Identification of a RIP1 Kinase Inhibitor Clinical Candidate (GSK3145095) for the Treatment of Pancreatic Cancer

Article abstract of DOI:10.1021/acsmedchemlett.9b00108

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been ad

Full text of DOI:10.1021/acsmedchemlett.9b00108

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Letter
Identification of a RIP1 Kinase Inhibitor Clinical Candidate
(GSK3145095) for the Treatment of Pancreatic Cancer
Philip A. Harris, Jill M. Marinis, John D Lich, Scott B. Berger, Anirudh Chirala, Julie A Cox, Patrick
M Eidam, Joshua N. Finger, Peter J. Gough, Jae U Jeong, James Kang, Viera Kasparcova,
Lara K Leister, Mukesh K. Mahajan, George Miller, Rakesh Nagilla, Michael T. Ouellette,
Michael A. Reilly, Alan R. Rendina, Elizabeth J. Rivera, Helen H. Sun, James H. Thorpe, Rachel
D Totoritis, Wei Wang, Dongling Wu, Daohua Zhang, John Bertin, and Robert W Marquis
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00108 • Publication Date (Web): 09 May 2019
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Identification of a RIP1 Kinase Inhibitor Clinical Candidate
(GSK3145095) for the Treatment of Pancreatic Cancer
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Philip A. Harris,*† Jill M. Marinis,† John D. Lich,† Scott B. Berger,† Anirudh Chirala,# Julie A.
Cox,‡ Patrick M. Eidam,† Joshua N. Finger,† Peter J. Gough,† Jae U. Jeong,† James Kang,† Viera
Kasparcova,† Lara K. Leister,† Mukesh K. Mahajan,† George Miller,#§ Rakesh Nagilla,† Michael T.
Ouellette,‡ Michael A. Reilly,† Alan R. Rendina,‡ Elizabeth J. Rivera,† Helen H. Sun,† James H.
‡
#
#
†
†
§
Thorpe, Rachel D. Totoritis, Wei Wang, Dongling Wu, Daohua Zhang, John Bertin and
Robert W. Marquis†
†Pattern Recognition Receptor DPU and ‡ Medicinal Science & Technology, GlaxoSmithKline, Collegeville Road,
Collegeville, Pennsylvania 19426, United States.
§ Medicinal Science & Technology, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
#S. Arthur Localio Laboratory, §Department of Cell Biology, New York University School of Medicine, 550 First Ave-
nue, New York, NY 10016
KEYWORDS RIP1, type III kinase inhibitors, pancreatic cancer
ABSTRACT: RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety
of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of
RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology
indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Com-
pound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-depend-
ent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor
suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical
studies for pancreatic adenocarcinoma and other selected solid tumors.
Receptor interacting proteins (RIP) 1 and 3 are known combination with PD-1 immunotherapy as a new option
to form a complex termed the necrosome that leads to a for the treatment of pancreatic cancer. We report in this
form of cell death called necroptosis and inflammatory Letter on the identification of a new clinical RIP1 inhibitor
cytokine production.1 Necroptosis can drive an inflamma- for pancreatic cancer and other solid tumors.
tory response through release of damage-associated mo-
The first reported RIP1 inhibitors discovered by Deg-
lecular patterns1 and the necrosome is also thought to terev et al., exemplified by indole-hydantoin 1 (see Figure
contribute to neuronal cell loss in neurodegenerative dis- 1), were shown by co-crystallized in the RIP1 kinase do-
eases.2 Several RIP1 inhibitors are now progressing in the main to occupy an allosteric lipophilic pocket at the back
clinic for the treatment of inflammatory diseases, such as of the ATP binding site.6,7 This type III binding mode, re-
psoriasis, rheumatoid arthritis and ulcerative colitis,3 as sulted in an excellent kinase selectivity profile.8 Our
well for CNS indications such as ALS and Alzheimer's dis- screening efforts against DNA-encoded libraries led to
ease.2 Additional functions of the RIP1 continue to be dis- the identification of a benzoxazepinone RIP1 inhibitor 2
covered. Recently Seifert and Miller have reported that (GSK’481), whose RIP1 co-crystal structure showed it oc-
the necrosome can also promote an immune suppression cupied the same allosteric binding pocket as 1.9 Subse-
in pancreatic tumors.4 Additionally, Miller’s lab, in collab- quent lead-optimization resulted in selection of develop-
oration with our group, have shown that RIP1 kinase in- ment candidate 3 (GSK2982772), which is currently under
hibition resulted in T cell differentiation towards a tumor evaluation in phase 2a clinical trials in psoriasis, rheuma-
suppressive phenotype leading to tumor-immunity in toid arthritis and ulcerative colitis patients.10 Recently,
mice and in models of human pancreatic cancer.5 Im- Yoshikawa et al. have disclosed a benzoxazepinone ana-
portantly, RIP1 kinase inhibition sensitized tumors to log 4 with a higher brain penetration and efficacy in a
checkpoint blockade, offering the potential for
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mouse experimental autoimmune encephalomyelitis assay and a human monocytic U937 cellular assay, but
(EAE) model of multiple sclerosis.11
suffered from high lipophilicity and suboptimal pharmaco-
kinetic (PK) profile (Table 1).9 The corresponding NH lac-
tam analog 7 possessed a better rat PK profile but at the
cost of lower RIP1 potency. This was a general trend ob-
served in this series: methylation of the amide increased
RIP1 potency by up to 10-fold, as the methyl group occu-
pies a small pocket on the face of the -sheet defined by
Leu90-Met92 and Ile43-Lys45.10 However, metabolic de-
methylation of the N-methyl lactam can increase turno-
ver, thus NH lactams tended to have better in vivo expo-
sures. Replacement of the isoxazole of both 2 and 7 with
a triazole (analogs 3 and 8) maintained comparable RIP1
potencies with significantly reduced lipophilicity and im-
proved rat oral exposures. Replacement of the benzoxa-
zepinone heterocycle for benzazepinone 9 and 10 showed
comparable in vitro potencies. Finally, introduction of
7,9-difluro substitution at the benzazepinone yielded an-
alogs 11 and 6. The increase in potency of 6 compared to
10 could be attributed to the two electron withdrawing
fluorines decreasing the pKa of the lactam nitrogen, thus
strengthening its H-bond to the backbone of Leu90 (Fig-
ure 2). The NH-lactam of 6 predictably increased rat ex-
posure compared to NMe-lactam 11. The optimal potency
and rat oral PK profile of benzazepinone 6 led to further
pre-clinical evaluation as detailed below.
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Figure 1. Structure of RIP1 kinase inhibitors.
We recently disclosed the efficacy of RIP1 inhibitor 5
(known as GSK’547) in an oncology mouse model of pan-
creatic cancer in both reducing tumor size and activating
an immune cell response.5 Co-treatment of 5 with either
a PD-1 antibody, to block immune down-regulation, or an
ICOS antibody, to stimulate immune signalling, further
reduced tumor size. Although 5 was an excellent tool for
exploring RIP1 inhibition in mouse oncology models, it
did not possess the required preclinical profile to support
development as a clinical lead, most notable suffering
from high turnover in human hepatocytes. Herein we de-
scribe the identification of a new RIP1 inhibitor clinical
candidate (S)-5-benzyl-N-(7,9-difluoro-2-oxo-2,3,4,5-tet-
rahydro-1H-benzo[b]azepin-3-yl)-1H-1,2,4-triazole-3-car-
boxamide 6 (GSK3145095), structurally related to benzox-
azepinone 3. Although 3 is in clinical development for
chronic immunoinflammatory disorders, development of
a separate RIP1 inhibitor for oncology indications with
their distinct safety tolerance and dosing criteria was re-
quired. The high RIP1 potency, mono-kinase selectivity
and excellent preclinical pharmacokinetic and developa-
bility profile of benzazepinone 6 led to its selection for
clinical development and it recently initiated phase 1 eval-
uation in pancreatic cancer patients.
Table 1. SAR summary of key compounds
c
Cpd ADP-Gloa
U937a
CHI
Rat AUC0-∞
μg.h/mL
IC50 (nM) IC50 (nM) log Db
2
7
1.6
32
7.9
200
6.3
100
10
5.9
5.1
0.38
2.2
2.3
3
1.0
32
3.8
3.0
3.8
3.1
We have recently detailed the lead optimization of the
DNA-encoded library derived hit benzoxazepinone 2 that
led to identification of 3, the first RIP1 inhibitor to enter
clinical evaluation.10 Since the SAR observations identi-
fied during the discovery of benzoxazepinone 3 also apply
to benzazepinone 6 only a brief synopsis of some of the
key modifications is warranted here. As a benchmark,
8
2.5
9
10
11
6
5.0
50
0.31
nd
63
6.3
6.3
6.3
10
4.3
3.4
0.16
1.1
benzoxazepinone 2 possessed excellent RIP1 in vitro po- aAssay protocols are described in Supporting Information; IC50
values are the average of at least two determinations. bCHI
tency, as measured by both an ADP-Glo biochemical
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(chromatographic hydrophobicity index) log D at pH 7.4 was
would reside, making them mutually exclusive, and ATP
competitive inhibition is indeed observed (see Support-
ing Information). The binding kinetics are similar to ben-
zoxazepinones recently profiled, with a moderate on-rate
constant (kon = 2.5 x 104 M-1s-1), accompanied by a slow off-
rate constant koff = 1.2 x 10-4 s-1 (t1/2 = 99 min), measured by
stopped-flow kinetics and fluorescence polarization com-
petitive binding, respectively (see Supporting Infor-
mation). In addition to efficacy against the immortalized
U937 human monocyte cell line, activity of 6 was also as-
sessed in primary neutrophils isolated from human whole
blood. As previously described, in this assay TNF is co-
incubated with both the caspase inhibitor QVD-Oph and
the SMAC mimetic RMT 5265, which block the apoptosis
and NF-κB pathways, respectively, driving the TNF re-
sponse down the necrosis pathway.10 Inhibitor 6 was able
to potently block this response as shown by determina-
tion of overall cell viability as measured by cellular ATP
levels (IC50 = 1.6 nM), cell death as measured by LDH re-
lease (IC50 = 0.5 nM) and RIP1-dependent inflammatory
cytokine MIP-1 production, either as absolute levels for
protein, or fold changes in mRNA expression (IC50 = 0.4
nM).
A human whole blood stimulation assay was previously
developed in which the necroptosis pathway is activated
through stimulation with TNF co-incubated with the
caspase inhibitor QVD-Oph or zVAD.fmk, and the SMAC
mimetic RMT 5265.10 In this assay benzazepinone 6 was
also shown to be very potent as measured by inhibition of
cytokine MIP-1β ((IC50 = 5 nM). In a similar monkey whole
blood stimulation assay, benzazepinone 6 exhibited an
IC50 of 16 nM.
A distinctive feature of RIP1 type III inhibitors is a sig-
nificant reduction in potency against non-primate RIP1.9
Compound 6 is over 380-fold less potent against non-pri-
mate RIP1 compared to primate RIP1 in biochemical as-
says (see Supporting Information) and shows a 340-fold
reduction in cellular potency for blockage of necrotic
death in mouse fibrosarcoma L929 cells (IC50 = 1.3 M)
compared to human U937 cells (IC50 = 6.3 nM). This re-
duced RIP1 mouse potency precludes evaluation of 6 in
rodent oncology models. However, 6 was evaluated in hu-
man ex vivo tumor cultures. For these studies, patient de-
rived organotypic spheroids (PDOTS) were prepared
from freshly resected tumors from pancreatic adenocar-
cinoma, colorectal, breast and gastric cancer patients, as
previously described.12 PDOTS were treated with 6 or ve-
hicle and following 3 days incubation, they were har-
vested and analysed by flow cytometry (see Figure 3).
PDOTS treated with 6 developed significantly more effec-
tor-memory T cells (CD44+) and immunogenic CD4+ T
cells (IFN+) compared to vehicle treated samples. A
trend toward increase CD8+ cytolytic T cells and TNF
expression was also observed (see Supporting
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5
6
7
8
calculated from the retention time (tR) observed in a fast gradi-
ent reverse-phase HPLC. cRat oral exposure at 2 mg/kg.
Co-crystallization of benzazepinone 6 was achieved
with the RIP1 construct (1–294, C34A, C127A, C233A, and
C240A). The resulting X-ray structure was refined to res-
olution of 2.87Å (see Supporting Information). In this
structure, benzazepinone 6 was observed to be buried
deep in the pocket between the N-terminal and C-termi-
nal domains with no interaction with the hinge residues,
making this a type III class of kinase inhibitor (see Figure
2). The triazole and benzyl functionality of 6 occupies the
same allosteric lipophilic pocket at the back of the ATP
binding site as Necrostatin-4. The benzoxazepine ring oc-
cupies space where the α-phosphate of ATP would reside,
so the inhibition is not strictly allosteric. The amide car-
bonyl attached to the triazole makes a direct hydrogen
bond interaction with the backbone amide NH of Asp156.
The triazole nitrogen makes a water mediated hydrogen
bond to the carbonyl oxygen of Met67. The lactam nitro-
gen shows a possible long hydrogen bond interaction to
the backbone carbonyl of Leu90 and the fluorine at the 9-
position of the benzazepinone occupies a pocket between
Met92 and Ile43. Details on co-crystallography are avail-
able in the Supporting Information section.
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Figure 2. Co-crystal structure of RIP1 (1–294, C34A, C127A,
C233A, and C240A) and benzazepinone 6.
The type III binding mode observed for this pharmaco-
phore in RIP1 results in complete kinase selectivity for
RIP1 as exemplified by the kinase profile of 6 against both
a P33 radiolabeled assay screen at Reaction Biology Corp
(359 kinases) and a competition binding assay KI-
NOMEscan at DiscoveRx Corp (456 kinases). In both as-
says benzazepinone 6 was tested at a concentration of 10
M and showed no inhibition of any kinase other than
RIP1, representing a >1500-fold selectivity window based
on the RIP1 ADP-Glo potency of 6.3 nM. Details are avail-
able in the Supporting Information section.
Although benzazepinone 6 is binding in an allosteric
pocket at the back of the ATP binding site, it is also par-
tially occupying the region where the α-phosphate of ATP
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Information); however, those markers failed to reach sta- kidney, and comparable concentrations (0.7-3 fold) in co-
tistical significance in this study. Patients with increased lon, heart and skin. In contrast, 6 had low brain penetra-
T-cell infiltration and activation have a better prognosis tion (6%) despite possessing moderate cell permeability
with longer survival and response to immunotherapy (6.7 x 10-6 cm/s), which is likely due to active extrusion
agents.13 This agrees with earlier results in the orthotopic from brain via the efflux drug transporter P-glycoprotein
mouse model of pancreatic cancer where RIP1 inhibition (P-gp) (see Supporting Information).
leads to an immunogenic tumor microenvironment.5
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Figure 3. Individual PDOTS were prepared from resected pa-
tient tumors and incubated for 3 days with vehicle or com-
pound 6 (0.5 nM in culture medium). Cells were labeled with
antibodies to detect CD3 (pan-T cells), CD4 (T helper cells),
CD44 (effector-memory cells), and IFN (marker of T cell ac-
tivation) then analyzed by flow cytometry. The percentage of
cells expressing the indicated marker in vehicle treated sam-
ples was set to 1 and the fold change in the presence of com-
pound 6 determined.
Figure 4. Proposed metabolic pathways of compound 6 in rat
(r), monkey (m) and human (h) hepatocytes.
Benzazepinone 6 displayed a good free fraction in
blood of rat (13%), dog (12%), monkey (12%) and human
(8.1%). In vitro evaluation of the metabolic stability of 6
in hepatocytes indicated moderate turnover in the rat and
low turnover in monkey and human (see Supporting In-
formation). A metabolite identification study in rat, mon-
key and human hepatocytes highlighted that 6 was me-
tabolized via both phase I and phase II biotransformation
pathways, as shown in Figure 4. The primary metabolic
pathways for 6 included hydroxylation and glucuronida-
tion. No human specific metabolites of 6 were detected in
these hepatocyte incubations, but trace levels of GSH
conjugates were detected only in rat and monkey, not in
human hepatocyte incubations. A pharmacokinetic eval-
uation of 6 in rat, dog and monkey demonstrated a low
clearance (≤ 35% liver blood flow) in all species (see Table
2). The volume of distribution was moderate along with a
relatively short to moderate terminal half-life. Good oral
bioavailability was observed across these preclinical spe-
cies. The tissue distribution of 6 in rat was evaluated fol-
lowing iv infusion over 4 hours (see Supporting Infor-
mation). This study showed that compared to blood 6 dis-
tributed at greater concentrations (7-8 fold) in liver and
Table 2. Pharmacokinetic Parameters of 6 Follow-
ing Administration to Rat, Dog and Monkey
Route
iva
Parameter
Rat
1.0
Dog
1.1
Monkey
0.93
dose (mg/kg)
Cl (mL/min/kg)
(%LBF)
27 ± 5
(35)
9.8 ± 1.8
(18)
6.4 ± 0.5
(15)
Vdss (L/kg)
1.8 ± 0.3
2.2 ± 0.8
2.1
1.1 ± 0.2
1.7 ± 0.02
2.0
1.8 ± 0.1
4.2 ± 0.6
1.9
t1/2 (h)
pob
dose (mg/kg)
Tmax (h)
0.83 ± 0.29 0.75 ± 0.43
1.5 ± 0.0
770 ± 99
4.4 ± 0.5
88 ± 13
Cmax (μg/mL)
AUC (μg·h/mL)
bioavailability (%)
320 ± 79
1.1 ± 0.2
84 ± 8
910 ± 130
2.5 ± 0.49
78 ± 3
aThe vehicle for iv studies was 20% Cavitron, 5% DMSO in PBS.
bThe vehicle for po studies was 6% Cavitron, 5% DMSO in PBS.
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ASSOCIATED CONTENT
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Allometric scaling and in vitro to in vivo extrapolations
Supporting Information
were used to generate predictions of human PK parame-
The Supporting Information is available free of charge
ters. These showed a high level of correlation and pre- on the ACS Publications website as a PDF. This contains
dicted 6 to have high bioavailability, low clearance, mod- details on preparation of compounds 2, 3, 6-11, in vitro as-
erate volume and a terminal half-life in the order of 3.3 says and RIP1 (1-375) preparation. The following data spe-
hours. The average clearance and volume values were cific to compound 6 is included: mode of inhibition, en-
then used to reconstruct a mono-exponential predicted zyme kinetics, ortholog inhibition profile, kinase selectiv-
human blood concentration time profile, as shown in Fig- ity profile, RIP1 co-crystallization, rat tissue distribution,
ure 5, for once and twice-daily dosing. Doses were scaled permeability and P-gp substrate evaluation, hepatocyte
and modelled to target maintaining concentrations above turnover and human PK/PD predictions and additional
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the human whole blood IC90 over 24 hours. Inhibitor 6 is
predicted to require doses of either 273 daily or 40 mg
twice daily to maintain 90% inhibition, with an AUC of
11.7 or 1.7 μg·h/mL, respectively. The predicted human
PK/PD was modelled using this predicted human PK pro-
file at these doses, along with human whole blood activ-
ity, maintaining 90% RIP1 inhibition levels over 24 hours,
as shown in the Supporting Information.
PDOTS evaluation for CD8+ T cells and TNF expression.
Accession Codes
Coordinates and structure factors for the cocrystal
structure of RIP1 (1−294, C34A, C127A, C233A, C240A) and
benzoxazepinone 6 have been deposited in the Protein
Data Bank with the accession number 6RLN.
AUTHOR INFORMATION
Corresponding Author
* E-mail: phaharris100@gmail.com.
Notes
The authors declare the following competing financial
interests: All authors, with the exception of George Mil-
ler, Wei Wang, Anirudh Chirala and Dongling Wu, are
current employees and stockholders of GlaxoSmithKline.
The human biological samples were sourced ethically,
and their research use was in accord with the terms of
the informed consents under an IRB/EC approved proto-
col.
All animal studies were conducted in accordance with
the GSK Policy on the Care, Welfare and Treatment of
Laboratory Animals and were reviewed the Institutional
Animal Care and Use Committee either at GSK or by the
ethical review process at the institution where the work
was performed.
Figure 5. Predicted once and twice daily human blood con-
centration time profile of benzazepinone 6 overlaid with the
human whole blood inhibition IC90 concentrations.
SUMMARY
The emerging biological understanding of the multiple
roles of RIP1 kinase has opened up a number of exciting
opportunities to investigate this target for drug interven-
tion in a number of diseases. Our recent findings that RIP1
signaling in macrophages regulates immune tolerance in
pancreatic cancer and confers resistance to immunother-
apy offers a strong rationale that RIP1 kinase inhibition
could help address this most significant unmet need in
oncology. Benzazepinone 6 was optimized from a DNA-
encoded library screen with both high in vitro RIP1 po-
tency and complete kinase selectivity. It has recently ini-
tiated phase 1 evaluation in pancreatic cancer patients,
the results of which will be reported in future publica-
tions.
ABBREVIATIONS USED
LDH, lactate dehydrogenase; MIP, macrophage inflam-
matory protein; PDOTS, patient derived organotypic
spheroids; RIP, receptor interacting protein; QVD-OPh,
(3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-1-oxo-2-
[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxopenta-
noic acid hydrate; SMAC, second mitochondrial-derived
activator of caspases; TNF, tumor necrosis factor;
zVAD.fmk, carbobenzoxy-valyl-alanyl-aspartyl-[O-me-
thyl]- fluoromethylketone.
AKNOWLEDGEMENTS
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(10)Harris, P. A.; Berger, S. B.; Jeong, J. U.; Nagilla, R.;
The authors are indebted to Nicolas Faucher, Nicolas
George, Frederic Donche and Alain Daugan from the for-
mer GSK group at Les Ulis, France for the design of the in
vivo tool inhibitor 5 (GSK’547).
1
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5
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Bandyopadhyay, D.; Campobasso, N.; Capriotti, C. A.; Cox,
J. A.; Dare, L.; Dong, X.; Eidam, P. M.; Finger, J. N.; Hoff-
man, S. J.; Kang, J.; Kasparcova, V.; King, B. W.; Lehr, R.;
Lan, Y.; Leister, L. K.; Lich, J. D.; MacDonald, T. T.; Miller,
N. A.; Ouellette, M. T.; Pao, C. S.; Rahman, A.; Reilly, M.
A.; Rendina, A. R.; Rivera, E. J.; Schaeffer, M. C.; Sehon, C.
A.; Singhaus, R. R.; Sun, H. H.; Swift, B. A.; Totoritis, R. D.;
Vossenkämper, A.; Ward, P.; Wisnoski, D. D.; Zhang, D.;
Marquis, R. W.; Gough P. J; Bertin, J. Discovery of a First-
in-Class Receptor Interacting Protein 1 (RIP1) Kinase Spe-
cific Clinical Candidate (GSK2982772) for the Treatment of
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