Structure-guided optimization of a novel class of ASK1 inhibitors with increased sp3 character and an exquisite selectivity profile

Article abstract of DOI:10.1016/j.bmcl.2020.127405

Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble and permeable, and exhibits a cellular EC₅₀ = 24 nM and K_d < 1 nM. Of the 350 kinases tested, 10 has an IC₅₀ ≤ 500 nM for only eight of them. This paper will describe the design hypotheses behind this series, key data points during the optimization phase, as well as a possible structural rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in <1percent of the >5000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this series.

Full text of DOI:10.1016/j.bmcl.2020.127405

Journal Pre-proofs
Structure-guided optimization of a novel class of ASK1 inhibitors with in‐
creased sp 3 character and an exquisite selectivity profile
Simone V. Bigi-Botterill, Anthony Ivetac, Erica L. Bradshaw, Derek Cole,
Douglas R. Dougan, Jacques Ermolieff, Petro Halkowycz, Ben Johnson,
Christopher McBride, Jason Pickens, Mark Sabat, Steven Swann
PII:
S0960-894X(20)30516-3
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127405
BMCL 127405
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 May 2020
6 July 2020
7 July 2020
Please cite this article as: Bigi-Botterill, S.V., Ivetac, A., Bradshaw, E.L., Cole, D., Dougan, D.R., Ermolieff, J.,
Halkowycz, P., Johnson, B., McBride, C., Pickens, J., Sabat, M., Swann, S., Structure-guided optimization of a
3
novel class of ASK1 inhibitors with increased sp character and an exquisite selectivity profile, Bioorganic &
Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127405
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Structure-guided optimization of a novel class of ASK1 inhibitors with increased
sp3 character and an exquisite selectivity profile
Simone V. Bigi-Botterill*a, Anthony Ivetacb, Erica L. Bradshawc, Derek Colea, Douglas R. Dougand,
Jacques Ermolieffa, Petro Halkowycza, Ben Johnsone, Christopher McBridef, Jason Pickensa, Mark Sabata,
and Steven Swanng
aMedicinal Chemistry & In Vitro Pharmacology, dStructural Biology & Biophysics, Gastroenterology Drug Discovery Unit, Takeda Research in California, 9625
Towne Centre Drive, San Diego, CA 92121, United States
bMirati Therapeutics, 9393 Towne Centre Drive #200, San Diego, CA 92121, United States
cQuantitative Translational Sciences, Takeda Research in California, 9625 Towne Centre Drive, San Diego, CA 92121, United States
eMedicinal Chemistry, Neuroscience Drug Discovery Unit, Takeda Research in California, 9625 Towne Centre Drive, San Diego, CA 92121, United States
f858 Therapeutics, 4757 Nexus Center Drive #150, San Diego, CA 92121, United States
gChemistry & Design, Silicon Therapeutics, 451 D Street #205, Boston, MA 02210, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated
Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and
JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based
Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of
ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the
hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the
broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble
and permeable, and exhibits a cellular EC50 = 24 nM and Kd < 1nM. Of the 350 kinases tested,
10 has an IC50 ≤ 500 nM for only eight of them. This paper will describe the design hypotheses
behind this series, key data points during the optimization phase, as well as a possible structural
rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic
cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in < 1% of
the > 5,000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this
series.
Keywords:
Mitogen-Activated Protein Kinase Kinase Kinase
(MAP3K);
Apoptosis Signal-Regulating Kinase-1 (ASK1);
Structure-Based Drug Design (SBDD);
Cardiac injury;
Kinome selectivity
2009 Elsevier Ltd. All rights reserved.
Kinases are amongst the most extensively studied protein
families based on the vast number of structures currently
available in the Protein Data Bank (PDB) and continue to be
attractive targets in a variety of diseases. In a 2016 analysis of
FDA-approved small molecule kinase inhibitors, Wu et al.
Figure 1. MAPKKK (MAP3K) signaling pathway. Inhibition
of ASK1 could attenuate cardiac inflammation.
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com

determined that of the 28 approved drugs, 25% were considered
“promiscuous” wherein they exhibited less than 20-fold
selectivity over more than ten off-targets. [1,2] Considering this
and the high structural similarity between the ATP-binding
pockets of different kinases, selectivity can be a challenge when
optimizing kinase inhibitors. For chronic, non-oncology
indications, a need for highly selective kinase inhibitors still
site confirmed the hydrogen bond to the hinge of the kinase
through the carbonyl of the iso-indolinone and the valine residue
(Val757) backbone N-H. The planarity of the hinge-binding
motif is maintained by the adjacent pyridine, while also
providing a flat trajectory for the isopropyl-triazole to sit and
hydrogen bond to the conserved catalytic lysine (Lys709). The
isopropyl group fills a small hydrophobic pocket. [10] To-date,
one of the most advanced clinical stage ASK1-targeting
Figure 2. (A) A crystal structure of 1 bound in the ASK1 ATP site to a resolution of 2.95 Å (PDB: 5UP3). Enzymatic/cellular potency is shown with ligand
efficiency (LE) and lipophilic ligand efficiency (LLE). (B) Deconstruction around Gilead Sciences Phase III inhibitor, Selonsertib or 2. A model of 2 (in orange)
was overlaid with the crystal structure of 1 (magenta). The figures were generated using Pymol®.
exists.
The Mitogen-Activated Protein Kinase Kinase Kinase
compounds is Selonsertib (2, Figure 2B), which in February 2019
failed to meet its primary endpoint in Phase III clinical study in
patients with NASH. However, 2 demonstrates efficacy against
cancer cells with overexpression of ATP-binding cassette (ABC)
transporters: ABC1 and ABCG2 [9, 11, 12].
(MAP3K) family mediates a wide array of cellular signaling
events and inflammatory responses. Within this pathway,
Apoptosis Signal-Regulating Kinase-1 (ASK1 or MAP3K5) is
activated by various extracellular stimuli including cytokines,
endoplasmic reticulum (ER) stressors, and reactive oxygen
species (ROS). In turn, ASK1 will phosphorylate its downstream
MAP Kinase Kinase (MKK) effectors, MKK3/6 and MKK4/7,
leading to activation of the p38- and JNK-pathways, respectively
(Figure 1). [3,4]
Also efficacious in a mouse ischemia/reperfusion model, 2 led
to a dose-dependent reduction in infarct size (31% for 10 mg/kg
(P < 0.001) and 60% for 30 mg/kg (P < 0.001)) and preservation
of left ventricular systolic function. [13,14] Although this
compound lacked a crystal structure and its profile was not
suitable for our purposes, it guided an integrated lead generation
strategy around public/in-house crystal structures and structure-
based drug design (SBDD). Despite the lack of structural data for
Although ASK1 signaling has primarily been of interest in the
field of immunology, there is evidence supporting its role in
several other therapeutic areas. [5] The function of ASK1 in the
pathophysiology of ischemia and heart failure has been
demonstrated where its overexpression can lead to cardiac
apoptosis, fibrosis, and hypertrophy. [6,7] More recently, the
pathogeneses of certain cardiovascular diseases, non-alcoholic
fatty liver disease (NAFLD), and non-alcoholic steatohepatitis
(NASH) have been linked through similar mechanisms
delineating ASK1 as a suitable target for therapeutic intervention
[8]. There has also been a renewed interest in targeting ASK1 for
cancer indications, specifically targeting cancer cells with
multidrug resistance (MDR) from over-expression of efflux
transporters [9].
To help validate the cardioprotective role of inhibiting ASK1,
our initial goal was to generate a potent and selective small-
molecule inhibitor to profile in key in vivo experiments. Several
parallel efforts in pursuit of novel, potent, and orally bioavailable
ASK1 inhibitors to interrogate therapeutic hypotheses around
ASK1 biology have been disclosed by Takeda, including the
discovery of a series containing an iso-indolinone core (1, Figure
2A). A co-crystal structure of 1 bound in the ASK1 ATP-binding
Figure 3. (A) Crystal structure of 6 bound in the ASK1 ATP site to a resolution
of 2.65 Å (PDB: 6XIH). (B) An overlay of the ASK1 crystal structure of 6 (in
turquoise) and a model of 2 (in orange). The figures were generated using
Pymol®.
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com

2, hypotheses regarding its potential binding mode supported an
integrated approach to novel lead discovery. Deconstruction of 2
illustrated the different contributions of the scaffold constituents
to its overall potency and is shown in Figure 2B. Pairing this
information with an overlay of the crystal structure of 1 and a
model of 2 highlighted the varying importance on activity of two
specific interactions with ASK1: the glycine in the solvent-
exposed region (Gly759) and Lys709. When the cyclopropyl
imidazole was removed, potency decreased by 18-fold, 3. From
modeling, it seemed possible that the cyclopropyl imidazole was
π-stacking over the N-H of Gly759 — an interaction Takeda
previously published in crystal structures from two different
ASK1 inhibitor series [15,16]. Removal of the isopropyl-triazole
(4) resulted in a 2000-fold loss in potency to an IC50 of 6.0 µM,
which was accompanied by an expected decrease in the ligand
efficiency (LE = -ΔG/HAC ~ -RTln(IC50)/HAC; the free energy
of binding divided by heavy atom count) and the lipophilic ligand
efficiency (LLE = pIC50-cLogD, where cLogD is the calculated
logarithm of the octanol/water partition coefficient measured at
pH of 7.4. [17,18]. Essentially, removing the hydrogen bond to
Lys709 removes most ASK1 activity.
Compound 6 showed improved selectivity compared to 1
when tested in a kinase selectivity mini-panel (Table 1), which
supported further optimization of this scaffold.
Table 1. Selectivity of 6 versus 1 against other MAP3K kinases in a binding
assay at a top concentration of 10 µM. Potencies are listed as pIC50 values.
Kinase
ASK1
6
1
6.6
7.9
6.7
PRKKA1 < 4.7
p38
EGFR
HER4
MER
RET
< 4.7 < 4.7
< 4.7 < 4.7
< 4.7 < 4.7
< 4.7
< 4.7
6.2
7.2
6.0
5.9
ROCK1 < 4.7
The severe drop in potency resulting from removal of the
isopropyl triazole of 4 focused optimization toward other regions
of the molecule. The co-planar, ring system served as an
attractive starting point because of the ability to manipulate and
mimic this type of shape— or lack thereof. Numerous strategies
were used to imitate this conformation while modulating physical
properties through ring modifications and substitutions,
specifically retaining the stabilized hinge-binding benzamide
while providing a vector towards Gly759.
ITK
< 4.7
ERBB4
EPHA3
GSK3
CDK2
< 4.7 < 4.7
< 4.7 < 4.7
6.2
5.5
7.4
7.2
An interesting compound that emerged from this exercise
incorporated a non-aromatic ether group adjacent to the hinge
amide (6, Figure 3A). This substitution showed only a moderate
decrease in potency relative to a comparator, 5. A co-crystal
structure of this compound bound to ASK1 was acquired and
confirmed the anticipated binding mode (Figure 3A). An overlay
of the crystal structure of 6 with the model of 2 showed the
inhibitors had a high degree of overlap, with the tetrahydropyran
sitting in similar chemical space to the phenyl group, with a clear
vector to grow towards Gly759 (Figure 3B).
Summarized in Figure 4A, replacement of the tetrahydropyran
with a morpholine ring (7), although less potent than 6, had a
higher LLE value representing a more compelling starting point
with a lower cLogD. Modeling of 7 alongside 2 suggested
extending from the morpholine nitrogen could provide a vector to
engage Gly759, thus providing an opportunity to increase
potency while also modulating the basicity of the amine in an
area of the active site where polarity is not typically tolerated.
The addition of the phenyl group provided a modest increase in
enzymatic potency to 130 nM (8). By adding a nitrogen to the
Figure 4. (A) Optimization to a nanomolar inhibitor. (B) A model of 10 in the ASK1 ATP site was generated using Pymol®. (C) Physiochemical property data,
absorption, and bioavailability (%F) for 10 (*data for racemate). Stability in human hepatocytes shown as hepatic extraction ratio (Eh) measured as hepatic
clearance/species-specific liver blood flow. Membrane permeability in LLC-PK1-MDR1 cell line shown as the movement from the apical compartment to the
basolateral compartment. Kinetic solubility shown as JP1(pH 1.2)/JP2(pH 6.5). Pharmacokinetic studies (PK) in male Sprague-Dawley rats: dosed at 1 mgkg-1 IV
(n=2, 20% β-cyclodextrin in 0.05 M methanesulfonic acid, pH 3) and 5 mgkg-1 PO (n=3, 20% β-cyclodextrin in 0.05 M methanesulfonic acid, pH 3).
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com

ring, clogD was lowered while the potency increased by 4-fold
(9), resulting in an almost two log unit increase in LLE. The
addition of a cyclopropyl further increased potency and, by
isolating the more active enantiomer, a novel lead compound (10)
was identified with a cellular EC50 = 24 nM and a Kd < 1nM.
Based on the model of 10 bound in the ASK1 active site (Figure
4B), and its cellular activity, it was concluded the pyridine was
stacking over Gly759.
group imparting increased selectivity for this target. The
structural basis for the selectivity seen in this series could be
borne from the conformational dynamics of the ASK1 pocket,
which may be able to form the cavity required to accept both flat
and globular rings.
Table 2. Occurrence of various aliphatic versus aromatic rings in the
“morpholine pocket” of the human protein kinases in the PDB.
Aliphatic Ring System
Tetrahydropyran
Morpholine
Count
1
0.7%
0.1%
The physiochemical properties and a subset of ADME
(absorption, distribution, metabolism, and elimination) properties
of 10 are summarized in Figure 4C. Overall, the physiochemical
properties of this molecule are favorable, including 3 aryl rings
and cLogD 2.1. Pharmacokinetic (PK) studies were performed in
rats where the inhibitor was well absorbed; however, its high
clearance led to low oral bioavailability (%F, see Supplementary
Data for full ADME and PK profiles). For Selectivity was
investigated across an InvitrogenTM panel of 350 kinases
(representing > 50% of the predicted human kinome). Compound
10 showed exquisite selectivity where only eight kinases were
inhibited with an IC50 ≤ 500 nM (< 20-fold selective). Full dose-
response curves at a top concentration of 30 µM were generated
for those eight kinases, and their IC50 data is shown boxed in
Figure 5. This data highlights that 10 is a highly selective
inhibitor of ASK1.
7
Piperidine
1
Piperazine
3
Cyclohexane
Aromatic Ring System
Phenyl
25
Count
1,717
66
34.6%
31.3%
Pyridine
Pyrimidine
116
In summary, a multi-prong lead generation strategy utilizing
deconstruction and SBDD resulted in a novel chemical series
with desirable drug-like properties and exquisite selectivity
across the broader kinome (Figure 6). Mining the PDB for
saturated, cyclic pharmacophores highlighted how unique a
morpholine is in that region of a kinase active site, appearing in <
1% of the 5,490 kinase structures analyzed. The utility of this
approach to parameterized searching of chemical space for novel
functional groups could be a useful strategy for kinase drug
discovery.
Figure 5. Selectivity profile of 10 against an InvitrogenTM panel of 350
kinases. The box highlights the eight kinases the inhibitor exhibited < 20-
fold selectivity over, with their potencies listed in the chart.
To help explain the impressive kinome selectivity of 10, we
examined the residues at positions lining the modeled morpholine
location. Of interest was the “GGSL” sequence containing the
Gly759 residue that, when engaged, appears to enhance ASK1
activity. However, this sequence was not found to be particularly
rare within the human kinome. Suggestive of a role for the sp3-
rich morpholine towards target selectivity, we mined all human
protein kinase crystal structures reported in the PDB using a 3D
search of all bound kinase ligands with a variety of aliphatic 6-
membered rings in that region of the ATP pocket (see
Supplementary Material).
Our results (Table 2) indicated that aliphatic ring systems are
particularly rare in this sub-region annexing the hinge, with the
morpholine appearing in < 1% of the 5,490 kinase structures
analyzed. By contrast, flat, aromatic, 6-membered rings are
significantly more prevalent— with phenyl, for example,
occurring in this sub-region in 31.3% of all kinase structures.
While this analysis is limited to the small region of chemical and
protein space covered by crystal co-complexes, one could
speculate that the ASK1 ATP pocket can accommodate both
aliphatic and aromatic groups at this location, with an aliphatic
Figure 6. A kinome dendrogram selectivity plot of 10 highlights the 8/350
kinases with IC50 ≤ 500 nM. The larger the circle that more active against that
kinase. Illustration reproduced courtesy of Cell Signaling Technology, Inc.
(www.cellsignal.com).
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com

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Acknowledgments
We would like to thank Charles E. Grimshaw for his help
generating selectivity data. We would also like to thank Steve
Woodhead and Tony Gibson for reviewing this manuscript. The
staff of the Berkeley Center for Structural Biology is gratefully
acknowledged for support of beamline 5.0.3 at the Advanced
Light Source. The Advanced Light Source is supported by the
Director, Office of Science, Office of Basic Energy Sciences, of
the U.S. Department of Energy under Contract No. DE-AC02-
05CH11231. This research was also resourced by the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357.
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Appendix A. Supplementary Data
Included are protocols for (1) synthetic procedures for 10,
including NMR and LC/MS spectra, (2) pharmacokinetic (PK)
evaluation, (3) mining the region-of-interest in the ATP-binding
site of kinases in the PDB using MOE® [19].
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Structure-guided optimization of a novel
class of ASK1 inhibitors with increased sp3
character and an exquisite selectivity profile
Leave this area blank for abstract info.
Simone V. Bigi-Botterill, Anthony Ivetac, Erica L. Bradshaw, Derek Cole, Douglas R. Dougan, Jacques
Ermolieff, Petro Halkowycz, Ben Johnson, Christopher McBride, Jason Pickens, Mark Sabat, and Steven Swann
O
O
N
O
N
N
N
N
N
N
H
N
N
N
N
N
H
N
N
N
O
N
N
O
N
1
7
10
IC50 14 nM
LE 0.45, LLE 5.9
cLogD 2.0
IC50 2.0 μM
LE 0.32, LLE 4.9
cLogD 0.9
IC50/EC50 17/24 nM
LE 0.33, LLE 5.4
cLogD 2.1
Declaration of interests
☒ The authors declare that they have no known
competing financial interests or personal
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com

relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
Simone V. Bigi 05/06/2020 Christopher McBride 05/06/2020
Derek C. Cole 05/06/2020 Douglas R. Dougan 05/06/2020
Jason C. Pickens 05/06/2020 Petro Halkowycz 05/07/2020
Ben R. Johnson 05/06/2020 Erica L Bradshaw 05/07/2020
Anthony Ivetac 05/06/2020 Mark Sabat 05/18/2020
Jacques Ermolieff 05/06/2020
Steve Swann 05/06/2020
*Corresponding author: Simone V. Bigi-Botterill; Tel.: +1-619-930-8125; e-mail: simone.bigi@takeda.com