Discovery of GSK8612, a Highly Selective and Potent TBK1 Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.9b00027

The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a noncanonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis, autophagy, and neuroinflammation. Herei

Full text of DOI:10.1021/acsmedchemlett.9b00027

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Letter
Discovery of GSK8612, a highly selective and potent TBK1 inhibitor
Douglas W. Thomson, Daniel Poeckel, Nico Zinn, Christina Rau, Katrin Strohmer, Anne
J Wagner, Alan P. Graves, Jessica Perrin, Marcus Bantscheff, Birgit Duempelfeld, Viera
Kasparcova, Joshi M. Ramanjulu, G. Scott Pesiridis, Marcel Muelbaier, and Giovanna Bergamini
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00027 • Publication Date (Web): 11 Mar 2019
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Discovery of GSK8612, a highly selective and potent TBK1 inhibitor
Douglas W. Thomson, †┴ Daniel Poeckel,†┴ Nico Zinn,† Christina Rau,† Katrin Strohmer,† Anne J.
Wagner,† Alan P. Graves,~ Jessica Perrin,† Marcus Bantscheff, † Birgit Duempelfeld,† Viera
Kasparcova,° Joshi M. Ramanjulu,° G. Scott Pesiridis,° Marcel Muelbaier,†* Giovanna Bergamini†*
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†Cellzome GmbH, A GlaxoSmithKline Company, Meyerhofstraße 1, 69117 Heidelberg, Germany.
°Pattern Recognition Receptor DPU, GlaxoSmithKline, Collegeville, PA, USA
˜Data and Computational Sciences, GlaxoSmithKline, Collegeville, PA, USA.
KEYWORDS. TBK1, kinase inhibitor, interferon, inflammation
ABSTRACT: The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a non-canonical member of the IkB
kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis,
autophagy and neuroinflammation. Herein, we report the discovery and characterization of a novel potent and highly selective
TBK1 inhibitor, GSK8612. In cellular assays, this small molecule inhibited toll-like receptor (TLR)3-induced interferon
regulatory factor (IRF)3 phosphorylation in Ramos cells and type I interferon (IFN) secretion in primary human
mononuclear cells. In THP1 cells, GSK8612 was able to inhibit secretion of interferon beta (IFN) in response to dsDNA and
cGAMP, the natural ligand for STING. GSK8612 is a TBK1 small molecule inhibitor displaying an excellent selectivity profile
and therefore represents an ideal probe to further dissect the biology of TBK1 in models of immunity, neuroinflammation,
obesity, or cancer.
TBK1 is a member of the noncanonical IKK family of
serine/threonine kinases and a central player in innate
immunity.1,2 Following ligation of TLR3 or TLR4, TBK1 is
activated via the adaptor protein TRIF, resulting in the
phosphorylation of its target protein IRF3. In a variety of cells
including conventional dendritic cells (DC) and macrophages,
IRF3 activation subsequently triggers the secretion of IFN, an
immunomodulatory chemokine.2–7 Through an autocrine loop
via type I IFN receptor (IFNAR) and induction of IRFs and
interferon-stimulated genes (ISGs), IFN typically primes
stimulated cells to produce high levels of IFNα.8,9
also been reported for TBK1.18 The discovery of TBK1
inhibitors has therefore gained attention from both industrial
and academic groups.19 In this context, the selective degradation
of TBK1 using proteolysis targeting chimeras (PROTACs) has
been recently explored.20 Further validation of the effects of
TBK1 inhibition in disease-relevant models is required to
improve the understanding of the biological function of this key
signalling mediator. To this end, the availability of potent and
selective chemical probes would greatly facilitate such target
validation efforts.21 We report here the discovery of GSK8612,
a highly potent and selective inhibitor for TBK1 and its activity
in cellular models.
TLR3 is naturally stimulated by viral double stranded (ds)RNA
or by its synthetic mimetic polyinosine-polycytidylic acid
(poly(I:C)), a suitable tool to trigger a type I IFN response in
vitro.8 Further, TBK1 is activated following dsDNA sensor
ligation via the signalling intermediate STING, likewise leading
to IRF activation and type I IFN expression.3,10 In turn,
poly(I:C) and IFNα promote STING expression in macrophages
and DCs, forming another mechanism of amplifying the IFN-
mediated antiviral immune response.10
The application of affinity enrichment based chemoproteomics
to the discovery of selective probes for PI3K,22 mTOR23 and
TNKS24, was previously described.25 Using these methods, the
kinase selectivity of two small molecules used to explore TBK1
biology, BX79526,27 and MRT6730728,29, was established
(supporting information table S1 and figure S1). BX795 and
MRT67307 were determined to have an average pKd of 7.7 and
7.1 respectively for TBK1 when profiled with kinobeads30 in a
mixture of cell lines and tissue extracts (optimized for a broad
protein kinase coverage). However, both molecules show
affinity for more than 20 other kinases within a 10-fold window
of their TBK1 binding affinity (supporting information table
S1 for full list of kinases). The inhibitor of nuclear factor kappa-
B kinase subunit  (IKK), a close homologue of TBK1 sharing
high sequence identity in the kinase domain, is among these 20
kinases. Of note, both molecules demonstrated highest affinity
for AP2-associated protein kinase 1 (AAK1), a protein involved
Misregulation of TBK1 activity is associated with autoimmune
disorders and has therefore been considered a target for the
treatment of (auto)inflammatory diseases.11–13 This concept is
however challenged by the observation that TBK1 inhibition
may have proinflammatory effects due to its regulatory function
in the non-canonical NF-B pathway.14–16 More recently, roles
for TBK1 in energy homeostasis and tumorigenesis have been
revealed, identifying TBK1 as potential target for obesity and
cancer.16,17 Key roles in autophagy and neuroinflammation have
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in neuropathic pain.31 Kinase inhibitors can be classified
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Assay
Value
119 µM
3.6
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according to their binding site (catalytic site or allosteric site)
and the form of the kinase bound (active or non-active and Asp-
Phe-Gly (DFG) loop in or out).32 The kinobeads are generated
by immobilization of kinase inhibitors that ligate the ATP
binding site of kinases. Therefore, molecules that demonstrate
competitive binding behavior with the kinobeads are concluded
to bind into the ATP site of kinases. In order to investigate to
which kinase state these TBK1 inhibitors bind, profiling
experiments with the kinobeads were performed using cell
extracts with and without enrichment of phosphorylated
kinases. The enrichment was achieved by treating Ramos cells
with the phosphatase inhibitor Calyculin A before cell lysis. In
the Calyculin A extracts, BX795 and MRT67307 were
determined to have an average pKd of 7.4 and 7.5 respectively,
compared to 6.8 and 6.6 in the control extracts (supporting
information table S1 and figure S2). This shows that BX795 and
MRT67307 may preferentially bind to the phosphorylated form
of TBK1 and the pKds determined against this form are within
the expected range considering the reported TBK1 inhibition
potencies in activity assays.26,28 Interestingly, in the two
different cell lysates no significant pKd value changes were
observed for other target kinases, such as BMP-2-inducible
protein kinase (BMP2K) and Serine/threonine kinase 17B
(STK17B), which cannot be readily explained.(Of note, AAK1
protein kinase was not detected in Ramos cells).
Solubility (CLND)
CHROM LogD (pH 7.4)
Artificial membrane
permeability
2.1x10-5 cm/s
Fraction bound in blood
(mouse, rat, human)
99.5%, 99.6%,
99.3%
Microsomal Clearance
(mouse, rat, human)
8.5, 2.1, 1.1
mL/min/g tissue
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Microsomal half life
(mouse, rat, human)
8.5, 34.2, 67.4
min
Determination of the physical chemical properties of GSK8612
(table 1) revealed that the low log D value translates into
aqueous solubility greatly exceeding the determined affinity for
TBK1 without being detrimental to its cellular permeability.
The low lipophilicity may also contribute to GSK8612’s low
microsomal clearance in human and rat, and low to medium
clearance in mouse. Nonetheless despite the low log D,
GSK8612 is highly protein bound in mouse, rat and human
blood.
Kinases are attractive and extensively explored drug targets33,34
and the 2,4-diaminopyrimidine core is a common structural
motif found in many kinase inhibitors, indeed both BX795 and
MRT67307 possess this structural feature. The majority of
kinase inhibitors that bind in the ATP binding pocket make two
conserved hydrogen bonding interactions with the hinge region
of the kinase backbone. GSK8612 was docked into a crystal
structure of TBK1 bound to MRT67307 (PDB 41WQ),35 and
the predicted binding mode is shown in figure S3 of the
supporting information. The model predicts that the N1 of the
pyrimidine nitrogen and the NH linking the pyrimidine and
pyrazole make the hinge region interactions with the backbone
NH and carbonyl of Cys89, respectively. It is further predicted
that the bromine sits toward a lipophilic pocket near Met86 and
the pyrazole may bind in two orientations projected towards the
solvent interface. The sulfonamide NH2 is predicted to make
two hydrogen bonds, one with the side chain of Asn140 and the
second with the side chain of Asp157, helping to explain the
high affinity imparted by this moiety. The combination of the
substituted pyrazole moiety on the N2-amine and the benzyl-
sulfonamide moiety on the N4-amine of the pyrimidine
conferred GSK8612 with high TBK1 affinity and good kinase
selectivity. This was revealed in the kinobead selectivity profile
of GSK8612 in extracts from a mixture of cell lines and tissue
(figure 2 and supporting information table S1 for a full list of
kinases). An average pKd of 8.0 was determined for GSK8612
against TBK1. Within 10-fold affinity with respect to TBK1, no
off-targets of GSK8612 could be identified. The highest
affinity off-target in our study was STK17B with an average
pKd of 6.2, and 100-fold selectivity was determined over the
close family member IKK (average pKd = 6.0). Binding of
GSK8612 to AAK1, the highest affinity target for both BX795
and MRT67307, showed a pKd of 5.1, 1000-fold lower than for
TBK1. Profiling of GSK8612 using the lipid kinase affinity
matrix in mixed cell extracts revealed that GSK8612 bound to
one lipid kinase, Phosphatidylinositol 4-kinase beta (PI4Kb),
though with only an average pKd of 5.3 (supporting information
table S1). The preference of GSK8612 to bind to activated or
non-activated TBK1 was investigated using extracts from
F
F
Br
N
N
N
F
N
N
N
H
H
O
S
H2N
O
GSK8612
Figure 1. Structure of TBK1 inhibitor GSK8612.
In an effort to discover new inhibitors of TBK1, the kinase
selectivity data of proprietary compounds was interrogated. That
revealed a series of 2,4-diaminopyrimidines with good affinity for
TBK1, and optimization of this series culminated in the discovery
of GSK8612 (figure 1). This molecule can be readily synthesized
by two sequential nucleophilic aromatic substitution reactions
(Scheme S1; full synthetic details are given in the supporting
information).
Table 1. Properties of TBK1 inhibitor GSK8612
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Ramos cells with and without Calyculin
A
treatment
secretion from THP-1 cells with low micromolar potency.
GSK8612 is highly selective TBK1 inhibitor, thus
1
(supporting information table S1 and figure S2). This revealed
that GSK8612 has lower affinity for phosphorylated TBK1. An
average pKd for TBK1 of 6.8 was determined in the extracts
from Calyculin A treated cells compared to 7.7 in the extracts
without Calyculin A treatment. In line with the affinity
determined in extracts from Calyculin A-activated cells,
GSK8612 inhibited recombinant TBK1 with an average pIC50
of 6.8 in a biochemical functional assay.36 Interestingly, the pKd
of the closest off-targets of GSK8612, such as IKK did not
show any significant change between Calyculin A treated and
control Ramos cells. Therefore, the actual selectivity of
GSK8612 for TBK1 in the cells will depend on the activation
state of TBK1.The effects of TBK1 inhibition with GSK8612
in live cells were then investigated. First, Ramos cells were
stimulated with the TLR3 ligand poly(I:C) and the
phosphorylation of IRF3 was measured by Western blot.
GSK8612 was able to inhibit phospho-IRF3 with an average
pIC50 of 6.0 (figure 3A), confirming effective inhibition of
TBK1 kinase activity in live cells. Next, the ability of GSK8612
to block TBK1-dependent functional responses was evaluated
also in primary cells. Type I IFN secretion was measured in
poly(I:C)-stimulated human peripheral blood mononuclear
cells (PBMCs).37 Since IFN was below the limit of detection
of the assay (MSD readout, data not shown) and IFNα could be
robustly detected by flow cytometry (CBA assay), the latter was
chosen as readout. GSK8612 inhibited the release of IFNα with
a pIC50 of 6.1, demonstrating sub-micromolar potency in
primary immune cells (figure 3B). Of note, IFNα secretion from
TLR3-stimulated PBMC is believed to result from a priming
effect by IFN, a well characterised IFN positive feedback
response.7,9,10 Accordingly, a blocking antibody against IFNAR
ablated poly(I:C)-triggered IFNα release in PBMC, confirming
IFNα secretion to be a secondary, though biologically relevant
downstream effect of TBK1 inhibition (supporting information
figure S4). Together, TBK1 inhibition interferes with the
activation of the TLR3-IFN axis and disrupts the IFN positive
feedback response, resulting in the abrogation of IFNα
secretion. It is also well known that TBK1 propagates biological
signaling of IFN downstream of DNA sensing by cGAS and
STING.38 To test whether GSK8612 inhibits this pathway,
THP-1 cells were stimulated with dsDNA containing virus
(bacmam) or the natural STING ligand cGAMP. GSK8612 was
able to completely inhibit secretion of IFN with a measured
pIC50 of 5.9 and 6.3 for the dsDNA virus and cGAMP
stimulated cells, respectively (figure 4).
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representing an ideal tool to further dissect the physiological
roles of TBK1 in biological models of immunity,
neuroinflammation,
obesity
and
cancer.
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Figure 2. Chemoproteomics based kinase selectivity of
GSK8612 determined using kinobeads in mixed HEK293, K-
562, HepG2, and placenta cell extracts and lipid kinobeads in
mixed HeLa, Jurkat, and K-562 cell extracts. pKd (shown on the
y-axis) were determined for 285 kinases.
In summary, the biological activity of GSK8612 was
demonstrated, resulting in inhibition of IRF3 phosphorylation
in Ramos cells, IFN secretion from human PBMCs and IFN
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IRF3 phosphorylation
IFN secretion
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log [M], GSK8612
log [M], GSK8612
Figure 3. Cell-based activity of GSK8612. A, GSK8612 inhibits IRF3 phosphorylation in Ramos cells. Western blot analysis reveals
inhibition of IRF3 phosphorylation (Ser396) by GSK8612 in poly(I:C)-stimulated Ramos cells with an average pIC50 = 6.0 (n=5). Western
blot densitometry data for pIRF3 were normalized to total IRF3 are displayed as percentage of maximal inhibition. B, GSK8612 inhibits
IFN secretion in human PBMC with an average pIC50 = 6.1 (n=3). IFN was measured in supernatants of poly(I:C)-stimulated human
PBMCs at 16 h by FACS-based Cytometric Bead Array (CBA) assay. Percent of maximal inhibition was derived from normalizing mean
fluorescence intensity (MFI) data to vehicle-treated, poly(I:C)-stimulated samples (0% inhibition).
B
A
Figure 4. GSK8612 inhibits secretion of IFN from THP-1 cells. A, GSK8612 inhibits secretion of IFN (pg/mL) in THP-1 cells stimulated
with dsDNA-containing virus (Baculovirus) with a pIC50 = 5.9 (n=3) or B, 60 µg/mL cGAMP with a pIC50 = 6.3 (n=3).
Synthesis and characterization of GSK8612, protocols for
selectivity profiling, cellular assays and in vitro physchem
properties determination (PDF)
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Figures on kinase selectivity of BX795 and MRT67307, docking
studies and inhibition of IFNα secretion by IFNAR-blocking
antibody (PDF)
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List of proteins identified and pKd determined in kinase-binding
profiling (Excel)
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Coordinates of docking models (pdb file)
AUTHOR INFORMATION
Corresponding Authors
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* E-mail: giovanna.2.bergamini@gsk.com
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Author Contributions
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┴ These authors contributed equally to manuscript
KS, JP, DP, VK, GSP contributed to the design, execution and
analysis of the cellular experiments; NZ, CR, BD contributed to
the design, execution and analysis of the proteomic experiments;
APG executed and analyzed computation experiments; DWT,
AJW, contributed to the chemical design and synthesis; MM led
the chemistry efforts; JMR, MB contributed to data interpretation;
DWT, MM, DP, NZ, AJW, GSP and GB wrote the manuscript;
GB designed and supervised the study. All authors have given
approval to the final version of the manuscript.
(7)
(8)
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Richez, C.; Yasuda, K.; Watkins, A. A.; Akira, S.; Lafyatis,
R.; van Seventer, J. M.; Rifkin, I. R. TLR4 Ligands Induce
IFN-alpha Production by Mouse Conventional Dendritic
Cells and Human Monocytes after IFN-beta Priming. J.
Notes
Human blood was obtained from healthy volunteers, and its
research use was in accordance with the terms of the informed
consent and IRB approved.
The authors declare the following competing financial interest(s):
The authors are employees or former employees of Cellzome
GmbH and GlaxoSmithKline, and the company funded the work.
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ACKNOWLEDGMENT
We thank Cellzome mass spectrometry department for help with
mass spectrometry sample preparation and mass spectrometry
instruments; also, the cell and tissue culture group for preparation
of cell extracts. We would like to thank colleagues within GSK who
performed the in vitro and in vivo DMPK experiments and the
determined the compounds physicochemical properties.
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ABBREVIATIONS
IKK, Inhibitor of nuclear factor kappa-B kinase; IFNα, interferon-
alpha; IFN, interferon-beta; IFNAR, interferon-α/β receptor; IL-
12, Interleukin 12; IRF3, Interferon regulatory factor 3; ISG,
interferon stimulated gene; poly(I:C), polyinosinic-polycytidylic
acid; TBK1, TANK-binding kinase 1; TLR, toll-like receptor.
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For table of contents only
Inhibition of cGAMP induced IFN secretion
-log [GSK8612]
F
F
F
Br
N
N
N
N
H
N
N
H
O
S
H2N
O
GSK8612
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