Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor

Article abstract of DOI:10.1002/anie.201913712

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe²⁺ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

Full text of DOI:10.1002/anie.201913712

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Image-based morphological profiling identifies a lysosomotropic,
iron-sequestering autophagy inhibitor
Authors: Herbert Waldmann, Luca Laraia, Guillaume Garivet, Daniel
J. Foley, Nadine Kaiser, Sebastian Müller, Sarah Zinken,
Thomas Pinkert, Julian Wilke, Dale Corkery, Axel Pahl, Sonja
Sievers, Petra Janning, Christoph Arenz, Yaowen Wu, and
Raphaël Rodriguez
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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
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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.201913712
Angew. Chem. 10.1002/ange.201913712
Link to VoR: http://dx.doi.org/10.1002/anie.201913712
http://dx.doi.org/10.1002/ange.201913712

10.1002/anie.201913712
Angewandte Chemie International Edition
Image-based morphological profiling identifies a lysosomotropic, iron-
sequestering autophagy inhibitor
Luca Laraia#, Guillaume Garivet#, Daniel J. Foley§, Nadine Kaiser§, Sebastian Müller, Sarah Zinken,
Thomas Pinkert, Julian Wilke, Dale Corkery, Axel Pahl, Sonja Sievers, Petra Janning, Christoph Arenz,
Yaowen Wu, Raphaël Rodriguez, Herbert Waldmann*
#joint first authors; §joint third authors.
Assist. Prof. Luca Laraia, Dr. Guillaume Garivet, Dr. N. Kaiser, Dr. Daniel J. Foley, MSc Sarah Zinken,
MSc Julian Wilke, Dr. Petra Janning, Prof. Dr. H. Waldmann
Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
*E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Dr. Guillaume Garivet, Dr. N. Kaiser, MSc Sarah Zinken, MSc Julian Wilke, Dr. Petra Janning, Prof.
Dr. H. Waldmann
Faculty of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund,
Germany
Dr. A. Pahl, Dr. S. Sievers,
Compound Management and Screening Center, Dortmund, Otto-Hahn-Str. 11, 44227 Dortmund,
Germany.
Assist. Prof. Luca Laraia – current address
Technical University of Denmark, Department of Chemistry, Kemitorvet 207, 2800 Kgs. Lyngby,
Denmark
Dr. Daniel J. Foley – current address
School of Physical and Chemical Sciences, University of Canterbury, Christchurch, NZ.
Dr. Dale Corkery, Prof. Yaowen Wu
Department of Chemistry, KB.A4, Linnaeus väg 10, (rum: A4.35.07), Umeå universitet, 901 87 Umeå,
Sweden
Thomas Pinkert, Prof. Dr. Christoph Arenz
Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2 (R 1´102), D-12489 Berlin
Dr. Sebastian Müller, Prof. Raphaël Rodriguez
Institut Curie, CNRS UMR 3666, INSERM U1143, PSL University Paris, Chemical Cell Biology
Group, 26 Rue d'Ulm, 75248 Paris Cedex 05, France
1
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10.1002/anie.201913712
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Graphical abstract:
Morphological profiling in the “cell painting” assay revealed that autoquin impairs lysosomal integrity,
iron homeostasis and autophagy.
Keywords: Autophagy, Cell painting, Lysosome, Proteomics, Target identification
2
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10.1002/anie.201913712
Angewandte Chemie International Edition
Abstract:
Chemical proteomics is widely applied in small molecule target identification. However, in general it
does not identify non-protein small molecule targets, and thus, alternative methods for target
identification are in high demand. We report the discovery of the autophagy inhibitor autoquin, and the
identification of its molecular mode of action using image-based morphological profiling in the cell
painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters
revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In
addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen
species and ultimately cell death. Such a mechanism of action would have remained challenging to
unravel by current methods including chemical proteomics. This work demonstrates the potential of the
cell painting assay to deconvolute modes of action of small molecules, warranting wider application in
chemical biology.
3
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10.1002/anie.201913712
Angewandte Chemie International Edition
Introduction
The identification of small molecules to probe biological systems is at the heart of chemical biology.
Target agnostic phenotypic screens represent a rapid way to identify bioactive small molecules in
physiologically relevant systems.[1] However, a major challenge with this approach is the subsequent
elucidation of molecular modes of action (MMOA) and target identification (ID) of bioactive
compounds.[2] Widely employed target ID techniques include affinity-based pull-down using
immobilised derivatives followed by mass spectrometric protein identification.[3] This typically requires
the synthesis of a suitably functionalised probe, which might be time consuming or even infeasible if
the compound of interest is highly complex. Emerging target identification strategies, which do not rely
on modifications of the hit compound, such as thermal proteome profiling, are powerful additions to
the target ID toolkit.[4] However, these techniques are restricted to small molecules that mediate their
activity through the targeting of proteins. In contrast, various drug classes target DNA,[5] RNA[6] and
lipids,[7] and the discovery of regulatory RNA-targeting small molecules has recently emerged as a new
field.[8] Therefore, the development of new methods, which enable the delineation of bioactive small
molecule modes of action not mediated by binding to a protein target, is in high demand.
Morphological profiling has recently emerged as a complementary strategy for small molecule target
identification. Monitoring changes in cellular morphology induced by a hit molecule and comparing
these to changes induced by a set of reference compounds with known modes-of-action and targets can
provide target hypotheses. Morphological profiles can be extracted from simple bright field images,[9]
and obtained from complex fluorescence-based high content screens where multiple subcellular
compartments are labelled with various fluorophores.[10] The multiplexed use of different fluorophores
has been established in the “cell painting” assay,[10-11] and has been proposed as a new strategy for
determining whether a compound displays bioactivity in a very broad setting.[12] In light of the promise
and the potential of this approach, we explored the use of the cell painting assay for MMOA
identification where other target identification methods had failed.[13]
Recently we identified oxautin-1 (1), a cinchona alkaloid-derived autophagy inhibitor containing an
oxazatwistane scaffold (Scheme 1a).[14] Autophagy is a cellular recycling process that degrades mis-
folded, aggregated and/or superfluous proteins and organelles. The inhibition of autophagy is
considered a potential anti-cancer strategy[15] making the identification of new small molecule
autophagy inhibitors and their targets an intensive area of ongoing research.[14, 16] Oxautin-1 was
predicted to inhibit both autophagosome biogenesis, and the fusion of autophagosomes and lysosomes,
but its MMOA had remained elusive. Given this unknown mode of action we embarked on the synthesis
and biological investigation of more readily accessible and structurally more diverse oxautin analogues.
We now report the discovery of the cinchona alkaloid derived autophagy inhibitor autoquin (2, Scheme
1a). Analysis of morphological changes induced by autoquin in the cell painting assay unraveled that
4
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10.1002/anie.201913712
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— like oxautin-1 — autoquin inhibits autophagy by indirect modulation of the activity of the lysosomal
enzymes sphingomyelinase and acid ceramidase, resulting in impaired lysosome-autophagosome
fusion. Deeper investigation revealed that autoquin also sequesters Fe2+ in lysosomes which results in
increased formation of lysosomal reactive oxygen species (ROS) and cell death.
Results and Discussion
The oxautins were synthesised by intramolecular cyclisation of quinidine (3) and cinchonine (4) but
respective analogues cannot be obtained from the cinchona alkaloids quinine (5) and cinchonidine (6)
due to their different configuration at the carbon α to the quinuclidine nitrogen, precluding cyclisation
and limiting further exploration of oxazatwistane analogues as autophagy inhibitors (Scheme 1a).[14] In
addition, the cyclisation to yield the oxazatwistane ring system in the oxautins requires somewhat harsh
conditions and removes two vectors for further functionalisation, thereby further limiting exploration
of SAR. Therefore, we investigated whether the oxazatwistane core was required for autophagy
inhibitory activity. To this end, the four major cinchona alkaloids quinidine, quinine, cinchonine and
cinchonidine were subjected to Borono–Minisci conditions[17] to selectively functionalise the C2
position and to evaluate the importance of the relative stereochemistry at the quinuclidine ring (Scheme
1b). In addition to the expected C2-functionalised derivatives, some reactions also delivered a minor
product corresponding to the C5- (compounds 9, from quinidine) or C7-functionalised scaffold (10,
from cinchonine, and 13, from cinchonidine). In addition, functionalisation of the C3 position had not
previously been investigated for the oxazatwistanes. Selective halogenation at C3[18] provided
intermediates that could be subjected to metal-catalysed cross coupling reactions (Scheme 1b, 14-15).
Suzuki reactions enabled the synthesis of 13 additional analogues (16a-l).
5
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10.1002/anie.201913712
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(a)
F
F
O
OH
OH
R
N
N
N
N
N
N
N
OH
N
OMe
OMe
R
3
5
R = OMe, quinidine,
R = OMe, quinine,
Oxautin-1 (1),
(b)
Autoquin (2),
IC50 = 0.56 0.15 µM
IC50 = 0.65 0.22 µM
4
R = H, cinchonine,
6
R = H, cinchonidine,
3
4
:
from or
OH
N
OH
OH
N
R2
R1
N
+
+
N
N
N
R1B(OH)2 (3.0 eq)
TFA (3.0 eq)
R
R
R
R3
R = H,
2, 7a-f
8a-g
R = OMe,
R = H,
9a-g
R = OMe,
(minor from
10a-d
(minor from
AgNO3 (0.6 eq)
3
)
4
)
3 6
-
K2S2O8 (2.0 eq)
CH2Cl2/H2O
rt, 6 - 24 h
5
6
:
from or
R
R
20-55%
N
R3
N
+
OH
OH
N
N
R1
11a-g
13a-c
R = OMe,
R = H,
12a-d
6
)
R = H,
(minor from
(c)
3
R2
B(OH)2 (2.05 eq)
1) MeLi (1 eq)
2) BF3.OEt2 (2.2 eq)
0°C, 15 min
Na2CO3 (10.0 eq)
E
R4
OH
OH
PdCl2dppf (0.05 eq)
N
N
3) TMPMg.LiCl (1.1 eq)
dioxane/H2O, 30 min,
µW, 100 °C
N
N
0°C, 40 min
E+
4)
(1.0 eq)
50-85 %
OMe
OMe
16a-l
14
, E = Br, 50%
15, E = I, 45%
Scheme 1. Synthesis of a cinchona alkaloid-derived compound library. (a) Molecular structures of previously
identified autophagy inhibitor oxautin-1, newly discovered inhibitor autoquin and the four most abundant
cinchona alkaloids; (b) synthesis of C2-functionalised derivatives using the Borono–Minisci reaction; (c)
synthesis of C3-functionalised derivatives using selective C-H activation followed by Suzuki coupling. See Table
1 for details of the R-groups investigated.
The resulting 49-membered compound collection was investigated for autophagy inhibition in MCF7
cells stably expressing EGFP-tagged LC3, a widely used autophagy marker.[19] Compounds that were
able to reduce EGFP-LC3 puncta formation upon autophagy induction by amino acid starvation using
[20]
Earle’s Balanced Salt Solution (EBSS) were classed as hits.
The direct oxautin-1[14] analogue 2
derived from quinidine but lacking the oxazatwistane ring, displayed very similar potency in the
autophagy assay, suggesting that the oxazatwistane ring was not essential for biological activity (Table
1, Entry 1). This compound, which we named autoquin, provided a benchmark against which all other
6
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10.1002/anie.201913712
Angewandte Chemie International Edition
compounds were assessed. Small variations at the C2 position resulted in a modest drop in activity
(Table 1, Entries 2 and 3), while removal of the p-F substituent completely abolished it (Entry 4).
Varying the position of substitution on the phenyl ring reduced or abolished activity (Entries 5-7).
Compounds with aryl substituents at C5 of the quinoline ring were generally inactive (Entries 8, 9, 11)
though a m-Cl substituent returned some activity (Entry 10). In general, cinchonine derivatives, lacking
the C6 methoxy group (Entries 12-21), were all less active than the quinidine-derived compounds,
confirming the importance of this residue for optimal activity. The p-F-C6H4 substituent at C2 retained
the highest levels of activity (Entry 12), observed for all the quinidine-derived compounds (Entry 1)
and the oxazatwistanes[14]. To assess the importance of the stereochemistry at the quinuclidine ring, a
small collection of C2 substituted derivatives of quinine and cinchonidine was synthesised and
evaluated (Entries 22-36). Although three quinine-derived analogues showed appreciable levels of
activity, they were all at least 6-fold less active than autoquin, and were not pursued further. Crucially,
the p-F-Ph substituted quinine and cinchonidine analogues (Entries 22 and 29) were significantly less
active than their quinidine and cinchonine-derived diastereomers (Entries 1 and 12). All compounds
with substituents at the C3 position on the quinoline ring were either less active than autoquin (Entries
39 and 40) or completely inactive (Entries 37-38 and 41-49).
Table 1. Structure activity relationships of cinchona alkaloid-derived autophagy inhibitors. IC50 data represents
the ability to inhibit autophagy induced by amino acid starvation using EBSS and is mean ± SD of three
independent experiments.
Entry
#
R
R1
R2
R3
R4
IC50 (µM)
1
2
OMe
p-F-C6H4
H
H
H
0.56 ± 0.15
(autoquin)
2
3
4
5
6
7
7a
7b
7c
7d
7e
7f
OMe
OMe
OMe
OMe
OMe
OMe
p-Cl-C6H4
p-Me-C6H4
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.44 ± 1.01
2.31 ± 0.07
>10
m-Br-C6H4
m-Cl-C6H4
m-Cl-p-F-
C6H3
7.48 ± 0.70
5.70 ± 0.39
>10
8
9
9a
9b
9c
OMe
OMe
OMe
H
p-F-C6H4
m-Cl-C6H4
m-Cl-p-F-
C6H3
H
H
H
H
H
H
>10
>10
H
10
H
5.40 ± 2.40
11
9d
OMe
H
Ph
H
H
>10
7
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12
13
14
15
16
17
18
8a
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
p-F-C6H4
p-Cl-C6H4
p-Me-C6H4
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.62 ± 0.19
Toxic
>10
>10
m-Br-C6H4
m-Cl-C6H4
m-Cl-p-F-
C6H4
>10
5.43 ± 0.13
>10
8g
19
20
21
22
23
24
25
26
27
28
10a
10b
10c
11a
11b
11c
11d
11e
11f
H
H
H
H
H
H
H
H
H
H
H
H
p-F-C6H4
H
H
H
H
H
H
H
H
H
H
>10
2.52 ± 0.32
>10
H
H
p-Cl-C6H4
H
H
m-Br-C6H4
OMe
OMe
OMe
OMe
OMe
OMe
p-F-C6H4
p-Cl-C6H4
p-Me-C6H4
Ph
H
H
H
H
H
H
H
3.30 ± 1.60
2.70 ± 1.30
>10
>10
m-Br-C6H4
m-Cl-C6H4
m-Cl-p-F-
C6H3
2.72 ± 1.31
na
11g
OMe
na
29
30
31
32
33
34
35
36
37
38
39
12a
12b
12c
12d
12e
13a
13b
13c
16a
16b
16c
H
H
p-F-C6H4
p-Cl-C6H4
p-Me-C6H4
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>10
5.84 ± 1.30
>10
H
H
H
H
H
H
H
>10
H
m-Br-C6H4
H
H
H
>10
H
p-F-C6H4
H
>10
H
H
p-Cl-C6H4
H
na
H
H
Ph
H
H
>10
OMe
OMe
OMe
H
p-F-C6H4
p-Cl-C6H4
p-NO2-
C6H4
>10
H
H
>10
H
H
3.87 ± 0.69
40
41
16d
16e
OMe
OMe
H
H
H
H
H
H
p-CF3-
C6H4
3.52 ± 0.69
>10
p-NHBoc-
C6H4
42
43
44
16f
16g
16h
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
4-py
>10
>10
>10
m-F-C6H4
m-F-p-F-
C6H4
45
46
47
16i
16j
16k
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
3,5-di-F-
C6H4
>10
>10
>10
m-Cl-p-F-
C6H4
3,4-di-
OMe-
C6H4
48
49
16l
14
OMe
OMe
H
H
H
H
H
H
3,5-di-
OMe-
C6H4
>10
>10
-Br
8
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Having established that the oxazatwistane scaffold was not essential for autophagy inhibition and that
the p-F-C6H4 substituent at C2 of the quinoline ring was best for autophagy inhibition, we proceeded to
validate autoquin as an autophagy inhibitor. As described above, autoquin showed a dose-dependent
inhibition of EGFP-LC3 puncta after 3 hours upon autophagy induction by amino acid starvation in the
primary screening assay (Figure 1a-b). Additionally, it increased the stability of the chaperone p62 to
autophagosome-mediated degradation as assessed by western blot (Figure 1c), suggesting that it is an
inhibitor of autophagic flux. However, autoquin also showed a dose dependent increase in LC3-II
levels, suggesting that it is also an inhibitor of autophagosome maturation, similarly to oxautin-1 (Figure
1c). This finding was further strengthened by using a tandem mCherry-EGFP-LC3 expressing cell line,
which enables the simultaneous monitoring of autophagosomes (green and red fluorescence) and
autolysosomes (red fluorescence only). Exposing fed cells to autoquin for 24 hours resulted in a marked
increase in autophagosomes, confirming its inhibitory effect on autophagosome maturation (Figure 1d).
Figure 1. Validation of autoquin as an autophagy inhibitor. (a) Effect of autoquin on MCF7 cells stably expressing
EGFP-LC3 upon autophagy induction by amino acid starvation using EBSS; n = 3, representative images, scale
bar = 100 µm; CQ = chloroquine (50 µM). (b) Quantification of the reduction in EGFP-LC3 puncta by autoquin.
Data points are mean ± sem of three independent experiments. (c) Effect of autoquin and analogues 17a and 20
(see Scheme 2 for structures and data) on LC3-II and p62 levels as assessed by western blot; n = 3, representative
images shown. (d) MCF7 cells stably expressing mCherry-EGFP-LC3 treated with vehicle or 5 µM autoquin for
9
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10.1002/anie.201913712
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24 h. Scale bars, 10 µm. (e) Autophagosomes (AP; yellow puncta) and autolysosomes (AL; red puncta) from (d)
were quantified and data represented as percentage of cell area. Bar graphs show mean ± SD from three
biologically independent experiments. Data points represent individual cells pooled from the three independent
experiments (n ≥ 23 cells per replicate). Significance was determined from biological replicates using a two-tailed,
unpaired t-test. ns = not significant, **p=0.0064.
Having validated autoquin as a bona fide autophagy inhibitor and confirmed its effect on
autophagosome maturation, we focused our attention on making smaller, more targeted modifications
to the autoquin scaffold to identify a suitable immobilisation point for affinity-based target enrichment.
Reduction of the alkene resulted in two derivatives with retained biological activity (17a-b), suggesting
that this may be a suitable position for further functionalisation (Scheme 2a and Figure 1c). C5-
substituted analogues (18a-b) were less active, as observed with the unsaturated analogues (Scheme 2a
and Table 1). Arylation of the vinyl group via Heck coupling produced three derivatives (19a-c) that,
though less active than autoquin, retained good potency levels. Oxidation of the hydroxyl group to a
ketone led to an inactive compound (20) while, interestingly, methylation of the hydroxyl group
produced a very active compound (21), suggesting that this position is critical for potency. To access a
derivative suitable for immobilisation and affinity-based target enrichment, autoquin was subjected to
an ene reaction with 2-(Boc-amino)ethanethiol to yield a compound (23) which was further elaborated
into a pull-down probe (25, Scheme 2b). A corresponding negative probe (24) lacking the p-
fluorophenyl group was also synthesized from 22. While the negative probe and its precursors were all
inactive, the final positive probe 25 was as well. The phenomenon by which the introduction of an
unprotected amino-PEG linker leads to loss of activity has been observed by us on several occasions,
and can be ascribed to a presumable loss of cell permeability. In this case the presence of two basic
amines makes it more likely that a doubly-charged molecule would interact with the cell membrane. As
the pulldown experiment is carried out in cell lysates and the intermediate 23 retained appreciable levels
of activity, we were continued with 25 as a positive probe.
10
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(a)
R
OH
OH
R
N
N
N
N
R
O
O
F
OH
18a,
R = F,
IC50 = 2.30 ± 0.50 µM
IC50 = 5.20 ± 1.02 µM
17a,
17b,
R = F,
R = Cl,
IC50 = 1.00 0.15 µM
IC50 = 1.61 0.15 µM
N
18b,
R = Cl,
N
O
F
F
O
O
19a,
19b,
19c,
R=H, IC50 = 2.84 ± 0.69 µM
R=4-F, IC50 = 5.56 ± 2.52 µM
R=3-OMe, IC50 = 4.99 ± 0.72 µM
N
N
N
N
O
O
21,
IC50 = 0.26 ± 0.07 µM
20,
inactive
2TFA
O
(b)
+
O
NH3
2
S
S
1. TFA
BocHN
O
N
2. Boc-NH-PEG(3)-CO2H,
TBTU, DIPEA, DMA, rt
3. TFA
H
SH
BocHN
80 °C
OH
OH
OH
R1
R1
R1
N
N
N+
H
N
N
N
OMe
= H, quinidine
autoquin (2)
OMe
OMe
R1
R1
R1
R1
22
R1
R1
24
= H, , 25%, inactive
= H, , 85%, inactive
23
25
= p-FC6H4, , 87%, inactive
= p-FC6H4,
= p-FC6H4, , 97%, IC50 = 7.28 2.0 µM
Scheme 2. SAR of the quinuclidine ring and pull-down probe synthesis. (a) autophagy inhibitory activity of
analogues with variations vicinal to the quinuclidine ring; variations highlighted in red; (b) synthesis and
autophagy inhibitory activities of pull-down probes for target identification experiments
To identify autoquin target proteins by affinity enrichment, the probes were immobilised on NHS-
activated magnetic beads and incubated with cell lysate. Proteins selectively enriched with the autoquin-
derived probe compared to the negative probe were considered to be hits. The only hit that was
conclusively identified in all replicates was Ferrochelatase (Supplementary Figure 1). Ferrochelatase
(FECH) is located in the mitochondria and catalyses the insertion of iron into protoporphyrin IX, the
last step in heme biosynthesis.[21] No known link between FECH and autophagy has previously been
reported. However, autophagy has been shown to play a role in mediating iron homeostasis through the
selective degradation of ferritin[22]. In an attempt to validate FECH as a target of autoquin, the pull-
down was repeated with non-immobilised autoquin as a competitor and analysed by western blot.
However, despite FECH enrichment by the positive probe (25) compared to the negative probe (24), no
competition was observed (Supplementary Figure 2a). Additionally, autoquin was not able to stabilise
FECH to thermal denaturation as assessed by isothermal dose response fingerprinting, even at very high
concentrations (Supplementary Figures 2b and 2c). Thus, FECH was not considered a direct functional
target of autoquin.
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Since chemical proteomics had not validated a target, autoquin was investigated in the multiparametric
image-based cell painting assay, which enables interrogation of a cellular system in its entirety.[10-12, 13]
Cell painting involves staining cells with markers for specific cellular compartments following
compound treatment. A vast array of phenotypes including cell shape, morphology, size and
fluorescence intensity and distribution can be assessed simultaneously for each treatment condition,
generating a set of fingerprints characteristic to a particular mode of action or target.[10-11, 23] The power
of this assay becomes apparent when a library of reference compounds of known bioactivity is included
in the screen and their fingerprints are compared with profiles recorded for novel compounds. In
principle, this can enable the discovery of bioactivity profiles that are different, and thus novel,
compared to a reference set, but also known modes of action can be revealed by similarity
assessment.[13]
Autoquin, oxautin-1 and several analogues were characterised in the cell painting assay, where their
effect on 579 parameters (see the Supporting Information for the delineation of the parameters) was
compared with the results obtained for a reference compound set comprising 3000 compounds with
known bioactivity (see the Supporting Information for details). To assess the similarity in the bioactivity
of the fingerprint profiles, “biological similarity” was employed (“BioSim”; see the Supporting
Information for determination of similarity). Furthermore an “induction” value (the fraction of
parameters (in %) that underwent significant changes (median absolute deviation (MAD) value upon
compound treatment of at least +/− three-fold of the median determined for the DMSO controls; see the
Supporting Information) was determined as measure for compound bioactivity. Compounds with an
induction value of >10% were considered bioactive in the cell painting assay. This analysis resulted in
the discovery of three annotated compounds with high similarity (>80%) in their bioactivity fingerprints
to autoquin (Figure 2a and Supplementary Figure 3). Although at first glance, perphenazine (reportedly
a non-selective G-protein coupled receptor ligand), loperamide (an opioid receptor agonist) and
toremifene (an estrogen receptor ligand) do not display obvious biological or indeed chemical
similarity, all three have been reported to be lysosomotropic compounds.[24] Lysosomotropic
compounds are typically hydrophobic with at least one basic nitrogen that, upon protonation, enables
them to be trapped in the lysosomes.[25] A comparison of their physicochemical properties revealed that
all compounds are likely to be fully protonated at pH 4-5, typically found in the lysosome
(Supplementary Table 1). This would favour a model in which they are able to pass cellular and
lysosomal membranes before being protonated and trapped in the lysosome. A potential lysosomotropic
profile had previously been suggested in the cell painting assay for structurally different compounds,
but was not further investigated.[11a] To confirm whether autoquin and oxautin were indeed
lysosomotropic, we assessed their ability to inhibit the accumulation of the lysosomal tracer Lysotracker
Red (LR) DND-99. A decrease in fluorescence intensity is often characteristic of a lysosomotropic
phenotype, if the lysosomal pH is increased. Both autoquin and oxautin-1 showed a dose-dependent
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decrease in lysosomal accumulation of LR after a 3 hour treatment, similarly to the known
lysosomotropes chloroquine and chlorpromazine (Figure 2b and 2c). Lysosomotropic compounds are
also often functional inhibitors of acid sphingomyelinase (FIASMAs), and other sphingolipid
hydrolases including acid ceramidase.[26] They do not directly interact with the hydrolases (therefore,
they are qualified as functional inhibitors) but rather affect the inner lysosomal membrane localisation
of acid sphingomyelinase and other sphingolipid hydrolases through a direct interaction with the
negatively charged lipid bis(monoacylglycerol)phosphate (BMP), resulting in the degradation of the
hydrolases[27] Both autoquin and oxautin-1 were tested in a fluorescence-based assay to monitor both
acid sphingomyelinase and acid ceramidase activity in a cell-based and cell-free system.[28] FIASMAs
characteristically inhibit hydrolase activity in cell-based assays, where intact lysosomes are present, but
not in cell-free systems. Both autoquin and oxautin-1 inhibited acid sphingomyelinase and ceramidase
activity in intact cells (Figure 2d), but not in lysates (Figure 2e), similarly to the control compound
desipramine.
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Figure 2. Autoquin is a lysosomotropic compound that acts as a functional inhibitor of acid sphingomyelinase.
(a) Cell painting profiles of autoquin and its most biosimilar compounds oxautin-1, perphenazine, loperamide and
toremifene. See Supplementary Figure 3 for representative images. (b) Representative fluorescence microscopy
images of MC7 cells treated with lysosomotropic compounds for 3 h and stained with Lysotracker DND-99; scale
bar = 110 µm. (c) Quantification of (b), n = 3, data is mean ± SD. (d) Product:substrate ratio of the acid
sphingomyelinase (left) and acid ceramidase (right) reaction in intact cells. n = 3, data is mean ± SD. (e)
Product:substrate ratio of the acid ceramidase reaction in cell lysates. n = 3, data is mean ± SD. Statistical
significance comparing treated samples to the DMSO control for (d) and (e) was assessed using the Students t-
test. *p < 0.05, **p < 0.01, ***p < 0.001.
In light of these findings, we re-evaluated the affinity isolation data and also took into consideration the
recently reported biological activity of the natural product salinomycin, which sequesters iron to the
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lysosomes, inhibiting autophagy and causing ferroptosis.[29] A similar phenotype has also been reported
for the lipophilic iron chelators di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and,
to a lower extent, desferrioxamine (DFO).[30] We speculated that the ability of autoquin to pull down
FECH might have been iron-dependent, and not specific to FECH. This hypothesis was strengthened
by reports that quinine and related cinchona alkaloids are able to chelate iron, contributing to their anti-
malarial effect.[31] To assess whether autoquin displayed a similar mechanism of action to salinomycin,
we evaluated its impact on lysosomal mass, lysosomal Fe2+ and ROS production. Autoquin significantly
increased lysosomal mass after 24 hours, as assessed by Lysotracker deep red (DR) staining (Figure 3a
and 3b). A decrease in lysotracker staining at early time points (<4 hours) due to increased pH is
generally observed for lysosomotropic compounds including autoquin (Figure 2b and 2c); however,
this effect is reversed at later time points (>24 hours) as cells adapt to protect themselves from lysosomal
stress.[32] Autoquin also significantly enhanced lysosomal Fe2+ levels (Figure 3a and 3c), as assessed by
the turn-on fluorescent probe RhoNox-M, and overall Fe2+ levels, as assessed by RhoNox-1
fluorescence (Figure 3d). The concomitant increase in lysosomal mass and Fe2+ levels also resulted in
a highly significant increase of lysosomal reactive oxygen species (ROS), as assessed by CellRox DR
(Figure 3e and Supplementary Figures 4a and 4b). Similarly to salinomycin, autoquin also induced lipid
peroxidation, as assessed by a BODIPY 581/591 undecanoic acid (C11) probe[33] (Supplementary
Figure 4d) and caused lipid membrane permeabilization (Supplementary Figure 4e). As salinomycin
had shown promising results in the selective targeting of breast cancer stem cells,[29, 34] a tumorigenic
cell subpopulation typically associated with resistance to chemotherapy and sustained tumor growth, it
was speculated that autoquin may display a similar profile and potential. Autoquin was selectively
cytotoxic against transformed human mammary epithelial HMLER CD44high/CD24low cells (HMLER
CD24low), an established model of human breast cancer stem cells, compared to a control isogenic cell
line (HMLER CD24high) (Supplementary Figure 4f). As such, autoquin represents a new lysosomotropic
compound which selectively targets breast cancer stem cells, and may hold promise in the study of anti-
cancer agents that target the lysosomes,[35] a field that has been growing steadily in recent years, with
multiple clinical trials focused on the approved anti-malarials chloroquine and hydroxychloroquine
(clinicaltrials.gov).
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Figure 3. Autoquin increases lysosomal mass and sequesters Fe2+ to the lysosomes in MCF7 cells, causing an
increase in lysosomal reactive oxygen species. (a) Fluorescence microscopy images showing the subcellular
localisation of Fe2+ (red) and lysosomes (green) by means of RhoNox-M and Lysotracker deep red (DR)
fluorescence respectively. (b) Flow cytometry quantification of Lysotracker DR intensity from cells treated
according to (a). (c) Flow cytometry quantification of RhoNox-M intensity from cells treated according to (a). (d)
Flow cytometry quantification of intracellular Fe2+ using an alternative turn-on fluorescent probe RhoNox-1. (e)
Fluorescence microscopy images showing the subcellular localization of ROS (red) by means of fluorogenic
reaction with CellROX deep red in MCF7 cells treated with autoquin (5 µM) for 24 h. Scale bar, 10 μm.
Colocalization with Lysotracker DND-26 (green) assessed by Pearson correlation coefficient (R). ****p < 0.0001,
unpaired Students t-test.
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Conclusion
We have employed the cell painting assay to identify the MMOA of autoquin, a cinchona-alkaloid
derived autophagy inhibitor. While affinity-based proteomic experiments proved inconclusive, image-
based profiling identified similarities in phenotypic profiles determined for autoquin and known
lysosomotropic compounds. This MMOA does not require a direct binding event between autoquin and
a target protein, which explains why mass spectrometry based proteomic approaches were inconclusive.
However, pull-down experiments suggested that perturbation of iron homeostasis may contribute to the
activity of autoquin, which was confirmed using markers for lysosomal ROS and lipid peroxidation.
This work showcases image-based profiling as an excellent complementary tool for mode-of-action and
target identification. Given that the lack of identification strategies for non-protein targets has been a
major drawback of mass spectrometry-based approaches, we envisage and indeed encourage the
chemical biology community to embrace the cell painting assay as an additional, alternative technique
for the discovery of modes-of-action and novel bioactivity. Crucially, this technique obviates the need
for functionalisation of the active molecule, making it particularly suitable for the identifying the
MMOA of complex, NP-like compounds. To further improve cell painting and increase its adoption,
significantly larger libraries of reference compounds will be required. Although truly novel modes-of-
action will still require proteomic experiments including pull-downs and/or thermal proteome profiling
to identify the molecular targets underlying an observed phenotype, the cell painting assay is a strong
addition to the chemical biologist’s tool-kit for tackling the challenge of target identification.
Acknowledgements
This work was supported by Max Planck Society to H.W., and by DFG grant (no.: SPP 1623), ERC
(ChemBioAP), Vetenskapsrådet (Nr. 2018-04585), and The Knut and Alice Wallenberg Foundation to
Y-W.W. L.L. was supported by a fellowship from the Alexander von Humboldt Stiftung. D.J.F. thanks
the European Commission for a Marie Skłodowska-Curie European Fellowship (Grant 794259). D.P.C.
is supported by a fellowship from the Canadian Institute of Health Research (MFE-152550). We
acknowledge the Biochemical Imaging Center (BICU) at Umeå University and the National
Microscopy Infrastructure, NMI (VR-RFI 2016-00968) for providing assistance in microscopy. Laura
Marongiu is acknowledged for support with the lysosomal hydrolase assays. R.R. and S.M. are funded
by the European Research Council (ERC) under the European Union’s Horizon 2020 research and
innovation programme (grant agreement No [647973]), the Fondation Charles Defforey-Institut de
France and Ligue Contre le Cancer (Equipe Labellisée). C.A. is grateful for financial support by the
DFG (AR 376/12-2).
Conflicts of interest
The authors declare no conflicts of interest.
17
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