Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8

Article abstract of DOI:10.1021/acsmedchemlett.6b00044

Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.

Full text of DOI:10.1021/acsmedchemlett.6b00044

Letter
pubs.acs.org/acsmedchemlett
Design and Development of a Series of Potent and Selective Type II
Inhibitors of CDK8
Philippe Bergeron,*,† Michael F. T. Koehler,† Elizabeth M. Blackwood,‡ Krista Bowman,§ Kevin Clark,∥
Ron Firestein, James R. Kiefer,§ Klaus Maskos,∇ Mark L. McCleland,# Linda Orren,∥
Sreemathy Ramaswamy,∥ Laurent Salphati,⊥ Steve Schmidt,∥ Elisabeth V. Schneider,∇,○ Jiansheng Wu,◊
and Maureen Beresini∥
‡
§
∥
†Department of Discovery Chemistry, Department of Translational Oncology, Department of Structural Biology, Department of
Small Molecule Biochemical Pharmacology, Department of Pathology, Department of Drug Metabolism, and ◊Department of
#
⊥
Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
∇Proteros Biostructures GmbH, Bunsenstr. 7a, D-82152 Martinsried, Germany
○Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany
̈
S
* Supporting Information
ABSTRACT: Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When
cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.
KEYWORDS: Sorafenib, CDK8, inhibitor, DMG-out
panel of 291 kinases and exhibited activity in a Wnt pathway-
dependent mouse tumor model.
n recent literature, CDK8 has been identified as playing a
Irole in the control of transcription regulating cellular
proliferation and differentiation. CDK8 is a cyclin-dependent
kinase that forms part of the mediator complex,1 which itself
regulates the transcriptional activity of RNA polymerase II. A
number of studies have shown that CDK8 modulates the
transcriptional output from distinct transcription factors
involved in oncogenic control. These factors include the
Wnt/β-catenin pathway, Notch, p53, and TGF-β.2,3
The means by which CDK8 activity accomplishes the
regulation of these various pathways remains an active area of
investigation. However, CDK8 has been found to be amplified
and overexpressed in colon cancer,4 acting through the Wnt
pathway and β-catenin, its key signaling molecule.5 Suppression
of CDK8 expression in colon cancer cell lines expressing high
levels of both CDK8 and β-catenin led to reduced proliferation
of these cell lines.6 Furthermore, in a survey of colorectal cancer
tissue samples, CDK8 was associated with β-catenin activation
and with reduced rates of survival.7 Two series of chemical
probes targeting CDK8 have recently been published.
CCT2515458,9 was identified and optimized through its
suppression of the Wnt pathway. It was shown to be a type I
inhibitor of CDK8 and CDK19 with excellent selectivity over a
We recently reported the independent development of
azabenzothiophene-derived CDK8 inhibitors that are potent
and selective and suppress phosphorylation of STAT1 at
Ser727 similarly to CCT251545.10 Both series of compounds
interact with the protein in the same fashion, principally
through a hydrogen bond from Lys52 to a carbonyl on the
ligand, a hydrogen bond accepted from the hinge residue
Ala100 to a pyridine nitrogen and through a cation−pi
interaction with Arg356.
As work on the azabenzothiophene inhibitors was underway,
we targeted the development of a potent and selective series of
type II kinase inhibitors. In many kinases, displacement of the
Asp-Phe-Gly (DFG) sequence that precedes the activation loop
defines type I and type II modes of inhibition, with type II
generally showing a displaced DFG motif from a buried
position in the protein to a DFG-out position, opening a “deep
pocket” that can be occupied by inhibitors. In the case of
CDK8, this trio of residues is Asp-Met-Gly (DMG). A DMG-
Received: January 28, 2016
Accepted: April 5, 2016
© XXXX American Chemical Society
A
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Letter
out class of inhibitors was sought to explore this mode of
inhibition. We began with Sorafenib (1),11 a known type II
kinase inhibitor. Sorafenib did not, however, meet the
selectivity requirements we had established for tool compounds
to study the role of CDK8. We used a Sorafenib-bound CDK8
structure (PDB: 3RGF) to model design ideas that could lead
to type II inhibitors.
Our initial efforts were aimed at altering the hinge binding
element of Sorafenib, and the 2,4-diaminopyrimidine seen in 2
appeared most favorable, making additional backbone hydrogen
bonds with the hinge residues Asp98 and Ala100 from its
amino groups. We hypothesized that these additional contacts
would improve CDK8 binding, as the pyrimidine ring nitrogen
was predicted to nearly superimpose with the pyridine ring
nitrogen in 1. We were encouraged to see that this modification
improved the CDK8 IC50 by about 2-fold in a TR-FRET based
binding assay (Table 1) and simultaneously improved the
rings) to drive this process. Lower values of the solubility index
predict improved solubility in aqueous buffer. The most
straightforward way to reduce the solubility index is to decrease
the number of aromatic rings, as it has been shown that
disrupting molecular planarity is beneficial for aqueous
solubility.13 We therefore sought to eliminate the central
phenyl ring in our initial set of compounds. In order to find a
suitable replacement, we utilized a pharmacophore similarity
search program called FastROCS,14 which performs a rapid
three-dimensional shape and pharmacophore match to a
database of conformers generated for an internal library of
110,000 compounds. We used a docked pose of compound 2 as
a query structure and examined the output for structures that
matched the pharmacophore of 2 both at the point of hinge
binding and the biaryl urea, which forces the DFG-out binding
mode, but did not have an aromatic ring linking the two
regions. Among those structures with the highest score, we
focused on the pyrrolidine 3 (Figure 2). The structure
Table 1. Potency Comparison between Sorafenib (1) and 2
Figure 2. Two-dimensional comparison of shape and volume between
compound 2 and the new pyrrolidine series (3).
kinase selectivity. When compound 2 was screened against a
panel of 220 kinases, only four of 220 kinases (1.8%) exhibited
>50% inhibition at 0.1 μM, with one of them being CDK8, an
improvement over Sorafenib, where 10 out 220 kinases (4.5%)
exhibited >50% inhibition at 0.1 μM (Figure 1).
Having improved our enzymatic potency and selectivity
through this alteration, we focused on improving the physical
properties of our molecules. We utilized a calculation of
compounds’ solubility index12 (SI = cLogD7.4 + aromatic
overlapped very well with compound 2, notably in the aromatic
urea region, which is essential for DFG-out binding, and had a
saturated pyrrolidine linker in place of a phenyl ring. Assayed as
a racemic mixture, 3 exhibited comparable potency to the input
compound 2.
We then synthesized one of the analogues in both
enantiomeric series, showing that the S isomer was more
potent (Table 2). While the solubility index improved, we did
not see a significant improvement in measured solubility.
However, we were pleased to see further improvement in
potency (Table 2).
In order to address the solubility issue, we focused our
attention on the 6-substituted triazolopyridazine, which was
predicted to act as the hinge binder in our molecules. Since the
docking protocol and the Sorafenib crystal structure we utilized
predicted that this bicycle only makes one contact with the
kinase hinge backbone, we attempted to achieve the same key
contacts using a single functionalized ring, again improving the
solubility index and hopefully the measured solubility.
Compounds 6−11 were therefore prepared and evaluated for
CDK8 inhibitory activity as well as kinetic solubility.
Replacement of the triazolopyridazine with a 2-
(trifluoromethyl)pyridine (6) or a pyrimidine (7) resulted in
compounds with greatly reduced activity and did not
significantly improve solubility. Addition of an amino group
at the 2-position of the pyrimidine (8) restored some activity
Figure 1. Kinase selectivity for compound 2 and Sorafenib (1)
measured at a concentration of 0.1 μM in a panel of 220 kinases. Each
vertical line represents a single kinase and is colored by the percent
inhibition seen. Selected potently inhibited kinases are highlighted.
B
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Table 2. Activity and Kinetic Solubility Data for 1-(1-
([1,2,4]Triazolo[4,3-b]pyridazin-6-yl)pyrrolidin-3-yl)-3-
ureas 4 and 5
Table 4. Potency, Kinetic Solubility, and Plasma Protein
Binding Data for (S)-1-(1-(2-(Methylamino)pyrimidin-4-
yl)pyrrolidin-3-yl)ureas 12−18
CDK8 IC50
(nM)
solubility
index
kinetic solubility
stereochemistry
(μM)
4
5
S
4.2
5.7
5.7
1.0
1.0
R
166
and improved solubility. Substitutions on the amino group
further improved potency, with methyl and ethyl being the
optimal groups discovered in an initial investigation (Table 3).
Table 3. Potency, Kinetic Solubility, and Plasma Protein
Binding Data for (S)-1-(4-Chloro-3-
(trifluoromethyl)phenyl)-3-(pyrrolidin-3-yl)ureas 6−11
Figure 3. Kinase selectivity for compound 2, measured at a
concentration of 0.1 μM in a panel of 220 kinases, and compound
12, measured at a concentration of 1 μM in a panel of 220 kinases.
Each vertical line represents a single kinase and is colored by the
percent inhibition seen. Selected potently inhibited kinases are
highlighted.
As a path forward, methylaminopyrimidine was selected due
to the combination of improved solubility and potency while
being less lipophilic than the ethylaminopyrimidine. We
synthesized a series of analogues using this headgroup, aiming
to further improve solubility and plasma protein binding by
removing the highly lipophilic CF3 substituent (Table 4).
While the solubility did improve through removal of this group
it became apparent that having the trifluoromethyl group in the
meta position of the phenyl urea was necessary for optimal
potency. All the compounds that were made lacking this group
were significantly weaker binders of CDK8, with one notable
exception being compound 12. In addition to having acceptable
potency and improved solubility, compound 12 exhibited
exquisite selectivity, inhibiting only CDK8 > 30% at 1 μM, in a
panel of 220 kinases (Figure 3).
the properties of our molecules while retaining this functional
group. Addition of a solubilizing group15 in the para position in
our series of molecules proved to be very beneficial for
solubility and plasma protein binding while retaining good
potency (Table 5).
Compound 20 was tested for kinase selectivity and showed
excellent selectivity with activity >30% against only one off-
target kinase (Flt3) in a panel of 220 kinases (Figure 4).
In order to understand the binding interactions we had
optimized more completely, we obtained a cocrystal structure
of compound 20 in complex with CDK8:Cyclin C at 2.4 Å
resolution (Figure 5 and Supporting Information). The
compound binds to the kinase active site with the DMG
motif in the “out” conformation, with the urea through
Having come to the conclusion that, while being detrimental
to the solubility and increasing plasma protein binding, the m-
CF3 was essential for potency, we sought other ways to improve
C
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Table 5. Potency, Kinetic Solubility, and Plasma Protein
Binding Data for (S)-1-(1-(2-(Methylamino)pyrimidin-4-
yl)pyrrolidin-3-yl)-3-(3-(trifluoromethyl)phenyl)ureas 19−
24
Figure 5. Crystal structure of compound 20 (gold carbons) at the
CDK8 active site. The DMG loop (center, D173 and M174 shown) is
in the “out” conformation. A network of three solvent molecules (red,
numbered spheres link the center of the compound to the protein).
Water 2 makes an additional H-bond to the backbone carbonyl of
D173 that was omitted for clarity. The hinge and C-helix (αC) are
labeled.
treated with compounds for 72 h in media containing fetal
bovine serum (FBS) at a concentration of either 0.625% or
10%. The lower concentration of FBS was used to mitigate the
effects of the low free fraction of some of our compounds.
Throughout the course of the experiment, the cells were in
growth phase, and yet we did not observe significant growth
inhibition (Table 6). Flavopiridol, a potent inhibitor of multiple
CDKs16 with known antiproliferative properties17 was used as a
positive control. With the Km for ATP measured at
approximately 300 μM using an ADP generation activity
assay (data not shown), the lack of antiproliferative activity
could not be explained by competition from endogenous ATP.
In addition, our compounds have moderate to good
permeability so poor cell penetration likely did not account
for lack of activity. Concerned that the compounds examined
were acting weakly on CDK8 in the cellular context, we
examined phosphorylation of S727 of STAT1, a known CDK8
substrate, under conditions of IFNγ stimulation using an in-cell
western assay.18 The results were similar to those obtained in
the proliferation assay. These potent inhibitors of CDK8/cyclin
C in biochemical assays showed only weak if any inhibition of
STAT1 phosphorylation in cells, while a control type I inhibitor
from a previous publication inhibited with nanomolar
potency.10 Our results corroborate the findings of Dale et al.
that type II inhibitors of CDK8 exhibit weak activity in cellular
assays (reporter, CETSA, and STAT1 phosphorylation) and
support their assertion that the inactive form of CDK8 that is
targeted by type II inhibitors is poorly accessible in cells.8
To investigate whether the observed weak antiproliferative
activity was due to CDK8 engagement, we used a set of HCT-
116 cell lines in which CDK8 alone or in combination with
CDK19 was targeted for genomic deletion using CRISPR guide
RNAs. These cell lines were originally designed and
demonstrated to validate STAT1 S727 as a substrate of
CDK8.10 We incubated wild type HCT-116 cells along with the
CDK8 and the CDK8/19 double deletion clones derived from
Figure 4. Kinase selectivity for compounds 12 and 20 measured at a
concentration of 1 μM in a panel of 220 kinases. Each vertical line
represents a single kinase and is colored by the percent inhibition seen.
Selected potently inhibited kinases are highlighted.
morpholine moieties nominally filling the space vacated by the
DMG loop. Two direct and two water-mediated hydrogen
bonds are formed between inhibitor and the kinase. The
methylpyrimidine group forms a single H-bond with the
backbone nitrogen of Ala100 (2.9 Å) on the hinge region. The
carbonyl oxygen of the central urea functionality forms the
second direct H-bond to the backbone nitrogen of Asp173 (2.9
Å) of the DMG motif. Both of the urea nitrogens of 20 form
hydrogen bonds with a solvent molecule, but neither nitrogen
directly interacts with Glu66, unlike what is observed in the
Sorafenib10 structure. This solvent molecule (water 1 in Figure
5) in turn indirectly links to the protein through H-bonds to
two other waters. This bridging of the inhibitor across two
water layers to the protein suggests that the urea group may not
be necessary for potency, opening room for further improve-
ment in physical properties by replacing the urea with a
carbonyl containing isostere.
Having generated highly potent and selective binders of
CDK8 we evaluated their antiproliferative effects in the
colorectal carcinoma HCT-116 cell line, which had previously
been shown to exhibit sharply reduced proliferation response to
CDK8 protein depletion using targeted shRNA.1 Cells were
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Table 6. Antiproliferative Activity on HCT-116 Cells and STAT1 S727 Phosphorylation IC50 of Compounds 9, 12, and 19−21
HCT116 EC50 10%
HCT116 EC50 0.625%
MDCK perm AB
MDCK perm BA
STAT1 S727
phosphorylation IC50 (μM)
CDK8 IC50
(nM)
compd
FBS (μM)
FBS (μM)
× 10−6 cm/sec
× 10−6 cm/sec
9
4.4
>10
8.2
2.9
4.9
>10
7.7
4.3
5.4
3.9
3.9
4.0
7.0
>10
10.1
18.4
20.5
17.4
14.7
1.5
12
19
20
21
4.1
4.5
4.2
>10
>10
4.8
12.4
0.3
12.2
4.2
4.0
Koehler
6.7
18.2
17.2
0.0023
et al.10
flavopiridol
0.034
0.059
them with varying concentrations of 21. As can be seen in
Figure 6, each of these cell lines showed very similar growth
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: bergeron.philippe@gene.com. Phone: (650) 467-
4532.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank members of the DMPK and purification groups
within Genentech Small Molecule Drug Discovery for
purification and analytical support. The crystallographic
experiments were performed on the X06SA beamline at the
Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland.
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Structure-guided modification of the well-known kinase
inhibitor Sorafenib led to a series of highly potent and selective
type II inhibitors of CDK8. Despite their lack of cellular
activity, we are confident that they are useful tool compounds
to investigate the biology of the CDK8 kinase domain.
Replacement of the phenyl ring linker by a saturated five-
membered ring improved potency, selectivity, and solubility.
Addition of a solubilizing group at the para position of the
phenyl urea greatly improved solubility and PPB while retaining
potency and selectivity. Similarly to what has been recently
reported,8 our series of type II inhibitors did not significantly
suppress phosphorylation of STAT1 at Ser727, while our series
of type I inhibitors showed strong activity.10
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00044. Coordinates and structure factors for the
crystallographic experiment are deposited at the Protein Data
Bank with accession code: 5HVY.
Synthetic schemes describing the preparation of 1−32
are provided along with kinase selectivity and crystallo-
graphic data (PDF)
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