Covalent-Allosteric Kinase Inhibitors

Article abstract of DOI:10.1002/anie.201502142

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-depende

Full text of DOI:10.1002/anie.201502142

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502142
German Edition: DOI: 10.1002/ange.201502142
Drug Design
Hot Paper
Covalent-Allosteric Kinase Inhibitors**
Jçrn Weisner, Rajesh Gontla, Leandi van der Westhuizen, Sebastian Oeck, Julia Ketzer,
Petra Janning, AndrØ Richters, Thomas Mühlenberg, Zhizhou Fang, Abu Taher,
Verena Jendrossek, Stephen C. Pelly, Sebastian Bauer, Willem A. L. van Otterlo, and
Daniel Rauh*
Abstract: Targeting and stabilizing distinct kinase conforma-
tions is an instrumental strategy for dissecting conformation-
dependent signaling of protein kinases. Herein the structure-
based design, synthesis, and evaluation of pleckstrin homology
(PH) domain-dependent covalent-allosteric inhibitors (CAIs)
of the kinase Akt is reported. These inhibitors bind covalently
to a distinct cysteine of the kinase and thereby stabilize the
inactive kinase conformation. These modulators exhibit high
potency and selectivity, and represent an innovative approach
for chemical biology and medicinal chemistry research.
of the cellular processes which comprise the hallmarks of
cancer.[8] Therefore, Akt dysregulation is directly associated
with neoplastic transformation and malignant progression as
well as increased resistance to chemo- and radiotherapy in
a variety of solid tumors, such as breast, prostate, and
colorectal cancer.[9] To this end, chemical modulation of
dysregulated Akt using selective small molecule inhibitors
provides a promising perspective for the treatment of patients
suffering from diverse forms of cancer.[8] Allosteric PH
domain-dependent Akt inhibitors, such as the clinical candi-
date MK-2206,[10] exhibit an exclusive binding mode. They
target a unique pocket at the interface of the regulatory PH
and the catalytically active kinase domain, thereby stabilizing
the inactive “PH-in” conformation (Figure 1).[11] Recently,
kinase-independent but conformation-dependent functions of
Akt were identified that promote cancer cell survival.[12]
Chemically modulating and interfering with such inter-
domain interactions is a major opportunity for understanding
kinase function beyond catalysis. X-ray structure analysis of
full-length Akt in complex with allosteric inhibitors Akti-1/2
(PDB code: 3O96)[13] and 12j (PDB code: 4EJN)[14] revealed
the presence of non-catalytic A-loop cysteines at positions
296 and 310, which are known to be redoxsensitive (Support-
ing Information, Figure S1).[15] Cys296 and Cys310 are located
in close proximity to these inhibitors and could be potentially
addressed by cysteine reactive probes.
O
ver the last two decades, kinases have evolved as central
targets for drug discovery in academic and industrial
research.[1] Dysregulated kinases in diseases such as cancer,
autoimmune disorders, neurodegenerative diseases, and dia-
betes have been addressed by a vast diversity of small-
molecule inhibitors.[2] Despite the highly conserved ATP-
binding site in kinases throughout the human kinome,
a number of orthosteric inhibitors has been identified and
approved by the FDA, including examples such as Erlotinib
and Gefitinib.[3] Furthermore, allosteric kinase inhibitors that
stabilize enzymatically inactive conformations[4] and covalent
inhibitors that irreversibly target rare non-catalytic cysteines
have recently been shown to be beneficial for increased
selectivity and potency.[5] These characteristics translate into
high efficacy and reduced off-target reactivity, which offer
significant benefits for targeted cancer therapies.[6]
We therefore set out to combine the outstanding selec-
tivity of PH domain-dependent Akt inhibitors[16] with phar-
macological and therapeutic benefits of targeted irreversible
modulators, which include superior drug-target residence
time and increased potency.[5c] Herein we report our efforts to
amalgamate these features and promote the design, synthesis,
The protein kinase Akt features a regulatory pleckstrin
homology (PH) domain, along with the catalytic kinase
domain, allowing for membrane attachment upon growth
factor stimulation and activation by phosphorylation by the
upstream kinase PDK1.[7] PI3K/Akt signaling mediates most
[*] J. Weisner,[+] Dr. R. Gontla,[+] Dr. A. Richters, Dr. Z. Fang,
Prof. Dr. D. Rauh
J. Ketzer, Dr. T. Mühlenberg, Prof. Dr. S. Bauer
Department of Medical Oncology, Sarcoma Center
West German Cancer Center
Technische Universität Dortmund
Fakultät für Chemie und Chemische Biologie
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
E-mail: daniel.rauh@tu-dortmund.de
University Duisburg–Essen, Medical School (Germany)
and
German Cancer Consortium (DKTK), Heidelberg (Germany)
L. van der Westhuizen, Dr. A. Taher, Dr. S. C. Pelly,
Prof. Dr. W. A. L. van Otterlo
Dr. P. Janning
Max-Planck-Institut für Molekulare Physiologie
Department of Chemistry and Polymer Sciences
Stellenbosch University, Matieland (South Africa)
Abteilung Chemische Biologie, Dortmund (Germany)
[+] These authors contributed equally to this work.
S. Oeck, Prof. Dr. V. Jendrossek
Institute of Cell Biology (Cancer Research)
Department of Molecular Cell Biology
[**] This work was co-funded by the German Federal Ministry for
Education and Research (NGFNPlus and e:Med) (Grant No. BMBF
01GS08104, 01ZX1303C) and by the Deutsche Forschungsgemein-
schaft (DFG).
University of Duisburg–Essen, Medical School (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502142.
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Figure 1. Representations of the structures of Akt in the active and inactive conformation. ATP-competitive inhibitor targeting the kinase domain
of Akt (left). Allosteric PH domain-dependent Akt inhibitors (blue) bind reversibly to an interface formed by the kinase domain and the PH
domain of Akt (middle). Covalent-allosteric PH domain-dependent inhibitors (green) irreversibly stabilize the enzymatically inactive ‘PH-in’
conformation. Structures of covalent-allosteric PH domain-dependent Akt inhibitors 1a and 2a and their reversible analogues 1b and 2b.
and biochemical and cellular characterization of PH domain-
dependent covalent-allosteric inhibitors (CAIs) as a novel
approach to target Akt and to probe its signaling and
functions beyond catalysis.
tively). These results were consistent with the fact that the
full-length Akt protein used in these studies had been
mutated for site-specific fluorescent labeling by replacing all
solvent-exposed cysteines with serines, including both A-loop
cysteines. We evaluated the potential influence of the Michael
acceptor and further assessed the inhibitory potencies of our
warhead-decorated probes in activity-based studies utilizing
activated full-length wtAkt1 (Table 1; Supporting Informa-
tion, Figure S3) as well as activated DPH-Akt1 (Supporting
Information, Figure S4). Whilst probe molecule 2a moder-
ately inhibited Akt kinase activity in comparison to reference
inhibitors MK-2206 and GSK690693 (372 Æ 48 nm vs. 7 Æ 1 nm
and 2 Æ 1 nm, respectively), probe compound 1a exhibited
a subnanomolar half-maximal inhibitory concentration (0.2 Æ
0.1 nm). In contrast, tenfold and fourfold higher concentra-
tions of 1b and 2b, respectively, were required to provoke
half-maximum inhibition of Akt1, suggesting the covalent
modification of Akt by 1a and 2a as reason for their higher
potency. Additionally, time-dependent IC50 determinations of
1a further indicated a covalent mode of action (Supporting
Information, Figure S5). Kinetic characterization of our
probe compounds by determination of kinact/Ki indicated
a significantly enhanced inhibition profile for 1a compared to
2a, with respect to both affinity and covalent complex
formation (Table 1; Supporting Information, Figure S6).
Moreover, superior inhibition properties were verified for
our covalent probes when compared to the clinical lead
compound MK-2206, which is currently being investigated for
the treatment of HER2-positive solid tumors and acute
myelogenous leukemia.[20]
Based on the previously described 1,6-naphthyridinone
scaffold[17] and an imidazo-1,2-pyridine core,[18] we designed
and synthesized chemically diverse probe compounds (1a and
2a) featuring Michael acceptors to target Cys296 or Cys310
(Figure 1). Additionally, we synthesized 1b and 2b as
reversible counterparts. All inhibitors were subjected to
kinetic analyses using the interface fluorescent labels in
kinases (iFLiK) system[19] that allows for the detection of PH
domain-dependent binders for Akt to determine their dis-
sociation rates with respect to fluorescently labeled full-
length Akt1 (Table 1; Supporting Information, Figure S2).
These results indicated similar binding affinities for probe
compound 1a and reference inhibitor MK-2206 (58 Æ 8 nm
and 69 Æ 13 nm, respectively), whereas 2a exhibited a signifi-
cantly lower binding affinity for Akt1 (795 Æ 176 nm). The
reversible counterparts 1b and 2b, however, possessed
comparable Kd values (62 Æ 12 nm and 797 Æ 180 nm, respec-
Table 1: Half-maximal inhibitory concentrations (IC50), kinact/Ki, and
dissociation constants (Kd) of reference compounds and CAIs.[a]
wtAkt1
iFLiK-Akt1
(E49C/C296S/
C310S/C344S)
Kd [nm]
Compound
IC50 [nm]
kinact/Ki [mmÀ1 sÀ1
]
MK-2206
GSK690693
1a
1b
2a
2b
6.5Æ0.8
2.3Æ0.3
0.2Æ0.1
7.5Æ2
–
–
69Æ13
n.r.
After evaluating the inhibitory effects of the compounds,
we utilized ESI-MS to investigate their respective binding
properties to Akt1. Treatment of recombinant wild-type Akt1
with probe compounds 1a and 2a resulted in mass increases
equivalent to the corresponding single labeled Akt1 (587 and
3.29Æ0.40
58Æ8
–
62Æ12
795Æ176
797Æ180
372Æ48
0.0023Æ0.0003
992Æ328
–
[a] n.r.=no response up to 100 mm.
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Furthermore, conserved cysteines analogous to Cys296 and
Cys310 most likely do not account for off-target activity as
deduced from the poor inhibition profile of PKCq (35%
inhibition), MAPKAPK2 (30%), and MELK (30%) as well
as 43 additional kinases harboring analogous cysteines
(< 30% inhibition). Therefore, probe compound 1a exhibited
an excellent selectivity profile, exclusively targeting Akt
isoforms without affecting kinases revealing high sequence
and structural homology.
We further characterized our most promising CAI 1a in
cellular studies using prostate cancer (PC3), breast cancer
(BT474), and gastrointestinal stromal tumor (GIST-T1) cell
lines, which possess genetic lesions (PC3: PTEN-/-, BT474:
PI3K mut, HER2 + ; GIST-T1: c-KIT mut) in the PI3K/Akt
pathway and display differences in basal phospho-Akt levels.
These cell lines were employed as model systems for Western
Blot analyses to investigate both the cell penetrating effect of
1a as well as its influence on cellular Akt1 and downstream
GSK3b phosphorylation states. Treatment with MK-2206
(reference compound) and with 1a induced sensitive dose-
dependent decreases of pAkt1 at both Thr308 and Ser473 in
PC3 and BT474 cancer cell lines; these results were correlated
with decreased phosphorylation of the Akt1 substrate GSK3b
(Supporting Information, Figure S8).
Figure 2. a) Deconvoluted mass spectra of Akt1 incubated with DMSO
(top), 1a (middle), and 2a (bottom), respectively. Mass differences in
relation to DMSO-treated control are displayed as D values and
demonstrate covalent modification of Akt1 with 1a and 2a. b) Kinome
dendrogram of 1a. At 1 mm compound concentration, only Akt1, Akt2,
and Akt3 were significantly inhibited (98%, 96%, and 83%) whereas
the remaining 97 kinases were inhibited by less than 40%. Kinase
profiling data were generated with the SelectScreen Profiling Service
from LifeTechnologies. The kinase dendrogram was adapted from
Kinome Render[22] and the illustration is reproduced with courtesy of
Cell Signaling Technology, Inc. (http://www.cellsignal.com).
Moreover, using c-KIT-dependent GIST-T1 cells, we
demonstrated the selectivity of 1a for Akt1 in a cellular
setting, whilst sparing further oncogenic protein kinases, such
as c-KIT and Erk1/2 (Figure 3). Therefore, 1a was demon-
strated to be a cell-permeable effector of Akt in various
cancer cell lines. These observations introduce the prospect of
utilizing our probe compounds for further medicinal chemis-
572 Da, respectively), as compared to control Akt1 treated
with DMSO (Figure 2). These results additionally corrobo-
rated the biochemical data and further substantiated the
anticipated covalent binding mode correlating with the
observed trends in activity-based studies (Table 1).
After tryptic digest of the 1a- and 2a-treated Akt and
ESI-MS/MS analysis, we identified Cys296 and Cys310 as the
modification sites for our probes (Supporting Information,
Figure S7). The respective mass signatures of both modified
cysteine-containing peptide fragments accounted for
Cys296_1a, as well as Cys310_1a. Analogous findings were
obtained for probe 2a. These results indicated that Cys296
was not preferentially targeted over Cys310 or vice versa by
our probes and further substantiated our hypothesis that both
cysteines located on the flexible activation loop are accessible
for covalent modification by suitable electrophile-decorated
inhibitors (Supporting Information, Figure S7).
We next investigated the selectivity of 1a for Akt1 over
other protein kinases with similar characteristics focusing on
a) members of the AGC kinase family, b) kinases featuring
a PH domain, and/or c) kinases containing cysteines on the
activation loop. One hundred different protein kinases
matching these criteria were assessed employing the
SelectScreen Kinase Profiling Services from Life Technolo-
gies (Supporting Information, Table S1).
Within the panel of kinases, solely the Akt isoforms 1–3
were significantly addressed (> 80% inhibition) at a concen-
tration of 1 mm of 1a (Supporting Information, Table S1).
MAP4K5, which was previously shown to be moderately
inhibited by a substrate-competitive phenylalanine-based Akt
inhibitor,[21] ranked fourth with 37% inhibition. Interestingly,
MAP4K5 contains neither an activation loop cysteine, nor
a PH domain and it does not belong to the AGC superfamily.
Figure 3. Covalent-allosteric inhibitor 1a impairs Akt1 phosphorylation
in cancer cells. Gastrointestinal stromal tumor cells (GIST-T1) exhibit
high basal pAkt1 levels related to constitutively active c-KIT (imatinib:
positive control for c-KIT inhibition). Treatment with CAI 1a reduces
cellular pAkt1 levels at low nanomolar concentrations correlating with
decreasing phosphorylation of downstream targets S6 and 4EBP1;
phosphorylation of other relevant protein kinases c-KIT and MAPK
(Erk1/2) is not affected by treatment with 1a. Two bands are visible for
c-KIT representing the mature glycosylated form and the immature
form. Regarding MAPK, the bands relate to Erk1 and Erk2.
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development of covalently modulating anticancer drugs.
In summary, we rationally designed, synthesized, and
characterized novel allosteric inhibitors that covalently
modify Akt at Cys296 and Cys310. Single labeling of the
protein with our covalently modulating inhibitors at the
desired cysteines was confirmed by mass spectrometry.
Further to promising biochemical inhibitory potency against
Akt and outstanding selectivity over other protein kinases
with homologous structure and similar sequence, 1a was
demonstrated to exhibit analogue impact as compared to the
current clinical lead candidate MK-2206 in cellular model
systems. Moreover, we demonstrated that targeting non-
catalytic cysteines in Akt is feasible by chemically diverse
methods that can likewise be used as functional probes for
Akt modulation in chemical and systems biology endeavors.
This proof-of-principle study illustrates the tremendous
potential of CAIs in the targeted therapy of patients suffering
from tumors with aberrantly activated Akt and, above all,
underlines the importance of defining and understanding
fundamental molecular mechanisms that induce, regulate,
and control cancer development.
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Received: March 6, 2015
Revised: April 13, 2015
Published online: June 25, 2015
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