Structure-Based Design of ASK1 Inhibitors as Potential Agents for Heart Failure

Article abstract of DOI:10.1021/acsmedchemlett.6b00481

Apoptosis signal-regulating kinase 1 (ASK1/MAP3K) is a mitogen-activated protein kinase family member shown to contribute to acute ischemia/reperfusion injury. Using structure-based drug design, deconstruction, and reoptimization of a known ASK1 inhibitor

Full text of DOI:10.1021/acsmedchemlett.6b00481

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Letter
Structure-Based Design of ASK1 Inhibitors
as Potential Agents for Heart Failure
Marion Lanier, Jason Pickens, Simone V. Bigi, Erica L. Bradshaw-Pierce, Alison Chambers, Derek C Cole,
Douglas R. Dougan, Jacques Ermolieff, Tony Gibson, Petro Halkowycz, Aki Hirokawa, Anthony David Ivetac,
Joanne Miura, Evan Nunez, Mark Sabat, John Tyhonas, Haixia Wang, Xiaolun Wang, and Steve Swann
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00481 • Publication Date (Web): 08 Feb 2017
Downloaded from http://pubs.acs.org on February 9, 2017
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Structure-Based Design of ASK1 Inhibitors as Potential Agents for
Heart Failure
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Lanier, Marion* ; Pickens, Jason ; Bigi, Simone V. ; Bradshaw-Pierce, Erica L. ; Chambers, Ali-
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son , Cole, Derek , Dougan, Douglas R. ; Ermolieff, Jacques ; Gibson, Tony ; Halkowycz, Petro ;
Hirokawa, Aki ; Ivetac, Anthony ; Miura, Joanne ; Nunez, Evan ; Sabat, Mark ; Tyhonas, John ;
Wang, Haixia ; Wang, Xiaolun ; Swann, Steve
Departments of Medicinal Chemistry, Drug Metabolism Pharmacokinetics, Structural Biology, Discovery Biolo-
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gy, Gastrointestinal Drug Discovery Unit, Takeda Pharmaceuticals, 10410 Science Center Drive, San Diego, CA 92121
KEYWORDS Apoptosis signal-regulating kinase 1 (ASK1), Structure-Based Drug Design (SBDD), cardiac injury
ABSTRACT: Apoptosis signal-regulating kinase 1 (ASK1 / MAP3K) is a mitogen-activated protein kinase family member
shown to contribute to acute ischemia/reperfusion injury. Using structure-based drug design, deconstruction, and re-
optimization of a known ASK1 inhibitor, a lead compound was identified. This compound displayed robust MAP3K path-
way inhibition and reduction of infarct size in an isolated perfused heart model of cardiac injury.
Apoptosis signal-regulating kinase 1 (ASK1) is a mito-
gen-activated protein kinase kinase kinase (MAP3K) fami-
ly member residing upstream of both Jun N-terminal ki-
nase (JNK) and p38. ASK1 is capable of activating JNK and
P38 via the phosphorylation of intermediate kinases.
ASK1 plays a role in the mammalian cell stress response
and the induction of apoptotic cell death. It also contrib-
utes to a range of systemic diseases including heart fail-
our internal database as well as public domain crystal
structures of small molecules in ASK1 and published
8
,9
pharmacophore models. Figure 1 illustrates the co-
crystal structure of 1 in hASK1 (pdb: 3VW6) and highlights
the key interactions between 1 and the ASK1 ATP binding
pocket. Binding to the kinase hinge is characterized by a
hydrogen bonding interaction with the backbone NH and
carbonyl of Val757. Furthermore, the imidazole of 1
reaches deep into the active site making an H-bond with
the side-chain amine of the catalytic lysine (Lys709). The
compound also extends towards the solvent exposed re-
gion with a t-butyl phenyl amide. The t-butyl group is
sandwiched between Arg705 and Lys769 and the phenyl
1
ure and acute ischemia/reperfusion injury, by reducing
structural and functional integrity of the mitochondria in
2-4
cardiac cells. ASK1-deficient mice display reduced levels
of cardiomyocyte apoptosis, hypertrophy and interstitial
5
fibrosis. Thus, selective inhibition of ASK1 represents an
6
attractive strategy for slowing or potentially reversing
harmful tissue changes associated with various forms of
heart failure.
ring makes a Π-NH interaction with Gly759. These in-
teractions recapitulate those highlighted in published
8
,9
pharmacophore models.
Based on this information, an
in-house model of 2 was generated (Figure 1). The triazole
ring of 2 (synthesis described in SI along with all other
compounds mentioned in the text) is able to interact with
Lys709 while the central benzamide moiety binds to the
hinge through Val757. The cyclopropyl imidazole moiety
at the opposite end likely makes the same interactions to
that of the t-butyl phenyl group in 1.
Using ASK1 structural information and deconstruc-
tion of known ASK1 inhibitors such as 1 and 2 (Figure 1),
our research team generated a novel, potent, and orally
bioavailable ASK1 inhibitor* with favorable physicochem-
ical properties to help further elucidate the role of ASK1 in
cardiac injury. Compound 1 was an early lead for Takeda’s
6
Ask1 inhibitor program. 2 (GS-4997, Gilead Sciences) is a
clinical stage ASK1 inhibitor, which has been evaluated as
In addition to identifying the key protein interactions
for ASK1 inhibitors, we prepared three molecules (3, 4, 5)
based on the deconstruction of 2 to evaluate the relative
contributions of the different binding elements to poten-
cy. Efficiency metrics such as lipophilic ligand efficiency
an experimental treatment for diabetic nephropathy and
7
kidney fibrosis.
Key structural features with respect to ligand interac-
tions within the ATP binding site were identified using
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LLE),
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(
ligand efficiency (LE), and group efficiency
(4) resulted in a concomitant 8-fold potency loss with a
negligible reduction in LE or LLE. Finally, truncation of
the isopropyl triazole ring (5) from the right hand side of
the molecule resulted in > 1000-fold loss in potency and a
significant drop in LLE. The calculated GE value for this
ring system is 0.56, highlighting the value of this moiety
on binding affinity for ASK1.
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(
GE) were calculated and are summarized in Table 1.
In light of the published data, structural analysis and
deconstruction data, subsequent efforts were directed
towards the development of a novel and more efficient
class of ASK1 inhibitor through the introduction of a more
efficient hinge-binding moiety while maintaining vectors
towards both Lys709 and Lys769 and/or Arg705.
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The model and deconstruction analysis of 2 suggest
that the benzamide group remains co-planar with the rest
of the core in the active site and that the ortho-fluorine
atom, in particular, shields the amide NH. A number of
hinge binders bearing a similar motif while maintaining a
planar character were explored (results not shown). From
this effort, we identified compound 6 (Figure 2), which
contains an isoindolinone core as the primary hinge bind-
er, but otherwise displays similar substitution to that seen
in 4. The pIC50 (7.9) and LLE (5.9) values for 6 were high-
er than 4 (pIC50 = 6.4 and LLE = 3.6), indicating that the
isoindolinone moiety makes a superior interaction with
the protein relative to the simple benzamide of
Figure 1: Known (labeled) key interactions between ASK1
protein and small molecule inhibitors. Overlay of the
crystal structure of 1 (orange) and model of 2 (blue).
Table 1. ASK1 potency and efficiency metrics for cpds 2-5.
a
Cmpd.
Structure
pIC50 LLE
LE
GE
(cLogP)
(group)
8
.3
2
5.3
0.35
-----
±
0.07
0
.19
7
±
.3
3
4
3.8
3.6
0.4
(cPr-
0.09
imidazole)
6
±
.4
0.54
(F, Me)
0.38
0.03
5
±
.2
0.56
(iPr-triazole)
5
2.1
0.28
0.01
LLE = pIC50-cLogP; LE = -RTln(pIC50)/Nheavy; GE =
a Geometric mean of at least two experiments unless otherwise stated.
ꢀ(ꢀG)/ꢀN
heavy
Removal of the cyclopropyl imidazole (3) from the
parent 2 resulted in a ten-fold loss in potency and reduc-
tion in LLE from 5.3 to 3.8. It may be noted that, when
comparing 3 to 2, although the imidazole ring system
contributes approximately 10-fold in potency with little
change in LE (0.4 compared to 0.35), the overall increase
in LLE (3.8 to 5.3) indicates this interaction may play a
key role in binding affinity. Removal of the methyl group
and fluorine atom to give an un-substituted benzamide
Figure 2: Crystal structure of 6 in ASK1
compound 4. The crystal structure of 6 bound to ASK1
was solved at 2.7 Å. Figure 2 illustrates a similar binding
mode to 2. The carbonyl and aryl hydrogen of the isoindo-
linone core interact with the NH and carbonyl of Val757
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respectively, while the triazole moiety interacts with A number of compounds were synthesized with a
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Lys709 as expected. Finally, the 6-position of the isoindo-
linone ring provided a vector towards the solvent.
substitution at the 6-position and containing an ether
oxygen atom as a hydrogen bond acceptor. Compounds
were tested in an ASK1 enzyme activity assay as well as a
cell-based assay, which monitors formation of phospho-
JNK. Table 3 lists key compounds from this effort. Using
this strategy, a number of compounds were identified that
exhibited pIC50 > 8 and high LLE values in the ASK1 en-
zyme assay and pEC50 > 7.5 in the cellular assay.
Based on the structural information obtained for 6,
we first sought to elaborate the isopropyl group on the
triazole ring. This substituent projects deep in the kinase
active site near the polar residues Asn808 and Ser821. Ac-
cordingly, we focused on introducing substituents capa-
ble of hydrogen bonding with those residues (summa-
rized in Table 2). Most compounds from this exercise
showed only a marginal increase in ASK1 inhibition.
However, compound 9 illustrates that the addition of a
terminal hydroxyl likely results in a positive interaction
with the protein, translating to an increase in LLE from
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Table 3. ASK1 enzymatic and cell potency, and LLE values for
cpds 15-19.
Cmpd.
a
Structure
pIC50
LLE
pEC50
(cLogP)
1
1
5
6
7.8±0.04
4.8
5.9
7.5
5
.9 to 6.6 (Compounds 6 and 9, respectively). The 1-
hydroxypropan-2-yl addition (9 and 10) resulted in the
highest LLE and therefore considered the best group to
get the chemotype in a desirable drug-like chemical
space.
8.1±0.04
8.2±0.05
7.8
8.2
17
6.1
7.0
Table 2. ASK1 potency and efficiency metrics for cpds 7-14.
a
LLE
(cLogP)
Cmpd.
Structure
pIC50
LE
1
8
8.1±0.09
8.3±0.06
7.9
8.2
7
8 (S)
(R)
R 7.3±0.03
S 7.7±0.03
4.4
4.7
0.40
0.42
19
6.4
9
(R)
R 8.0±0.14
S 7.5±0.03
6.6
6.1
0.43
0.41
10 (S)
a Geometric mean of at least two experiments unless otherwise stated.
Co-crystal structures with ASK1 were solved for two
11
7.4±0.02
6.0
0.39
key compounds (17 and 19). The crystal structure of 17
(
Figure 3a) recapitulates the general binding features of
the truncated compound 6. The observed hydrogen bond
between the morpholine oxygen and the backbone NH of
Gly759 confirmed our design hypothesis. The structure of
1
1
2
3
7.9±0.01
7.7±0.01
5.7
5.9
0.43
0.4
1
9 illustrates an additional interaction between the termi-
nal hydroxyl group and the side chains of Asn808 and
Ser821. The increased potency and LLE value for 19 rela-
tive to the des-hydroxy analog 15 can be attributed to this
favorable interaction.
1
4
6.1±0.01
4.7
0.32
a Geometric mean of at least two experiments unless otherwise stated.
Compound 19 showed overall good in vitro ADME
properties (human and rat liver microsome clearance <8.3
and <16.6 ml/min/kg respectively; Papp (A-B)/(B-A)
In parallel, we made modifications at the 6-position
of the isoindolinone ring to target the solvent region. In
contrast to other known ASK1 inhibitors that contain ar-
omatic rings in this portion of the active site, we targeted
Gly759 by extending into the region with aliphatic groups
bearing hydrogen bond-accepting substituents. It was
considered that this would allow us to maintain a low
LogP and good physicochemical properties.
5
4.9/262.3 nm/sec at 10uM in MDR1-LLC-PK1; soluble at
1
55 uM at pH 1, details in SI). To further evaluate 19,
screening was conducted against 356 kinases (screened at
1 micromolar at Invitrogen then full dose response curves
for kinases with inhibition greater than 95% at 1 mi-
cromolar). Good kinase selectivity was observed with only
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2 kinases having less than 30 fold selectivity (Figure 4).
displayed suitable properties for broader pharmacological
evaluation. Compound 19 was later shown to reduce in-
farct size in the Langendorff perfused ex-vivo heart model.
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None of them are highly expressed in the heart.
Compound 19 was subsequently evaluated in a low-
dose rat pharmacokinetic (PK) study. From this, an esti-
mated dose required to provide at least eight hours of
>
50% ASK1 inhibition in a rat PD model of cardiac ische-
mia was calculated (Figure 5a). Accordingly, compound 19
was tested at 2.25 mg/kg in the Langendorff perfused
heart model to determine the effect of ASK1 inhibition on
cardiac infarct size. Cardiac infarct size was measured in
isolated heart preparations at 1 and 8 hours after dosing
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(
Figure 5b). Oral administration of 19 resulted in a 42%
reduction in infarct size after one hour and a 24% reduc-
tion after 8 hours (Figure 5b) suggesting that sustained
ASK1 inhibition may provide significant tissue protection
against the effects of myocardial ischemia.
Figure 4: Overall kinase selectivity for Compound 19.
With an appropriate tool molecule in hand it will be pos-
sible to test additional hypotheses surrounding ASK1 in-
hibition in relation to harmful tissue changes associated
with chronic heart failure and fibrotic disease.
Figure 3. Crystal structures of compounds 17 (a) and 19 (b)
bound to ASK1 kinase domain.
In summary, a combined structure-based and compound
deconstruction approach was used to rapidly produce a
novel series of potent ASK1 inhibitors with ideal physio-
chemical properties and efficiencies. Following an initial
deconstruction exercise, compound 6 was identified as a
prototype for an efficient hinge-binding series. Subse-
quent elaboration led to compounds such as 19, which
Figure 5. (a) Simulated rat pharmacokinetics and simulated
ASK1 inhibition for 19 at 2.25mpk. (b) Ex-vivo profiling of 19 in
cardiac ischemia / reperfusion injury.
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Mitochondrial Function of Thioredoxin-interacting Pro-
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■
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:xxxxx
It contains the following:
I/ Synthetic procedures for all the compounds listed in the
manuscript
3
. Shiizaki, S.; Naguro, I.; Ichijo, H. Activation mecha-
nisms of ASK1 in response to various stresses and its sig-
nificance in intracellular signaling. Adv. Biol. Regul. 2013,
5
3, 135-144.
II/ Protocols for in vitro assays (binding assay, functional
assay, ADME assays)
4. Tibbles, L. A.; Woodgett, J. R. The stress-activated pro-
tein kinase pathways. Cell. Mol. Life Sci. 1999, 55, 1230-
1254
III/ pharmacological evaluation (PK, Langendorff Perfused
Heart Model)
IV/ Crystal structures information (ASK1_1 (PDB), ASK1_6
0
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9
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5. Hattori, K.; Naguro, I.; Runchel, C.; Ichijo, H. The roles
of ASK family proteins in stress responses and diseases.
Cell Commun. Signaling 2009, 7, 9-19.
6. Terao, Y.; Suzuki, H.; Yoshikawa, M.; Yashiro, H.;
Takekawa, S.; Fujitani, Y.; Okada, K.; Inoue, Y.; Yamamo-
to, Y.; Nakagawa, H.; Yao, S.; Kawamoto, T.; Uchikawa, O.
Design and biological evaluation of imidazo[1,2-a] pyri-
dines as novel and potent ASK1 inhibitors. Bioorg. Med.
Chem. Lett. 2012, 22, 7326-7329.
7. Lin, J. H.; Zhang, J. J.; Lin, S. L.; Ghertow, G. M. Design
of a phase 2 clinical trial of an ASK1 inhibitor, GS-4997, in
patients with diabetic kidney disease. Nephron. 2015, 129,
29–33.
(
PDB), ASK1_17 (PDB), ASK1_19 (PDB))
Accession Codes
Coordinates and structure factors are available from the Pro-
tein Data Bank with accession codes X, Y, and Z for ASK1_1,
ASK1_6, ASK1_17 and ASK1_19 respectively.
■
AUTHOR INFORMATION
Corresponding Author
E-mail: marion.lanier@takeda.com
*
Tel: +1-858-731-3650
8
. Starosyla, S. A.; Volynets, G. P., Bdzhola, V. G.; Golub,
Notes
A. G.; Protopopov, M. V.; Yarmoluk, S. M. ASK1 pharma-
cophore model derived from diverse classes of
inhibitors. Bioorg. Med. Chem. Lett. 2014, 24, 4418-4423.
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
9
. Singh, O.; Shillings, A.; Craggs, P.; Wall, I.; Rowland, P.;
Skarzynski, T.; Hobbs, C. I.; Hardwick, P.; Tanner, R.;
Blunt, M.; Witty, D. R.; Smith, K. J. Crystal structures of
ASK1-inhibtor complexes provide a platform for structure-
based drug design. Protein Sci. 2013, 22, 1071-1077.
We would like to thank Steve Bowlin for running the HR-MS,
Georgia Kefala, Irena Levin, Weston Lane and Gyorgy Snell
for their help getting the crystal structures. The staff of the
Berkeley Center for Structural Biology is gratefully acknowl-
edged for support of beam line 5.0.3 at the Advanced Light
Source. The Advanced Light Source is supported by the Di-
rector, Office of Science, Office of Basic Energy Sciences, of
the U.S. Department of Energy under Contract No. DE-
AC02-05CH11231. This research used also resources of the
Advanced Photon Source, a U.S. Department of Energy
1
0. LLE = pEC50 – cLogP or pIC50 –cLogP. Leeson, P. D.;
Springthorpe, B. The influence of drug-like concepts on
decision-making in medicinal chemistry. Nature Rev.
Drug Discov. 2007, 6, 881–890.
1
1. LE = pIC50 x (-1.4) /HAC with HAC is Heavy Atom
Count. Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand effi-
ciency: a useful metric for lead selection. Drug Discovery
Today 2004, 9, 430–431.
(
DOE) Office of Science User Facility operated for the DOE
Office of Science by Argonne National Laboratory under
Contract No. DE-AC02-06CH11357
12. GE=ꢀG/ꢀHAC = ꢀ(-RTlnKi)/ꢀHAC with HAC is Heavy
Atom Count. Verdonk M. L., Rees D. C. Group efficiency:
a guideline for hits-to-leads chemistry. ChemMedChem
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ACS Medicinal Chemistry Letters
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