Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy

Abstract

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

Full text

RESEARCH PAPER
Identification of a novel compound that simultaneously impairs the ubiquitin-
proteasome system and autophagy
Tatiana A. Giovannucci
a
, Florian A. Salomons
a
, Henriette Stoy
a
, Laura K. Herzog
a
, Shanshan Xu
a
, Weixing Qian
b
,
Lara G. Merino
a
, Maria E. Gierisch
a
, Martin Haraldsson
c
, Alf H. Lystad
d
, Hanna Uvell
b
, Anne Simonsen
d,e
, Anna-
Lena Gustavsson
c
, Michaela Vallin
c
, and Nico P. Dantuma
a
a
Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Stockholm, Sweden;
b
Laboratories for Chemical Biology Umeå, Chemical
Biology Consortium Sweden (CBCS), Umeå University, Umeå, Sweden;
c
Chemical Biology Consortium Sweden (CBCS), Science for Life Laboratory,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
d
Department of Molecular Medicine, Institute of
Basic Medical Sciences and Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Blindern,
Oslo, Norway;
e
Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo, Norway
ABSTRACT
The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic
systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the
timely destruction of aberrant proteins, these complementary pathways keep the intracellular
environment free of inherently toxic protein aggregates. Chemical interference with the UPS or
autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which,
owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems.
Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation
by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like
molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises
autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation
of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is
independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinosi-
tol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS
impairment but failed to restore autophagy, indicating that activation of stress responses does not
allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small
molecule that simultaneously impairs the two main proteolytic systems for protein quality control
provides a starting point for the development of a novel class of proteostasis-targeting drugs.
ARTICLE HISTORY
Received 17 May 2021
Revised 18 September 2021
Accepted 28 September 2021
KEYWORDS
Autophagy; compound
screen; inhibitor;
proteostasis; stress response;
ubiquitin-proteasome
system
Introduction
Intracellular protein homeostasis, i.e., proteostasis, depends
on efficient recycling of defective proteins [1]. Protein recy-
cling must be temporally and spatially regulated, and per-
formed in an orchestrated manner, to safeguard that only
designated proteins are degraded while leaving other proteins
unharmed. The ubiquitin-proteasome system (UPS) and
macroautophagy (herein referred to as autophagy) are the
main proteolytic systems responsible for eliminating aberrant
and misfolded proteins that are intercepted by protein quality
control systems.
The UPS is a complex system that involves a large number
of proteins and is characterized by two key players: the small
protein modifier ubiquitin, which tags proteins designated for
degradation, and the proteasome, a multi-subunit proteolytic
complex that executes the destruction of these proteins [2].
The compartmentalized nature of the proteasome requires
proteins to be unfolded in order to be translocated into the
proteolytic chamber. This requirement renders difficult-to-
untangle, aggregated proteins resistant to degradation by the
UPS [3]. Consequently, a failure in the timely degradation of
misfolded proteins typically gives rise to the appearance of
insoluble protein aggregates [4].
Autophagy, on the other hand, is less sensitive to size
restrictions as it does not require unfolding of substrates [5].
This pathway comprises the capturing of intracellular consti-
tuents in autophagosomes, which are double-membrane vesi-
cles that fuse with lysosomes, where their content is
hydrolyzed. Ubiquitin-like proteins belonging to the Atg8
family play a central role in the formation and maturation
of autophagosomes through their conjugation to phosphati-
dylethanolamine (PE) [6]. In addition to facilitating the degra-
dation of large protein complexes, protein aggregates, or
dysfunctional organelles, autophagy is also an important cata-
bolic pathway for replenishing cellular nutrients, particularly
under challenging conditions of nutrient shortage [7].
Treatment of cells with specific proteasome inhibitors
results in the accumulation of misfolded proteins that preci-
pitate in intracellular aggregates [4]. Also, malfunctioning of
autophagy causes impaired clearance of aggregation-prone
proteins, increasing the load of protein aggregates, which
may ultimately lead to cell death [8,9]. Aggregates are actively
CONTACT Nico P. Dantuma nico.dantuma@ki.se Department of Cell and Molecular Biology, Karolinska Institutet, Biomedicum, 7A, Solnavägen 9, Stockholm
S-171 65, Sweden
Supplemental data for this article can be accessed here
AUTOPHAGY
2022, VOL. 18, NO. 7, 1486–1502
https://doi.org/10.1080/15548627.2021.1988359
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

sequestered in aggresomes, which are subcellular, membrane-
less structures that serve as a deposit for aggregation-prone
proteins [10,11]. By creating a physical barrier, aggresomes
confine the impact of these proteins, which are inherently
toxic for eukaryotic cells [12]. As such, the presence of aggre-
somes is indicative for insufficient protein quality control by
the UPS and/or autophagy and intimately linked to loss of
proteostasis and induction of proteotoxic stress.
Proteotoxic stress is a common condition in malignant
cells [13]. The constant challenge of keeping the negative
consequences of proteotoxic stress in check increases the
sensitivity of cancer cells to drugs that interfere with protec-
tive mechanisms aimed at restoring proteostasis, such as the
UPS and autophagy [14,15]. The increased load of misfolded
proteins in malignant cells as well as their altered metabolic
state generates a therapeutic window at which limiting the
activity of these proteolytic systems has lethal consequences
for cancer cells without causing substantial collateral damage
to healthy, untransformed cells. Hence, in the face of consti-
tutive proteotoxic stress, curtailing the destruction of mis-
folded proteins in malignant cells causes uncontrolled
accumulation of aberrant proteins that pollute their intracel-
lular environment, resulting in induction of cell death due to
an unresolvable loss of proteostasis [16].
The UPS in particular has emerged as a powerful target for
a new class of cancer therapeutics, resulting in the development
of highly potent proteasome inhibitors that are currently used as
a first-line treatment for certain hematological malignancies
[17]. Inhibitors of autophagy, such as the lysosomotropic agent
hydroxychloroquine, which inhibits lysosomal degradation, are
also being explored in various clinical trials for their potential in
cancer treatment [18]. While both the UPS and autophagy have
great potential as targets for anti-cancer drugs, inhibiting only
one of these systems leaves space for adaptive responses between
these partially overlapping proteolytic pathways [19]. Indeed,
inhibition of autophagy is accompanied by induction of the
UPS [20], while UPS inhibition results in activation of protein
degradation by autophagy [21]. Accordingly, stimulation of
autophagy makes cells less sensitive to the toxic effect of UPS
inhibition [22], and combined inhibition of the UPS and auto-
phagy increases induction of apoptosis in cancer cells as com-
pared to targeting only one of these proteolytic systems [23].
Here, we report on a cell-based, high-content screening
campaign for small molecules that inhibit the UPS. This
resulted in the identification of a novel aminothiazole,
CBK79, which not only caused a dramatic inhibition of the
UPS but also impaired degradation of long-lived proteins by
autophagy. The unique features of this compound cause a loss
of proteostasis, which, despite activation of stress responses,
cannot be resolved and eventually leads to cell death.
Simultaneously impairing these complementary systems for
eliminating aberrant proteins is expected to severely impair
the ability of cells to cope with misfolded proteins, which
provides a likely explanation for the activation of stress
responses and pronounced toxicity observed in CBK79-
treated cells. We propose that CBK79 provides an attractive
active compound for the development of global disruptors of
proteostasis with therapeutic potential.
Results
High-content screening for compounds that impair the
ubiquitin-proteasome system
A human melanoma cell line (MelJuSo) that stably expresses
a yellow fluorescent protein (YFP)-based reporter substrate of
the ubiquitin-proteasome system (UPS) was used in a semi-
automated, high-content screen for small molecules that impair
degradation of proteins by the UPS [24]. The YFP-based repor-
ter substrate ubiquitin
G76V
-YFP (Ub-YFP) consists of an
N-terminal ubiquitin moiety that functions as a ubiquitin fusion
degradation (UFD) signal, targeting the reporter for proteasomal
destruction (Figure 1A) [25]. Prior to degradation, the
N-terminal ubiquitin moiety must be modified by polyubiquitin
chains rendering the UFD signal strictly dependent on polyubi-
quitination [26]. Quantitative analysis of the levels of the repor-
ter substrate in cells treated with experimental small molecules
readily enabled identification of compounds that impair the
cell’s capacity to degrade UPS substrates [27]. Compounds that
directly or indirectly impair any of the critical steps required for
efficient ubiquitin-dependent degradation of proteins – from the
initial activation of ubiquitin until the final hydrolysis in the
proteasome – will be identified as hits in this phenotypic screen.
A small-molecule screening campaign was performed using
a ChemBridge compound set available at the Chemical Biology
Consortium Sweden (CBCS), consisting of 17,500 compounds
that have been selected to cover a large chemical space. Each 384-
well plate contained 32 controls (8 positive controls: 100 nM
epoxomicin; 24 negative controls: 0.1% DMSO) with the
remaining wells, each containing a library compound at a final
concentration of 10 µM (Figure 1B). The average z’-factor of the
plates was 0.54, confirming the robustness of the screening
procedure. Compounds were considered as potential hits if
they caused an increase in the mean fluorescent intensity that
was larger than the mean fluorescence intensity of the epoxomi-
cin control after subtraction of 3 times its standard deviation.
From 17,500 compounds, 70 compounds showed increase
YFP signal in the primary screen. The images of potential hits
were visually inspected in order to distinguish compounds that
caused intracellular fluorescence, consistent with accumulation
of Ub-YFP, from compounds that gave rise to extracellular
fluorescence, indicative for autofluorescent substances. Forty-
three compounds were excluded at this point. The remaining
27 compounds were analyzed for dose-dependent effects on YFP
fluorescence in both the parental MelJuSo cells and Ub-YFP
MelJuSo cells. The effect on cell viability in the latter was also
inferred by the effect of increasing concentrations of compound
on the cell count. Out of the 27 compounds, only 4 compounds
(ChemBridge IDs: 5676202, 6050076, 5128401 and 7869981) did
not give a fluorescent signal in the parental cell line and
increased the fluorescence intensity of the Ub-YFP-expressing
MelJuSo cells, while also having a negative impact on the cell
count (Table S1). These four hit substances were further vali-
dated by weighing in dry substances and making new compound
stocks, which were used for analysis of dose-dependent effects
on YFP fluorescence (Fig. S1).
Next, we applied several computational methods to identify
and remove potentially problematic compounds from
AUTOPHAGY 1487

a medicinal chemistry perspective. Compound 5128401 violated
the Lipinski rule of 5 [28] and was therefore excluded (Fig. S1).
The remaining three hit compounds did not violate Lipinski’s
rule of 5, with compound 7869981 being the only compound
that passed the PAINS filter [29] and had a drug-like profile
according to the REOS criteria [30]. Based on the dose-response
curves and these chemical criteria, we decided therefore to focus
our attention on compound 7869981: 3-methyl-N-[4-(pyridine-
2-yl)-1,3thiazol-2-yl]-pyridin-2-amine (CBK267272) as this
compound had a drug-like profile, had the lowest molecular
weight and caused a dose-dependent increase in reporter levels
that was accompanied by loss of cell viability.
To further define the chemical space of this compound, we
analyzed 31 structural analogues that were used in a limited
structure-activity relationship (SAR) analysis (Table S2).
Repositioning of the nitrogen atoms in the two pyridine
rings resulted in loss of UPS inhibition, suggesting that this
feature was critical for its inhibitory activity (Figure 1(C, D);
Table S2). However, small substituents were accepted in the
pyridine rings, which allowed us to look for more potent
analogues. From the structural analogues, we selected 3-
methyl-N-[4-(3-methylpyridin-2-yl)-1,3-thiazol-2-yl]pyridin-
2-amine (CBK288679, which we will refer to as CBK79) for
further characterization based on its increased potency to
Figure 1. A high-content screen for inhibitors of the UPS. (A) Schematic representation of the ubiquitin-fusion degradation (UFD) signal used for the screen. MelJuSo
cells stably express ubiquitin fused to YFP (Ub-YFP). A point mutation in glycine 76 to valine (G76V) disrupts the GG motif, hindering cleavage by deubiquitinating
enzymes and therefore serving as a degradation signal for the UFD pathway. The rapid turnover of the protein by the proteasome provides cells with low basal YFP
levels, which will be elevated upon blockade of ubiquitin-dependent degradation. (B) Workflow of the screen. 17,500 compounds were screened in an automated
manner in 384-well plates. An automated analysis was performed to find compounds that elevated YFP over a predefined threshold based on the wells treated with
DMSO (negative controls). CBK267272 was selected for further study. More information can be found in Fig. S1 and Table S1. (C) MelJuSo Ub-YFP cells were treated
for 6 h with 29 of the structural analogues of CBK267272 at a final concentration of 20 µM. Nuclei were stained with Hoechst 33342 and cells were directly imaged
live with an automated widefield microscope. Data are represented as scatter plots, where each dot represents the mean YFP nuclear intensity per cell of one
experiment. The mean ± SD of three independent experiments is shown. More information can be found in Table S2. (D) Summary of the findings of the SAR. (E)
Chemical structures of the initial hit compound CBK267272 and the selected optimized compound after SAR, CBK288679 (hereafter referred to as CBK79). (F)
Representative maximal intensity projections of MelJuSo Ub-YFP cells treated for 6 h with CBK267272 or CBK79 (5 µM). DMSO 0.1% was used as negative control. The
nuclei were counterstained with Hoechst 33342 and cells imaged live with an automated widefield microscope. Scale bar: 20 µm. (G) Dose-response curves
performed with MelJuSo Ub-YFP cells. Cell viability was assessed after the indicated timepoints. Data are represented as mean ± SD of three independent
experiments (except for the 48 h timepoint, which corresponds to two independent experiments). Non-linear curve fitting is depicted in red. The half-maximal
inhibitory concentration (IC
50
) upon CBK79 treatment for each timepoint is shown (95% confidence intervals [CI]).
1488 T. A. GIOVANNUCCI ET AL.

induce reporter accumulation and cell death (Figure 1(E, F);
Table S1). The MelJuSo Ub-YFP cell line displayed IC
50
values
of 2.38 µM (95% confidence interval [CI] 1.72–4.64), 0.38 µM
(95% CI 0.26–0.57) and 0.22 µM (95% CI 0.18–0.35) when
analyzed 24, 48 and 72 h after administration of CBK79,
respectively (Figure 1G). CBK79-inflicted cell death was cas-
pase-independent as this was not prevented by administration
of the pan-caspase inhibitor Q-VD-OPh (Fig. S2).
CBK79 causes accumulation of ubiquitin-dependent and -
independent proteasome substrates
Administration of CBK79 to MelJuSo Ub-YFP cells resulted
in a steep dose-dependent increase in YFP fluorescence that
was readily detectable by fluorescence microscopy
(Figure 2A). Quantitative analysis showed that the observed
EC
50
for inhibition of Ub-YFP degradation was 2.2 µM (95%
CI 1.3–4.9) when assessed 6 h after administration of CBK79
(Figure 2B). An increase in Ub-YFP levels was already detect-
able by western blotting 1 h after administration of the com-
pound (Figure 2C). The levels of polyubiquitinated proteins
also increased with similar kinetics, indicative for a general
defect in the degradation of ubiquitinated proteins in CBK79-
treated cells (Figure 2C). To determine if the accumulation
was caused by a delay in proteasomal degradation, we wanted
to test if CBK79 inhibits the turnover of Ub-YFP. As the
steady-state levels of Ub-YFP are very low and difficult to
detect, we treated Ub-YFP-expressing cells briefly with the
reversible proteasome inhibitor bortezomib to allow accumu-
lation of the Ub-YFP reporter to detectable levels. After wash-
ing away the reversible proteasome inhibitor and
administration of cycloheximide to switch off protein synth-
esis, we analyzed the clearance of the accumulated Ub-YFP in
the absence or presence of CBK79. This experiment con-
firmed that the clearance of Ub-YFP was inhibited in the
presence of CBK79, consistent with an inhibitory effect of
CBK79 on proteasomal degradation (Figure 2D).
Importantly, the kinetics of reporter accumulation were com-
parable to the increase in the levels of TP53/p53 (tumor
protein p53) (Figure 2C) and HIF1A/HIF-1α (hypoxia indu-
cible factor 1 subunit alpha) (Figure 2E), two endogenous
substrates for ubiquitin-dependent proteasomal degradation
[31,32]. The accumulation of endogenous substrates and ubi-
quitin conjugates confirms that the Ub-YFP substrate reliably
reports on the overall status of the UPS.
To test whether the inhibitory effect of CBK79 is confined
to ubiquitin-dependent proteasomal degradation, we took
advantage of a reporter substrate that is targeted for protea-
somal degradation by a motif derived from ornithine decar-
boxylase (ODC). The ODC degradation signal targets for
proteasomal degradation without requiring polyubiquitina-
tion of the substrate [33]. Although the compound only had
a marginal effect on ODC degradation under short incuba-
tions, 24 h treatment with CBK79 resulted in increased levels
of the ubiquitin-independent proteasome substrate that were
comparable to the levels observed in proteasome inhibitor-
treated cells (median YFP levels = 2.1 a.u in DMSO vs 91.6 in
CBK79 and 99.4 in epoxomicin) (Figure 2F; Fig. S3A).
A possible explanation for the observed inhibition of
ubiquitin-dependent and -independent substrates could be
inhibition of the proteolytic activity of the proteasome.
However, administration of CBK79 caused only a 16%
decrease in the chymotrypsin-like activity of the proteasome,
indicating that proteasome inhibition is not the primary cause
for the accumulation of UPS substrates (Fig. S3B). Notably, it
has been shown that as much as 80% reduction in chymo-
trypsin-like activity is required to cause a general accumula-
tion of proteasome substrates [25,34]. Together, these data
show that CBK79 causes global impairment of proteasomal
degradation of ubiquitin-dependent and ubiquitin-
independent substrates without substantially inhibiting the
enzymatic activity of the proteasome.
CBK79 inhibits autophagy
In subsequent analysis of the cellular response to CBK79, we
found that administration of CBK79 to human osteosarcoma
(HOS) cells expressing green fluorescent protein (GFP)-
tagged MAP1LC3B/LC3B (microtubule-associated protein 1
light chain 3 beta) caused the formation of cytosolic GFP-
LC3B foci, which is indicative for an effect on autophagy
(Figure 3A). The formation of LC3B foci was dose-
dependent (Figure 3B) and time-dependent (Figure 3C) and
coincided with the generation of lipidated LC3B (LC3B-II)
(Figure 3D). An increase in LC3B-II can be caused by either
an induction or inhibition of autophagy [35]. To distinguish
between these two possibilities, we measured the autophagic
flux by flow cytometry using a U2OS cell line that stably
expresses LC3B carrying a tandem fluorescent tag consisting
of monomeric red fluorescent protein (mRFP) and GFP.
Because GFP fluorescence is quenched in the acidic lysosomal
compartment, in contrast to mRFP, the mRFP:GFP ratio can
be used as a readout for the autophagic flux [35]. Notably,
CBK79 caused a 4.4-fold reduction in the autophagic flux that
was comparable to the reduction observed after administra-
tion of the autophagy inhibitor bafilomycin A
1
(BafA1)
(Figure 3(E, F)), which blocks autophagy by inhibiting the
vacuolar-type H
+
-translocating ATPase (V-ATPase) [35].
Analysis of the degradation of long-lived proteins, which are
primarily degraded by autophagy, confirmed a significant
inhibition in the turnover of this pool of proteins in CBK79-
treated cells although to a lesser extent than observed with
BafA1 administration (Figure 3G). We conclude that CBK79
not only impairs the UPS but also inhibits degradation of
long-lived proteins by autophagy.
CBK79 induces non-canonical lipidation of LC3B
Since the autophagic flux measured by the tandem-tagged
LC3B was more severely impaired than the degradation of
long-lived proteins, we had a closer look at the status of LC3B
in CBK79-treated cells. As expected, co-treatment of CBK79
with MTOR (mechanistic target of rapamycin kinase) inhibi-
tor (KU-0063794), which induces autophagy, caused an addi-
tional increase in the levels of lipidated LC3B, consistent with
CBK79 obstructing the delivery of LC3B to the lysosomal
compartment (Figure 4(A, B)). Accordingly, co-treatment
with MTOR inhibitor also further increased the number of
AUTOPHAGY 1489

GFP-LC3B puncta (Figure 4(C, D)). Surprisingly, whereas
treatment of cells with BafA1 caused an accumulation of
lipidated LC3B that was comparable to the effect of CBK79,
co-treatment of CBK79 with BafA1 prevented the appearance
of LC3B-II and the formation of LC3B puncta (Figure 4
(A-D)). However, administration of chloroquine (CQ),
a weak base that inhibits autophagy by neutralizing the acidic
lysosomal pH [35], did not prevent lipidation of LC3B,
ZsGreen-ODC
CBK79 (µM)
)sruoh(0
CBK79
Ubiquitin
100
20
25
50
75
37
0.5 1 2 4 6
0 (hours)0.5 1 2 4 6
TP53
50
Ub-YFP
37
ACTB
HIF1A
100
75
ACTB
a b
c
e
d
f
Ub-YFP
ACTB
t0
DMSO
EPX
CBK79
+ CHX (4 h)
0
200
400
600
800
DMSO
EPX
CBK79
YFP intensity/cell (a.u)
YFP-positive cells (%)
EC50 = 2.2 µM
(95% CI = 1.3 - 4.9)
CBK79 (µM)
DMSO
Hoechst/YFP
5 10 20
0 5 10 15 20
0
20
40
60
80
100
MelJuSo Ub-YFP
CBK79
37
****
****
kDa
kDa
kDa
Figure 2. CBK79 causes accumulation of ubiquitin-dependent and -independent proteasome substrates. Representative images of MelJuSo Ub-YFP cells treated for
6 h with CBK79 at the indicated concentrations. DMSO at 0.1% was used as negative control. The nuclei were counterstained with Hoechst 33342 and cells imaged
live with an automated widefield microscope. Scale bar: 20 µm. (B) Dose-response experiments performed with MelJuSo Ub-YFP cells. Cells were treated for 6 h with
a range of compound concentrations. Nuclei were stained with Hoechst 33342 and cells were directly imaged live with an automated widefield microscope. Data
were pooled from three independent experiments and are represented as mean ± SD. Non-linear curve fitting is depicted in green. The half-maximal effective
concentration (EC
50
) upon CBK79 treatment is shown (2.2 µM, 95% confidence interval 1.3–4.9). (C) MelJuSo Ub-YFP cells were treated with CBK79 (10 µM) and
harvested at the indicated timepoints. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Beta-actin (ACTB) is shown as loading control.
Representative blots from one of three independent experiments are shown. (D) MelJuSo Ub-YFP cells were pre-treated for 3 h with the reversible proteasome
inhibitor bortezomib (25 nM) to increase the levels of YFP substrate before the treatment. Samples were taken directly after pretreatment (t0). The remaining wells
were co-treated with cycloheximide (CHX, 50 µg/ml) and either DMSO 0.1%, epoxomicin (EPX, 100 nM) or CBK79 (10 µM) and harvested after 4 h (CHX 4 h). Cell
lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of two independent experiments are shown. (E) Cell lysates
from (C) were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of three independent experiments are shown. (F) MelJuSo
ZsGreen-ODC cells were treated for 16 h with CBK79 (2.5 µM). The proteasome inhibitor epoxomicin (100 nM) was included as positive control. Nuclei were
counterstained with Hoechst 33342 and the cells imaged live with an automated widefield microscope. The nuclear YFP intensity per cell was quantified using
MetaXpress. Frequency and distribution of the YFP intensity per cell after background substraction (determined as the nuclear YFP average intensity of all DMSO-
treated cells) are shown as violin plots. n = 1308 cells (DMSO); n = 297 cells (CBK79) and n = 207 cells (proteasome inhibitor, epoxomicin 200 nM) from
a representative experiment (of two independent experiments). Black lines within each distribution represent the median; colored lines represent the upper and
lower interquartile range limits. Significant differences are based on adjusted p-values (Kruskal-Wallis [H = 1075, df = 2, p < 0.0001] with Dunn’s multiple
comparisons test). ****p < 0.0001.
1490 T. A. GIOVANNUCCI ET AL.

suggesting that the effect of BafA1 is not caused by preventing
acidification of the lysosomes but likely relates to its ability to
act as a V-ATPase inhibitor (Figure 4(A, B)).
Lipidation of LC3 that is BafA1 sensitive has also been
reported for other inhibitors of autophagy and has been
attributed to induction of a non-canonical pathway for lipida-
tion of LC3 [36,37]. Canonical lipidation of LC3 to double-
membrane phagophores requires the ULK1 (unc-51 like auto-
phagy activating kinase 1) complex and the class III phospha-
tidylinositol 3-kinase (PtdIns3K) complex I that produces
phosphatidylinositol-3-phosphate (PtdIns3P). The PtdIns3P-
binding protein WIPI2B (WD repeat domain, phosphoinosi-
tide interacting 2 beta) further recruits the ATG12–ATG5-
ATG16L1 complex, which functions as an E3 ligase that
determines the site of LC3 lipidation [5]. In contrast, the
ULK1 and PtdIns3K complexes are dispensable for the non-
canonical pathway of LC3 lipidation [38].
To explore if CBK79 induces non-canonical LC3 lipida-
tion, cells were co-treated with CBK79 and 3-methyladenine
(3-MA), a compound that blocks canonical lipidation of LC3
by inhibiting the PtdIns3K complex [39]. 3-MA did not inhi-
bit CBK79-induced LC3B lipidation and formation of GFP-
LC3B puncta in HOS (Figure 4(A-D)) and HeLa cells (Fig.
S4A, B), indicating that CBK79 induces LC3B lipidation in
a PtdIns3P-independent, BafA1-sensitive manner, character-
istic for non-canonical lipidation. Consistently, an increase in
lipidated LC3B and LC3B puncta was also observed upon
CBK79 treatment of HeLa cells that lack the ULK1 complex
component ATG13 (Figure 4E, Fig. S4C). Importantly, HeLa
cells deficient for the ATG16L1α and β isoforms, and hence
lacking LC3 ligase activity, did not show LC3B lipidation nor
LC3B puncta formation upon CBK79 treatment, confirming
that the formation of LC3B puncta and appearance of LC3B-II
is due to actual lipidation of LC3B (Figure 4F, Fig. S4D).
Figure 3. CBK79 inhibits the autophagic flux. (A) Representative images of HOS GFP-LC3B cells. Cells were treated for 4 h with DMSO 0.1%, CBK79 (10 µM) or
epoxomicin (EPX, 100 nM). Nuclei were counterstained with Hoechst 33342 and the cells imaged live with an automated widefield microscope. Scale bar: 20 µm. (B)
Dose-response experiments performed with HOS GFP-LC3B cells. Cells were treated for 4 h with a range of compound concentrations. Nuclei were stained with
Hoechst 33342 and cells were directly imaged live with an automated widefield microscope. Single-cell measurements of GFP-LC3B puncta from a single experiment
are shown (n ≥ 193 cells/condition). Data are shown as box plots with median and 5–95 percentiles. (C) HOS GFP-LC3B cells were treated with CBK79 (10 µM) for the
indicated timepoints. Nuclei were stained with Hoechst 33342 and cells were directly imaged live with an automated widefield microscope. Single-cell measurements
of GFP-LC3B puncta from a single experiment are shown (n > 200 cells/condition). Data are shown as box plots with median and 5–95 percentiles. (D) HOS GFP-LC3B
cells were treated with CBK79 (10 µM) and harvested at the indicated timepoints. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Beta-
actin (ACTB) is shown as loading control. Representative blots from one out of >3 independent experiments are shown. (E) Analysis of autophagic flux in U2OS mRFP-
GFP-LC3B cells treated with DMSO 0.1%, bafilomycin A
1
(BafA1, 100 nM) or CBK79 (10 µM) for 4 h, washed briefly in saponin (0.05%) and analyzed by flow cytometry.
Autophagic flux was determined as the ratio of mean mRFP- and mean GFP-fluorescence. A representative histogram from one of three independent experiments is
shown. (F) Data from (E) were normalized to BafA1. Data are presented as the mean ± SD of three independent experiments (unpaired, two-tailed t-test, t(4) = 12.60,
p < 0.0002). (G) Long-lived protein degradation assay in U2OS cells treated with DMSO 0.1%, bafilomycin A
1
(BafA1, 100 nM) or CBK79 (10 µM) for 4 h. The
percentage of long-lived protein degradation was quantified. Data are presented as the mean ± SD of three independent experiments, each performed in technical
duplicates. Significant differences are based on adjusted p-values of multiple comparisons against DMSO (one-way ANOVA [F
2,6
= 42.74, p = 0.0003] with Dunnett’s
multiple comparisons test). **p = 0.0052; ***p = 0.0002.
AUTOPHAGY 1491

To further explore if the effect of CBK79 on autophagy was
confined to non-canonical lipidation of LC3B, we treated
ATG16L1-deficient HEK293 cell lines that ectopically
expressed either GFP-tagged wild-type ATG16L1 or
a C-terminally truncated ATG16L1 (ATG16L1[1–249]) that
can execute canonical but is deficient for non-canonical LC3
lipidation [40]. In line with non-canonical lipidation being the
primary cause for the increase in LC3B-II in response to
a
c
ef
hg
HOS GFP-LC3B
GAPDH
CBK79
15
kDa
LC3B-I
LC3B-II
CBK79
HEK293 ATG16L1 KO + eGFP-ATG16L1β
CBK79
HEK293 ATG16L1 KO + eGFP-ATG16L1[1-249]
15 15
15
LC3B-I
LC3B-II
LC3B-I
ACTB
ACTB
CBK79
CBK79
HeLa ATG13 KO HeLa ATG16L1 KO
GAPDH
ACTB
GAPDH
ATG16L1
50
ACTB
ATG16L1
100
b
BafA1 DMSO MTORi
BafA1
+ MTORi 3-MA
CBK79
HOS GFP-LC3B
Hoechst/GFP-LC3B
ns
****
****
****
****
DMSO
CBK79
BafA1
BafA1
MTORi
BafA1+MTORi
3-MA
MTORi
BafA1+MTORi
3-MA
GFP-LC3B puncta/cell
DMSO
BafA1
MTORi
BafA1+MTORi
CQ
3-MA
BafA1
MTORi
BafA1+MTORi
CQ
3-MA
CBK79
LC3B-II:GAPDH
relative to DMSO
single treat.
CBK79 co-treat.
d
0
10
20
30
40
LC3B-I
LC3B-II LC3B-I
LC3B-II
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
MTORi
CQ
3-MA
BafA1
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
MTORi
CQ
3-MA
BafA1 ++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
MTORi
CQ
3-MA
BafA1
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
MTORi
CQ
3-MA
BafA1 ++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-+
+
MTORi
CQ
3-MA
BafA1
0
5
10
15
15
kDa
kDa
kDa
kDa
Figure 4. CBK79 induces non-canonical lipidation of LC3B. (A) HOS GFP-LC3B cells were treated with either DMSO 0.1% or CBK79 (10 µM) in co-treatment with the
indicated autophagy modulators for 4 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of five
independent experiments are shown. MTOR inhibitor (“MTORi”, Torin-1); CQ = chloroquine; 3-MA: 3-methyladenine. (B) Band intensities were measured using ImageJ.
LC3B-II band was normalized to the loading control (GAPDH) and the levels are displayed relative to DMSO. Data are shown as average ± SD of five independent
experiments. (C) Representative images from HOS GFP-LC3B cells treated with the indicated compounds for 4 h. Nuclei were stained with Hoechst 33342 and cells
were directly imaged live with an automated widefield microscope. Scale bar: 20 µm. (D) Quantification of the GFP-LC3B puncta per cell from (C). Data are shown as
box plots with median and 5–95 percentiles (n > 200 cells/condition). Significant differences are based on adjusted p-values between relevant conditions (Kruskal-
Wallis [H = 1256, df = 9, p < 0.0001] with Dunn’s multiple comparisons test). ns > 0.9999; ****p < 0.0001. (E) HeLa ATG13 knockout (ATG13 KO) cells, or (F) HeLa
ATG16L1 knockout (ATG16L1 KO) cells were treated with either DMSO 0.1% or CBK79 (10 µM) in co-treatment with the indicated autophagy modulators for 4 h. Cell
lysates were analyzed by immunoblotting with the indicated antibodies. Beta-actin (ACTB) is shown as loading control. Representative blots from one of three
independent experiments are shown. (G) HEK293 ATG16L1 knockout cells stably rescued with eGFP-tagged full length ATG16L1β were treated with either DMSO 0.1%
or CBK79 (5 µM) in co-treatment with the indicated autophagy modulators for 3 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies.
Representative blots from one of three independent experiments are shown. (H) HEK293 ATG16L1 knockout cells stably rescued with eGFP-tagged ATG16L1[1–249]
were treated with either DMSO 0.1% or CBK79 (5 µM) in co-treatment with the indicated autophagy modulators for 3 h. Cell lysates were analyzed by
immunoblotting with the indicated antibodies. Representative blots from one of three independent experiments are shown.
1492 T. A. GIOVANNUCCI ET AL.

CBK79, we found that the accumulation of LC3B-II was
strongly reduced in cells deficient for non-canonical autopha-
gy (ATG16L1[1–249]) as compared to those rescued with full-
length ATG16L1 (Figure 4(G, H)). A small 3-MA-sensitive
increase in lipidated LC3B was still observed upon CBK79
administration to ATG16L1[1–249]-expressing cells, suggest-
ing that a minor fraction of the total increase in LC3B-II can
be attributed to canonical lipidation (Figure 4H). Altogether,
these data show that the increase in lipidated LC3B upon
CBK79 administration is primarily mediated by an unconven-
tional route that bypasses the requirement of the ULK1 and
PtdIns3K complexes, but that still depends on the ATG12–
ATG5-ATG16L1 conjugation complex.
CBK79 induces proteotoxic stress
Insufficient clearance of misfolded proteins can cause proteo-
toxic stress, which in turn can lead to the activation of
adaptive responses aimed at resolving the critical situation
[41]. One such important adaptive response is the sequestra-
tion of inherently toxic aggregation-prone proteins in
a cytosolic, non-membranous subcellular structure known as
the aggresome [42]. Aggresomes are formed by active trans-
port of protein aggregates to the perinuclear region, where
they are surrounded by a cage consisting of the intermediate
filament VIM (vimentin) [10,11]. Immunostaining of CBK79-
treated cells indeed showed the formation of ubiquitin-
positive aggresomes that contained the ubiquitin receptor
SQSTM1/p62 (sequestosome 1) and were surrounded by
VIM, confirming that administration of CBK79 causes loss
of proteostasis (Figure 5(A, B); Fig. S5). Moreover, staining of
lysosomes using a LAMP1 (lysosomal associated membrane
protein 1) antibody showed positioning of lysosomes around
the VIM cage (Figure 5(A, B)), an occurrence previously
described in aggresomes caused by treatment with proteasome
inhibitor [43].
In addition to minimizing the toxic effects of aggregation-
prone proteins, proteotoxic stress can also trigger cellular
responses to reduce the levels of aberrant proteins by inhibit-
ing the synthesis of new proteins [44]. Puromycin labeling
was used to analyze the effect of CBK79 on protein synthesis
[45], which revealed that the initial inhibition of the UPS and
autophagy was followed by an overall reduction in synthesis
of proteins that became prominent 16 h after administration
of the compound (Figure 5C). Inhibition of protein synthesis
was accompanied by reduced levels of the Ub-YFP reporter
substrate and the endogenous substrate TP53/p53, implying
that inhibition of protein synthesis successfully reduced the
load of UPS substrates (Figure 5D).
Administration of puromycin in combination with com-
pounds that inhibit the UPS has been shown to result in the
induction of stress granules, which are small cytosolic gran-
ules that contain naked mRNAs and RNA binding proteins
[46]. Immunostaining for the RNA binding protein and stress
granule marker G3BP1 (G3BP stress granule assembly fac-
tor 1) showed that while administration of either puromycin
or CBK79 did not induce the formation of stress granules,
these structures were formed when cells were exposed to
puromycin and CBK79 at the same time (Figure 5(E, F)).
Thus, like other compounds that disturb proteostasis,
CBK79 induces stress granules in puromycin-sensitized cells.
Notably, CBK79 administration strongly reduced the puro-
mycin signal in immunostainings (Figure 5E) and gave rise to
fewer puromycin-positive foci than cells that had been incu-
bated with puromycin in the absence of CBK79 (Figure 5G),
which is consistent with the decrease in puromycin signal
observed by western blotting. We conclude that CBK79 acti-
vates cellular stress responses, indicative for induction of
proteotoxic stress.
CBK79 induces the heat shock response
The heat shock response plays an important role in adequate
handling of proteotoxic stress as it induces molecular chaper-
ones that prevent protein aggregation, assist in (re-)folding of
proteins or redirect terminally misfolded proteins for degra-
dation [47]. HSF1 (heat shock transcription factor 1), which is
the central transcriptional regulator of the heat shock
response, accumulates in the nucleus when the levels of mis-
folded proteins exceed a critical threshold [48]. In agreement
with induction of a stress response, administration of CBK79
resulted in an increase in nuclear HSF1 (Figure 6A). CBK79
treatment also resulted in accumulation of HSF1 in nuclear
stress bodies in a time-dependent fashion (Figure 6(B, C)),
arguing that its ability to disturb proteostasis is not confined
to the cytosol but also involves the nuclear compartment [49].
Throughout the study, we used relatively high concentrations
of CBK79 (5–10 µM) when investigating its effect on the UPS
and autophagy whereas we detected effects on cell viability at
lower concentrations upon extended exposure. Therefore, we
determined the effect of CBK79 on the response to misfolded
proteins (by detecting HSPA1A/Hsp70 [heat shock protein
family A (Hsp70) member 1A]), the UPS (Ub-YFP and
TP53/p53) and autophagy (LC3B-II) in a dose-response
experiment. This revealed that induction of HSPA1A/Hsp70
as well as accumulation of Ub-YFP, TP53/p53 and LC3-II can
already be detected when MelJuSo cells had been exposed for
6 h to 2.5 µM CBK79 (Figure 6(D, E)). A significant reduction
in the autophagic flux was also already evident after a 4 h
incubation with 2.5 µM CBK79 (Figure 6(F, G)). We conclude
that CBK79 inflicts UPS and autophagy impairment and
induces proteotoxic stress already after a short exposure to
2.5 µM CBK79.
Thermal preconditioning prevents inhibition of the UPS
while autophagy impairment persists
Activation of the heat shock response by exposing cells to
a brief heat shock renders them resistant to conditions that
cause proteotoxic stress, a phenomenon known as thermoto-
lerance [47]. When MelJuSo cells were exposed for 30 min to
43°C prior to administration of CBK79, the accumulation of
the Ub-YFP reporter substrate was strongly reduced, while
preconditioning had little effect on stabilization of Ub-YFP in
cells treated with proteasome inhibitor (Figure 7A).
Moreover, the increase in ubiquitin conjugates in response
to CBK79 was less pronounced in preconditioned cells, show-
ing that this effect is not limited to the reporter substrates but
AUTOPHAGY 1493

Figure 5. CBK79 induces proteotoxic stress. (A) Representative images of HOS GFP-LC3B cells treated with DMSO 0.1% or CBK79 (10 µM) for 4 h. Cells were fixed and
immunostained using antibodies against VIM, SQSTM1, ubiquitin or LAMP1. Scale bar: 20 µm. (B) Line scans at the aggresome sites are shown to visualize the spatial
distribution of the indicated proteins (red curves) compared to VIM (gray curves). Intensities are normalized to percentages were 0% = minimum intensity value and
100% = maximum intensity value. (C) MelJuSo Ub-YFP cells were treated with DMSO 0.1% or CBK79 (10 µM) for the indicated timepoints. Two samples were treated
for 16 h and then treated with fresh compound solution for the indicated timepoints (“16 h+”). Fifteen minutes before harvesting, puromycin (5 µg/ml) was added to
the cells to monitor its incorporation into newly synthesized proteins. Cycloheximide (CHX, 50 µg/ml) was included as control. An untreated sample without
puromycin was added (“NP”) as a technical control. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Beta-actin (ACTB) is shown as
loading control. Representative blots from one of two independent experiments are shown. (D) MelJuSo Ub-YFP cells were treated with DMSO 0.1% or CBK79 (10 µM)
for the indicated timepoints. Two samples were treated for 16 h and then treated with fresh compound solution for the indicated timepoints (“16 h+ re-addition”).
Cell lysates were analyzed by immunoblotting with the indicated antibodies. Representative blots from one of three independent experiments are shown. (E)
Representative confocal images of MelJuSo Ub-YFP cells treated with DMSO 0.1% or CBK79 (10 µM), alone or in co-treatment with puromycin for 2 h. Cells were fixed
and immunostained using antibodies against G3BP1 and puromycin. Scale bar: 20 µm. (F) MelJuSo Ub-YFP cells were pre-treated with DMSO or CBK79 (10 µM) for
30 min and then co-treated with puromycin (5 µg/ml) for 2 h. After nuclei counterstaining with Hoechst 33342, cells were imaged in an automated manner with
a widefield fluorescent microscope. The number of G3BP1 foci per cell were quantified using CellProfiler. Pooled data from three independent experiments
(DMSO = 1471 cells; DMSO+puromycin = 1521 cells; CBK79+ puromycin = 1467 cells) are shown as box plots with median and 5–95 percentiles. Significant
differences are based on adjusted p-values (Kruskal-Wallis [H = 1029, df = 2, p < 0.0001] with Dunn’s multiple comparisons test). ****p < 0.0001. (G) Cells in (F) were
1494 T. A. GIOVANNUCCI ET AL.

analyzed for the number of puromycin foci per cell. Pooled data from three independent experiments (DMSO = 1457 cells; DMSO+puromycin = 1515 cells; CBK79
+puromycin = 1472 cells) are shown as box plots with median and 5–95 percentiles. Significant differences are based on adjusted p-values (Kruskal-Wallis [H = 1383,
df = 2, p < 0.0001] with Dunn’s multiple comparisons test). ****p < 0.0001.
Figure 6. CBK79 induces the heat shock response. (A) HOS GFP-LC3B cells were treated with DMSO 0.1% (8 h), CBK79 10 µM (0 to 8 h; a representative image for the
2 h timepoint is shown or epoxomicin (EPX, 100 nM) for 8 h. Cells were fixed and immunostained with an HSF1 antibody. HSF1 nuclear foci are marked with white
arrows. Scale bar: 20 µm. (B) The percentage of cells with HSF1 foci from one of two independent experiments are shown. (C) The number of HSF1 foci per cell were
quantified using CellProfiler. Data from one of two independent experiments (n > 200 cells/condition) are shown as box plots with median and 5–95 percentiles. (D)
MelJuSo Ub-YFP cells were treated with DMSO 0.1% (0) or the indicated concentrations of CBK79 for 6 h. Cell lysates were analyzed by immunoblotting with the
indicated antibodies. Beta-actin (ACTB) is shown as loading control. Representative blots from one of three independent experiments are shown. (E) MelJuSo Ub-YFP
cells were treated with DMSO 0.1% (0) or the indicated concentrations of CBK79 for 48 h. Cell lysates were analyzed by immunoblotting with the indicated
antibodies. Representative blots from one of three independent experiments are shown. (F) Analysis of autophagic flux in U2OS mRFP-GFP-LC3B cells treated with
DMSO 0.1%, bafilomycin A
1
(BafA1, 100 nM) or CBK79 at the indicated concentrations for 4 h, washed briefly in saponin (0.05%) and analyzed by flow cytometry.
Autophagic flux was determined as the ratio of mean mRFP- and mean GFP-fluorescence. A representative histogram from one of three independent experiments is
shown. (G) Data from (F) were normalized to BafA1. Data are presented as the mean ± SD of three independent experiments. Significant differences are based on
adjusted p-values of multiple comparisons to the DMSO control condition (one-way ANOVA [F
4,10
= 4.860, p = 0.0195] with Dunnett’s multiple comparisons test).
*p = 0.0388; **p = 0.0064; ns p (0.62) = 0.3356; ns p (1.25) = 0.1791.
AUTOPHAGY 1495

also applies to endogenous proteasome substrates (Figure 7B).
Interestingly, the CBK79-induced appearance of LC3B puncta
(Figure 7C) and non-canonical lipidation of LC3B
(Figure 7D) were still evident after thermal preconditioning.
Thus, while our data suggest that proteotoxic stress contri-
butes to the accumulation of UPS substrates, the effect of
CBK79 on autophagy is disconnected from UPS impairment
and cannot be counteracted by activation of cellular stress
responses.
Discussion
Although efficient and tightly regulated intracellular degrada-
tion is essential for the viability of practically all cells, the
increased dependency of malignant cells on these proteolytic
systems gives rise to a therapeutic window where curtailing
their capacity is lethal for cancer cells without causing
substantial harm to other cells [16]. In various studies, the
critical roles of the UPS and autophagy in intracellular protein
degradation have been explored as therapeutic targets for
cancer treatment [18,50]. This has led to the development of
clinically approved proteasome inhibitors for the treatment of
multiple myeloma and mantle cell lymphoma [51]. On the
other hand, chloroquine and its derivative hydroxychloro-
quine, two FDA-approved drugs that target lysosomal degra-
dation, are being tested in various clinical trials [18]. Our data
on the novel compound CBK79 shows that this active mole-
cule has unique features as it inhibits both ubiquitin-
dependent and -independent degradation of short-lived pro-
teins by the UPS, as well as the degradation of long-lived
proteins by autophagy.
Interference with both proteolytic pathways is an excep-
tional feature although not unprecedented. A small molecule
inhibitor of the ATPase activity of the ubiquitin-selective
ba
dc
CBK79DMSO
Ub-YFP
HSPA1A
ACTB
ACTB
Ubiquitin
100
20
25
50
75
75
37
37
heat shock
6 h0 h
Untr.
+
-+
-+
-
kDa
LC3B-II
ACTB
15
CBK79DMSO
heat shock
6 h0 h
Untr.
+
-+
-+
-
kDa
DMSO
Controlheat shock
CBK79
HOS GFP-LC3B
Hoechst/GFP-LC3B
MelJuSo Ub-YFP
CBK79 XPEOSMD
Controlheat shock
Hoechst/YFP
Figure 7. Thermal preconditioning prevents inhibition of the UPS while autophagy impairment persists. (A) Maximum intensity projections of MelJuSo Ub-YFP
primed with a 30-min heat shock (43°C), recovered for 8 h, and treated with CBK79 (10 µM), epoxomicin (EPX, 100 nM) or DMSO 0.1% for 6 h. Nuclei were
counterstained with Hoechst 33342 and cells imaged in an automated widefield microscope. Scale bar: 20 µm. (B) MelJuSo Ub-YFP were primed with a 30-min heat-
shock (43°C), recovered for 8 h, and treated with DMSO 0.1% or CBK79 (10 µM) for 6 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies.
Beta-actin (ACTB) is shown as loading control. Representative blots from one of three independent experiments are shown. (C) Maximum intensity projections of HOS
GFP-LC3B cells primed with a 30-min heat shock (43°C), recovered for 8 h, and treated with DMSO 0.1% or CBK79 (10 µM) for 4 h. Nuclei were counterstained with
Hoechst 33342 and cells imaged in an automated widefield microscope. Scale bar: 20 µm. (D) HOS GFP-LC3B cells were treated as in (C). Cell lysates were analyzed by
immunoblotting with the indicated antibodies. Representative blots from one of two independent experiments are shown.
1496 T. A. GIOVANNUCCI ET AL.

unfoldase VCP/p97 (valosin containing protein) impaired
both the UPS and autophagy resulting in a more rapid induc-
tion of apoptosis as compared with proteasome inhibitor [52].
Earlier studies showed that this AAA-ATPase by means of its
unfolding activity is involved in ubiquitin-dependent protea-
somal degradation of a diverse set of substrates [53], as well as
the maturation of autophagosomes [54,55], providing an
explanation for the dual activity of this inhibitor. Consistent
with its behavior as a ubiquitin-selective complex, inhibition
was, however, restricted to ubiquitin-dependent proteasome
substrates [52]. This stands in contrast to the activity of
CBK79 as this also impairs degradation of the ubiquitin-
independent substrate ZsGreen-ODC, suggesting a different
mode of action. It is also noteworthy that UPS inhibition by
another VCP/p97 inhibitor was accompanied by stimulation
of autophagy [56], implying that the ability of VCP/p97 inhi-
bitors to target both pathways may depend on the nature of
the inhibitory compound.
While it is clear that the simultaneous impairment of these
partially compensatory proteolytic systems will severely chal-
lenge the capacity of cells to properly execute protein quality
control, the precise mode of action of CBK79 responsible for
this dual effect remains to be clarified. A possible cause for the
UPS impairment is an acute overload of this proteolytic
system with misfolded proteins, which is supported by our
observation that thermal preconditioning can prevent CBK79-
inflicted UPS impairment. This is in line with several studies
that show that various proteotoxic stress conditions can com-
promise the UPS [24,34,57]. Overwhelming the pool of pro-
teasomes with misfolded proteins, many of which may be
hard to degrade due to their propensity to aggregate, is there-
fore likely to contribute to the observed UPS defect. This may
also explain the inhibition of a ubiquitin-independent sub-
strate as these substrates equally depend on sufficient protea-
somal capacity for their degradation. As proteasomes, due to
the size constraints of the entrance pore, cannot handle pro-
tein aggregates, they may be more susceptible to the negative
effects of protein aggregates than autophagy [34]. Consistent
with this model, thermal preconditioning did not relieve the
inhibitory effect of CBK79 on autophagy, suggesting that
preventing protein aggregation is of less relevance for auto-
phagy inhibition. This does not exclude the possibility that
proteotoxic stress plays a role in CBK79-induced inhibition of
autophagy, as critical autophagic proteins may be subject to
misfolding rendering them nonfunctional. While the increase
in molecular chaperones in thermally preconditioned cells
might prevent their aggregation, it may be less successful in
restoring their functionality in autophagy.
Interestingly, we found that the formation of LC3B
puncta in CBK79-treated cells was primarily facilitated
by non-canonical lipidation of LC3B as it was indepen-
dent of the ULK1 and PtdIns3K complexes that are cri-
tical for the formation of autophagosomes. Although
surprising, other autophagy inhibitors have also been
shown to display broad, poorly characterized unconven-
tional types of LC3 lipidation, suggesting that this is
a more common feature among autophagy inhibitors
[36,37]. Importantly, the reduced autophagic flux and
impaired degradation of long-lived proteins unequivocally
shows that this proteolytic pathway is inhibited in
CBK79-treated cells.
Except for proteasome inhibitors, attempts to clinically
exploit drugs that target proteostasis have been with limited
success so far. The novel compound presented in this study
shows that it is possible to target both the UPS and autophagy
causing a global collapse of intracellular protein quality control.
To what extent the accompanying loss of proteostasis caused by
CBK79 is a cause or consequence of inhibition of these proteo-
lytic systems remains to be elucidated. However, regardless of
the sequence of events that underlies the inhibition of intracel-
lular protein degradation, our study illustrates the potential of
disturbing proteostasis by simultaneously interfering with these
two central processes in protein quality control.
Materials and methods
Cell culture
MelJuSo Ub-YFP cells have been previously described [24].
HOS parental and GFP-LC3B were obtained from Gerald
McInerney [58]. U2OS mRFP-GFP-LC3 were obtained from
Jeff MacKeigan [59]. HeLa TREx FlpIn WT were a kind gift
from A. Tighe and S.S. Taylor (University of Manchester, UK)
and used to generate the ATG13 and ATG16L1 knockout cell
lines, as described below. HEK293 WT cells were obtained
from Tamotsu Yoshimori (Osaka University, Japan) and were
used to generate ATG16L1 knockout cell lines reconstituted
with either eGFP-ATG16L1β or eGFP-ATG16L1[1–249] as
described in [40]. All cells were cultured in DMEM
+GlutaMAX (Life Technologies, 31,966–021) supplemented
with 10% fetal bovine serum (Life Technologies, 10,270–106)
in a humidified incubator at 37°C and 5% CO
2
. All cell lines
were routinely tested for Mycoplasma infection.
To induce autophagy, cells were treated with 250 nM Torin
1 (Tocris, 4247) or with 500 nM KU-0063794 (Sigma-Aldrich,
SML0382). Autophagy was blocked using 10 mM 3-methyla-
denine (3-MA; Sigma-Aldrich, 189,490), 100 nM bafilomycin
A
1
(BafA1; Enzo Life Sciences, BML-CM110) or 10 µM chlor-
oquine (CQ; Sigma-Aldrich, C6628).
CRISPR-Cas9 knockout of human ATG16L1 and ATG13
Knockouts of ATG16L1 and ATG13 were generated in HeLa
TREx FlpIn cells using sgRNA guides designed using the
CRISPR Design Tool through MIT (crispr.mit.edu). The
following guides were selected: ATG16L1 guide 4 (5`-TTA
CGT GGC TGC TCT GCT GA), ATG16L1 guide 5 (5`-GCC
ACA TCT CGG AGC AAC TG) and ATG13 guide 1 (5`-
TTT ACC CAA TCT GAA CCC GT). The sgRNA guides
were synthesized (Sigma-Aldrich) and cloned into the
pSpCas9 (BB)-2A-Puro (pX459) V2.0 plasmid (Addgene,
62,988; deposited by Feng Zhang). Cells were transfected
using XtremeGene9 (Roche, obtained via Sigma-Aldrich,
6,365,787,001) according to the manufacturer’s protocol.
After 24 h, cells were treated with selection medium con-
taining 2.0 μg/ml puromycin and fresh selection medium
was provided every 2–3 days. After 7 days puromycin resis-
tant cells were seeded as single cell/well in a 96-well plate
AUTOPHAGY 1497

through serial dilution. Individual clones were isolated and
expanded before knockouts were confirmed via western
blotting.
Chemical screen
For the primary screen, MelJuSo Ub-YFP cells were seeded in
black with clear bottom 384-well plates (Falcon, 35–3962) by
distributing 30 µl/well using Matrix WellMate™ (Thermo
Fisher Scientific; Waltham, MA, USA) and incubated over-
night at 37°C in a 5% CO
2
atmosphere. The following day,
cells were exposed to compounds at a final concentration of
10 µM. The compound stocks (in DMSO) were first diluted in
DMEM at a concentration of 20 µM, to then distribute 30 µl/
well to achieve the desired final concentration using the
Biomek 384-well NX robot (Beckman Coulter). Epoxomicin
(Enzo Life Sciences, BML-PI127) was added to specific wells
(8 wells per plate) as a positive control at a final concentration
of 100 nM. DMSO controls (24 wells per plate) at 0.1%
concentration were used as negative controls. The cells were
incubated with the compounds for 19–20.5 h before Hoechst
33342 (Life Technologies, H3570) was added at a final con-
centration of 2 µg/ml. Plates were imaged using the high-
content screen microscope ArrayScan VTi (Thermo Fisher
Scientific) coupled to Cellomics. Images were automatically
taken using 10x magnification and two randomized images/
well were taken. Using Cellomics, an algorithm was created to
first segment the nuclei based on the Hoechst staining, and
then measure the mean YFP intensity per cell including the
Hoechst area + 2 pixels. The final measurements obtained
were total cell count/well and mean YFP intensity/well calcu-
lated by the sum of all the YFP-intensities per cell divided by
the total number of nuclei. The z’-factor was used as
a measurement of quality control. For each plate of the screen,
the z’-factor was calculated based on the YFP readout using
the following formula:
z0¼13σposctrl þ3σnegctrl
 
μposctrl μnegctrl
 
where σposctrl is the standard deviation of the positive control
(epoxomicin), σnegctrl is the standard deviation of the negative
control (DMSO) and μposctrl μnegctrl is the difference between
the mean value for the positive and negative controls. The
average z’-factor of the screen was 0.5387.
Compound library
The compound library was provided by the Laboratories for
Chemical Biology Umeå (LCBU), a part of the Chemical
Biology Consortium Sweden (CBCS). This chemical collec-
tion, named LCBU primary screening set, was constructed by
selecting and purchasing compounds from ChemBridge
representing a diverse selection biased toward lead-like and
drug-like properties with regards to molecular weight, hydro-
gen bond donors/acceptors and LogP. The compounds were
stored at 1 or 5 mM concentration in DMSO in desiccators at
room temperature (RT).
Filtering of hit compounds
In order to identify and remove potentially problematic com-
pounds, the hits from the primary screen were assessed for
Lipinski’s rule of 5 violations and passed through filters built
in the analytics platform KNIME (KNIME AG) by Evert
Homan (Karolinska Institutet) to detect unwanted substruc-
tures and known PAINS (https://hub.knime.com/evert.
homan_scilifelab.se/spaces/Public/latest/~
MrK3gq6WBD5fAfgy/).
Long-lived protein degradation assay
Cells were added at a density of 8.8 × 10
4
cells/ml in a final
volume of 400 μl. Cells were labeled by the addition of 0.125
μCi/ml L-[
14
C] valine (Perkin Elmer, NEC291EU050UC) to
the medium for 24 h, followed by two washes and 16 h chase
in medium containing 10 mM nonradioactive L-valine
(Sigma-Aldrich, V0513), to allow degradation of short-lived
proteins. Subsequently, cells were washed and subjected to the
indicated treatments in medium containing 10 mM nonra-
dioactive L-valine for 4 h. Radioactivity in the acid-soluble and
acid-insoluble fractions was measured using Ultima Gold LSC
cocktail (Perkin Elmer, obtained via Sigma-Aldrich, L8286)
and the liquid scintillation counter Tri-Carb 3100 T (Perkin
Elmer; Waltham, MA, USA).
GFP-LC3B puncta assay
For analysis of living cells, HOS GFP-LC3B cells were seeded
and treated in a 96-well imaging plate (BD Falcon™, 35–3219).
Hoechst 33342 (12 μg/mL in 1 x PBS [Statens
Veterinärmedicinska Anstalt, 992442]) was added to the cells
at a final concentration of 2 μg/mL and incubated for 30 min.
Prior to imaging, medium was replaced by Leibovitz’s L-15
medium (Life Technologies, 11415–064) and four to six sites
per well imaged without delay using an ImageXpress auto-
mated widefield microscope (Molecular Devices) equipped
with a 20x objective. Analysis of immunolabeled cells was
done similarly. The number of cytoplasmic GFP-LC3B puncta
per cell was quantified using CellProfiler Software 2.1.1.
(Broad Institute).
Ub-YFP levels by microscopy
For the analysis of the Ub-YFP reporter in living cells,
MelJuSo cells stably expressing the Ub-YFP reporter were
seeded and treated in a 96-well plate. Hoechst 33342 (12 μg/
mL in 1 x PBS) was added to the cells at a final concentration
of 2 μg/mL and incubated for 30 min. Four to nine sites per
well were imaged using an ImageXpress automated widefield
microscope (Molecular Devices) equipped with a 20x objec-
tive. The YFP intensity per cell and the percentage of YFP-
positive cells were quantified using the MetaXpress software.
The in-built pipeline Multiwavelength Scoring analysis was
performed. Briefly, nuclei segmentation was based on the
Hoechst staining, with minimum intensity and nuclei size
thresholds defined by the user tailored to each experiment.
The mean YFP intensity per cell reported throughout the
1498 T. A. GIOVANNUCCI ET AL.

manuscript represents the mean YFP intensity per cell in the
nucleus. Based on a tailored threshold defined by the user, the
percentage of YFP positive cells was also obtained.
Flow cytometry analysis of autophagic flux
For analysis of autophagic flux by flow cytometry, U2OS
mRFP-GFP-LC3B cells were seeded and treated in 6-well
plates. Cells were harvested with trypsin and washed with
PBS. Extraction of non-autophagosome associated mRFP-
GFP-LC3B was done by briefly washing with 0.05% saponin
(Sigma-Aldrich, 47036) in PBS [58]. Using the BD FACSAria
III (BD Biosciences; Franklin Lakes, NJ, USA), 30,000 events
were captured per experiment and cellular mRFP- and GFP-
intensities measured using the 488-nm and 561-nm lasers.
Data were analyzed using FlowJo (Treestar Inc.). Autophagic
flux was determined as the ratio between mean intensities of
mRFP and GFP.
Immunofluorescent labeling
Cells were seeded on 18-mm coverslips or in 96-well imaging
plates and treated as indicated. Cells were fixed in 4% paraf-
ormaldehyde in 1 x PBS for 20 min; permeabilized using 0.2%
Triton X-100 (Sigma-Aldrich, T8787) or 0.2% saponin in 1
x PBS for 15 min (in the case of endogenous LC3B staining)
and incubated with 100 mM glycine in 1 x PBS for 10 min.
Unspecific binding was blocked using 3% BSA (Sigma-
Aldrich, A7030) in 1 x PBS for 30 min. All steps were per-
formed at RT. The following primary antibodies were diluted
in BSA 3% in PBS and incubated overnight at 4°C: anti-LC3B
(MBL Life Science, PM036), anti-LAMP1 (Santa Cruz
Biotechnology, sc-20011), anti-VIM/vimentin (Sigma-
Aldrich, AB5733), anti-SQSTM1/p62 (Santa Cruz
Biotechnology, sc-25575), anti-G3BP1 (Invitrogen, PA5–
29455), anti-puromycin (Sigma-Aldrich, MABE343), anti-
ubiquitin (FK2, conjugated ubiquitin; Enzo Life Sciences,
PW8810), anti-HSF1 (Cell Signaling Technologies, 4356).
Goat anti-mouse or goat/donkey anti-rabbit IgG coupled to
Alexa Fluor 488, 546 or 647 (Life Technologies, A-11029,
A-11030, A-21235, A-11035, A-27040) were diluted 1:500 in
BSA 3% in PBS. Nuclear staining was performed using
Hoechst 33342 1:5000 in PBS for 10 min. After washing
with PBS, the plate was imaged using the ImageXpress micro-
scope (Molecular Devices; San Jose, CA, USA) with a 20x
objective or mounted and imaged in the LSM880 confocal
microscope and Zeiss ZEN software (Zeiss; Oberkochen,
Germany) with a 63x objective.
The number of puromycin foci in the cytoplasm per cell,
the number of G3BP1 foci in the cytoplasm per cell and the
HSF1 foci in the nucleus per cell were quantified using
CellProfiler Software 2.1.1. (Broad Institute). ImageJ (https://
imagej.nih.gov/ij/) was used for measuring intensities in line
scans and for the processing of representative images.
Cell viability assay
Cells were seeded into clear-bottom, white 96-well plates at
4500 (MelJuSo) cells per well. Sixteen h after seeding (ca 60%
confluency), cells were treated in technical triplicates with the
indicated compound concentrations in a 9-point serial dilu-
tion (unless otherwise indicated in the figure legends, starting
from 10 µM, 2-fold serial dilutions were performed). DMSO
at 0.1% was used as control. After 24, 48 or 72 h of incuba-
tion, an ATP-based cell viability assessment was performed
using CellTiter-Glo (Promega, G7573) following the manu-
facturer’s instructions. Briefly, the plate was taken out of the
incubator approximately 30 min before adding the CellTiter-
Glo reagent. To aid cell lysis, the plate was placed in the plate
reader FLUOStar OMEGA (BMG Labtech; Ortenberg,
Germany) and orbital shaking at 500 rpm was performed
for 2 min. Afterward, the plate was incubated 10 min in the
dark before the luminescent signals were detected. Three wells
with medium + CellTiter-Glo without cells were used as
blanks, and the average luminescent signal of these wells
was subtracted from all the values.
Western blotting
Equal amounts of cells were lysed in 1X SDS sample buffer
(0.3 M Tris-HCl, pH 6.8, 2% SDS, 17.5% glycerol [Sigma-
Aldrich, G5516], bromophenol blue [Sigma-Aldrich, B0126])
containing 10% NuPAGE reducing agent (Thermo Fisher
Scientific, NP0004) and lysates were boiled at 95°C for 5 min.
Cell protein extracts were resolved by Bis-Tris polyacrylamide
gel electrophoresis gels (Thermo Fisher Scientific, 4–12% gradi-
ent gels [NP0323] or 12% gels [NP0343] for LC3B blots) and run
in either 1X MOPS (Thermo Fisher Scientific, NP0001) or for
LC3B blots in 1X MES buffer (Thermo Fisher Scientific,
NP0002). Proteins were transferred onto PVDF 0.45 µm or
nitrocellulose membranes (GE Healthcare, 10600023) in a Tris-
glycine transfer buffer (25 mM Tris, 192 mM glycine) containing
20% methanol. After blocking in Tris-buffered saline (TBS;
Statens Veterinärmedicinska Anstalt 303252), 5% nonfat milk
containing 0.1% Tween-20 (Sigma-Aldrich, P9416), membranes
were incubated with primary antibodies, washed with TBS-0.1%
Tween-20 and incubated with secondary HRP-linked antibodies
(GE Healthcare, NA934V and NA931V). Detection was per-
formed by enhanced chemiluminiscence (Amersham ECL
reagents; GE Healthcare, RPN2106) on Medical X-ray films
(Super XR, Fujifilm). Alternatively, secondary antibodies
coupled to near-infrared fluorescent dyes (LI-COR, 926–68070
and 926–68071) were used, and membranes scanned with an
Odyssey scanner (LI-COR, Lincoln, NE, USA) and analyzed
with Image Studio Lite analysis software version 5.2 (LI-COR).
The following antibodies were used: anti-GFP (Abcam,
ab290), anti-HSPA1A/Hsp70 (C92F3A-5; Enzo Life Sciences,
ADI-SPA-810), anti-GAPDH (Abcam, ab9485), anti-ACTB/β-
actin (Abcam, ab8226), anti-TP53/p53 (DO-1; Santa Cruz
Biotechnology, sc-126), anti-HIF1A/HIF1α (GeneTex,
GTX127309), anti-LC3B (Sigma-Aldrich, L7543), anti-
ATG16L1 (MBL Life Science, PM040).
Proteasome activity assay
After treatments, cells were trypsinized and lysed (25 mM
HEPES pH 7.2 [Thermo Fisher Scientific, 15630–056],
50 mM NaCl, 1 mM MgCl
2
[Sigma-Aldrich, M8266], 1 mM
AUTOPHAGY 1499

ATP [Sigma-Aldrich, A1852], 1 mM DTT, 10% glycerol, 1%
Triton X-100) for 30 min at 4°C. Ten μg protein in 100 μl
reaction buffer were mixed with 100 μM Suc-Leu-Leu-Val-Tyr
-AMC (Affiniti, P802). In one well, 500 nM epoxomicin (Enzo
Life Sciences, BML-PI127) was added additionally. Samples
were analyzed in a microplate reader FLUOStar OPTIMA
(BMG Labtech; Ortenberg, Germany) at 380 nm/440 nm
every min for 1 h.
Statistical analysis
Statistical analyses were performed using GraphPad Prism
version 8.3. To test for Gaussian distribution, the
D’Agostino & Pearson or Shapiro-Wilk normality test (for
smaller sample sizes) were used. If the normality test was
passed, data were analyzed by Student’s unpaired two-tailed
t-test (two groups) or by ANOVA (more than two groups). If
the data were not normally distributed, statistical analysis was
performed using the nonparametric Mann-Whitney test (two
groups) or Kruskal-Wallis test for multiple comparisons, with
Dunnett’s or Tukey’s test to adjust for multiple comparisons.
Data are shown as mean ± SD (standard deviation), unless
stated otherwise, as indicated in each figure legend. The
following p-values were considered significant: *p ≤ 0.05;
**p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
EC
50
or IC
50
values
Dose-response curves were fitted in GraphPad Prism by
a non-linear regression analysis using a four-parameter logis-
tic curve fit to estimate IC
50
(cell viability) or EC
50
(Ub-YFP
accumulation) values. In the case of the IC
50
, bottom values
were constrained to 0.
General synthetic procedures for compounds
All reactions were carried out using dry solvents and anhy-
drous condition, unless otherwise stated. All of solvents were
dried with PPT’s solvent purification systems. Liquid chroma-
tography/mass spectrometry (LC/MS) was carried out with
either an Agilent system (Santa Clara, CA, USA) or
a Waters system (Milford, MA, USA), as explained below.
Agilent 1260 binary LC system equipped with an Agilent
EC-C18 column (3.0 x 50 mm, 2.7 µm), eluted with a linear
gradient of CH
3
CN in H
2
O, both of which contained trifluor-
oacetic acid (0.1%). A flow rate of 0.7 mL/min was used and
detection was performed at 214 and 254 nm. Low resolution
mass spectra (LRMS) data were obtained on an Agilent 6130
Quadrupole LC/MS using positive and negative electrospray
ionization.
Waters LC system equipped with an Xterra C-18 column
(50 x 19 mm, 5 µm, 125 Å), eluted with a linear gradient of
CH
3
CN in H
2
O, both of which contained formic acid (0.2%).
A flow rate of 1.5 mL/min was used and detection was
performed at 214 and 254 nm, and with positive and negative
electrospray mass analysis. Low resolution mass spectra
(LRMS) data was obtained on a Water micromass ZQ 2000
using positive and negative electrospray ionization.
Flash column chromatography was performed on silica gel
(60 Å, 230–400 mesh). Preparative HPLC was performed using
VP 250/21 NUCLEODUR C-18, HTEC, 5 µm column on
a GILSON 333/334 Prep-Scale system with a flow rate of 20 mL/
min, detection at 254 nm, and CH
3
CN/H
2
O eluent system.
1
H and
13
C NMR spectra were recorded at 298° K with
a Brucker DRX-400 spectrometer at 400 MHz and 100 MHz, or
Brucker DRX-600 spectrometer at 600 MHz and 150 MHz,
respectively, and calibrated using the residual peak of the solvent
as internal standard CDCl
3
(CHCl
3
δ
H
7.26 ppm, CDCl
3
δ
C
77.16
ppm) or DMSO
d6
(DMSO
d5
δ
H
2.50 ppm, DMSO
d6
δ
C
39.52 ppm).
Specific information for each compound is provided in the
supplementary material.
Acknowledgments
We thank the members of the Dantuma lab for helpful input. We thank
Prof. Mikael Elofsson (Umeå University) and Dr. Magdalena Otrocka
(Chemical Biology Consortium Sweden) for help and support. This work
was supported by the Swedish Research Council (N.P.D.; 2016–02479),
the Swedish Cancer Society (N.P.D.; CAN 2018/693), the Novo Nordisk
Foundation (N.P.D; Exploratory Pre-Seed Grant 1–2019,
NNF19OC0056441), Karolinska Institutet (N.P.D.) and the Research
Council of Norway through its Centres of Excellence funding scheme
(A.S.; Project: 262652).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the Cancerfonden [CAN 2018/693]; Novo
Nordisk Fonden [1-2019, NNF19OC0056441]; Vetenskapsrådet [2016-
02479]; Research Council of Norway [262652].
ORCID
Tatiana A. Giovannucci http://orcid.org/0000-0001-8978-6318
Anne Simonsen http://orcid.org/0000-0003-4711-7057
Nico P. Dantuma http://orcid.org/0000-0002-6090-4170
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