Identification of RIPK3 Type II Inhibitors Using High-Throughput Mechanistic Studies in Hit Triage

Article abstract of DOI:10.1021/acsmedchemlett.9b00065

Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.

Full text of DOI:10.1021/acsmedchemlett.9b00065

Subscriber access provided by BOSTON COLLEGE
Letter
Identification of RIPK3 Type II in-hibitors using
high-throughput mechanistic studies in hit triage
Amy C Hart, Lynn Abell, Junqing Guo, Michael E. Mertzman, Ramesh Padmanabha,
John E Macor, Charu Chaudhry, Hao Lu, Kevin O'Malley, Patrick J Shaw, Carolyn A.
Weigelt, Matthew Pokross, Kevin Kish, Kyoung Soon Kim, Lyndon Cornelius, Andrew
E. Douglas, Deepa Calambur, Ping Zhang, Brian Carpenter, and William J Pitts
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00065 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 6, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Identification of RIPK3 Type II inhibitors using high-throughput
mechanistic studies in hit triage
Amy C. Hart*, Lynn Abell†, Junqing Guo, Michael E. Mertzman, Ramesh Padmanabha, John E. Macor†, Charu
Chaudhry, Hao Lu†, Kevin O’Malley, Patrick J. Shaw, Carolyn Weigelt, Matthew Pokross, Kevin Kish, Kyoung
S. Kim, Lyndon Cornelius, Andrew E. Douglas, Deepa Calambur, Ping Zhang, Brian Carpenter and William J.
Pitts
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bristol-Myers Squibb Research & Development, P.O. Box 4000, Princeton, NJ 08543, USA
RIPK3, Type II kinase inhibitor, kinase, structure-based drug design (SBDD), high-throughput screening (HTS), necroptosis
Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this
signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the
hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3 which was confirmed by
protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an
enhancement in selectivity against key off target kinases.
Necroptosis is a programmed form of cell death and has been
associated with a variety of diseases, including ischemia
reperfusion injury, neurodegenerative disorders, pancreatic
cancer and autoimmune diseases such as inflammatory bowel
disease (IBD).1,2,3,4,5 Upon stimulation of death receptors (such
as the family of TNF receptors), signaling can initiate a
necroptotic cell death process. This involves formation of a
necrosome which includes receptor interacting protein kinases
1 and 3 (RIPK1, RIPK3) in a cytosolic complex. RIPK3
phosphorylates the pseudokinase mixed-lineage kinase like
(MLKL) which can subsequently induce cell death.
Necroptotic cell death leads to the release of inflammatory
damage-associated molecular patterns (DAMPs) into the
extracellular environment and contributes to inflammation
through activation of the Toll-like receptor pathway.3 Unlike
RIPK3 and MLKL, RIPK1 can participate both in necroptosis
and death receptor-mediated apoptosis. RIPK1, RIPK3 and
pseudokinase MLKL have been the focus of drug discovery
efforts in academia and industry.6,7,8,9,10,11
RIPK3 which target an inactive conformation of the kinase
from a high throughput screen (HTS). To assist in the
identification of such inhibitors, biophysical screening was
incorporated at the hit selection and triage phase.
NH2
N
N
NH2
N
HN
N
OH
F
S
S
S
F
H
N
N
S
N
N
N
N
O
O
F
1
2
3
S
NH
N
HN
N
O
O
O
S
O
N
N
O
4
5
Figure 1. Reported RIPK3 inhibitors
A common issue with pursuing kinase targets is kinome
selectivity.16 Off-target kinase activities can lead to several
challenges including off-target toxicology and opposing or
confounding pharmacology to the target of interest. One
potential way to mitigate issues related to kinome selectivity is
to target the inactive form of the kinase. When targeting the
inactive form of a kinase, both ATP competitive and non-
competitive binders can be found targeting Type II, III or IV
pockets.17,18,19,20 In addition to affecting kinome selectivity,
kinase inhibitors targeting inactive conformations may differ
from Type I inhibitors in other ways. Enhanced residence times
are common in non-Type I inhibitors.21 The additional
conformational changes required to access the pockets distal or
allosteric to the ATP binding site is a likely contributing factor.
Inhibitors targeting some inactive conformations (primarily
In 2013, the first X-ray crystal structures for RIPK3 were
reported.12 As is common in kinases, RIPK3 was crystallized in
its active form. To date, only Type I RIPK3 inhibitors have been
reported13 (Figure 1) and they tend to suffer from poor RIPK
family selectivity, notably with respect to RIPK2 activity.14,15
In contrast, the co-crystal structure of RIPK3 and MLKL with
AMP-PNP indicated that RIPK3 was adopting an inactive
conformation in the complex. Key findings included a shifting
of the C-helix along with partial unwinding of the helix.
Significant movement and realignment in the hydrophobic
spine was also noted. Based on these findings, we were
intrigued by the potential opportunity to identify inhibitors of
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
Type III or Type IV) of the kinase may not be fully competitive
After running the HTS with an HTRF assay on the RIPK3
full length kinase, TD studies were run on a subset of
with ATP leading to another source of differentiation.19,22
1
2
3
4
5
6
7
8
9
compounds meeting potency requirements.
With the
Numerous kinases that can adopt an inactive conformation
have been identified to date.17 Several methods have been
developed to further identify kinases which may be able to
adopt a Type II DFG-out conformation, including labeling
approaches24,25 and predictive computational models.25 From
an inhibitor standpoint, a characteristic pharmacophore for
Type II kinase inhibitors has been established.17 Screening
inhibitors designed on the pharmacophore has identified
establishment of a baseline expectation from the described
validation runs, HTRF time dependency for RIPK3 hits at 5 and
60 min led to the identification of a small subset of compounds
which may be slow binding (Figure 2). Specifically 44
compounds out of 2571 tested showed a ≥ 5-fold shift in IC50 at
5 and 60 min.
kinases capable of adopting
a
DFG-out inactive
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
conformation.17,26
In this report we disclose the use of high throughput
mechanistic assays in the hit triage stage to enable identification
of non-Type I RIPK3 kinase inhibitors. For the follow-up
mechanistic studies, we needed to consider what assays would
be compatible with the HTS platform and automation. At the
outset of the project, we considered several potential screening
assays and deemed that
a homogenous time-resolved
fluorescence assay (HTRF) would be most suitable for this
kinase program.27 Although consideration of a RIPK3 construct
was taken into account, we found that using the active,
commercial full length enzyme was well-suited to the HTRF
platform, demonstrating improved probe binding over the
kinase domain construct (data not shown). After studying
multiple pre-incubation times for the complex, the pre-
incubation time for the HTS screen was increased to 60 min to
accommodate any inhibitors with a slow on-rate. The complex
was found to reach equilibrium within 45 min, with minimal
changes beyond 60 min. Taking into account some of the
reported attributes of non-Type I inhibitors, assays for time
dependency (TD) and mode of inhibition (MOI) were deemed
to be the most pertinent mechanistic studies to incorporate early
in our screening paradigm. Use of the HTRF platform allowed
for the option of utilizing probe displacement as opposed to
ATP displacement as a tool for the studies.
Figure 2. Analysis of high-throughput TD studies from the HTS.
Compounds in the yellow box (44) had a shift of ≥ 5 in the RIPK3
IC50 at 5 and 60 min.
Multiple non-traditional kinase chemotypes were identified
which represented interesting lead molecules. We were
gratified to see that this screening methodology did identify a
known Type II DFG-out kinase inhibitor, in addition to Type I
kinase chemotypes (Figure 4).28,29 For example, compound 6
demonstrated exquisite binding potency for RIPK3 in the HTS
assay along with superb selectivity over RIPK1 and RIPK2. As
a key proof of concept to the incorporation of mechanistic tools
at the hit triage stage, compound 6 demonstrated a greater than
6 fold shift in the TD assay. A shift of 2 in the TD assay was
observed for compound 7, along with potent inhibition of
RIPK2. Structurally similar compounds from the HTS shared
similar profiles (data not shown). Two structurally related Type
II DFG-out inhibitors, 8 and 9, were identified through internal
deck-mining (Table 1). We were pleased to find that while
some binding potency was lost, the excellent RIPK family
selectivity could be maintained.
While time dependent inhibition is not
a defining
characteristic for inhibitors targeting Type II conformations, it
is found frequently enough to be suggestive of a Type II
inhibitor.21 For the initial TD studies, several methods were
evaluated based on probe or inhibitor displacement. Initially
probe displacement of the inhibitor at a single concentration
wherein the output would be a percent shift in the displacement
at 5 and 60 min was explored. This was based on forming an
initial complex of the inhibitor, kinase and antibody then
treating with the probe at 30 fold the probe Kd. An alternative
approach was to pre-form a complex consisting of the kinase,
probe and antibody. The inhibitor could then be added to
displace the probe and diluted to determine an 11-point
concentration response curve (CRC) at 5 and 60 min. Based on
the results observed with a small test set of internal compounds,
inhibitor displacement of the probe at 5 and 60 min in CRC
format would be utilized for the time dependency studies.
O
NH
N
HN
O
O
N
N
H
F
NH
O
H2N
F
O
O
6
7
N
Type II DFG-out kinase inhibitor
HTS RIPK3 IC50 = 0.3 nM
HTS RIPK2 IC50 = 2400 nM
HTS RIPK1 IC50 = 310 nM
Type I kinase inhibitor
HTS RIPK3 IC50 = 270 nM
HTS RIPK2 IC50 = 50 nM
HTS RIPK1 IC50 = 17000 nM
H2N
N
Initial exploration of MOI assays to identify compounds
which may bind in a pocket more removed from the ATP
binding site proved challenging. Assays were attempted at both
low ATP and high ATP concentrations and low probe and high
probe concentrations in an effort to bin compounds as
‘competitive binders’ or ‘mixed type binders’ (uncompetitive or
non-competitive). The high hit rate in the initial study precluded
the use of this assay in guiding a selection of chemotypes.
Figure 3. Type II and Type I kinase chemotypes from the HTS.
HTRF assay data acquired at 60 min for all kinases shown.
While 8 and 9 are known Type II DFG-out inhibitors in c-
Met kinase,30,31 it is possible for an inhibitor targeting an
inactive conformation in one kinase to target the active
conformation in
a
different kinase.17 As no inactive
conformation inhibitors have been reported for RIPK3
previously, we were interested in building a model for a DFG-
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
out conformation in RIPK3 to help guide structure design to
and death receptor-mediated apoptotic signaling.3 By targeting
exclusive RIPK3 inhibition, we were seeking a profile devoid
of the liabilities which may be found with immunosuppression
(RIPK2) or prevention of cell death (RIPK1).
1
2
3
4
5
6
7
8
improve kinase selectivity of our inhibitors; however, there
were a number of challenges associated with building such a
model. Based on the co-crystal structure with MLKL, RIPK3
is known to have a very short C-helix which is susceptible to
unwinding and would preclude the formation of a DFG-out
conformation.12 In order to identify a conformation that may
accommodate a DFG-out inhibitor, a model was constructed in
which the activation loop from a RIPK2 DFG-out crystal
structure32 was merged with the RIPK3 Type I/active
conformation crystal structure. This provided a reasonable
docking mode to use for evaluation of these compounds as
putative RIPK3 Type II inhibitors. Ultimately this binding
mode was confirmed by X-ray protein crystallography in a
murine kinase domain construct bound with 9, demonstrating
for the first time that RIPK3 can adopt this unreported
conformation (Figure 5). The azaindole core engaged in
hydrogen bond interactions with Met98 at the hinge. The DFG
motif flipped outward, exposing the DFG backbone which was
able to form hydrogen bond interactions with the pyridone
oxygen at Asp161. The pyridone oxygen also locked the
conformation of the back pocket group through hydrogen
bonding with the amide, further rigidifying the structure. The
DFG flip revealed an available distal pocket that is filled by the
pyridone and its substituent.
RIPK2 selectivity, a particular issue for most of the reported
RIPK3 Type I inhibitors, was not a concern for the RIPK3 Type
II inhibitors. Mono- and bi-dentate hinge binders surveyed
showed a moderate degree of RIPK family selectivity (Table 1).
Notably, 9 and 10 demonstrated 64- and 38-fold selectivity over
RIPK1. Over 180-fold selectivity for RIPK2 was observed in
all of the examples. In an effort to understand the importance
of the hydrogen bonding interaction of the pyridone oxygen
with the DFG backbone in the crystal structure and the internal
amide, biphenyl analog 12 was prepared. The inability of 12 to
inhibit RIPK3 suggests a strong dependence on the presence of
the hydrogen bond network of the pyridone exists in this class
of inhibitors.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1a,b. Preliminary SAR
O
R1
F
NH
O
R
RIPK3 RIPK2 / RIPK1
(IC50 M) (IC50 M)
c-Met
(IC50 M)
Example
R
R1
,
,
,



N
0.016d
0.18
3.0d / 0.60d,e
> 15 / 1.5d
1.8 / 0.18
NTc
8
O
O
O
O
H2N
N
F
F
F
F
F
N
N
N
9
0.0057
0.0025
0.018
4.5
N
H
N
O
10
11
12
0.0029
0.4
N
N
H
> 15 / > 15d
> 15 / > 15
N
> 15
N
aHTRF assay protocol details are provided in the Supporting
Information. bAssay results are the average of at least two replicates
c
and IC50 determination occurred after 60 min incubation. Not
d
e
tested in this assay. Assay results are N of 1. Full length construct
used in assay instead of kinase domain.
As can be seen in Table 1, the selected compounds showed
potent binding to c-Met kinase in addition to RIPK3. As the
crystal structure for 9 in c-Met kinase had already been
obtained,31 we were able to compare the protein crystal
structures of c-Met (human) and RIPK3 (murine) and define
spaces in which optimization targeting improved kinome
selectivity could be realized (Figure 6). In both kinases, the
inhibitor binds to an inactive conformation. The azaindole
moiety is engaged in hydrogen bonding at the hinge, and the
amide and pyridone form a hydrogen bond network with the
protein backbone of the kinases at the DFG motif of the
activation loop. The 4-fluorophenyl moiety occupies the distal
pocket created by the DFG flip. Of note, the smaller gatekeeper
residue present in RIPK3 (Thr95 vs Leu1157 in c-Met kinase)
could provide an opportunity to increase bulk around the phenyl
linker moiety and thereby improve selectivity over c-Met
kinase.
Figure 4: X-ray crystal structure of compound 9 bound to
the catalytic domain of murine RIPK3. The carbons of 9 are
colored in pink and the carbons for RIPK3 are colored in purple.
Oxygens are colored red, nitrogens blue and sulfurs yellow.
Hydrogen bonds are indicated with dashed lines. PDB code
6OKO
With Type II DFG-out inhibitors reported for both RIPK2
and RIPK1,6 initial concern focused on RIP family selectivity.
RIPK2 is part of the NOD signaling cascade, an innate immune
pathway.14 The benefit of RIPK2 kinase inhibition is still
somewhat controversial given that the RIPK2 kinase dead
mutant (D146N) suggests that RIPK2 kinase activity and
RIPK2 autophosphorylation were not linked to NOD2
signaling.33 RIPK1 is an important kinase in both necroptotic
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
O
N
1
2
NH
O
F
A
O
N
3
4
5
R
RIPK3 RIPK2 / RIPK1
(IC50 M) (IC50 M)
c-Met
(IC50 M)
6
7
8
9
Example
R
H
A
,
,
,



F
> 15 / > 15c
13 / > 15c
0.018
0.5c
11
0.40
0.32
0.18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
13
14
15
> 15 / > 15d
> 1.5
> 15 / > 15c
1.8 / 0.18c
0.68 / 0.20c
12
H
0.050
0.0029
0.0080
O
F
0.0025
0.0063
10
16
N
H
O
O
O
Figure 5: X-ray crystal structure of 9 in murine RIPK3
(magenta, PDB code 6OKO) overlaid with 9 in human c-Met
kinase (green, PDB code 3CE3). Crystal structures of compound 9
are bound to the kinase catalytic domains. Oxygens are colored red,
nitrogens blue and sulfurs yellow. Hydrogen bonds are indicated
with dashed lines.
N
H
17
18
0.088
3.1 / 11
0.30
1.1
N
H
0.0091
> 10 / 5.5
N
H
Initial work focused on improving selectivity of 11 versus c-
Met kinase via modification of the A-ring that binds near the
gatekeeper residue (Table 2). Incorporation of methyl groups
ortho to the oxygen yielded 13. While only a small
improvement in RIPK3 potency was observed, a significant
decrease in c-Met kinase potency indicated that changes to the
linker phenyl group were having the desired effect. Placing the
methyl groups on the same side of the phenyl linker (14) further
improved selectivity against c-Met kinase (> 10 fold). Further
increasing the bulk to napthyl (15) was effective in both
improving RIPK3 potency and increasing selectivity over c-
Met kinase by > 150-fold; however 15 also demonstrated potent
binding to p38 kinase (data not shown).
To improve c-Met kinase selectivity in the bidentate hinge
binder, the direct replacement of the A-ring fluorine in 10 with
a methyl group (16) was explored. While this modification
failed to improve c-Met kinase selectivity, incorporation of
dimethyl substitution (17 and 18) yielded more promising
results. Placement of the dimethyl groups on the same side of
the phenyl ring (18) maintained the majority of RIPK3 potency
while increasing selectivity over c-Met kinase to 100-fold.
Consistent with other results in this series, especially the
disubstitued analogs, 18 was exquisitely selective amongst the
RIPK family members. More extensive screening across the
kinome revealed that 18 inhibited roughly 40 kinases (out of
246 tested) with an IC50 < 1 M (data in supplemental
information) which may impact its ability to serve as a
useful tool to probe RIPK3 pharmacology.
aHTRF assay protocol details are provided in the Supporting
Information. bAssay results are the average of at least two replicates
c
and IC50 determination occurred after 60 min incubation. Assay
results are N of 1. dFull length construct used in assay instead of
kinase domain.
The synthesis of 8 and 9 has been previously described.30,31
Modifications to the phenyl linker and the hinge binder are
detailed in Scheme 1. Reaction of p-nitrophenols 20 with 4-
halopyridine or 2,4-dichloropyridine (19) provided the nitro
intermediate 21. While most analogs were made using
nitrophenols, use of the aniline was also tolerated. Reduction
of the nitro group and coupling with previously reported 1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid31
led to final monodentate hinge binding compounds exemplified
by
23.
Palladium-mediated
couplings
with
cyclopropanecarboxamide yielded bidentate hinge binding
compounds exemplified by 24.
Scheme 1. Synthesis of hinge binder and linker analogsa
Table 2a,b. Hinge binder and phenyl linker SAR
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
NO2
†John Macor, currently at Sanofi, 153 2nd Ave., Waltham, MA
02451
NO2
1
2
3
4
5
6
7
Cl
a
b
O
R1
R1
Author Contributions
+
The manuscript was written through contributions of all authors.
R
N
OH
R
N
Notes
19
20
21
The authors declare no competing financial interest.
R = Br, Cl, H
R = Br, Cl, H
ACKNOWLEDGMENT
8
O
9
Special thanks to Randi Brown, Sarah Malmstrom and Alan Futran
for assay support.
N
NH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NH
O
c
F
O
R1
ABBREVIATIONS
O
N
R1
DAMP, damage-associated molecular patterns; IBD, inflammatory
bowel disease; RIPK, receptor interacting protein kinase; MLKL,
mixed-lineage kinase like; AMP-PNP, adenylyl-imidodiphosphate;
ATP, adenosine triphosphate; DFG, aspartic acid, phenylalanine,
glycine; HTS, high-throughput screen; HTRF, homogenous time-
resolved fluorescence; CRC, concentration response curve; TD,
time dependency; MOI, mode of inhibition; Met98, methionine 98;
Asp161, aspartic acid 161; Thr95, threonine 95; Leu1157, leucine
1157; c-Met, a receptor tyrosine kinase; TNF, tumor necrosis
factor; NOD, nucleotide binding oligomerization domain; iPr2NEt,
diisopropylethylamine; NMP, N-methyl-2-pyrrolidinone; EtOAc,
R
N
23
22
R
R = Br, Cl, H
R = Br, Cl, H
23
R = Cl, Br
R = H,
d or e
O
24
HN
aReagents and conditions: (a) iPr2NEt, NMP, microwave, 210 °C,
33-94%; (b) SnCl2, EtOAc, 80 °C, 24-33%; (c) 1-(4-fluorophenyl)-
2-oxo-1,2-dihydropyridine-3-carboxylic acid, BOP, Et3N, DMF,
22-87%; (d) cyclopropanecarboxamide, Pd2(dba)3, Xantphos,
Cs2CO3, dioxane, 100 °C, 7-20%; (e) H2(g), Pd/C, MeOH, HCl, 6-
61%.
ethyl
yloxy)tris(dimethylamine)phosphonium
Et3N, triethylamine; DMF,
acetate;
BOP,
(benzotriazol-1-
hexafluorophosphate;
dimethylformamide;
dba,
dibenzylideneacetone; Xantphos, 4,5-bis(diphenylphosphino)-9,9-
dimethylxanthene; MeOH, methanol.
In conclusion, the use of mechanistic screening to assess HTS
hits can assist in the triaging of chemotypes with alternate
binding modes. Time dependency studies on the HTS hits led
to the identification of a putative Type II kinase inhibitor for
RIPK3. An X-ray structure co-crystal of compound 9 with
RIPK3 demonstrated that the kinase can adopt a previously
unreported DFG-out conformation. Structure-based drug
design considerations aided the optimization effort to identify
compounds with improved selectivity over c-Met kinase (e.g.
18). Importantly, RIPK3 Type II kinase inhibitors were found
to have excellent RIPK family selectivity, although broader
kinome selectivity needs further optimization.
REFERENCES
1
Grootjans, S.; Vanden Berghe, T.; Vandenabeele, P. Initiation and
execution mechanisms of necroptosis: an overview. Cell Death Differ.
2017, 24, 1184-1195.
2
Conrad, M.; Angeli, J.P.F.; Vandenabeele, P.; Stockwell, B.R.
Regulated necrosis: disease relevance and therapeutic opportunities.
Nat. Rev. Drug Discov. 2016, 15, 348-366 and references therein.
3 Silke, J.; Rickard, J.A.; Gerlic, M. The diverse role of RIP kinases in
necroptosis and inflammation. Nat. Immunol. 2015, 16, 689-697 and
references therein.
4
Wang, W.; Marinis, J.M.; Beal, A.M.; Savadkar, S.; Wu, Y.; Khan,
M.; Taunk, P.S.; Wu, N.; Su, W.; Ahson, A.; Kurz, E.; Chen, T.; Yaboh,
I.; Li, F.; Gutierrez, J.; Diskin, B.; Hundeyin, M.; Reilly, M.; Lich, J.D.;
Harris, P.A.; Mahajan, M.K.; Thorpe, J.H.; Massau, P.; Mosley, J.E.;
Leinwand, J.; Kochen Rossi, J.A.; Mishra, A.; Aykut, B.; Glacken, M.;
Ochi, A.; Verma, N.; Kim, J.I.; Vasudevaraja, V.; Adeegbe, D.;
Almonte, C.; Bagdatlioglu, E.; Cohen, D.J.; Wong, K.-K.; Berton, J.;
Miller, G. RIP1 Kinase drives macrophage-mediated adaptive immune
tolerance in pancreatic cancer. Cancer Cell 2018, 34, 757-774.
ASSOCIATED CONTENT
Supporting Information
Full experimental details for key compounds, biological protocols
and screening protocols. The Supporting Information is available
free of charge on the ACS Publications website.
5
Werba, G.; Seifert, L.; Miller, G. Necroptotic cell death – an
unexpected driver on pancreatic oncogenesis. Cell Cycle 2016, 15,
2095-2096.
6
Bryan, M.C.; Rajapaksa, N.S. Kinase inhibitors for the treatment of
Experimental details, screening protocols and crystallography
details (PDF)
immunological disorders: recent advances. J. Med. Chem. 2018 doi:
10.1021/acs.jmedchem.8b00667 and references therein.
7 Yoshikawa, M.; Saitoh, M.; Katoh, T.; Seki, T.; Bigi, S.V.; Shimizu,
Y.; Ishii, T.; Okai, T.; Kuno, M.; Hattori, H.; Watanabe, E.; Saikatendu,
K.S.; Zou, H.; Nakakariya, M.; Tatamiya, T.; Nakada, Y.; Yogo, T.
Discovery of 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine
derivatives as potent, orally available, and brain penetrant receptor
interacting protein 1 (RIP1) kinase inhibitors: analysis of structure-
kinetic relationships. J. Med. Chem. 2018, 61, 2384-2409.
AUTHOR INFORMATION
Corresponding Author
* Tel: +1-609-252-3472. Email. amy.hart@bms.com
Present Addresses
8 Patel, S.; Hamilton, G. Inhibitors of RIP1 and methods of use thereof.
PCT Int. Appl. 2018 WO 2018/109097 A1.
†Lynn Abell, currently at Agios, 88 Sidney Street, Cambridge,
MA, 02139
†Hao Lu, currently at EMD Serono, 45A Middlesex Turnpike,
Billerica, MA, 01821
9
Delehouzé, C.; Leverrier-Penna, S.; Le Cann, F.; Comte, A.;
Jacquard-Fevai, M.; Delalande, O.; Desban, N.; Baratte, B.; Gallais, I.;
Faurez, F.; Bonnet, M.C.; Hauteville, M.; Goekjian, P.G.; Thuillier, R.;
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
23
Favreau, F.; Vandenabeele, P.; Hauet, T.; Dimanchi-Boitrel, M.T.
Simard, J.R.; Rauh, D. FliK: a direct-binding assay for the
identification and kinetic characterization of stabilizers of inactive
kinase conformations. Methods Enzymol. 2014, 548, 147-171.
Bach, S. 6E11, a highly selective inhibitor of receptor-interacting
protein kinase 1, protects cells against cold hypoxia-reoxygenation
injury. Sci. Rep. 2017, 7, 12931.
1
2
3
4
5
6
7
8
24
Ranjitikar, P.; Perera, B.G.K.; Swaney, D.L.; Hari, S.B.; Larson,
10
Yan, B.; Liu, L.; Huang, S.; Ren, Y.; Wang, H.; Yao, Z.; Lin, L.;
E.T.; Krishnamurty, R.; Merritt, E.A.; Villén, J; Maly, D.J. Affinity-
based probes based on Type II kinase inhibitors. J. Am. Chem. Soc.
2012, 134, 19017-19025.
Chen, S.; Wang, X.; Zhang, Z. Discovery of a new class of highly
potent necroptosis inhibitors targeting the mixed lineage kinase
domain-like protein. Chem. Comm. 2017, 53, 3637-3640.
11 Lessene, G.L.; Wilks, A.F.; Murphy, J.M.; Garnier, J.-M.; Czabotar,
P.E.; Hildebrand, J.M.; Lucet, I.; Silke, J.H.; Feutrill, J.T.; Cuzzupe,
A.N.; Sharma, P. Methods for inhibiting necroptosis. PCT Int. Appl.
2015 WO 2015/172203 A1.
25
Ung, P.M.-U.; Schlessinger, A. DFGModel: predicting protein
kinase structures in inactive states for structure-based discovery of
Type-II inhibitors. ACS Chem. Biol. 2015, 10, 269-278.
26
Dietrich, J.; Hulme, C.; Hurley, L.H. The design, synthesis and
9
evaluation of 8 hybrid DFG-out allosteric kinase inhibitors: a structural
analysis of the binding interactions of Gleevec®, Nexavar®, and
BIRB796. Bioorg. Med. Chem. 2010, 18, 5738-5748.
12
Xie, T.; Peng, W.; Yan, C.; Wu, J.; Gong, X.; Shi, Y. Structural
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
insights into RIP3-mediated necroptotic signaling. Cell Rep. 2013, 5,
27
70-78.
Jia, Y. Current status of HTRF(®) technology in kinase assays.
13
Hart, A.C.; Shaw, P.J. Targeting Necroptosis for Therapeutic
Expert Opin. Drug Discov. 2008, 3, 1461-1474.
28 Borzilleri, R.M.; Cornelius, L.A.M.; Schmidt, R.J.; Schroeder, G.M.;
Kim, K.S. Monocyclic heterocycles as kinase inhibitors U.S. Patent
2008, 7459562 B2.
Intervention in Inflammatory Diseases. In Medicinal Chemistry
Reviews; Desai, M.J., Ed.; ACS: Washington DC, 2016; Vol. 51, pp
101-114.
29
14 Haile, P.A.; Votta, B.J.; Marquis, R.W.; Bury, M.J.; Mehlmann, J.F.;
Singhaus, Jr., R.; Charnley, A.K.; Lakdawala, A.S.; Convery, M.A.;
Lipshutz, D.B.; Desai, B.; Swift, B.; Capriotti, C.A.; Berger, S.B.;
Mahajan, M.K.; Reilly, M.A.; Rivera, E.J.; Sun, H.H.; Nagilla, R.;
Beal, A.M.; Finger, J.N.; Cook, M.N.; King, B.W.; Ouellette, M.T.;
Totritis, R.D.; Pierdomenico, M.; Negroni, A.; Stronati, L.; Cucchiara,
S.; Ziólkowski, B.; Vossenkämper, A.; MacDonald, T.T.; Gough, P.J.;
Bertin, J.; Casillas, L.N. The identification and pharmacological
characterization of 6-(tert-butylsulfonyl)-N-(5-fluoro-1H-indazol-3-
yl)quinolin-4-amine (GSK583), a highly potent and selective inhibitor
of RIP2 kinase. J. Med. Chem. 2016, 59, 4867-4880.
For advancement in the Type I kinase chemotype, see Hart, A.C.;
Pitts, W.J.; Mastalerz, H.; Guo, J.; Brown, G.D. Preparation of
substituted dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-ones as RIPK3
inhibitors PCT Int. Appl. 2016, WO 2016100166.
30
Schroeder, G.M.; An, Y.; Cay, Z.-W.; Chen, X.-T.; Clark, C.;
Cornelius, L.A.M.; Dai, J.; Gullo-Brown, J.; Gupta, A.; Henley, B.;
Hunt, J.T.; Jeyaseelan, R.; Kamath, A.; Kim, K.; Lippy, J.; Lombardo,
L.J.; Manne, V.; Oppenheimer, S.; Sack, J.S.; Schmidt, R.J.; Shen, G.;
Stefanski, K.; Tokarski, J.S.; Trainor, G.L.; Wautlet, B.S.; Wei, D.;
Williams, D.K.; Zhang, Y.; Zhang, Y.; Fargnoli, J.; Borzilleri, R.M.
Discovery
of
N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-
15 http://lincs.hms.harvard.edu/db/datasets/20131/main
fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (BMS-777607), a selective and orally
efficacious inhibitor of the Met kinase superfamily. J. Med. Chem.
2009, 52, 1251-1254.
16
Davis, M.I.; Hunt, J.P.; Herrgard, S.; Ciceri, P.; Wodicka, L.M.;
Pallares, G.; Hocker, M.; Treiber, D.K.; Zarrinkar, P.P. Comprehensive
analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2011, 29,
1046-1051.
31
Kim, K.S.; Zhang, L.; Schmidt, R.; Cai, Z.-W.; Wei, D.; Williams,
17 Zhao, Z.; Wu, H.; Wang, L.; Liu, Y.; Knapp, S.; Liu, Q.; Gray, N.S.
Exploration of Type II binding mode: a privileged approach for kinase
inhibitor focused drug discovery? ACS Chem. Biol. 2014, 9, 1230-
1241.
D.K.; Lombardo, L.J.; Trainor, G.L.; Xie, D.; Zhang, Y.; An, Y.; Sack,
J.S.; Tokarski, J.S.; Darienzo, C.; Kamath, A.; Marathe, P.; Zhang, Y.;
Lippy, J.; Jeyaseelan, Sr., R.; Wautlet, B.; Henley, B.; Gullo-Brown, J.;
Manne, V.; Hunt, J.T.; Fargnoli, J.; Borzilleri, R.M. Discovery of
pyrrolopyridine-pyridone based inhibitors of Met kinase: synthesis, X-
ray crystallographic analysis, and biological activities. J. Med. Chem.
2008, 51, 5330-5341.
18 Fang, Z.; Grütter, C.; Rauh, D. Strategies for the selective regulation
of kinases with allosteric modulators: exploiting exclusive structural
features. ACS Chem. Biol. 2013, 8, 58-70.
19 Garuti, L.; Roberti, M.; Bottegoni, G. Non-ATP competitive protein
kinase inhibitors. Curr. Med. Chem. 2010, 17, 2804-2821.
20 Alton, G.R.; Lunney, E.A. Targeting the unactivated conformations
of protein kinases for small molecule drug discovery. Expert Opin.
Drug Discov. 2008, 3, 595-605.
32 Canning, P.; Ruan, Q.; Schwerd, T.; Hrdinka, M.; Maki, J.L.; Saleh,
D.; Suebsuwong, C.; Ray, S.; Brennan, P.E.; Cuny, G.D.; Uhlig, H.H.;
Gyrd-Hansen, M.; Degterev, A.; Bullock, A.N. Inflammatory signaling
by NOD-RIPK2 is inhibited by clinically relevant Type II kinase
inhibitors. Chem. Biol. 2015, 22, 1174-1184.
33 Goncharov, T.; Hedayati, S.; Mulvihill, M.M.; Izrael-Tomasevic, A.;
Zobel, K.; Jeet, S.; Gedoroba, A.V.; Eidenschenk, C.; deVoss, J.; Yu,
K.; Shar, A.S.; Kirkpatrick, D.S.; Fairbrother, W.J.; Deshayes, K.;
Vucic, D. Disruption of XIAP-RIPK2 association blocks NOD2-
mediated inflammatory signaling. Mol. Cell 2018, 69, 551-565.
21
Roskoski, Jr., R. Classification of small molecule protein kinase
inhibitors based upon the structures of their drug-enzyme complexes.
Pharmacol. Res. 2016, 103, 26-48 and references therein.
22
Palmieri, L.; Rastelli, G. -C helix displacement as a general
approach for allosteric modulation of protein kinases. Drug Discov.
Today 2013, 18, 407-414 and references therein.
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
For Table of Contents Use Only:
8
9
Identification of RIPK3 Type II in-hibitors using high-throughput mechanistic studies in hit triage.
Amy C. Hart*, Lynn Abell†, Junqing Guo, Michael E. Mertzman, Ramesh Padmanabha, John E. Macor†, Charu
Chaudhry, Hao Lu†, Kevin O’Malley, Patrick J. Shaw, Carolyn Weigelt, Matthew Pokross, Kevin Kish, Kyoung
S. Kim, Lyndon Cornelius, Andrew E. Douglas, Deepa Calambur, Ping Zhang, Brian Carpenter and William J.
Pitts
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment