Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors

Article abstract of DOI:10.1016/j.tet.2018.07.021

Autophagy is a catabolic process, which mediates degradation of cellular components and has important roles in health and disease. Therefore, small molecule modulators of autophagy are in great demand. Herein, we describe a phenotypic high-content screen for autophagy inhibitors, which led to the discovery of a dimethoxypyridine-based class of autophagy inhibitors, which derive from previously reported, natural product-inspired MAP4K4 inhibitors. Comprehensive structure-activity relationship studies led to a potent compound, and biological validation experiments indicated that the mode of action was upstream or independent of mTOR.

Full text of DOI:10.1016/j.tet.2018.07.021

Tetrahedron xxx (2018) 1e7
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors
Lucas Robke a , Tiago Rodrigues , Peter Schr o€ der , Daniel J. Foley ,
,
b
c
a
a
c
,
d
a,
e
,
**
, Herbert Waldmann a
, b, *
Gonçalo J.L. Bernardes , Luca Laraia
Max Planck-Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227, Dortmund, Germany
Department of Chemistry and Chemical Biology of the Technical University Dortmund, Otto-Hahn-Str. 6, 44227, Dortmund, Germany
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av Prof Egas Moniz, 1649-028, Lisboa, Portugal
Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW, Cambridge, UK
a
b
c
d
e
Technical University of Denmark, Department of Chemistry, Kemitorvet, Building 207, DK-2800, Kgs. Lyngby, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2018
Received in revised form
Autophagy is a catabolic process, which mediates degradation of cellular components and has important
roles in health and disease. Therefore, small molecule modulators of autophagy are in great demand.
Herein, we describe a phenotypic high-content screen for autophagy inhibitors, which led to the dis-
covery of a dimethoxypyridine-based class of autophagy inhibitors, which derive from previously re-
ported, natural product-inspired MAP4K4 inhibitors. Comprehensive structure-activity relationship
studies led to a potent compound, and biological validation experiments indicated that the mode of
action was upstream or independent of mTOR.
4
July 2018
Accepted 9 July 2018
Available online xxx
Keywords:
Autophagy
©
2018 Elsevier Ltd. All rights reserved.
Phenotypic screening
High-throughput screening
Natural products
Medicinal chemistry
1
. Introduction
infections and aging. Although primarily a protective pathway,
autophagy can also be involved in cell death. Due to its involvement
in these (patho)physiological processes, autophagy is an essential
mechanism for the development, homeostasis and survival of cells.
Autophagy plays a crucial role in the degradation of protein ag-
gregates, which cause several neurodegenerative diseases,
including Alzheimer's, Huntington's and Parkinson's diseases [4e6].
Additionally, there is evidence that autophagy is involved in the
prevention of cancer by degrading toxic metabolites in the cell [7,8].
However, it is also reported to promote the survival of cancer cells
under conditions of nutrient deprivation [9e13]. Consequently,
whereas an upregulation of autophagy might serve as a preventative
strategyagainst cancer, autophagy inhibition is a potential approach
for cancer therapy after its onset. Since many unanswered questions
remain regarding the dual role of autophagy in physiology and
pathophysiology, there is a strong interest in a deeper under-
standing of its mechanisms. Selective autophagy modulators are
valuable tools to study this process at different stages of disease
Macroautophagy (hereafter autophagy) is a highly conserved
cellular process in eukaryotes, which mediates the degradation of
cellular components within specialised subcellular compartments
(
autophagosomes) [1e3]. Autophagosomes derive from the phag-
ophore, a double membrane structure that engulfs cellular com-
ponents that are to be recycled. The autophagosomes subsequently
fuse with lysosomes to form autophagolysosomes, in which
degradation carried out by lysosomal enzymes takes place. This
multistep process is tightly regulated by upstream signaling and is
modulated by growth factors, the concentration of amino acids and
the energy level of the cell. The purpose of this degradation is not
only to compensate for a temporary lack of nutrients but also to
eliminate dispensable, long-lived proteins, protein aggregates and
organelles. Moreover, autophagy has been linked to microbiological
[
14e17]. In this regard, phenotypic screening offers a useful starting
*
Corresponding author. Max-Planck-Institute of Molecular Physiology, Dort-
mund, Germany.
* Corresponding author. Technical University of Denmark, Department of
point for the identification of new biologically active compounds by
representing a disease relevant system [18e21].
*
Chemistry, Kemitorvet, Building 207, DK-2800, Kgs. Lyngby, Denmark.
E-mail addresses: luclar@kemi.dtu.dk (L. Laraia), herbert.waldmann@mpi-
dortmund.mpg.de (H. Waldmann).
Herein we report the identification and validation of
dimethoxypyridine-containing autophagy inhibitors (DMPs)
https://doi.org/10.1016/j.tet.2018.07.021
0
040-4020/© 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

2
L. Robke et al. / Tetrahedron xxx (2018) 1e7
Fig. 1. Structural development of an autophagy inhibitor from a neuritogenic MAP-kinase inhibitor. Dimethoxypyridine 2 represents a structural precursor for the neuritogenic
MAP-kinase inhibitor 1. By varying the substitution pattern of 2 and maintaining the alcohol adjacent to the pyridine, autophagy inhibitors (including 3) were identified.
identified through a phenotypic screen. The DMPs were originally
synthesised in the context of a biology-oriented synthesis (BIOS)
accumulates at autophagosomes under starvation conditions,
which can be visualised as fluorescent puncta. The puncta are
detected by automated fluorescence microscopy and quantified
using automated image analysis [25]. Incubating the cells with
Earle's balanced salt solution (EBSS) induces autophagy through
amino acid starvation. Chloroquine (CQ), which is also included in
the starvation medium, inhibits the fusion of autophagosomes with
lysosomes, preserving eGFP fluorescence by inhibiting its degra-
dation by the autolysosomes. This increases the dynamic range of
the assay [29].
[
22,23] effort to synthesise pyridones based on the natural product
Militarone, which were found to be MAPK inhibitors [24]. We
describe a comprehensive SAR analysis, detailing the requirements
for activity, in addition to biological validation experiments.
2
. Results
2.1. Phenotypic screening for autophagy inhibitors
As part of our ongoing programme to identify autophagy in-
hibitors [30e32], we identified a 2,4-dimethoxypyridine (DMP)
based compound class, which inhibited starvation-induced auto-
phagy (Fig. 1). These compounds are synthetic precursors of a
Militarinone-inspired, neuritogenic compound collection. The
finding that synthetic precursors have different biological activity
underlines the utility of the BIOS concept [33]. We sought to explore
the SAR of this compound class and optimised them for autophagy
inhibition, leading to a compound we termed DMP-1 (Fig. 1).
For monitoring and quantifying autophagy, MCF7 cells stably
transfected with green fluorescent protein tagged light chain 3
eGFP-LC3) were employed, as developed by Balgi et al. [25e27]
Upon autophagy induction, the ubiquitin-like cytosolic protein LC3
is conjugated to phosphatidylethanolamine (PE) and consequently
recruited to the autophagosomal membrane [28]. Whereas eGFP-
LC3, and by association the fluorescence signal, is distributed
throughout the cytoplasm without autophagy induction, eGFP-LC3
(
ꢁ
2
ꢁ
Scheme 1. General scheme for the synthesis of derivatives of DMP-1 and other analogues starting from precursor 4. a) s-BuLi, THF, ꢀ78 C, 1 h, then R NCO, ꢀ78 C / rt, overnight;
1
ꢁ
2
ꢁ
b) R B(OH)
CH Cl
2
, Pd(PPh
3
)
4
, dppf, Na
2
3
CO , PhMe/EtOH,
D
, 16 h; c) s-BuLi, THF, ꢀ78 C, 1 h, then R CHO, ꢀ78 C / rt, overnight; d) MnO
2 2 2
, CH Cl , rt, 2 d or DesseMartin periodinane,
, NaOtBu, PhMe, 80 C, overnight. dppf ¼ 1,1 -Bis(diphenylphosphino)ferrocene, THF ¼ tetrahydrofuran, dba ¼ dibenzylidineacetone.
3
4
ꢁ
0
2
2
, rt, 2 h; e) R R NH, tBuXPhos, Pd
2
dba
3
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

L. Robke et al. / Tetrahedron xxx (2018) 1e7
3
Table 1
Inhibition of starvation-induced autophagy determined for selected analogues with varying R1 substituents. Where no compound number is stated and the IC50 data is
replaced by a dashed line, the compound has not been synthesised. IC50 data is provided as the mean ± SD, n ꢂ 3. Where no SD is given, n ¼ 2. For a full list of analogues and
activities see Supplementary Tables 1e6.
Entry
1
R1
Alcohol
Alcohol IC50
[m
M]
Ketone
Ketone IC50 [mM]
12
1.3 ± 0.5
e
2
3
DMP-1 (3)
1.9 ± 0.8
2.2 ± 2.4
13
15
6.5 ± 3.2
14
4.1
4
5
6
7
16
18
20
22
2.5 ± 0.5
3.8 ± 1.0
3.9
17
19
21
3.9 ± 0.4
e
>10
e
4.4 ± 0.7
8
9
23
25
4.6 ± 0.6
5.5 ± 1.4
24
8.8 ± 0.1
e
10
11
12
26
28
30
6.5 ± 1.6
6.6 ± 1.0
7.0 ± 0.6
27
29
31
4.4 ± 1.6
8.3 ± 0.8
7.3 ± 2.1
13
32
9.2 ± 1.0
e
1
1
1
4
5
6
33
34
35
>10
>10
>10
e
e
e
17
36
>10
37
>10
1
1
8
9
Br
38
40
>10
>10
39
41
>10
3.0
20
42
>10
e
(
continued on next page)
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

4
L. Robke et al. / Tetrahedron xxx (2018) 1e7
Table 1 (continued )
Entry
R1
Alcohol
Alcohol IC50
[m
M]
Ketone
Ketone IC50
4.2
[mM]
2
1
2
43
>10
44
2
45
>10
e
2
.2. Synthesis
with respect to the naphthalene on 3, whilst retaining hydropho-
bicity also reduced the potency, suggesting that there are favoured
orientations for the hydrophobic interactions (Table 2, entries 5e7).
Aromatics substituted with non-lipophilic heteroatoms were
generally less active (Table 2, entries 8 and 9). Amides bearing
phenyl or benzyl substituents proved to be inactive (Table 2, entries
10 and 11). Any further attempts to include more polar substituents
In addition to the 2,4-dimethoxypyridines that were prepared in
the original study [24], further compounds of this class were syn-
thesised, leading to >200 analogues (Scheme 1). The synthesis of
compounds 5e11 began from 2,4-dimethoxypyridine, which was
reacted with bromine in acetic acid to give the versatile precursor
2
3
,5-dibromo-2,4-dimethoxypyridine 4, which was subsequently
at R was not successful (Supplementary Tables 1e5).
used in the synthesis of most DMP analogues (Scheme 1). Bromine-
lithium exchange with s-BuLi, followed by the addition of either an
isocyanate or aldehyde electrophile, gave the amides 5 and the
secondary alcohols 7 respectively. Subsequent SuzukieMiyaura
couplings yielded the analogues of type 6 and 8. Oxidation of the
secondary alcohol of compounds 7 or 8 with DesseMartin peri-
In final SAR studies, further variations of substituents on and
around the pyridine ring were investigated. The synthesis of the
different scaffold variations is detailed in Supplementary Fig. 1. The
two methoxy groups were both required for optimal activity, with
the 2-methoxy contributing to a greater extent than the 4-methoxy
substituent (Table 3, entries 2e4). The pyridine nitrogen was
dispensable for activity but was retained, as it was expected to
increase solubility (Table 3, entry 5). Surprisingly, the presence of a
2
odinane or MnO gave the ketones 10 and 9 respectively. In order to
access products with different substituents at the 5-position of the
pyridine ring, BuchwaldeHartwig couplings were used to access
products of type 11.
5
methoxy group at R rendered the compound inactive (Table 3,
entry 6).
Overall, we generated a library that allowed us to thoroughly
explore the SAR around the dimethoxypyridine scaffold (Fig. 2). We
selected DMP-1 (3) as a representative inhibitor to further validate
this class of compounds as inhibitors of autophagy due to its
favourable potency, solubility and ease of synthesis.
2
.3. Structure activity relationship (SAR)
1
2
SAR was primarily investigated at the R and R variable groups
of the 2,4-dimethoxypyridines, along with further variation around
the pyridine scaffold (see later). In each case, generally both the
alcohol and ketone analogues at position 3 of the parent 2,4-
dimethoxypyridines were investigated. Initially, variation at R
was investigated, whilst R was fixed with a 2-naphthalenyl sub-
stituent (Table 1). Substitution at R was found to be crucial for
activity, and nitrogenous heterocycles bearing hydrophobic sub-
stituents provided the most active compounds (Table 1, entries
2.4. Biological validation
1
2
DMP-1 inhibited starvation-induced autophagy potently and
dose-dependently, as assessed by the reduction in LC3 puncta in
our primary screening assay (Fig. 3A and B). To further validate
DMP-1 as an autophagy inhibitor, we studied its effect on the key
autophagy markers: LC3 lipidation and p62 degradation. After the
induction of autophagy, LC3 is lipidated with phosphatidyletha-
nolamine to form LC3-II [28], an effect that should be reversed by
an autophagy inhibitor that does not target autophagosome-
lysosome fusion. DMP-1 inhibited LC3 lipidation dose-
dependently as assessed by western blot analysis (Fig. 3C) p62
(also known as sequestome 1) acts as a chaperone to target proteins
for degradation by the autophagic machinery, where it is also
degraded. Autophagy inhibitors inhibit the degradation of p62
when autophagy is induced. DMP-1 inhibited the degradation of
p62, confirming its ability to inhibit autophagic flux (Fig. 3C).
As autophagy is a cytoprotective mechanism that is activated in
conditions of cellular stress, autophagy inhibition is reported to
render cells more sensitive to the effects of starvation [34]. In line
with this expectation, DMP-1 selectively inhibited the growth of
starved MCF7-eGFP-LC3 cells compared to fed cells as assessed by a
WST-1 proliferation assay (Fig. 3D). It has been reported that
autophagy inhibition causes cells to die via apoptosis [35]. MCF7
cells treated with DMP-1 showed apoptotic cell death under fed as
well as under starved conditions, as assessed by live cell imaging of
a caspase 3/7 selective probe, which releases a DNA intercalating
dye that labels the nuclei of apoptotic cells (Fig. 3E). In order to
delineate the mode of action of the DMPs further, their ability to
inhibit autophagy induced by the pharmacological inhibition of
mTOR by Rapamycin was assessed in MCF7-eGFP-LC3 cells. Neither
1
1
e4). For the 1-N-benzylpyrazole-substituted analogues investi-
gated (Table 1, entries 1, 2, 7 and 9), substitution on the benzyl
component significantly influenced potency. Phenols exhibited
slightly reduced potency (Table 1, entries 5e6). Some substituted
phenyls were active in the same concentration range as the phenols
(Table 1, entries 8 and 11e12), whilst others were inactive (entry
17). However, smaller and/or more polar substituents than the 1-N-
benzylpyrazole ring system were not sufficient for activity (Table 1,
entries 14e16 and 19e22). The analogue where the aromatic ring
substituent at R1 was replaced with a Br (Table 1, entry 18) was
inactive.
2
Next, we turned our attention to varying the R group of the 2,4-
dimethoxypyridines, whilst keeping the 1-benzyl-1H-pyrazol-4-yl
1
2
substituent constant at R . Favourable substituents at R were
mostly bulky and lipophilic (Table 2, entries 1e7), and were linked
to the pyridine either by a hydroxymethyl group, a ketone, or an
amide. The hydroxymethyl group seems to be favoured in terms of
potency when compared to the carbonyl (Table 2, entry 1). This
trend was found to be consistent throughout the analogue series
studied, with the exception of two pairs of compounds (Table 1,
entries 19 and 21). Small, fluorine-containing aromatics retained
2
activity (Table 2, entries 2e3). Efforts to reduce the size of R by
replacing the naphthalene ring system in the lead compounds
(Table 1, entry 1e2) with a phenyl acetylene (Table 2, entry 4) led to
2
a slightly less active analogue. Varying the size and orientation of R
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

L. Robke et al. / Tetrahedron xxx (2018) 1e7
5
Table 2
2
Inhibition of starvation-induced autophagy determined for selected analogues with variations at R . Where no compound number is stated and the IC50 data is replaced by a
dashed line, the compound has not been synthesised. IC50 data is provided as the mean ± SD, n ꢂ 3. Where no SD is given, n ¼ 2. For a full list of analogues and activities see
Supplementary Tables 1e6.
entry
1
R2
Alcohol
Alcohol IC50
[m
M]
Carbonyl
Carbonyl IC50 [mM]
DMP-1 (3)
1.9 ± 0.8
46
6.5 ± 3.2
2
3
4
5
47
48
50
2.2 ± 2.0
2.4 ± 0.6
3.3 ± 2.5
e
e
49
6.4 ± 0.6
e
51
53
4.0 ± 1.0
6
52
4.2 ± 1.4
>10
7
8
54
55
5.5 ± 1.5
5.9 ± 1.8
e
56
3.5 ± 0.3
9
57
10 ± 6.0
e
1
0
1
e
e
58
59
>10
>10
1
DMP-1 nor any of its analogues inhibited Rapamycin-induced
autophagy, which suggests that they act upstream or indepen-
dently of mTOR (data not shown).
To identify the molecular target(s) of DMP-1, we employed a
newly developed machine learning approach using random forest
technology and the CATS2 topological pharmacophore descriptors
[
36,37]. The built regression random forest models use an ensemble
of decision trees to provide a consensus affinity value prediction
)] for a range of >1000 human drug
Table 3
[pAffinity ¼ - log(IC50 or K
D
Inhibition of starvation-induced autophagy determined for selected analogues with
variation on and around the pyridine ring. IC50 data is provided as the mean ± SD,
n ꢂ 3. For a full list of analogues and activities see Supplementary Tables 1e6.
targets extensively curated from ChEMBL [38]. Through these
models, the cannabinoid receptor 1 (CB1) was predicted as
macromolecular target for DMP-1 (pAffinity ¼ 6.8) with high con-
fidence. Indeed, testing of DMP-1 in a CB1 functional assay (Cerep,
France) revealed moderate antagonist effect (IC50 ¼ 3.1 ± 0.08
¼ 0.32 M; Fig. 4), yet fully in line with our regression model
experimental pAffinity ¼ 6.5). A radioligand displacement assay
further confirmed binding of DMP-1 to CB1 (IC50 ¼ 4.3 M,
¼ 3.2 M, nHill ¼ 1.1).
CB1 has previously been linked to autophagy [39], and CB1
mM;
K
(
B
m
m
K
i
m
R3
R4
R5
Entry
Compound
X
IC50 [mM]
antagonists have been suggested to weakly induce autophagy,
which is contradictory to our findings. We tested a CB1 antagonist
(Rimonabant) and an inverse agonist (Ipinabant) for their ability to
inhibit starvation-induced autophagy. Rimonabant weakly inhibi-
1
2
3
4
5
6
DMP-1 (3)
OMe
H
OMe
H
OMe
H
OMe
OMe
H
H
OMe
OMe
H
H
H
H
H
OMe
N
N
N
N
CH
N
1.9 ± 0.8
3.7 ± 1.5
6.2 ± 1.2
6.2 ± 2.2
1.8 ± 0.8
>10
60
61
62
63
64
ted starvation-induced autophagy (IC50 ¼ 4.1 ± 0.7
mM), while Ipi-
nabant did not. Considering the fact that both compounds target
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

6
L. Robke et al. / Tetrahedron xxx (2018) 1e7
Fig. 2. Summary of the explored SAR of the dimethoxypyridine compound class in starvation-induced autophagy.
Fig. 3. Phenotypic validation of DMP-1 as an autophagy inhibitor. A: Fluorescence microscopy images of the starvation-induced autophagy screen for inhibition of eGFP-LC3
accumulation. Fed ¼ DMSO control incubated in full media (MEM) as positive control. Starved ¼ autophagy was induced by amino acid withdrawal (EBSS). DMP-1 reverts the
phenotype in a dose dependent manner. Scale bar ¼ 50
mm. B: Dose-dependent inhibition of amino acid starvation induced eGFP-LC3 accumulation by DMP-1. Data is mean ± SD,
n ꢂ 3; representative graph shown. C: Inhibition of LC3 lipidation and p62 degradation by DMP-1 in MCF7-eGFP-LC3 cells. Starvation-induced autophagy induces lipidation of LC3-I
to LC3-II and degradation of p62. DMP-1 inhibits both effects in a dose-dependent manner. n ꢂ 3, representative blot shown. D and E: DMP-1 induces cell death in starved cells by
means of apoptosis. D: Treatment of MCF7-eGFP-LC3 cells under starved conditions (EBSS) or fed conditions (MEM) with DMP-1. Under starvation conditions survival is reduced.
Cytotoxicity was assessed by means of a WST-1 assay. Data points are mean ± SD, n ꢂ 3, representative graphs shown. E: DMP-1 dose dependently induces apoptosis in starved cells.
Apoptosis was assessed by using a selective caspase 3/7 probe. The experiment was performed with an Incucyte Zoom live-cell imaging device. Noc. ¼ Nocodazole (10
presented as a percent of the DMSO control. Data points are mean ± SD, n ꢂ 3, representative bars shown.
mM). Data are
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021

L. Robke et al. / Tetrahedron xxx (2018) 1e7
7
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2018.07.021.
References
[
[
[
[
1] D.C. Rubinsztein, T. Shpilka, Z. Elazar, Curr. Biol. 22 (2012) 34.
2] D.J. Klionsky, Science 290 (2000) 1717e1721.
3] P. Boya, F. Reggiori, P. Codogno, Nat. Cell Biol. 15 (2013) 713e720.
4] Z. Berger, B. Ravikumar, F.M. Menzies, L.G. Oroz, B.R. Underwood,
M.N. Pangalos, I. Schmitt, U. Wullner, B.O. Evert, C.J. O'Kane, et al., Hum. Mol.
Genet. 15 (2006) 433e442.
[
[
5] B. Ravikumar, Hum. Mol. Genet. 11 (2002) 1107e1117.
6] J.L. Webb, B. Ravikumar, J. Atkins, J.N. Skepper, D.C. Rubinsztein, J. Biol. Chem.
278 (2003) 25009e25013.
[7] H.-Y. Chen, E. White, Canc. Prev. Res. 4 (2011) 973e983.
Fig. 4. DMP-1 is
a
cannabinoid
¼ 0.32 M, n ¼ 2. Control: AM281, IC50 ¼ 28 nM. Binding assay:
M, nHill ¼ 1.1, 2. Control: R(þ)-WIN-55,212-2,
1
receptor antagonist. Functional assay:
[8] E. White, C. Karp, A.M. Strohecker, Y. Guo, R. Mathew, Curr. Op. Cell Biol. 22
(2010) 212e217.
[9] R. Mathew, C.M. Karp, B. Beaudoin, N. Vuong, G. Chen, H.-Y. Chen, K. Bray,
A. Reddy, G. Bhanot, C. Gelinas, et al., Cell 137 (2009) 1062e1075.
10] K. Degenhardt, R. Mathew, B. Beaudoin, K. Bray, D. Anderson, G. Chen,
C. Mukherjee, Y. Shi, C. G eꢀ linas, Y. Fan, et al., Canc. Cell 10 (2006) 51e64.
IC50 ¼ 3.1 ± 0.08
IC50 ¼ 4.3 M,
IC50 ¼ 0.53 M.
m
M; K
¼ 3.2
B
m
m
K
i
m
n
¼
m
[
[
[
11] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646e674.
12] A.C. Kimmelman, Gend. Dev. 25 (2011) 1999e2010.
CB1 with nanomolar potency, but only show moderate autophagy-
modulating capabilities, it is unlikely that the ability of DMP-1 to
inhibit autophagy stems only from its ability to inhibit CB1. Addi-
tionally, DMP-1 was also tested in a full kinase panel at Life Tech-
nologies (Thermo Fischer). No kinase was inhibited more than 30%
[13] K.S. Choi, Exp. Mol. Med. 44 (2012) 109e120.
[
14] D.C. Rubinsztein, P. Codogno, B. Levine, Nat. Rev. Drug Discov. 11 (2012)
09e730.
15] B. Levine, G. Kroemer, Cell 132 (2008) 27e42.
7
[
[16] C.B. Lebovitz, L. DeVorkin, D. Bosc, K. Rothe, J. Singh, M. Bally, X. Jiang,
R.N. Young, J.J. Lum, S.M. Gorski, Autophagy 11 (2015) 1949e1952.
at a concentration of 5 mM, including the MAP4K4, the target of
[17] D.C. Rubinsztein, J.E. Gestwicki, L.O. Murphy, D.J. Klionsky, Nat. Rev. Drug
Militarinone derivative 1. Further efforts to elucidate the molecular
target(s) of DMP-1 are currently ongoing and will be reported in
due course.
Discov. 6 (2007) 304e312.
[18] J.G. Moffat, J. Rudolph, D. Bailey, Nat. Rev. Drug Discov. 13 (2014) 588e602.
[19] D.C. Swinney, J. Anthony, Nat. Rev. Drug Discov. 10 (2011) 507e519.
[
20] A. Gough, T.Y. Shun, D. Lansing Taylor, M. Schurdak, Methods 96 (2016)
1
2e26.
3
. Conclusion
[21] C.J. O'Connor, L. Laraia, D.R. Spring, Chem. Soc. Rev. 40 (2011) 4332e4345.
[
[
[
[
22] M. Kaiser, S. Wetzel, K. Kumar, H. Waldmann, Cell. Mol. Life Sci. 65 (2008)
186e1201.
1
We reported the discovery, development and biological valida-
23] S. Wetzel, R.S. Bon, K. Kumar, H. Waldmann, Angew. Chem. Int. Ed. 50 (2011)
10800e10826.
24] P. Schr o€ der, T. F o€ rster, S. Kleine, C. Becker, A. Richters, S. Ziegler, D. Rauh,
K. Kumar, H. Waldmann, Angew. Chem. Int. Ed. 54 (2015) 12398e12403.
25] A.D. Balgi, B.D. Fonseca, E. Donohue, T.C.F. Tsang, P. Lajoie, C.G. Proud, I.R. Nabi,
M. Roberge, PLoS One 4 (2009), e7124.
[26] J.V. Peppard, C. Rugg, M. Smicker, C. Dureuil, B. Ronan, O. Flamand, L. Durand,
B. Pasquier, Curr. Chem. Genomics Transl. Med. 8 (2014) 3e15.
27] G. Konstantinidis, S. Sievers, Y.-W. Wu, Methods in Molecular Biology,
tion of 2,4-dimethoxypyridine-based autophagy inhibitors. This
compound class was developed from Militarinone-inspired
MAP4K4 inhibitors [24], demonstrating the versatility of the BIOS
principle for the development of biologically active small mole-
cules. The most potent compound, DMP-1, inhibited starvation but
not rapamycin-induced autophagic flux, suggesting that it acts
upstream or independently of mTOR. Computational target iden-
tification correctly identified the CB1 receptor as a molecular target
of DMP-1, however this could not be validated using clinically
approved CB1 antagonists or inverse agonists, highlighting the
great challenge that target ID can pose in phenotypic screening
campaigns. Nonetheless, DMP-1 and analogues thereof expand the
toolkit of autophagy inhibitors available to study this highly com-
plex process and may provide new starting points for drug dis-
covery programmes in cancer or neurodegenerative diseases.
[
Humana Press, 2018.
[28] N. Mizushima, T. Yoshimori, Y. Ohsumi, Annu. Rev. Cell Dev. Biol. 27 (2011)
07e132.
1
[
29] J. Zhou, S.-H. Tan, V. Nicolas, C. Bauvy, N.-D. Yang, J. Zhang, Y. Xue, P. Codogno,
H.-M. Shen, Cell Res. 23 (2013) 508e523.
[30] L. Laraia, K. Ohsawa, G. Konstantinidis, L. Robke, Y.-W. Wu, K. Kumar,
H. Waldmann, Angew. Chem. Int. Ed. 56 (2017) 2145e2150.
[
[
31] L. Robke, L. Laraia, M.A. Carnero Corrales, G. Konstantinidis, M. Muroi,
A. Richters, M. Winzker, T. Engbring, S. Tomassi, et al., Angew. Chem. Int. Ed.
56 (2017) 8153e8157.
32] L. Robke, Y. Futamura, G. Konstantinidis, J. Wilke, H. Aono, Z. Mahmoud,
N. Watanabe, Y.-W. Wu, H. Osada, L. Laraia, et al., Chem. Sci. 9 (2018)
3
014e3022.
Acknowledgements
[33] L. Laraia, H. Waldmann, Drug Discov. Today 23 (2017) 75e82.
[
[
34] J. Liu, H. Xia, M. Kim, L. Xu, Y. Li, L. Zhang, Y. Cai, H.V. Norberg, T. Zhang,
T. Furuya, et al., Cell 147 (2011) 223e234.
35] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Nat. Rev. Mol. Cell Biol. 8
(2007) 741e752.
This research was supported by the Max-Planck-Gesellschaft.
L.R. is grateful to the Boehringer Ingelheim Fonds for a fellowship.
L.L. is grateful to the Alexander von Humboldt Foundation for a
fellowship. T.R. is a Marie Skłodowska-Curie Fellow (Grant 743640).
D.J.F. is grateful to the European Commission for a Marie Skło-
dowska-Curie European Fellowship (Grant 794259). G.J.L.B. ac-
knowledges Fundaç ~a o Ci e^ ncia Tecnologia (iFCT), Royal Society
[36] T. Rodrigues, M. Werner, J. Roth, E.H.G. da Cruz, M.C. Marques, P. Akkapeddi,
S. Lobo, A. Koeberle, F. Corzana, E.N. da Silva Júnior, O. Werz, G.J.L. Bernardes,
Chem. Sci. (2018). Accepted.
[
37] M. Reutlinger, C.P. Koch, D. Reker, N. Todoroff, P. Schneider, T. Rodrigues,
G. Schneider, Mol. Inf. 32 (2013) 133e138.
38] A.P. Bento, A. Gaulton, A. Hersey, L.J. Bellis, J. Chambers, M. Davies, F.A. Krüger,
Y. Light, L. Mak, S. McGlinchey, et al., Nucleic Acids Res. 42 (2014)
D1083eD1090.
[
(
URF) and the European Research Council (ERC StG TagIt) for
funding.
[39] C. Hiebel, T. Kromm, M. Stark, C. Behl, J. Neurochem. 131 (2014) 484e497.
Please cite this article in press as: L. Robke, et al., Discovery of 2,4-dimethoxypyridines as novel autophagy inhibitors, Tetrahedron (2018),
https://doi.org/10.1016/j.tet.2018.07.021