Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype Targeting Lipid Kinase VPS34

Article abstract of DOI:10.1002/anie.201703738

Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small-molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy-induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

Full text of DOI:10.1002/anie.201703738

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Phenotypic Identification of a Novel Autophagy Inhibitor
Chemotype Targeting Lipid Kinase VPS34
Authors: Herbert Waldmann, Lucas Robke, Luca Laraia, Marjorie A.
Carnero Corrales, Georgios Konstantinidis, Makoto Muroi,
André Richters, Michael Winzker, Tobias Engbring, Stefano
Tomassi, Nobumoto Watanabe, Hiroyuki Osada, Daniel Rauh,
Yaowen Wu, and Julian Engel
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703738
Angew. Chem. 10.1002/ange.201703738
Link to VoR: http://dx.doi.org/10.1002/anie.201703738
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10.1002/anie.201703738
Angewandte Chemie International Edition
Phenotypic Identification of a Novel Autophagy Inhibitor Chemotype
Targeting Lipid Kinase VPS34
Lucas Robkea,b, Luca Laraiaa, Marjorie A. Carnero Corralesa,b, Georgios
Konstantinidisc, Makoto Muroid,e, André Richtersb, Michael Winzkera,b, Tobias
Engbringb, Stefano Tomassib, Nobumoto Watanabee,f, Hiroyuki Osadad,e,
Daniel Rauhb, Herbert Waldmanna,b*, Yao-Wen Wuc, Julian Engelb
a: Max-Planck-Institute of Molecular Physiology, department of Chemical
Biology, Otto-Hahn-Str. 11, 44227 Dortmund (Germany); b: Faculty of
Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 4a,
44227 Dortmund (Germany); c: Chemical Genomics Centre of the Max-Planck-
Society, Otto-Hahn-Str. 15, 44227 Dortmund (Germany); d: Chemical Biology
Research Group, RIKEN CSRS, 2-1, Hirosawa, Wako, Saitama 351-0198
(Japan), e: RIKEN-Max Planck Joint Research Division for Systems Chemical
Biology, RIKEN CSRS, 2-1, Hirosawa, Wako, Saitama 351-0198 (Japan); f:
Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, 2-1, Hirosawa,
Wako, Saitama 351-0198 (Japan).
* Herbert.Waldmann@mpi-dortmund.mpg.de
Keywords: Autophagy, VPS34, kinase inhibitor, aminopyrimidine, privileged
scaffold, phenotypic assay,
1
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10.1002/anie.201703738
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Autophagy is a critical regulator of cellular homeostasis and metabolism.
Interference with this process is considered a new approach for the
treatment of disease, in particular cancer and neurological disorders.
Therefore, novel small molecule autophagy modulators are in high
demand. We describe the discovery of autophinib, a potent autophagy
inhibitor with a novel chemotype. Autophinib was identified by means of
a phenotypic assay monitoring formation of autophagy-induced puncta
indicating accumulation of lipidated cytosolic protein LC3 on the
autophagosomal membrane. Target identification and validation revealed
that autophinib inhibits autophagy induced by starvation or Rapamycin
by targeting the lipid kinase VPS34.
Autophagy is a conserved eukaryotic pathway which mediates the degradation
of cellular components within specialized subcellular compartments, termed
autophagosomes (SI-Figure 1).[1] Autophagy plays a crucial role in the
degradation of protein aggregates involved in several neurodegenerative
diseases,[2] and promotes the survival of cancer cells.[3] Novel small molecule
autophagy modulators may offer new opportunities for drug discovery[4–6] and
to decipher the underlying molecular mechanisms of this important cellular
process.[7]
Target-agnostic cellular assays monitoring changes in phenotype followed by
target identification and validation offer unbiased opportunities to discover
novel chemotypes and cellular targets linked to the biological process of
interest by means of phenotypic changes.[8]
2
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10.1002/anie.201703738
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Herein we report the discovery of a potent autophagy inhibitor by means of a
phenotypic screening approach monitoring small molecule induced impairment
of autophagy.[9] The compound, termed autophinib, targets the lipid kinase
Vacuolar Protein Sorting 34 (VPS34)[10] which is a promising target for selective
autophagy modulation.[11]
To identify novel autophagy inhibitors, we employed a previously described
cellular assay.[9,13] In brief, autophagy was induced in MCF7 cells stably
expressing eGFP-LC3 (enhanced green fluorescent protein tagged light
chain 3; MCF7-LC3) by amino acid starvation using Earle’s balanced salt
solution (EBSS), or by inhibition of mTOR using Rapamycin. Accumulation of
eGFP-LC3 on autophagosomes is visualized and quantified as formation of
puncta by means of automated microscopy and image analysis.[13]
Screening of ca. 160.000 compounds identified a class of aminopyrimidines
which dose-dependently inhibited autophagosome formation (e.g. 1; Figure 1
and SI-Table 1). Compounds active under both starvation- and rapamycin-
induced autophagy were of particular interest, since they are expected to act
downstream of mTOR in canonical autophagy regulation or in non-canonical
autophagy independent of mTOR.
Autophagy inhibitor 1 and analogues thereof were previously developed as
inhibitors of the protein kinase TBK1.[12] However, TBK1 inhibition would result
in inhibition of autophagosome maturation, increased autophagosome and
eGFP-LC3 puncta formation and, therefore, cannot explain the activity of the
hit class in the screen.[14]
3
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Figure 1: Phenotypic screen for inhibition of LC3 accumulation. A: Dose-
dependent inhibition of amino acid starvation induced eGFP-LC3 accumulation
by 1. B: Dose dependent inhibition of Rapamycin induced eGFP-LC3
accumulation by 1. C: Fluorescence microscopy images of the starvation
induced autophagy screen. D: Fluorescence microscopy images of the
Rapamycin-induced autophagy screen. E: Structure of compound 1. Scale
bar = 50 µm. Data is mean ± SD, n ≥ 3, representative graphs shown.
For target identification, development of a structure-activity relationship (SAR)
and identification of more potent inhibitors, 46 analogues were synthesized as
shown in SI-Figure 2. Introduction of small substituents lacking amino groups
4
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10.1002/anie.201703738
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at the 6-position (R3) led to an increase in inhibitory activity as compared to
compound 1 (Table 1 and SI-Tables 1-5). This finding further supported the
notion that TBK1 was not the target responsible for autophagy inhibition, as the
protonated piperazine moiety generates a key salt bridge with the Asp157 in
TBK1 which leads to enhanced potency toward TBK1.[12] Other smaller amine-
containing inhibitors, (e.g. Table 1, entry 6) were also less potent in comparison
to the chloro- or methyl-substituted analogues. Based on these observations a
chloro-substituent at the 6-position was chosen for further compound
improvement.
Replacement of the pyrazole in the 4-position led to loss of activity (Table 1,
entries 9 - 12). By analogy to findings of Rauh et al.,[15] this finding suggests
that the pyrazole may be involved in a key hydrogen bond to the kinase hinge
region. A methyl group on the pyrazole was most advantageous for activity,
whilst the larger tert-butyl group (Table 1, entry 7) led to a significant drop in
cellular activity.
At the 2-position (R1) an ether-oxygen is favored for activity as replacement by
a thioether (SI-Table 3, entry 2) or an amine (Table 1, entry 18) decreased
activity. Replacement of the nitro group at R1 led to compounds with a
sulfonamide (Table 1, entry 13) and a nitrile group (Table 1, entry 15) at the
para position respectively, for which activity was only slightly lower. Replacing
the phenyl group by a pyridine, however, significantly reduced activity (SI-
Table 5, entry 24).
5
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Table 1: Inhibition of autophagy and VPS34 determined for selected
analogues. Starvation = starvation induced autophagy assay; Rapamycin =
Rapamycin induced autophagy assay; VPS34 = in vitro assay for VPS34
activity. Inactive = no inhibition at a test concentration > 10 µM. Data is mean
± SD, n ≥ 3 for all autophagy IC50 values; n ≥ 2 for all in vitro VPS34 IC50 values.
For a full list of analogues and activities see SI-Tables 1 - 5.
starvation
IC50 [µM]
rapamycin
IC50 [µM]
VPS34
IC50 [µM]
entry
nr.
R1
R2
R3
Cl
Autophinib
1
2
3
4
5
6
0.09 ± 0.04
0.07 ± 0.04
0.19 ± 0.11
0.39 ± 0.07
0.23 ± 0.11
0.45 ± 0.16
0.04 ± 0.01
0.04 ± 0.02
0.07 ± 0.03
0.13 ± 0.06
0.16 ± 0.06
0.28 ± 0.12
0.019
0.023
0.078
0.146
0.121
0.285
(2)
3
4
1
5
6
Me
CF3
7
8
7
8
Cl
Cl
1.40 ± 0.70
0.13 ± 0.03
2.00 ± 0.80
0.10 ± 0.02
0.627
0.036
9
9
Cl
Cl
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
10
10
6
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10.1002/anie.201703738
Angewandte Chemie International Edition
11
12
13
14
15
16
17
18
11
12
13
14
15
16
17
18
Cl
Cl
2.30 ± 1.20
Inactive
Inactive
Inactive
Inactive
0.061
0.020
0.049
0.110
0.214
0.310
Inactive
Cl
0.45 ± 0.19
0.17 ± 0.06
0.13 ± 0.08
0.38 ± 0.20
0.20 ± 0.09
0.34 ± 0.16
0.42 ± 0.17
0.12 ± 0.03
0.09 ± 0.08
0.28 ± 0.13
0.47 ± 0.27
0.44 ± 0.26
Cl
Cl
Cl
Cl
CF3
Based on the SAR investigation compound 2, which we termed autophinib, was
chosen for further in-depth characterization (SI-Figure 3). Autophinib inhibited
LC3 lipidation to form LC3-II in a dose-dependent manner in starved MCF7-
LC3 cells (Figure 2A). Consistent with inhibition of autophagic flux, autophinib
also inhibited p62 degradation by autophagy dose-dependently in MCF7-LC3
cells (Figure 2A).[16]
7
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Figure 2: Phenotypic validation of Autophinibautophinib as an autophagy
inhibitor. A: Inhibition of LC3 lipidation and p62 degradation by autophinib in
MCF7-LC3 cells. n ≥ 3, representative blot shown. B: Autophinib induces cell
death in starved MCF7 cells.Cytotoxicity was assessed by means of the
CellTox™ green dye binding to DNA due to cytotoxicity. C: Autophinib dose
dependently induces apoptosis in starved cells. Apoptosis was assessed by
using a selective caspase 3/7 probe. The experiments were performed with an
Incucyte Zoom instrument. Data are presented as a ratio of green fluorescent
area to cellular area as assessed by phase contrast (mean ± SD, n ≥ 3,
representative graph shown). D: Autophinib inhibits formation of PI3P positive
vesicles in HEK293A cells stably expressing eGFP-WIPI-2. E: Quantification of
the eGFP-WIPI-2 assay shown in D. Autophinib reverts the phenotype (starved,
10 µM, p = 0.0002) showing a similar effect as wortmannin (Wort., 500 nM) and
the opposite effect of bafilomycin A1 (Baf. A1, 50 nM). Scale bar = 10 µm.
Statistical analysis was performed using student’s t-test (*** = p≤0.001). Data
are shown with mean values ± 95% CI, N ≥ 25.
As expected for an autophagy inhibitor, autophinib enhanced cell death of
starved cells as compared to fed cells (Figure 2B),[4,6] which occurred via the
induction of apoptosis (Figure 2C).
Since autophinib inhibited Rapamycin induced autophagy and thus acts
downstream or independently of mTOR, we performed an image based flux
assay quantifying the number of autophagosomes as well as autolysosomes
(SI-Figure 4). Autophinib inhibits the accumulation of autophagosomes under
9
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10.1002/anie.201703738
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fed as well as starved conditions, but does not affect the number of
autolysosomes. Thus it is not a late stage autophagy inhibitor but an inhibitor
of autophagosome formation. PI3P is essential to initiate autophagosome
biogenesis and is mainly generated by the kinase VPS34. PI3P recruits effector
protein WIPI2, which is important for the formation of autophagosomes.[17]
Therefore, eGFP-WIPI2b serves as a sensor for autophagosomal PI3P.[18]
Image analysis of HEK293A cells, stably transfected with a construct for eGFP
fused to WIPI2b, showed an increasing number of fluorescent vesicles upon
starvation, which was reduced upon treatment with autophinib (Figure 2D and
E, see SI-Figure 5 for the full image set).
For target identification autophinib was profiled on a proteome-wide level by
comparing it to compounds with known modes of action and targets (Figure 3A,
SI-Tables 6-8). The ChemProteoBase method employs image based proteome
analysis by means of 2-D difference gel electrophoresis (DIGE) in comparison
to reference compounds.[19] Since no compound with similarity > 0.7 was
identified in this analysis, the mechanism of action of autophinib is suggested
to be novel. Despite a low cosine value, several kinase inhibitors emerged at
the top of the list such as the PI3K inhibitor LY294002 (SI-Table 7). This finding
in combination with the potent effect of autophinib on the formation of PI3P
containing puncta and structural similarity to known kinase inhibitors led us to
investigate inhibition of the PI3 kinase VPS34 by autophinib.
Indeed, VPS34 was inhibited with IC50 values of 19 ± 5 nM and 51 ± 8 nM at
ATP concentrations of KM (10 µM) and 100 µM respectively (Figure 3B)
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10.1002/anie.201703738
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suggesting an ATP competitive mode of action. Activity in the phenotypic assay
correlated strongly with in vitro potency against VPS34 (Table 1).
Figure 3: Target identification and engagement for autophinib. A: Hierarchical
cluster analysis of the ChemProteoBase. HeLa cells were treated with 30ꢀμM
autophinib for 18ꢀh, and proteomic analysis was done by the 2-D DIGE. Cluster
analysis was performed using quantitative data of the 296 common spots
(x-axis) derived from autophinib and those of 41 reference compounds. In the
heat map, log-fold (natural base) of the normalized volume is shown on the
colored scale. B: Dose dependent biochemical inhibition of VPS34 by
autophinib at different ATP concentrations. C: Identification of VPS34 as target
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of autophinib with ATP-biotin probes and readout via Western blot (ActiveX
assay). Competition of the ATP probes with autophinib validates binding of
autophinib to VPS34 in cell lysate. Data is mean ± SD, n ≥ 3, representative
graph / blot shown.
Autophinib did not inhibit other phosphatidylinositol kinases and mTOR at a
concentration of 1 µM (SI-Table 9). Remarkably, it also did not inhibit TBK1, the
target of the parent compound 1. Selectivity was further assessed in a panel of
>460 kinases (SI-File 1) from which 45 were inhibited ≥ 80% at a concentration
of 1 µM. However, most of them could be excluded as being not relevant for
autophagy by testing known, selective inhibitors in the autophagy assay (SI-
Table 10). Among 27 investigated inhibitors, the most potent autophagy
inhibitory activity was observed for the two recently reported VPS34 inhibitors:
SAR405 and VPS34-IN-1.[20,21]
VPS34 engagement by autophinib in a complex biological medium was
demonstrated by means of an ActiveX assay[22],[23], which employs a reactive
desthiobiotin-labeled ATP-probe. Incubation of the probe with cell lysate
covalently labels ATP-binding proteins, including kinases, which are then
captured by streptavidin-agarose-beads, eluted and analyzed. In order to
identify ATP binding targets of a small molecule the lysate is preincubated with
the compound prior to incubation with the ATP-probe to block the small
molecule binding site. Bound target proteins then do not bind to the ATP-probe
and are not detected. Autophinib dose-dependently inhibited the covalent
binding of the ATP probe to VPS34 Figure 3C). This result further supports the
notion that autophinib is an ATP competitive VPS34 inhibitor.
12
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An isothermal dose-response fingerprint (ITDRF)[24] confirmed target
engagement of autophinib in cell lysate, which stabilized VPS34 towards heat
induced aggregation (SI-Figure 6).
In summary, we report the discovery of a new autophagy inhibitor chemotype
by means of phenotypic screening. Autophinib targets VPS34 but not other lipid
kinases, mTOR and TBK1. Autophinib appears to be ATP-competitive, targets
VPS34 in cell lysate and induces cellular phenotypes consistent with VPS34
inhibition.
Wortmannin and 3-methyladenine (3-MA) have been used widely to study the
effect of PIK3 inhibition. However, both non-selectively inhibit all isoforms of the
PIK3 family.[25] In addition it has been observed that conditional knockout of
VPS34 shows severe differences to 3-MA or wortmannin treatment, indicating
that these inhibitors display VPS34-independent effects.[26] Recently more
potent and selective alternatives have been developed.[20,21] However, an
expanded toolkit to study autophagy and VPS34 activity would be very
desirable for basic research and potential drug discovery applications.
Autophinib distinguishes itself by its ready synthetic accessibility (3 steps,
overall yield: 45%), combined with high in vitro potency, selectivity and cellular
activity. It promises to be an excellent tool compound for the study of VPS34
biology and autophagy. Our results provide further proof for the utility of
unbiased, target agnostic phenotypic screens as a fruitful strategy for the
discovery of potent autophagy inhibitors in particular and of new bioactive
compounds in general.
13
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Acknowledgements
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. M. A. C. C. is grateful to
the Swiss National Science Foundation and the Fonds der Chemischen
Industrie for a fellowship. Y.-W. W. acknowledges funding from European
Research Council (ChemBioAP) and Behrens-Weise-Stiftung. We thank Miho
Tanaka for ChemProteoBase analyses. We thank Sharon Tooze for the gift of
WIPI2 cells.
14
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Phenotypic
discovery
drug
Lucas Robke, Luca Laraia,
Marjorie A. Carnero Corrales,
Georgios Konstantinidis, Makoto
Muroi, André Richters, Michael
Winzker,
Tobias
Engbring,
Stefano Tomassi, Nobumoto
Watanabe, Hiroyuki Osada,
Daniel
Rauh,
Herbert
Waldmann*, Yao-Wen Wu,
Julian Engel
__________ Page – Page
Phenotypic Identification of a
Novel
Autophagy
Inhibitor
Chemotype Targeting Lipid
Kinase VPS34
PIK(3)C(3)ing autophagy apart: Phenotypic screening
followed by target identification reveals a novel autophagy
inhibiting chemotype that inhibits the lipid kinase PIK3C3
(VPS34) with high potency, disrupting autophagosome
biogenesis.
17
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