2, −NH2,
−Cl, −F or −CF3, as well as −OMe. We therefore synthesized
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Scheme 1. Synthetic Procedure for the Preparation of Tozasertib Derivativesa

a
Reagents and conditions: (i) mCPBA, DCM, 0 °C−room temperature (RT), 72 h, 86%; (ii) substituted phenol and (a) NaH, THF, 0 °C, 30−240
min or (b) NaOAc, tBuOH, 90 °C, 40 min, 73−95% (9c−f: crude); (iii) 5-methyl-1H-pyrazol-3-amine, DIPEA, DMF, NaI (optional), 85 °C, 4−12
h, 69−83% (12c−f: 13−28%, 2 steps); (iv) 1-methylpiperazine, 110 °C, 15−30 min, 48−84%; (v) 5% Pd/C, NH4HCO2, EtOH, 95 °C, 1 h, 69%;
(vi) Boc2O, Et3N, MeOH, RT, 12 h, 56−66%; (vii) 5% Pd/C, NH4HCO2, EtOH, 95 °C, 30 min, 82−87%; (viii) propionyl chloride in THF, DIPEA,
THF, 0 °C, 30−60 min, crude; (ix) 25% TFA in DCM, RT, 45−60 min, 58−93% (2 steps).

a small focused library of tozasertib derivatives for subsequent
use in inhibition and SAR studies.
Synthesis of a Focused Library of Tozasertib
Derivatives. We synthesized a compound collection of
tozasertib derivatives (15a−k) following a general procedure
(Scheme 1). Initially, 4,6-dichloro-2-(methylthio)pyrimidine 5
was oxidized using mCPBA in order to generate an appropriate
leaving group leading to the corresponding sulfone 6 in good
yield (86%). Subsequent introduction of a para-modified
phenyl by nucleophilic aromatic substitution resulted in
intermediates 9a−j. Due to the substitution pattern of the
applied phenols, we observed differing reaction times and
correspondingly different yields in the range 45−95%. In the
initial effort to alter the position of phenyl substituents, we
introduced a 2-nitrophenol group (7, 27%) and a 3-nitrophenol
group (8, 84%). One of the 2,4-connected chlorines was
substituted with 5-methyl-1H-pyrazol-3-amine using DIPEA in
DMF resulting in intermediates 10, 11, and 12a−j in yields of
70−80%. Finally, we introduced the methylpiperazine moiety at
the pyrimidine’s 6-position by refluxing the respective precursor
in 1-methylpiperazine, forming compounds 13, 14, and 15a−j
in good yields (74−84%). To generate the p-amino substituted
analogue, we reduced the respective p-nitro intermediate (15a)
to the corresponding amine (15k) using Pd/C and NH4HCO2
in moderate yield (69%).
In a continued synthetic approach, we introduced propyl-
amine moieties in the ortho, meta, and para positions by starting
from the corresponding nitro-substituted intermediates (13, 14,
15a) of which the secondary pyrazole amine was first Boc
protected using Boc2O (yield: 55−67%). We reduced the nitro
group to obtain the corresponding amines 19, 20, and 21
(Yield: 60−83%), which were then converted into propiony-
lamides by SN2 substitution using propionyl chloride.
Successive deprotection using 25% TFA in DCM led to the
desired compounds (22, 23, 24) in moderate to good yield
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(58−92%, two steps). Our focused library, comprised of 16
derivatives, was subsequently applied to biochemical character-
izations with respect to TBK1 inhibition utilizing the activity-
based assay systems. Detailed synthetic procedures are
described in the Supporting Information Methods.
Activity-Based In Vitro Characterization and SAR
Studies. To investigate structure−activity relationships, we
conducted analyses by applying serial dilutions (0−20 μM) of
each synthesized compound to active TBK1. The observed
inhibitory effects were shown to be dependent on the position
of derivatization as well as the size of the introduced substituent
and its electron withdrawing or donating properties (Table 1).
In particular, we demonstrated weak inhibition when the
terminal cyclopropylamide moiety was replaced by an open
chain propylamide (24, IC50 = 2.5 μM). Studies comparing the
connection positions (ortho, meta) illustrated that decorations
at the para site revealed the most significant inhibitory effects.
Propylamide substitution in the meta and para locations led to
remarkably reduced inhibitory effects exhibiting IC50 values of
>10 μM and no inhibition, respectively. Further structural
improvements were therefore focused on the derivatization of
the para-phenyl position. Replacing the terminal propylamide
with a tert-butyl group did not improve the inhibitory effects
(15b, 2.7 μM), while a decrease in the spatial arrangement of
the terminal functional group, for example, introducing
isopropyl (15c) or methyl (15d) substituents, led to improved
inhibitory activity (IC50 = 0.34 μM and 0.38 μM, respectively).
Introduction of a methoxy group (15e) as well as halogen
substitutions as in the case of 15f (p-F) and 15g (p-Cl) slightly
decreased the inhibitory activity, leading to IC50 values in the
range 0.65−1.2 μM. Strong electron donating effects as for
amino substitution (15k, 0.73 μM) did not improve the
inhibition. In contrast, electron withdrawing groups led to
consistent inhibitory properties as compared to isopropyl and
methyl substitution (15c,d) as demonstrated for trifluorometh-
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yl (15h, 0.29 μM). Notably, nitro decoration (15a) induced a
strong beneficial effect in terms of inhibition toward TBK1
possessing an IC50 value of 0.06 μM corresponding to a 100-
fold improvement in inhibition versus the initial screening hit
tozasertib. Taking into account the most potent compound
(15a) of our focused library, we likewise explored different
nitro attachment positions as was done for the cyclo-
propylamide decoration (22 vs 23 vs 24) and observed analog
adverse inhibitory effects as illustrated before (13 vs 14 vs 15a,
IC50 = 2.4, 1.4, and 0.06 μM, respectively).
Evaluation of 15a, 15c, and 15f in RAW Macrophages.
Since the potential oncogenic role of TBK1 is not proven, we
set out to utilize qPCR-coupled analyses of IFN-β expression in
macrophages in order to investigate the inhibitory effect of 15a
on the relevant TBK1-associated signaling cascade in a cellular
setup. The expression of IFNs correlates with the affection of
the TBK1 pathway to trigger antiviral and TLR-mediated
immune responses and therefore monitoring IFN expression
levels by qPCR represents a powerful tool to scrutinize the
impact of our TBK1 inhibitors in cell-based studies as was done
before.33 Remarkably, we did not find compound 15a to
significantly affect the IFN-pathway in RAW macrophages,
whereas 15c and 15f were illustrated to reveal more dramatic
impact on the respective signaling cascade. In detail, LPS
stimulated cells were shown to increase IFN-expression to
about 10-fold as compared to unstimulated cells (Figure 5).
Figure 5. qRT-PCR-based examination of IFN-pathway interference
in RAW macrophages rendered by compounds BX795, 15a, 15c, and
15f at 1 μM compound concentration (ctrl: unstimulated; DMSO:
LPS-induced).
Notably, for 15a, we observed a 6-fold increased mRNA level of
IFN-β at 1 μM compound concentration. The respective
pathway was apparently still induced and therefore 15a was
expulsed from representing a potent cellular modulator of
TBK1 and its associated signaling cascade. In contrast, 15c and
15f, previously highlighted as moderate TBK1 inhibitors in
activity-based analyses, were capable of blocking the pathway
activation more dramatically. In detail, 15c merely led to a 2-
fold increase in mRNA levels as compared to unstimulated cells
though 15f likewise impaired the pathway activation ending up
into 4-fold IFN-β expression above control level. Hereby, we
confirmed that our inhibitors significantly impact the TBK1
associated signaling cascade on a cellular level. We can only
speculate on the reasons for the converse result of 15a
representing the most potent inhibitor in activity-based assays,
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and coincidently, it does not significantly affect the IFN-β
pathway in cellular studies. On the contrary, 15f and 15c as less
effective inhibitors illustrate the opposed effect. One explan-
ation might be justified in their different chemical properties
(nitro- vs isopropyl- vs fluorine-substituents), which might
interfere with cell penetration of the compound. However,
further analyses and studies need to be performed to deeper
investigate these opposing results and the specific properties of
the designed inhibitors.
Activity-Based Profiling and Docking Studies. To
investigate the selectivity of 2,4,6-substituted pyrimidine-based
inhibitor 15a, we analyzed its inhibitory activity with respect to
a panel of 80 representative kinases at a screening
concentration of 1 μM (Supporting Information Table S1,
SelectScreen Kinase Profiling Service from Life Technologies).
We identified 25 kinases that were significantly inhibited in
their catalytic activity by more than 70%. Tyrosine kinases such
as Abelson kinase (99% inhibition) and four representatives of
the fibroblast growth factor receptor (FGFR) and vascular
endothelial growth factor receptor (VEGFR) families were
most effectively inhibited (>95%). Notably, the ten prime
targets of 15a within this panel are represented by tyrosine
kinases, and GSK3β (91% inhibition) is the most effectively
inhibited Ser/Thr kinase followed by Aurora A (88%). TBK1
was likewise demonstrated to be efficiently inhibited by 15a
(79%), but the vast diversity of addressed targets illustrates that
selective inhibition of TBK1 is challenging. Remarkably, PDK1
(33% inhibition) as well as IKKε (13% inhibition), which
represent prime targets of the known TBK1 inhibitor BX795,
were not significantly impaired.
In addition to profiling studies, we further investigated the
binding mode of 15a within the catalytic domain of TBK1 by
performing docking studies using the free available receptor−
ligand docking server DockThor (Supporting Information
Figure S5).34 These analyses support our initially proposed
binding mode based on the superposition of tozasertib in
complex with Aurora A and TBK1. According to our
assumption that the pyrazole moiety accounts for the formation
of hydrogen interactions to the backbone amides within the
hinge region a similar binding mode was observed. However,
the docking studies solely revealed the formation of two instead
of three hydrogen bond interactions to the peptide backbone of
Glu87 and Phe88. In addition, we identified a vice versa
orientation of the piperazine and para-nitrophenyl moieties
most likely due to the formation of favorable ionic interactions
of the piperazine with Asp157 and due to the lack of an
aromatic amino acid within the glycine-rich loop that allows for
flexible ligand rearrangement (see Supporting Information
Figure S5 for detailed information).
Conclusions. In the present study, we report the successful
identification of TBK1 inhibitors by activity-based screening
endeavors. Compilations of industry provided compounds
(GSK and ROCHE) were scrutinized for inhibitory activity
with respect to TBK1. Since this collection was comprised of
molecules that were already subject to drug development
endeavors, we derived a plethora of compounds with
remarkable inhibitory effects and were able to retrace SARs
among the identified scaffolds. Among these, oxindole 77 (IC50
= 0.003 μM) from ROCHE and imidazopyrimidine 28 (IC50 =
0.14 μM) from GSK were shown to constitute the most potent
representatives of each library.
In addition, two independently compiled small molecule in-
house libraries afforded 2,4,6-substituted pyrimidine scaffolds
DOI: 10.1021/cb500908d
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ACS Chemical Biology
(tozasertib and Scaffold I) that were considered to serve as
basis for further rational design approaches to improve these
core structures with respect to their inhibitory activity toward
TBK1. Consecutive validation studies and optimization of
tozasertib resulted in the linear synthesis of a small focused
library of derivatives (13−15a−k and 22−24). We paid
particular attention to the derivatization of the para-phenyl
position, which was found to be crucial for modulating TBK1
activity in vitro. Candidates in our focused library were then
tested for enhanced inhibitory effects resulting in distinct SARs.
Notably, the nature of the para-connected functional group
with respect to its spatial arrangement combined with electron
withdrawing and, in turn, electron donating properties was
pivotal for the evoked inhibitory effect. We illustrated the ability
of terminal tert-butyl moieties to exhibit weak inhibition (15b,
2.7 μM), which was significantly improved by decreasing the
spatial arrangement with isopropyl and methyl derivatives for
improved potency (15c and 15d; 0.34 and 0.38 μM,
respectively). The most significant perturbation of TBK1
revealed an inhibitory effect within the midnanomolar range
(IC50 = 0.06 μM) and was elicited by nitro substituted
compound 15a providing evidence for the advantageous effect
of electron withdrawing functional groups.
Ultimately, we conducted cellular studies using RAW
macrophages and methods of qPCR to investigate the
compounds impact on the expression of interferons as a
marker for the TBK1-pathway modulation. Significant down-
regulation of the IFN-β expression was confirmed for
compounds 15c and 15f as the copy number of IFN-β was
decreased, at 1 μM compound concentrations, to approximately
2- and 4-fold, respectively, as compared to the relative
expression in nonstimulated macrophages.
Consecutive profiling studies revealed a plethora of kinases
that is significantly inhibited by the most potent compound 15a
and underline the need for further design approaches to tune
the specificity of our 2,4,6-substituted pyrimidine-based
inhibitors. Docking studies further illuminated the potential
binding mode of 15a and provide a substantial basis for broader
design endeavors.
In conclusion, our activity-based screening procedure
allowed us to identify compounds that inhibited TBK1.
These compounds were then optimized by using methods of
medicinal chemistry. Potential use of these compounds to
decipher TBK1 related signaling or to provide starting materials
for subsequent drug design approaches is anticipated for our
optimized derivatives.
■ METHODS
Reagents and Materials. Unless otherwise noted, all reagents and
solvents were purchased from Acros, Fluka, Sigma-Aldrich, or Merck
and used without further purification. Small volume (20 μL fill
volume) white round-bottom 384-well plates were obtained from
Greiner Bio-One GmbH (Solingen, Germany). All supplies for the
TBK1 HTRF assay kit were purchased from CisBio (Bagnols-sur-
Cèze, France). Axitinib, bosutinib, dovitinib, erlotinib, K252a,
sunitinib, tofacitinib, and tozasertib were purchased from LC
Laboratories (Woburn, MA).
Activity-Based Assay for IC50 Determination and Screening.
IC 50 determinations for TBK1 (Millipore, #14-628M, Lot#
D9SN044N-B) were performed with the KinEASE-STK assay from
Cisbio according to the manufacturer’s instructions. A biotinylated
substrate peptide (STK3 from Cisbio) was phosphorylated by TBK1.
After completion of the reaction, an antiphosphoserine/-threonine
antibody labeled with europium cryptate and streptavidin labeled with
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Articles
the fluorophore XL665 were added. FRET between europium cryptate
and XL665 was measured to quantify the phosphorylation of the
substrate peptide. ATP concentrations were adjusted to 30 μM while a
substrate concentration of 1 μM was used. Kinase and inhibitor were
preincubated for 30 min before the reaction was started by addition of
ATP and substrate peptide. A Tecan infinite M1000 plate reader was
used to measure the fluorescence of the samples at 620 nm (Eu-
labeled antibody) and 665 nm (XL665 labeled streptavidin) 60 μs after
excitation at 317 nm. The quotient of both intensities for reactions
made with eight different inhibitor concentrations was fit to a Hill
four-parameter equation to determine IC50 values. Each reaction was
performed in duplicate, and at least three independent determinations
of each IC50 were made.
For screening purposes, we utilized the Cisbio system HTRF
KinEASE-STK as stated above and TBK1 from Millipore (#14-628,
Lot# D9SN044N-A). Robotics assisted compound transfers (2 × 6.6
nL for 13.2 μM screening concentration) were processed using a
PinTool system from Tecan, or compounds were manually transferred
where appropriate (10 μM screening concentration). At least 8 data
points for positive and negative controls were included for each assay
plate using BX795 (13.2 μM or 10 μM) as 0% and DMSO as 100%
TBK1 kinase activity control. The assay readout was performed using a
Tecan infinite M1000 plate reader, and a Z′-factor of 0.81 was
determined for the entire screen.
qRT-PCR in RAW Macrophages. RAW 264.7 macrophages were
preincubated for 1 h with DMSO or TBK1 inhibitors as indicated and
then stimulated with 100 ng/mL LPS (Invivogen) for 2 h. Hereafter,
RNA was extracted using Quiageńs RNeasy Mini Kit. cDNA was
prepared from 500 ng RNA using the Superscript III reverse
transcriptase (Invitrogen #18064). qRT-PCR was performed using a
7300 Real-Time PCR System (Applied Biosystems) and Power SYBR
Green PCR Master Mix (Applied Biosystems) with primers for mouse
GAPDH and Interferon-β (both at 60 °C) as described by Orzalli et
al.33 ΔCt-values were determined using the 7300 System Software
(Applied Biosystems) using GAPDH as reference control and gene
expression was calculated by the ΔΔCt-method.35
■
ASSOCIATED CONTENT
*
S Supporting Information
Detailed experimental procedures for compound synthesis,
experimental screening workflows, cluster analysis of screening
hits from Collection II, superposition of Aurora A and TBK1,
qPCR validation studies, docking studies, profiling studies as
well as IC50 data for identified screening hits derived from GSK-
and ROCHE-libraries. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 (0)231−755 7080. Fax: +49 (0)231−755 7082.
E-mail: daniel.rauh@tu-dortmund.de.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was cofunded by the German federal state North
Rhine Westphalia (NRW) and the European Union (European
Regional Development Fund: Investing in Your Future), the
German Federal Ministry for Education and Research
(NGFNPlus and e:Med) (Grant No. BMBF 01GS08104,
01ZX1303C). R.T. is funded and supported by CAPES and
FAPERJ.
■
ABBREVIATIONS
Akt, protein kinase B; CCL5, C−C motif chemokine 5; FGFR,
fibroblast growth factor receptor; GSK3β, glycogen synthase
DOI: 10.1021/cb500908d
ACS Chem. Biol. 2015, 10, 289−298
ACS Chemical Biology
kinase-3 β; HER2, receptor tyrosine-protein kinase erbB-2;
HPIP, hematopoietic PBX-interaction protein; IL-6, intereukin
6; MDA5, melanoma differentiation-associated protein 5;
NAP1, NFκB-activating kinase-associated protein 1; NFκB,
nuclear factor NFκB p105 subunit; PBX, pre-B-cell leukemia
transcription factor; PKC, protein kinase C; RIG-I, retinoic
acid-inducible gene 1 protein; RTK, receptor tyrosine kinase;
SAR, structure−activity relationship; SINTBAD, TBK1-binding
protein 1; TANK, TRAF family member-associated NFκB
activator; TBK1, TANK binding kinase 1; TNF, tumor necrosis
factor; TRAF, TNF receptor associated factor; qRT-PCR,
quantitative real-time polymerase chain reaction; VEGFR,
vascular endothelial growth factor receptor; ZEB1, zinc finger
E-box-binding homeobox 1
■
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