Controlling the Covalent Reactivity of a Kinase Inhibitor with Light

Article abstract of DOI:10.1002/anie.202103767

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acryla

Full text of DOI:10.1002/anie.202103767

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: Controlling the Covalent Reactivity of a Kinase Inhibitor with Light
Authors: Martin Reynders, Apirat Chaikuad, Benedict-Tilman Berger,
Katharina Bauer, Pierre Koch, Stefan Laufer, Stefan Knapp,
and Dirk Trauner
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202103767
Link to VoR: https://doi.org/10.1002/anie.202103767

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
Controlling the Covalent Reactivity of a Kinase Inhibitor with
Light
Martin Reynders,[a,b] Apirat Chaikuad, Benedict-Tilman Berger, Katharina Bauer, Pierre Koch,
[c]
[c]
[d]
[d,e]
Stefan Laufer, Stefan Knapp, Dirk Trauner[a]*
[
d]
[c,f]
[
a]
Department of Chemistry, Silver Center for Arts and Science, New York University
00 Washington Square East, New York, NY 10003 (USA)
E-mail: dirktrauner@nyu.edu
b] Martin Reynders
Martin Reynders, Prof. Dr. Dirk Trauner
1
[
Department of Chemistry, Ludwig Maximilians University of Munich
Butenandtstr.5–13, 81377 Munich (Germany).
[c]
Dr. Apirat Chaikuad, Dr. Benedict-Tilman Berger, Prof. Dr. Stefan Knapp
Buchmann Institute for Molecular Life Sciences
Johann Wolfgang Goethe-University
60438 Frankfurt am Main (Germany)
and
Institute for Pharmaceutical Chemistry
Johann Wolfgang Goethe-University
60438 Frankfurt am Main (Germany)
and
Structural Genomics Consortium Frankfurt
6
[
0438 Frankfurt am Main (Germany)
d] Katharina Bauer, Prof. Dr. Pierre Koch, Prof. Dr. Stefan Laufer
Department of Pharmaceutical / Medicinal Chemistry, Eberhard-
Karls-University Tübingen, Auf der Morgenstelle 8, D-72076
Tübingen (Germany) and Tuebingen Center for Academic Drug Discovery
[
e]
Prof. Dr. Pierre Koch
Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy,
University of Regensburg
93040 Regensburg (Germany)
[
f] Prof. Dr. Stefan Knapp
German Cancer Network (DKTK), Frankfurt/Mainz site
60438 Frankfurt am Main (Germany)
Supporting information for this article is given via a link at the end of the document.
Abstract: Covalent kinase inhibitors account for some of the most
successful drugs that have recently entered the clinic and many
others are in preclinical development. A common strategy is to target
cysteines in the vicinity of the ATP binding site using an acrylamide
electrophile. To increase the tissue selectivity of kinase inhibitors, it
could be advantageous to control the reactivity of these electrophiles
with light. Here, we introduce covalent inhibitors of the kinase JNK3
that function as photoswitchable affinity labels (PALs). Our lead
compounds contain a diazocine photoswitch, are poor non-covalent
inhibitors in the dark, and becomes effective covalent inhibitors after
irradiation with visible light. Our proposed mode of action is supported
by X-ray structures that explain why these compounds are unreactive
in the dark and undergo proximity-based covalent attachment
following exposure to light.
and neratinib, are now clinically approved (Figure 1A).[1] Most of
them feature an acrylamide “warhead” that is poorly reactive
towards water and other weak nucleophiles but undergoes rapid
bioconjugation following non-covalent binding and placement into
the trajectory of a suitable cysteinate anion.[2,3]
To target covalent inhibitors to kinases expressed in specific cells
and tissues, one could take advantage of the temporal and spatial
precision that light affords. This could be done, for instance, with
a caging group that masks the part of the molecule that binds
noncovalently (Figure 1B).[4,5] Alternatively, the covalent reactivity
could be concealed with a photoreactive group that delivers a
suitable electrophile upon irradiation. Although caging groups for
electrophiles, such as α-haloketones,[ have been reported, they
have not been applied, to the best of our knowledge, to covalent
kinase inhibitors. A third alternative for the design of light
activatable covalent inhibitors is shown in Figure 1C. It involves a
photoswitch that keeps the electrophile away from the nucleophile
in the dark form but allows for proximity-based rate enhancement
6]
The development of protein kinase inhibitors is one the most
active areas of medicinal chemistry. Early on, selectivity amongst
the more than 500 members of the human kinome was
considered a major challenge since the ATP binding site of protein
kinases is highly conserved. In recent years, however, a wide
range of selective inhibitors have emerged that have put such
concerns largely to rest. One strategy that has proven to be
particularly successful is the covalent engagement of native
cysteines that are present in the vicinity of the ATP binding site
with suitable electrophiles. Several covalent kinase inhibitors that
target the “cysteinome”, such as afatinib, ibrutinib, osimertinib,
and
efficient
bioconjugation
after
photoisomerization.
Photoswitchable tethered ligands (PTLs) and photoswitchable
affinity labels (PALs) have been reported for the optical control of
a wide variety of enzymes and receptors that feature engineered
[7]
cysteines or native electrophiles, respectively.
Non-covalently binding photoswitchable kinase inhibitor have
been explored, but their development has proven to be
1
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
challenging.[4,8–13] This prompted us to focus on covalent inhibitors, our JNK inhibitors represents a compelling strategy to further
which have advantages in terms of residency time and selectivity.
Amongst these, compounds that target the cysteinome have
proven to be most effective, due to the nucleophilicity of this
residue and its occurrence close to the ATP binding site in a
variety of important kinases.[
We report here the development of covalent inhibitors for the MAP
kinase JNK3 that function as PALs targeting a native cystine close
to the ATP binding site (Cys154). Our lead compound contains a
diazocine photoswitch, is a comparatively weak noncovalent
inhibitor in the dark, and becomes an effective covalent inhibitor
of JNK3 upon irradiation with 400 nm light.
improve their utility.
Our photoswitchable inhibitors are based on the known ligand 1,
which combines a pyridinylimidazole hinge-binding motif with a
diarylamide spacer that connects to
a cysteine-reactive
3]
acrylamide (Figure 2A). It is important to preserve the
pyridinylimidazole moiety binding the ATP-pocket, since
incorporation of a photoswitch into this crucial motif would
negatively influence potency and selectivity of our inhibitor.[
Therefore, we replaced the central diarylamide of 1, which does
23]
not engage in hydrogen bonding, as shown by a recent structure,
with an isosteric azobenzene[
.
17]
The MAP kinase c-Jun N-terminal kinase 3, abbreviated as JNK3,
is mainly expressed in the central nervous system, testes, and
pancreatic β-cells, in contrast to its ubiquitously expressed
This afforded the photoswitchable JNK inhibitor 2, which was
designed to be reactive in the dark adapted trans state and remain
a non-covalent inhibitor when irradiated to preferentially adopt the
cis form. To ensure that the photoswitchable inhibitor would be
unreactive in the dark and would become active only after
irradiation, we next implemented a sign-inversion approach,[
replacing the azobenzene moiety with a diazocine photoswitch.
Diazocines are known to be more stable in the bent cis form and
isomerize to the thermodynamically less stable elongated trans
[
14]
isoforms JNK1 and JNK2. JNK1-3 are key signaling enzymes
in the cellular stress response and JNK3 has been targeted for
the treatment of neurodegenerative diseases, including
Alzheimer's, Huntington's and Parkinson's disease, and type 2
diabetes.[
24]
14,15]
Cl
[25]
form upon isomerization with violet-blue light. This strategy led
F
O
N
to the light-activated JNK3 inhibitor 3, which is shown both in its
cis form and its trans form in Figure 2A. Molecular docking
confirmed that both isomers should be able to bind to the ATP-
pocket of JNK3 but only the metastable trans form would be able
to reach and covalently bind to Cys154. In addition to this, we
designed a version with an (E)-dimethylaminocrotyl amide
electrophile, which is known to improve solubility and reactivity,
yielding inhibitor 4. The half-life, absorption spectra and
photostationary state (PSS) are strongly dependent on
azobenzene substituents, which are in part dictated by the
requisite pharmacophore and the appended electrophile.
Therefore, we also explored a version with a meta-meta
substitution pattern in the diazocine, i.e. compound 5. Finally, we
designed a control compound 6, which bears a propionamide and
is therefore not cysteine-reactive.
A
HN
Cl
N
HN
H
N
H
N
N
N
N
O
O
O
N
O
N
afatinib
neratinib
O
N
2
H N
H
N
H
N
N
N
N
O
O
N
N
N
N
N
O
N
osimertinib
ibrutinib
O
B
C
h
covalent
The representative synthesis of inhibitor 3 is shown in Figure 1C.
It started with ethylene dianiline 7, which underwent oxidative
cyclization to afford diazocine 8.[ Slow addition of the oxidant
favors the desired intramolecular Baeyer-Mills reaction and
avoids overoxidation (see Supporting Scheme 1).[
Desymmetrization through Buchwald coupling with tert-butyl
carbamate yielded 9, which underwent deprotection and acylation
attachment
26]
26]
covalent
h
1
2
h
attachment
or k T
B
to afford acrylamide 10. Another Buchwald coupling with the
Figure 1. A) Representative FDA-approved covalent kinase inhibitors binding
irreversibly to specific cysteine residues. B) Photoactivation through caging. C)
Photoactivations through photoswitching.
[18,23]
known pyridinylimidazole ligand 11
yielded the desired
photoswitchable inhibitor 3. Covalent inhibitors 4 and 5, as well as
the propionamide control compound 6 were prepared in a similar
fashion (see Supporting Information).
With compound 2-6 in hand, we turned to their photophysical
characterization (Figure 2D, S1). In keeping with the general
photophysical behavior of substituted diazocines, the absorption
bands are not well separated and in particular, amino substituted
diazocines show only partial cis-to-trans isomerization at the
The development of reversible and selective JNK inhibitors that
bind non-covalently has proved to be difficult.[16] However, JNK3
features Cys154 in the vicinity of the ATP binding site that has
been exploited for the covalent attachment of inhibitors with
improved kinome selectivity.[
17,18]
So far, no JNK inhibitor has
4
00 nm PSS.[26] However, this is not a functional flaw for a PAL
progressed to clinical testing. Pharmacological intervention is
further complicated by the nature of JNK signaling, where the time
course of either kinase activity or inhibition is crucial in controlling
since the PSS is a dynamic equilibrium and continued pulse
irradiation will therefore enrich the covalently bound inhibitor over
time. The meta-meta-substituted diazocine 5 showed lower
extinction coefficients than the corresponding para-para
substituted diazocines 3 and 4 (Figure S1).
the cellular outcome, which can range from cell survival to
apoptosis.[19,20,15] Given the importance of spatiotemporal control
of the JNK pathway,[
21,22]
implementation of light-sensitivity into
2
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
F
F
F
F
N
N
N
N
N
N
SMe
N
SMe
O
SMe
azostere
azobenzene
SMe
N
400 nm
N
A
N
N
N
>
500 nm
or
N
O
sign-inversion
HN
HN
HN
HN
O
H
N
H
N
k T
B
HN
N
N
N
H
N
H
N
O
O
1
2
N
N
cis 3
trans 3
F
F
F
N
N
N
N
N
SMe
SMe
N
SMe
B
N
N
N
N
H
N
H
N
H
N
N
HN
HN
HN
N
O
O
O
N
N
N N
4
5
6
NH2
BocNH , Cs CO ,
XantPhos Pd G3 (0.05 eq)
H
N
2
2
3
Br
Br
CH COOOH
Br
3
Boc
Br
C
Br
AcOH, RT, 20 h
(75%)
1,4-Dioxane
N
N
N , 100 °C 14 h
2
N N
7
H N
2
(45%)
8
9
F
N
N
F
(
1:1)
SMe
1
) TFA/CH
Cl
Cl
2 2
2
)
N
N
1
1
SMe
N
H
N
Cs CO , BrettPhos
O
2
3
H
N
DIPEA (5 eq)
Br
Pd G3
HN
N
CH Cl , 0 °C to RT,
O
1,4-dioxane
2
(36%)
2
2
O
12h
N
N
N , 115 °C 2 h
NH2
11
N
N
(69%)
1
0
3
D
0
.5
1
.0
.8
.6
.4
.2
.0
2
2
Dark
0.8
0.03
3
Dark
400 nm
0
0
0
0
0
0.4
0.3
0.2
0.1
0.0
3 400 nm
3
0
0
0
.6
.4
.2
t1/2 (37 °C) = 26.5 min
0
0
.02
.01
2
t1/2 (37 °C) = 0.0137 min
0.0
0.00
0.00
4
00
500
600
700
0.02
0.04
0.06
0.08
400
500
λ (nm)
600
700
0
50
100
t (min)
t (min)
λ (nm)
Figure 2. Design and synthesis of the covalent photoswitchable inhibitors. A) Azologization of the known covalent inhibitor 1 and sign inversion to yield diazocine
. B) Additional photoswitchable JNK3 inhibitors prepared. C) Synthesis of diazocine 3. D) Absorption spectra of inhibitors 2 and 3 in the dark or after 400 nm
irradiation for 3 minutes in DMSO and thermal characterization of 2 and 3 in a 1:1 mixture of DMSO and PBS (pH 7.4).
3
Relaxation to the thermodynamically favored cis state could be
achieved via irradiation with longer wavelengths (500- 600 nm) or
thermally. The latter occurs on a timescale of minutes to hours in
DMSO or mixtures of DMSO and PBS (Figure 2D, S1). In
aqueous environments, amino substituted azobenzenes such as
min reaction time. As expected, the parent inhibitor 1 did not
respond to 400 nm irradiation. No light-dependency was
observed for its azostere 2, which had reduced potency compared
to 1. The lack of response to irradiation was in line with the fast
relaxation of a red-shifted p-amino substituted azobenzene and
the reactivity of the acrylamide in the thermodynamically stable
trans form of the azobenzene.
2
relax within seconds whereas the corresponding diazocines are
substantially more stable in their trans states.[
24,27]
Following their photophysical characterization, the inhibitors were
tested for their ability to inhibit JNK3 in vitro (Figure 3, Supporting
Table 1). This was determined through measuring the
phosphorylation of the immobilized kinase substrate ATF-2 at
different inhibitor concentrations after incubation for 50 minutes.
Kinase activity was determined with an ELISA assay, following a
previously published method.[ Photoswitches were either pulse-
irradiated every 5 s for 100 ms or kept in the dark during the 50
Photoswitchable inhibitor 3, which incorporates a para, para-
substituted diazocine, was a weak inhibitor of JNK3 in the dark
(IC50 = 646 nM) but showed much stronger inhibition at all tested
concentrations after pulse-irradiation with 390 nm light (IC50 = 21.4
nM). Compound 4, which bears a dimethylaminocrotyl amide as
electrophilic warhead, showed improved solubility but showed
JNK3 inhibition with similar potency in the dark (IC50 = 507 nM)
and after irradiation (IC50 = 31.9 nM). The meta, meta-substitution
28]
3
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
pattern on the diazocine in 5 gave similar results compared to 3,
with a pronounced increase in JNK3 inhibition upon isomerization
to its more potent trans form after 400 nm irradiation. Interestingly,
once isomerization and potential covalent attachment took place,
inhibition by compounds 3 and 5 was found to be irreversible. Use
of an inactivating 565 nm pulse after the activating light pulse did
not restore kinase activity (Figure S2).
Figure 3. In vitro characterization. Quantitative ELISA assay of differential ATF-
2
phosphorylation by JNK3 for 50 min at 37 C in the dark or with pulsed
irradiation (100 ms every 5 s) and inhibitors: A) 1 B) 2 C) 3 D) 4 E) 5 F) 6.
Thus, it appears that binding to the ATP binding site was too tight
to pull the pyridinylimidazole from the active site. Indeed, non-
covalent inhibitors that contained this motif did bind to JNK3 and
p38α in the low nanomolar concentration range.[23] Alternatively,
covalent attachment of the inhibitor could inhibit kinase function
even if the ligand does not occupy the ATP-pocket. The non-
covalent, propionamide control compound 6 only showed a small
increase in JNK3 inhibitory activity upon 400 nm irradiation. This
small activity difference was probably due to an increased steric
clash of the cis isomer with the glycine-rich loop which was in
agreement with a non-covalent mechanism of inhibition where
isomerization only changes positioning of the propionamide.
We demonstrated cellular target engagement of the inhibitors
using the NanoBRET tracer displacement assay[29] and
determined the IC50s to be in the high nanomolar to low
micromolar range (Figure S3). The observed improvement in
potency after 400 nm irradiation was not as prominent as in the in
vitro assay. However, the potent cellular activity of the inhibitors
harboring the diazocine moiety demonstrated target engagement,
suggesting that these inhibitors represent lead structures for
further development, including in vivo studies.
Figure 4. X-ray analysis of 4 bound to JNK3. A) Structural overlay of 1 and 4
covalently bound to JNK3. B) The bent cis isomer cannot reach Cys154 and
only binds non-covalently. C) The extended isomer binds covalently to Cys154.
To confirm covalent labelling, we performed mass spectrometry
of JNK3 and inhibitor 4 incubated under pulsed 400 nm irradiation
(
Figure S4). A mass shift corresponding to the molecular weight
of 4 was observed, demonstrating covalent adduct formation in a
:1 ratio.
1
To gain further insight into the mode of action of our compounds,
we carried out structural studies using X-ray crystallography. To
this end, we soaked crystals of JNK3 with inhibitor 4, which
showed improved solubility, whilst pulse-irradiating with 400 nm
light. We collected several datasets after different irradiation
schedules and observed clear electron density that supported a
4
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
[
[
1]
2]
R. Roskoski, Pharmacological Research 2019, 144, 19–50.
Q. Liu, Y. Sabnis, Z. Zhao, T. Zhang, S. J. Buhrlage, L. H. Jones, N. S.
Gray, Chemistry & Biology 2013, 20, 146–159.
non-covalent as well as covalent binding mode of 4 (Figure 4A, B
and C).
Both isomers have the ATP-mimetic pyridinylimidazole moiety in
the same position which was interacting with the hinge region, the
adenine pocket, and the hydrophobic pocket in a similar manner
to the same moiety of the starting compound 1 (Figure 4A).
However, the electrophilic warhead reached Cys154 only in the
elongated trans isomer but not in the bent cis form present in the
dark (Figure 4 and Supporting Figure S5). This enabled covalent
adduct formation, which was not feasible with the cis isomer. As
such, inhibitor 4 operated in the manner indicated in Figure 1C.
[3]
A. Chaikuad, P. Koch, S. A. Laufer, S. Knapp, Angewandte Chemie
International Edition 2018, 57, 4372–4385.
C. L. Fleming, M. Grøtli, J. Andréasson, ChemPhotoChem 2019, 3,
318–326.
D. Kolarski, A. Sugiyama, G. Breton, C. Rakers, D. Ono, A. Schulte, F.
Tama, K. Itami, W. Szymanski, T. Hirota, B. L. Feringa, J. Am. Chem.
Soc. 2019, 141, 15784–15791.
A. Kobori, T. Yamauchi, Y. Nagae, A. Yamayoshi, A. Murakami,
Bioorganic & Medicinal Chemistry 2012, 20, 5071–5076.
P. Leippe, J. Koehler Leman, D. Trauner, Biochemistry 2017, 56, 5214–
5220.
[
[
4]
5]
[6]
[
[
[
7]
8]
9]
R. Ferreira, J. R. Nilsson, C. Solano, J. Andréasson, M. Grøtli, Scientific
Reports 2015, 5, 9769.
D. Wilson, J. W. Li, N. R. Branda, ChemMedChem 2017, 12, 284–287.
In summary, we developed covalent kinase inhibitors that can be
activated by irradiation and function as photoactivated affinity
labels. Notably, our approach has advantages over caged
inhibitors, since it largely retains the properties of the parent drug,
whereas a caging group adds significant molecular weight and
can negatively impact cellular uptake and distribution. Additionally,
the uncaging reaction generates byproducts, whereas our
inhibitors just undergo a reversible isomerization. While we were
unable to show reversibility on target, an unbound inhibitor can
still revert to its inactive form, whereas an uncaged inhibitor will
always remain active.
[10] M. W. H. Hoorens, M. E. Ourailidou, T. Rodat, P. E. van der Wouden, P.
Kobauri, M. Kriegs, C. Peifer, B. L. Feringa, F. J. Dekker, W. Szymanski,
European Journal of Medicinal Chemistry 2019, 179, 133–146.
[11] M. Schehr, C. Ianes, J. Weisner, L. Heintze, M. P. Müller, C. Pichlo, J.
Charl, E. Brunstein, J. Ewert, M. Lehr, U. Baumann, D. Rauh, U.
Knippschild, C. Peifer, R. Herges, Photochem. Photobiol. Sci. 2019, 18,
1398–1407.
[12] L. Heintze, D. Schmidt, T. Rodat, L. Witt, J. Ewert, M. Kriegs, R. Herges,
C. Peifer, International Journal of Molecular Sciences 2020, 21, 8961.
[
13] D. Schmidt, T. Rodat, L. Heintze, J. Weber, R. Horbert, U. Girreser, T.
Raeker, L. Bußmann, M. Kriegs, B. Hartke, C. Peifer, ChemMedChem
2018, 13, 2415–2426.
[
[
14] E. T. Coffey, Nat Rev Neurosci 2014, 15, 285–299.
15] G. Solinas, B. Becattini, Molecular Metabolism 2017, 6, 174–184.
[16] P. Koch, M. Gehringer, S. A. Laufer, J. Med. Chem. 2015, 58, 72–95.
[
17] T. Zhang, F. Inesta-Vaquera, M. Niepel, J. Zhang, S. B. Ficarro, T.
Machleidt, T. Xie, J. A. Marto, N. Kim, T. Sim, J. D. Laughlin, H. Park, P.
V. LoGrasso, M. Patricelli, T. K. Nomanbhoy, P. K. Sorger, D. R. Alessi,
N. S. Gray, Chemistry & Biology 2012, 19, 140–154.
Our most successful inhibitor incorporates a new class of
photoswitch, viz. diazocines, a moiety that is increasingly used to
convert compounds that are constitutively inactive in the dark into
light-activated ones. X-ray crystallography provides further insight
[
[
18] F. Muth, A. El-Gokha, F. Ansideri, M. Eitel, E. Döring, A. Sievers-Engler,
A. Lange, F. M. Boeckler, M. Lämmerhofer, P. Koch, S. A. Laufer, J.
Med. Chem. 2017, 60, 594–607.
19] J.-J. Ventura, A. Hübner, C. Zhang, R. A. Flavell, K. M. Shokat, R. J.
Davis, Molecular Cell 2006, 21, 701–710.
into the mode of action of a photoswitchable inhibitor bound to its
target.[
30,31]
Our strategy could be easily transferred to other
[20] C. H. Nijboer, H. J. C. Bonestroo, J. Zijlstra, A. Kavelaars, C. J. Heijnen,
Neurobiology of Disease 2013, 54, 432–444.
21] A. Zeke, M. Misheva, A. Reményi, M. A. Bogoyevitch, Microbiol. Mol.
Biol. Rev. 2016, 80, 793–835.
kinase targets that harbor a cysteine in positions close to the ATP
binding site once suitable inhibitors and linkers have been
developed and may therefore serve as a prototype for future
photoswitchable covalent kinase inhibitors.
[
[22] R. M. M.-F. de Mera, L.-L. Li, A. Popinigis, K. Cisek, M. Tuittila, L.
Yadav, A. Serva, M. J. Courtney, Nat Commun 2017, 8, 1–18.
[
[
[
23] F. Muth, M. Günther, S. M. Bauer, E. Döring, S. Fischer, J. Maier, P.
Drückes, J. Köppler, J. Trappe, U. Rothbauer, P. Koch, S. A. Laufer, J.
Med. Chem. 2015, 58, 443–456.
24] J. B. Trads, K. Hüll, B. S. Matsuura, L. Laprell, T. Fehrentz, N. Görldt, K.
A. Kozek, C. D. Weaver, N. Klöcker, D. M. Barber, D. Trauner,
Angewandte Chemie International Edition 2019, 58, 15421–15428.
25] R. Siewertsen, H. Neumann, B. Buchheim-Stehn, R. Herges, C. Näther,
F. Renth, F. Temps, J. Am. Chem. Soc. 2009, 131, 15594–15595.
Acknowledgements
M.R. and D.T. thank the German Research Foundation (DFG) for
financial support (SFB749). Nuclear magnetic resonance spectra
were acquired using the TCI cryoprobe supported by the NIH
[26] M. S. Maier, K. Hüll, M. Reynders, B. S. Matsuura, P. Leippe, T. Ko, L.
Schäffer, D. Trauner, J. Am. Chem. Soc. 2019, 141, 17295–17304.
[
[
[
27] K. Hüll, J. Morstein, D. Trauner, Chemical Reviews 2018, 118, 10710–
10747.
28] M. Goettert, S. Luik, R. Graeser, S. A. Laufer, Journal of Pharmaceutical
and Biomedical Analysis 2011, 55, 236–240.
29] J. D. Vasta, C. R. Corona, J. Wilkinson, C. A. Zimprich, J. R. Hartnett,
M. R. Ingold, K. Zimmerman, T. Machleidt, T. A. Kirkland, K. G. Huwiler,
R. F. Ohana, M. Slater, P. Otto, M. Cong, C. I. Wells, B.-T. Berger, T.
Hanke, C. Glas, K. Ding, D. H. Drewry, K. V. M. Huber, T. M. Willson, S.
Knapp, S. Müller, P. L. Meisenheimer, F. Fan, K. V. Wood, M. B.
Robers, Cell Chemical Biology 2018, 25, 206-214.e11.
(OD016343). We thank Silke Bauer and Jens Strobach for
assistance in the in vitro assays. S.K., A.C. and B.-T.B. are
grateful for support by the German cancer network DKTK as well
as the SGC, a charity a registered charity (number 1097737) that
receives funds from AbbVie, Bayer Pharma AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman Institute
for Innovation, Genome Canada, Innovative Medicines Initiative
[
[
30] V. Arkhipova, H. Fu, M. W. H. Hoorens, G. Trinco, L. N. Lameijer, E.
Marin, B. L. Feringa, G. J. Poelarends, W. Szymanski, D. J. Slotboom,
A. Guskov, J. Am. Chem. Soc. 2021, 143, 1513–1520.
31] S. Pospich, F. Küllmer, V. Nasufović, J. Funk, A. Belyy, P. Bieling, H.-D.
Arndt, S. Raunser, Angewandte Chemie International Edition 2021, 60,
8678–8682.
(EU/EFPIA), Janssen, Merck KGaA Darmstadt Germany, MSD,
Novartis Pharma AG, Ontario Ministry of Economic Development
and Innovation, Pfizer, São Paulo Research Foundation-FAPESP,
Takeda, and Wellcome. The authors thank staffs at BESSY and
Diamond synchrotron for their assistance during data collection.
Keywords: covalent inhibitor • photopharmacology •
photoswitch • photoswitchable affinity label • kinase inhibitor •
JNK3
5
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.202103767
COMMUNICATION
Entry for the Table of Contents
To increase the tissue selectivity of kinase inhibitors, we introduce the combination of covalent inhibitors, targeting a cysteine in the
vicinity of the ATP binding site of the c-Jun N-terminal kinase 3 (JNK3) and diazocine photoswitches. X-ray crystallography shows
that our lead inhibitor is a poor non-covalent inhibitor in the dark and undergoes proximity-based covalent attachment following
irradiation with visible light.
Institute and/or researcher Twitter usernames: @DirkTrauner, @xmr128
6
This article is protected by copyright. All rights reserved.