Neuritogenic Militarinone-Inspired 4-Hydroxypyridones Target the Stress Pathway Kinase MAP4K4

Article abstract of DOI:10.1002/anie.201501515

Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2

Full text of DOI:10.1002/anie.201501515

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201501515
German Edition: DOI: 10.1002/ange.201501515
Biological Activity
Neuritogenic Militarinone-Inspired 4-Hydroxypyridones Target the
Stress Pathway Kinase MAP4K4**
Peter Schrçder, Tim Fçrster, Stefan Kleine, Christian Becker, Andrꢀ Richters, Slava Ziegler,
Daniel Rauh, Kamal Kumar, and Herbert Waldmann*
Abstract: Progressive loss and impaired restoration of neuro-
nal activity are hallmarks of neurological diseases, and new
small molecules with neurotrophic activity are in high demand.
The militarinone alkaloids and structurally simplified ana-
logues with 4-hydroxy-2-pyridone core structure induce pro-
nounced neurite outgrowth, but their protein target has not
been identified. Reported herein is the synthesis of a milita-
rinone-inspired 4-hydroxy-2-pyridone collection, its investiga-
tion for enhancement of neurite outgrowth, and the discovery
of the stress pathway kinase MAP4K4 as a target of the
discovered neuritogenic pyridones. The most potent 4-hy-
droxy-2-pyridone is a selective ATP-competitive inhibitor of
MAP4K4 but not of the other stress pathway related kinases, as
proven by biochemical analysis and by a crystal structure of the
inhibitor in complex with MAP4K4. The findings support the
notion that MAP4K4 may be a new target for the treatment of
neurodegenerative diseases.
as the identification of their target proteins are of major
current interest.
Frequently, natural products command this kind of
[
4]
bioactivity. Since their underlying scaffolds define biologi-
cally prevalidated structures in chemical space, compound
collections inspired by them may be valuable sources for the
discovery of novel bioactive compounds endowed with the
[
5]
same or similar activity.
In this respect the militarinones 1 and 2 and related
[6,7]
alkaloids,
like farinosone A (3; Figure 1) are of particular
S
evere neurological diseases like Alzheimerꢀs disease (AD),
Parkinsonꢀs disease (PD), and amyotrophic lateral sclerosis
ALS) are characterized by progressive loss and impaired
restoration of neuronal activity leading to reduced neuronal
(
[1]
complexity and function. These diseases impose a large and
increasing burden on society, and it has been estimated that
[
1a]
by 2050 AD will affect 1 in 85 persons.
Despite the
importance and the advances made in understanding the
molecular basis of neurodegenerative disorders, current
treatments mostly achieve symptomatic benefits but do not
[
1–3]
target the root cause of the diseases.
Therefore, the
discovery of new small-molecule classes with neurotrophic
or neuroprotective activity which may restore neuronal
function and viability and prevent neuronal decline, as well
[*] Dr. P. Schrçder, Dipl.-Biol. T. Fçrster, Dr. S. Ziegler, Dr. K. Kumar,
Prof. Dr. H. Waldmann
Max-Planck-Institut fꢀr Molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Straße 11, 44227 Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Figure 1. Selected alkaloids having 4-hydroxy-2-pyridone structures and
the core scaffolds 5 and 6 used for library design and synthesis.
Dr. P. Schrçder, Dipl.-Biol. T. Fçrster, M. Sc. S. Kleine,
M. Sc. C. Becker, Dr. A. Richters, Prof. Dr. D. Rauh, Dr. K. Kumar,
Prof. Dr. H. Waldmann
Technische Universitꢁt Dortmund
Fakultꢁt fꢀr Chemie und Chemische Biologie
Otto-Hahn-Straße 6, 44221 Dortmund (Germany)
interest. These fungal metabolites induce pronounced neurite
[6]
outgrowth from neuronal precursor PC-12 cells. This kind of
[7b]
bioactivity is conserved in structurally simplified analogues,
and the side-chain of the natural products has only a minor
[7c]
influence on neuritogenic activity. However, the protein
target of these militarinone analogues which induce neurite
outgrowth has not yet been identified.
[
**] This work was funded by the European Union Seventh Framework
Programme under grant agreement no. HEALTH-F2-2009-241498
(
“EUROSPIN” project) and ERC grant agreement no. 268309.
Herein we report on the synthesis of a militarinone-
inspired 4-hydroxy-2-pyridone collection, its phenotypic
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501515.
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investigation for enhancement of neurite outgrowth, and the
discovery of the stress pathway serine/threonine kinase
MAP4K4 as a target of the newly discovered neuritogenic
and other related coupling products with unsatisfying yields,
a different strategy was developed and it employs regiose-
lective lithiation of 7 at C3 as the first key step (Scheme 1b).
Treatment of 7 with sBuLi and subsequent reaction with
different aromatic and (cyclo)aliphatic aldehydes gave the
secondary alcohols 12a–h in moderate to good yields. Suzuki
couplings with boronic acids or boronic acid pinacol esters
generated the pyridines 9e–u and the corresponding ketones
10e–v in viable yields. This strategy allowed successful
introduction of a broader range of substituents compared
with that of the initial synthesis sequence. For final demethy-
lation either AlCl /NaI or BBr /TBAI was employed to yield
4
-hydroxy-2-pyridones.
Militarinone-A-related neuritogenic alkaloids frequently
embody the 4-hydroxy-2-pyridone scaffold 5, which is sub-
stituted at the 3- and 5-positions, as the characteristic
[6,7]
structural core,
and was therefore chosen as a target for
compound collection synthesis (Figure 1). To determine
whether the 4-hydroxy-2-pyridone structure is required for
bioactivity, we also envisaged synthesizing the corresponding
2
,4-dimethoxypyridines 6. Initially the desired compounds
3
3
were synthesized by means of a regioselective Suzuki
the desired pyridones 11e–u. In addition, the intermediate
pyridines 9 and 10, supporting a TBS-protected phenyl
moiety, were desilylated to deliver the alcohols 13a–h and
ketones 14a–e (see the Supporting Information for further
details of the synthesis). In total 21 pyridones and 38 pyridines
were synthesized.
[
8]
coupling employing the dibromopyridine 7 and p-TBSO-
substituted phenylboronic acid (Scheme 1a). The resulting
monobromopyridine 8 was lithiated at C3, and after nucle-
ophilic addition to different aldehydes, the resulting secon-
dary alcohols 9a–d were oxidized to the corresponding
ketones 10a–d. Demethylation and simultaneous desilylation
For evaluation of their neurotrophic potential the com-
pounds were subjected to phenotypic screening in human
neuroblastoma SH-SY5Y cells to monitor neurite out-
by means of treatment with AlCl and NaI yielded the desired
3
pyridones 11a–d. Since the initial Suzuki coupling yielded 8
[
9]
growth. To this end, upon differentiation for 72 hours in
a serum-free N2 medium supplemented with retinoic acid
(RA), SH-SY5Y cells were treated with the respective
compounds at concentrations of 1 mm and 10 mm for
9
1
6 hours. 0.1% DMSO was used as a vehicle control, while
% DMSO served as a negative control because of its
retracting properties (see Figure S1 in the Supporting Infor-
[
10]
mation). Brain-derived neurotrophic factor (BDNF) was
used as a positive control (Figure 2). Cells were stained for a-
tubulin and DNA to visualize neurites and cell nuclei prior to
high-content analysis.
Gratifyingly, three pyridones embodying a p-hydroxyphe-
nol substituent significantly enhanced neurite outgrowth in
SH-SY5Y cells at a concentration of 10 mm (Figures 2 and 3).
The compound 11e was the most active and increased the
relative neurite outgrowth, defined as total neurites length
per cell, by 74% compared to that of the 0.1% DMSO
control. The pyridones 11 f and 11a exhibited a relative
neurite outgrowth enhancement of 54 and 33%, respectively.
Apparently, the active small molecules display structural
similarity to farinosone A (3) which also contains a 4-
hydroxy-2-pyridone scaffold and bears a 4-hydroxyphenyl
moiety at C5 (Figure 1).
Pyridones with residues, which are bulkier than cyclo-
pentyl or longer than n-pentyl, at C3 were either inactive or
cytotoxic. For instance, 11b and 11i (see Figure S2a), having
longer alkyl chains (n-nonyl and n-heptyl, respectively), were
cytotoxic at 10 mm and were inactive at a concentration of
1
1
mm. Replacing the alkyl chains with aromatic rings yielded
1d and 11s and abolished the activity, thus demonstrating
that only limited hydrophobic bulk is tolerated at this position
for neuritogenic activity (Figure 2). Notably, replacing the
phenol substituent by a p-fluorophenyl group (compare 11g
and 11h with 11 f and 11e) led to the loss of neuritogenic
properties. The 2,4-dimethoxypyridines 14a,b and 14d,
embodying the same C3- and C5-appendages as those in the
active pyridones 11 f, 11e, and 11a, were either inactive or
cytotoxic at 10 mm (see Figure S2), thereby clearly highlight-
Scheme 1. Strategies for the synthesis of functionalized pyridones and
pyridines. DMP=Dess–Martin periodinane, TBAF=tetra-n-butylam-
monium fluoride, TBAI=tetra-n-butylammonium iodide, TBS=tert-
butyldimethylsilyl.
2
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In addition, the natural product
pyridovericin (4), which is sim-
ilar in structure to the neurite
outgrowth
inducing
com-
pounds identified by us, inhib-
[6d]
its tyrosine kinases.
These findings and struc-
tural similarities suggested that
the
4-hydroxy-2-pyridones
might target kinases. To inves-
tigate this possibility we pro-
filed 11a and 11e for the inhib-
ition of 67 kinases involved in
neurite
outgrowth
(see
[
12]
Table S1). Both compounds
almost completely inhibited
the kinase MAP4K4 (mito-
gen-activated protein kinase
kinase kinase kinase 4, also
termed HPK/GCK-like kinase
HGK or MEKKK4) at a con-
centration of 20 mm (see
Table S1). The siRNA-medi-
ated knock-down of MAP4K4
[12]
induces neurite outgrowth,
and therefore MAP4K4 inhib-
ition also might bear a similar
consequence. The dimethoxy-
pyridines 14a,b and 14d, bear-
ing the same substitution pat-
tern as the active pyridones
Figure 2. Influence of militarinone-inspired small molecules on neurite outgrowth in SH-SY5Y cells. Upon
initiation of differentiation, SH-SY5Y cells were incubated with the compounds at a concentration of 10 mm
or 0.1% DMSO and 200 ngmL BDNF as controls for 96 h. Cells were fixed, permeabilized, and stained
for DNA (blue) and microtubules (green) using DAPI and an anti-a-tubulin antibody and a secondary
antibody coupled to Alexa 488. Scale bar: 50 mm. Representative images (a) and the calculated relative
neurite outgrowth (b) are shown. Data are mean values Æs.d. (n=3). Mean neurite outgrowth values
were normalized to the mean neurite outgrowth of cells that were treated with 0.1% DMSO. Statistical
significance was analyzed by one-sample t-test (**: p<0.01, *: p<0.05). c) Structures of active and
inactive pyridones.
À1
(
11a and 11e,f), neither pro-
moted neurite outgrowth nor
inhibited MAP4K4 (see Fig-
ure S2 and Table S2). Further-
more, 11d,s and 11g,h, which
did not induce neurite out-
growth, were only very weak
ing the importance of the core pyridone scaffold for neuro-
trophic activity. At 1 mm the pyridines were inactive. These
data show that the 4-hydroxy-2-pyridone core in conjunction
with a 4-hydroxyphenyl residue at C5 is mandatory for
neurotrophic activity. Substitution at C3 is variable to
a limited extent and strongly influences the neuritogenic
properties. These observations led us to conclude that the
pharmacophore-determining neuritogenic activity is defined
by the 3-acyl-4-hydroxy-5-(4-hydroxyphenyl)pyridin-2(1H)-
one 15 (Figure 3). The close structural similarities of the
identified neuritogenic compounds (11a and 11e,f) indicate
a distinct mode of action.
inhibitors of MAP4K4 (see Table S2). The most active
pyridones, 11a, 11e, and 11 f, inhibited MAP4K4 with
IC50 values in the range of 1.9–3.6 mm and the potency of
MAP4K4 inhibition directly correlated with degree of
neuritogenic activity (Figure 3b).
This observed parallel in structure–activity correlation for
inhibition of MAP4K4 and neurite outgrowth induction
strongly supports the notion that the biological activity of
the 4-hydroxy-2-pyridones may be mediated by MAP4K4
inhibition. In comparison, biological evaluation of 1 revealed
activation of MAP and Akt kinase pathways as well as
[6e]
a potentiation of NGF-mediated SAPK/JNK1 activation.
Neuroprotection and neurite outgrowth have been linked
This finding suggests a different mode of action for 11e.
Further enzymatic analysis revealed an increase in IC50 values
for MAP4K4 inhibition by 11e with increasing ATP concen-
trations (Figure 4a,b). These findings are indicative of an
ATP-competitive mode of inhibition.
To characterize the binding of 11e to the kinase in detail,
the compound was co-crystallized with the kinase domain of
human wild-type MAP4K4. The crystal structure clearly
revealed that 11e binds in a type I fashion (Figure 4c). The
[
1c,11–13]
to the modulation of different kinases.
An RNAi-based
genetic screen in SH-SY5Y cells identified kinases which are
essential for growth-cone collapse, and either neurite retrac-
[12]
tion or neurite outgrowth. Moreover, kinase inhibitors can
promote neurite outgrowth from primary rat hippocampal
[
13]
cells.
Notably, the GSK3 inhibitor SB415286 possesses
neuritogenic and neuroprotective properties, and clearly
[14]
shares structural features with 11a,e and 11 f (Figure 3).
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Figure 3. Scaffold structures of 4-hydroxypyridone neurite outgrowth
enhancers and correlation between neurite outgrowth and MAP4K4
inhibition. a) The potential pharmacophore 15, 11a, and GSK3 inhib-
itor SB 415286. b) Correlation between neurite outgrowth and inhib-
ition of MAP4K4. Neurite outgrowth was determined in SH-SY5Y cells
at a concentration of 10 mm after treatment for 96 h. Mean neurite
outgrowth was normalized to the mean neurite outgrowth of cells that
were treated with 0.1% DMSO. Data are mean values (n=3) Æs.d.
pyridone core forms two hydrogen bonds to the kinase hinge
region, thus addressing the peptide backbone of Glu106 and
Cys108. The glycine-rich loop is folded over the inhibitor such
that the side-chain of Tyr36 partly shields the 4-hydroxy-2-
pyridone core of the ligand from the solvent, thereby
stabilizing inhibitor binding. Similar binding characteristics,
ligand-induced structural plasticity of the glycine-rich loop,
and binding interactions with the conserved Tyr36 have also
[15]
been reported for the Ser/Thr kinase p38a. The 4-hydroxy-
phenyl group forms an additional hydrogen bond to Lys54,
adjacent to the glycine-rich loop. This particular hydrogen
bond seems to be mandatory for an effective inhibition of
MAP4K4 and explains why the corresponding fluorinated
compounds 11g and 11h fail to inhibit MAP4K4 and there-
fore do not induce neurite outgrowth. In addition, the n-
pentyl chain of 11e resides in the hydrophobic front pocket
which is limited in space by the surrounding helices. Therefore
propyl (11 f) and cyclopentyl (11a) groups are accommo-
dated, however, spatially more demanding residues such as
aromatic moieties (11d,s) and longer alkyl chains are not. Our
structural observations within the MAP4K4 binding cleft are
consistent with findings of Crawford et al. on various
Figure 4. 11e is an ATP-competitive inhibitor of MAP4K4. a,b) Dose-
response data for MAP4K4 inhibition by 11e in presence of different
concentrations of ATP. MAP4K4 enzymatic activity was determined by
means of Z’-Lyte Kinase Ser/Thr 7 Assay. a) Dose-response curves.
Data are mean values (n=3) Æs.d. Data were fitted using four-
parameter Hill equation. b) Estimated IC50 values for MAP4K4 inhib-
ition by 11e at different ATP concentrations. c) The compound 11e
complexed with wild-type MAP4K4 (PDB code: 4RVT). Experimentally
determined electron density of 11e at 2.4 ꢂ resolution is displayed
(
2F ÀF map contoured at 1s). Hydrogen-bond interactions are
o
c
displayed by red dotted lines. The kinase domain is in the active DFG-
in conformation and compound 11e is caged by the glycine-rich loop
and flanked by Tyr36. Direct hydrogen-bond interactions are formed
between 11e and the hinge region by addressing the peptide backbone
of Glu106 and Cys108 as well as the side chain of Lys54 adjacent to
the glycine-rich loop. Color code: hinge region (orange), helix C
[16]
quinazoline-based inhibitors in complex with MAP4K4.
(turquoise), DFG motif (pink), glycine-rich loop (blue), 11e (yellow
Wang et al. reported on selective 4-amino-pyridopyrimidines
as MAP4K4 inhibitors and disclosed complex structures
revealing binding modes which translate into fairly different
structural conformations as the ligands are not strictly caged
sticks).
a hallmark of many neurodegenerative diseases and can lead
[
17]
[18]
by the glycine-rich loop.
to activation of this apoptotic cascade,
and the use of
Rubin et al. recently demonstrated that the structurally
unrelated kinase inhibitor kenpaullone inhibits the JNK-
dependent apoptotic cascade by simultaneously blocking
GSK3 and MAP4K4 activity and thereby increases survival
selective JNK inhibitors may circumvent the activation of this
pathway. JNK isoforms share high structural homology and
selective inhibition of JNK3 over JNK1 and JNK2 is desirable
because of the importance of JNK1 and JNK2 in neurogenesis
[3d]
[1c,18a]
of motor neurons.
Prolonged over-activation of JNKs is
and learning.
However, selective inhibition of JNK3 over
4
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JNK1 and JNK2 using competitive inhibitors is challenging.
Thus, the inhibition of an upstream kinase of the apoptotic
cascade, for example, MAP4K4, TAK, and MKK4, could
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inhibitor class which may serve as a starting point to develop
novel approaches for the treatment of neurodegenerative
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neuritogenic compounds selectively inhibit MAP4K4 kinase
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Keywords: alkaloids · biological activity · drug discovery ·
natural products · neurological agents
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Published online: && &&, &&&&
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!

Angewandte
Chemie
Communications
Biological Activity
No stress: Reported herein is the syn-
thesis of a militarinone-inspired 4-hy-
droxy-2-pyridone collection, its investiga-
tion for enhancement of neurite out-
growth, and the discovery of the stress
pathway kinase MAP4K4 as a target of the
discovered neuritogenic pyridones. The
findings support the notion that MAP4K4
may be a new target for the treatment of
neurodegenerative diseases.
P. Schrçder, T. Fçrster, S. Kleine,
C. Becker, A. Richters, S. Ziegler, D. Rauh,
K. Kumar, H. Waldmann*
&&&& — &&&&
Neuritogenic Militarinone-Inspired 4-
Hydroxypyridones Target the Stress
Pathway Kinase MAP4K4
Angew. Chem. Int. Ed. 2015, 54, 1 – 7
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!