Computationally Empowered Workflow Identifies Novel Covalent Allosteric Binders for KRASG12C

Article abstract of DOI:10.1002/cmdc.201900727

Due to its frequent mutations in multiple lethal cancers, KRAS is one of the most-studied anticancer targets nowadays. Since the discovery of the druggable allosteric binding site containing a G12C mutation, KRAS^G12C has been the focus of attention in oncology research. We report here a computationally driven approach aimed at identifying novel and selective KRAS^G12C covalent inhibitors. The workflow involved initial enumeration of virtual molecules tailored for the KRAS allosteric binding site. Tools such as pharmacophore modeling, docking, and free-energy perturbations were deployed to prioritize the compounds with the best profiles. The synthesized naphthyridinone scaffold showed the ability to react with G12C and inhibit KRAS^G12C. Analogues were prepared to establish structure-activity relationships, while molecular dynamics simulations and crystallization of the inhibitor-KRAS^G12C complex highlighted an unprecedented binding mode.

Full text of DOI:10.1002/cmdc.201900727

Communications
doi.org/10.1002/cmdc.201900727
ChemMedChem
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Computationally Empowered Workflow Identifies Novel
G12C
Covalent Allosteric Binders for KRAS
[a]
[a]
[a]
[a]
[a]
Jérémie Mortier,* Anders Friberg, Volker Badock, Dieter Moosmayer, Jens Schroeder,
[a]
[a]
[a]
[a]
[a]
Patrick Steigemann, Franziska Siegel, Stefan Gradl, Marcus Bauser, Roman C. Hillig,
Hans Briem, Knut Eis, Benjamin Bader, Duy Nguyen,* and Clara D. Christ*
[a]
[a]
[a]
[a]
[a]
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Due to its frequent mutations in multiple lethal cancers, KRAS is
one of the most-studied anticancer targets nowadays. Since the
found predominantly in a mutated isoform (45%), whereas
NRAS mutations are infrequent (in 7.5% of CRC) and HRAS
[4]
discovery of the druggable allosteric binding site containing a
mutations have not been detected. RAS proteins act as
molecular switches alternating between an active, GTP-bound
state and an inactive, GDP-bound state. Activated by guanine
nucleotide exchange factors (GEFs), RAS in its GTP-bound state
G12C
G12C mutation, KRAS
has been the focus of attention in
oncology research. We report here a computationally driven
G12C
approach aimed at identifying novel and selective KRAS
[5]
covalent inhibitors. The workflow involved initial enumeration
of virtual molecules tailored for the KRAS allosteric binding site.
Tools such as pharmacophore modeling, docking, and free-
energy perturbations were deployed to prioritize the com-
pounds with the best profiles. The synthesized naphthyridinone
scaffold showed the ability to react with G12C and inhibit
interacts with a number of effectors. The return to the inactive
state is driven by GTPase-activating proteins (GAPs), which
down-regulate active RAS by accelerating the weak intrinsic
[
6]
GTPase activity by up to five orders of magnitude. For
oncogenic RAS mutants, however, the GAP activity is impaired
or greatly reduced, resulting in permanent activation, which is
G12C
[7]
KRAS . Analogues were prepared to establish structure-
the basis of oncogenic RAS signaling.
activity relationships, while molecular dynamics simulations and
Mutation G12C in KRAS was recently identified to be
potentially druggable by allele-specific covalent inhibitors
targeting the Cys12 side chain in vicinity to an inducible
allosteric pocket, called the switch-II pocket (also known as
G12C
crystallization of the inhibitor-KRAS
unprecedented binding mode.
complex highlighted an
[8]
pocket 2). Occupation of this pocket with a covalently bound
inhibitor results in a protein locked in an inactive GDP-bound
state. Locked in this conformation, the mutated KRAS cannot
return to an active GTP-bound state and activity of the G12C
mutant is thereby shut down. Starting from Shokat’s ground-
[
1]
First identified in Kirsten rat sarcoma (KRAS) virus, mutations
[2]
in the KRAS gene have widespread prevalence in cancers. In
982, abnormally activated RAS genes were found in human
cancers, marking the first discovery of mutated genes in this
disease. The frequent mutation of RAS in three of the four
most lethal cancers (lung, colon, and pancreatic cancers) in the
United States has spurred intense interest and effort in
developing RAS inhibitors. Overall, RAS mutations have been
detected in 9–30% of all tumor samples sequenced, with the
specific RAS isoform generally differing according to cancer
type. In pancreatic ductal adenocarcinoma and lung adenocar-
cinoma, there is a KRAS mutation frequency of 98% and 31%,
respectively. In colon and rectal carcinoma (CRC), KRAS is also
1
G12C
[8a]
breaking work on covalent KRAS
inhibitors, Araxes pro-
[3]
gressed with various lead series, disclosing their strategy for the
G12C
optimization of covalent KRAS
inhibitors. Compound ARS-
[9]
853 can be regarded as an in vitro tool compound, whereas
quinazoline derivative ARS-1620 has been used as an in vivo
chemical probe to investigate KRAS
[4]
G12C
[8b]
biology.
The first
[
10]
Araxes patent containing ARS-1620 was published in 2015,
and all inhibitors reported since then consist of minor structural
variations of ARS-1620 (Figure 1A). To the best of our knowl-
edge, three companies have announced clinical trials so far: i)
[11]
Amgen with AMG-510 in 2018, ii) Mirati Therapeutics with
[12]
[13]
MRTX849 in 2019, and iii) Araxes Pharma with ARS-3248.
[
a] Dr. J. Mortier, Dr. A. Friberg, Dr. V. Badock, Dr. D. Moosmayer,
Dr. J. Schroeder, Dr. P. Steigemann, Dr. F. Siegel, Dr. S. Gradl, Dr. M. Bauser,
Dr. R. C. Hillig, Dr. H. Briem, Dr. K. Eis, Dr. B. Bader, Dr. D. Nguyen,
Dr. C. D. Christ
We report here a computationally driven methodology
developed to identify novel chemical matter able to modulate
G12C
KRAS
activity through allosteric binding. With a view to
Bayer AG
Research & Development, Pharmaceuticals
Müllerstrasse 178, 13342 Berlin (Germany)
E-mail: jeremie.mortier@bayer.com
generate chemical novelty while conserving the binding mode
and potency of ARS-1620, we deconstructed the molecule into
four fragments: i) the acryloyl warhead, ii) the bridge piperazine,
iii) the quinazoline core, and iv) the fluorophenol head group
(Figure 1B). A computer-aided scaffold hopping workflow was
developed for the core fragment and the bridge, while the
reactive acrylamide warhead and the fluorophenol head group
were conserved (see the Supporting Information). The gener-
duy.nguyen@bayer.com
clara.christ@bayer.com
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.201900727
©
2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
6
ated library includes almost 7×10 compounds consisting of all
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G12C
Figure 1. A) Inhibitors known to bind to the switch-II pocket of KRAS . B) Fragmentation of the inhibitor structure before enumeration, including a depiction
of the nature and size of the used fragment libraries. C) Enumeration and prioritization workflow.
possible building block combinations. None of these 7 million
bridge fragment was evaluated by visual inspection and
compounds substituted at a position with poor electrophilicity
were filtered out. Then, to allow for a rapid synthesis of the de
novo designed compounds, the commercial availability of the
required building blocks was evaluated. Eventually, a set of
132 compounds with tractable synthetic chemistry was priori-
tized.
[
14]
was found in the ChEMBL database, thus indicating the high
novelty of the generated chemical matter. Five exact matches
[15]
[10]
were found in SureChEMBL, all from the Araxes patent.
Although the ARS compound series had been patented
[10]
when our project was initiated, a binding mode had not been
reported. Hence, one compound later confirmed as ARS-1620
G12C
was modeled into the switch-II pocket of KRAS
(PDB entry
At this stage of the project, ARS-1620 had been successfully
[8a]
G12C
4
LV6)
using Cys12 as an anchor point, and then using
synthesized and co-crystallized with KRAS
in-house, confirm-
molecular dynamics (MD) for refinement. The resulting trajecto-
ries allowed for the identification of a favored binding mode
from which key interactions were extracted and compiled in
one pharmacophore model (Figure S1 in the Supporting
Information). Using Phase, the 7×10 compound library was
screened and, from the compounds that matched all pharmaco-
phore features, the 10 with the best alignment were retained
Figure 1C). In subsequent covalent docking, 10 compounds
were prioritized using MM/GBSA scoring, which balances
ing the binding mode hypothesis previously used to generate
the pharmacophore model (Figure S1). This allowed us to
progress with the previously prioritized 132 compounds, and
binding affinity estimates were calculated using free-energy
perturbations (FEP), a computationally expensive method that
takes into account protein flexibility. Four compounds with
calculated relative ΔG in the range to this of ARS-1620 were
prioritized.
[16]
6
[18]
5
4
(
One of the most synthetically accessible compounds, 1,6-
naphthyridin-5(6H)-one (1), was prepared (Table 1; Scheme S1).
Structurally close to 1, isoquinolin-1(2H)-one 2 derivatives were
also considered for the exploration of structure-activity relation-
[17]
computational efficiency and accuracy. To discard structures
with low synthetic accessibility, the nucleophilicity of the
position on the core aromatic fragment covalently bound to the
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G12C
Table 1. Compound structures, KRAS
binding measured by mass spectrometry (MS), and biochemical IC50 measurements.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
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0
1
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2
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7
1
2
3
G12C
WT
Cmpd
R
R
R
W
C
X
Y
Z
MS assay
% binding]
IC50 KRAS
[μM]
IC50 KRAS [μM]
[
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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4
4
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5
5
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5
5
5
5
1
H
H
CH
N
80
2.8
>20
2
3
4
H
H
H
H
H
H
C
C
C
CH
CH
CH
CH
CH
CH
35
34
0
4.7
>20
>20
>20
5.0
>20
5
6
H
H
H
H
C
C
CH
CH
CH
CH
60
20
8.0
>20
>20
>16
7
Cl
H
H
H
H
C
C
C
C
CH
CH
CH
CH
CH
CH
CH
CH
51
0
14.6
>20
>20
>20
>20
>20
>20
>20
8
H
9
H
0
10
CH3
11
1
1
H
H
H
H
C
C
N
N
CH
CH
CH
59
0
4.90
>20
1.3
>20
>20
>20
12
13
OH
–
CH
CH
73
ships (SAR). To measure the proportion of inhibitor reacting
with Cys12 of the KRAS
ratio was quantified by mass spectrometry (MS). Then, the
inhibition potency of these compounds towards KRAS
evaluated in biochemical assays measuring the activation of
G12C
G12C
WT
[19]
mutant, the bound/unbound protein
GDP-bound KRAS
or KRAS by SOS1.
Naphthyridinone 1 decorated with a fluorophenol head
group and bridged to an acrylamide warhead through a
pyrrolidine emerged as a promising novel KRAS
G12C
was
G12C
inhibitor
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with a bound fraction of 80% and IC value of 2.8 μM, while no
inhibition of KRAS was observed up to a concentration of
5
0
Table 2. Measurements of binding constant of the reversible binding
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
WT
G12C
event (K), maximum potential rate of KRAS
I
inactivation (kinact), and
cellular activity.
2
0 μM (Table 1). Quinazolinone 11 and isoquinolinone 2 (with
G12C
one nitrogen atom less than 1) displayed a KRAS -bound
fraction of 59% and 35%, respectively. Their measured KRAS
Cmpd
K
I
[μM]
kinact [1/s]
kinact/K
I
[1/(M*s)]
Cellular IC50 [μM]
G12C
1
2
1
39.3
4.4
32.2
0.00105
0.00024
0.00159
26.72
55.05
49.38
>30
>30
22
IC50 values were below 5 μM (4.9 μM and 4.7 μM, respectively).
A series of boronic acids were coupled to the corresponding
bromo-substituted isoquinolinone to yield four analogues 3–6
3
(Table 1, also see the Supporting Information). While a complete
loss of activity was observed when the fluorophenol moiety
was replaced by a quinolinyl (6) or a difluorophenyl (4) group,
conserving the ortho-fluorine and replacing the hydroxy group
The MS covalent binding assay revealed that 12 is unable to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
G12C
react with Cys12 of KRAS
(Table 1); this, indicates that the
hydroxy group flanking the isoquinolinone core might be
inadequate for optimal binding. However, inversing the intra-
molecular hydrogen bond allows for the fluorophenol moiety
to be conserved: 2,6-naphthyridinone 13 reacts with Cys12 of
by a para-fluoro substituent (3) or an ortho-ethyl group (5)
G12C
allows for the inhibitor to modulate KRAS
activity in the low
micromolar range (5.0 μM and 8.0 μM, respectively). Also,
alternatives to the pyrrolidine bridge were investigated, with
the 4- and 3-piperidinyl derivatives (8 and 9, respectively) both
being inactive. The isoquinolinone variant 10 decorated with a
methyl on the core nitrogen shows a similar inability to bind
G12C
KRAS , with a bound fraction of 73%, and shows a slightly
improved IC50 value (1.3 μM) compared to the analogue with no
intramolecular hydrogen bond (2, IC =4.7 μM). The binding
5
0
constant of the reversible binding event (K) and the maximum
I
G12C
KRAS , highlighting the importance of the hydrogen-bond
potential rate of inactivation (kinact) were measured for com-
[
20]
donor at this position.
pounds 1, 2 and 13. These results indicate a weak kinact
,
and a
Crystallization trials were initiated and a crystal structure of
kinact/K ratio in the same range for the three compounds
I
G12C
3
in complex with KRAS /GDP was determined at high
(Table 2). Finally, cellular activity was detected for compound
G12C
resolution (2.0 Å). As suggested by MD (see the Supporting
Information), the covalent inhibitor does exhibit a different
binding mode compared to ARS-1620. The most obvious
difference is the rotation of the isoquinolinone core, which
allows direct hydrogen bonding between the ligand and the
backbone atoms of Gly10. In contrast, the carbonyl oxygen of
the covalent warhead is coordinated by Lys16 in a very similar
fashion as the respective moiety in ARS-1620. Also, the terminal
difluorophenyl head group is essentially in the same subpocket
and orientation as that of the fluorophenol in ARS-1620.
Consequently, the isoquinolinone core and the head group are
closer to a coplanar orientation (146°), unlike ARS-1620 (70°), as
illustrated in Figure 2A–F. In a retrospective analysis of the
trajectories resulting from the FEP calculations, a similar rotation
of the core fragment was detected for 1, explaining why this
scaffold was selected in the initial screening campaign (Fig-
ure S3). This result mirrors the SAR, demonstrating a novel
mode of binding for this series.
13 (IC =22 μM) and the crystal structure of KRAS
in
5
0
complex with 13 confirmed its binding mode (Figure 2H). These
results provide the foundation for further optimization, aiming
for sub-micromolar cellular activity.
Interestingly, free-binding-energy calculations using
[21]
FEP+
could not accurately anticipate the effect of this
intramolecular hydrogen bond. The relative ΔG of 12 and 13
was greatly overestimated, predicting an improvement of about
100-fold in binding affinity compared to 2. On the other hand,
the involvement of protein dynamics and free energy calcu-
lations in our workflow was key to the identification of a
scaffold with a binding mode unprecedented since the
G12C
discovery of the KRAS
allosteric pocket. With the presented
computer-aided approach coupled with a stepwise experimen-
tal validation, we have reported here the design of a novel
G12C
chemical series binding to KRAS
with high potential for the
development of pioneering KRAS-targeted anti-cancer treat-
ments.
In the isoquinolinone series, quantum chemical calculations
suggested that adding decorating groups at the ortho positions
of the phenyl moiety energetically disfavors a coplanar Acknowledgements
orientation (Figure 2G). Similarly, adding a chloro substituent to
the isoquinolinone core (7) was suggested to have a negative
effect on the binding affinity. However, introducing one nitro-
gen atom at the same position of the core scaffold should allow
for the formation of an intramolecular hydrogen bond with the
fluorophenol head group. The energy landscape of the dihedral
between the 2,6-naphthyridin-1(2H)-one core (13) and the
fluorophenol head group indicates that an energy minimum is
reached at a dihedral angle between 150° and 210° (Figure 2G),
We thank Anja Wegg, André Hilpmann, Christina Gomez, and
Vivian Bell for technical support, the staff at the Helmholtz-
Zentrum Berlin and DESY (Hamburg, Germany), a member of the
Helmholtz Association HGF, for access to synchrotron radiation
and support during data collection, and moloX GmbH for data
collection services. We thank Robert Abel, Hege Beard, Daniel
Cappel, Joseph Goose, Thomas Steinbrecher and Lingle Wang for
technical support and helpful discussions. We thank also Dr. K.
Greenfield for proofreading and editing this manuscript.
the range of the active conformation. As these results highlight
the potential of stabilizing a coplanar orientation with an
intramolecular hydrogen bond, 12 and 13 were synthesized
and tested.
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G12C
Figure 2. Co-crystal structures of KRAS
with compound 3 (A, PDB entry: 6TAM) and ARS-1620 (C), and an overlay of both ligands (B). Extracting these
particular conformations, the head group of compound 3 (D) is compared to that of ARS-1620 (F), with a superimposition of both (E); in the case of 3, the
isoquinolinone core rotates, replacing the water molecule observed in the co-crystal structure with ARS-1620 and allowing a direct interaction with Gly10. G)
Comparison of the energy landscape of the dihedral of fragments from compounds 2, 13, and a virtual analogue with Cl and F atoms flanking the
isoquinolinone core (* indicates the position of the compound fragmentation for the QM study). H) Co-crystal structure of KRAS
entry: 6TAN).
G12C
with compound 13 (PDB
[
6] K. Scheffzek, M. R. Ahmadian, W. Kabsch, L. Wiesmuller, A. Lautwein, F.
Schmitz, A. Wittinghofer, Science 1997, 277, 333–338.
Conflict of Interest
[
[
7] K. M. Haigis, Trends Cancer 2017, 3, 686–697.
8] a) J. M. Ostrem, U. Peters, M. L. Sos, J. A. Wells, K. M. Shokat, Nature
2013, 503, 548–551; b) M. R. Janes, J. Zhang, L.-S. Li, R. Hansen, U.
Peters, X. Guo, Y. Chen, A. Babbar, S. J. Firdaus, L. Darjania, J. Feng, J. H.
Chen, S. Li, S. Li, Y. O. Long, C. Thach, Y. Liu, A. Zarieh, T. Ely, J. M.
Kucharski, L. V. Kessler, T. Wu, K. Yu, Y. Wang, Y. Yao, X. Deng, P. P.
Zarrinkar, D. Brehmer, D. Dhanak, M. V. Lorenzi, D. Hu-Lowe, M. P.
Patricelli, P. Ren, Y. Liu, Cell 2018, 172, 578–589; c) B. J. Grant, S. Lukman,
H. J. Hocker, J. Sayyah, J. H. Brown, J. A. McCammon, A. A. Gorfe, PloS
one 2011, 6, e25711.
All authors are current or former employees of Bayer AG.
Keywords: free-energy perturbation (FEP) · KRAS · medicinal
chemistry · molecular dynamics · virtual library design
[9] M. P. Patricelli, M. R. Janes, L. S. Li, R. Hansen, U. Peters, L. V. Kessler, Y.
Chen, J. M. Kucharski, J. Feng, T. Ely, J. H. Chen, S. J. Firdaus, A. Babbar,
P. Ren, Y. Liu, Cancer Discovery 2016, 6, 316–329.
[10] L. Li, J. Feng, T. Wu, P. Ren, Y. Liu, Y. Liu, Y. O. Long (Araxes Pharma LLC),
WO2015054572, 2015.
[
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COMMUNICATIONS
Discovery of a novel covalent
Dr. J. Mortier*, Dr. A. Friberg, Dr. V.
Badock, Dr. D. Moosmayer, Dr. J.
Schroeder, Dr. P. Steigemann, Dr. F.
Siegel, Dr. S. Gradl, Dr. M. Bauser,
Dr. R. C. Hillig, Dr. H. Briem, Dr. K. Eis,
Dr. B. Bader, Dr. D. Nguyen*, Dr. C. D.
Christ*
G12C
KRAS
inhibitor with an unprece-
dented mode of binding in the
switch-II allosteric pocket. This is the
outcome of a computationally driven
workflow based on the enumeration
of novel chemical matter and
stepwise prioritization. The most
promising compounds were synthe-
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– 7
sized, biochemically tested, and co-
Computationally Empowered
G12C
crystallized with KRAS
.
Workflow Identifies Novel Covalent
G12C
Allosteric Binders for KRAS