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Expert Opinion on Drug Discovery
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iedc20
Targeting Autophagy In Disease: Recent Advances
In Drug Discovery
Dasol Kim , Hui-Yun Hwang & Ho Jeong Kwon
To cite this article: Dasol Kim , Hui-Yun Hwang & Ho Jeong Kwon (2020): Targeting Autophagy
In Disease: Recent Advances In Drug Discovery, Expert Opinion on Drug Discovery, DOI:
10.1080/17460441.2020.1773429
To link to this article: https://doi.org/10.1080/17460441.2020.1773429
Published online: 16 Jun 2020.
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REVIEW
Targeting Autophagy In Disease: Recent Advances In Drug Discovery
Dasol Kim
a
*, Hui-Yun Hwang
a
* and Ho Jeong Kwon
a,b
a
Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University,
Seoul, Republic of Korea;
b
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea
ABSTRACT
Introduction: Small molecules targeting autophagy have been highly implicated as new therapeutic
agents to treat diseases of interest. With the increasing demand for autophagy-targeting drugs, this review
attempts to provide an efficient strategy to explore major autophagy-based human disease interventions
with newly explored mechanisms using small molecules and promising therapeutic approaches.
Areas covered: Introduced in this review are direct links and applications among autophagy pathways,
their modulators, and phenotypic diseases, along with recent approaches. Autophagy-related diseases,
machinery, and compounds are introduced to guide the appropriate investigation of autophagy in the
pharmaceutical industry. The authors then provide their expert perspectives on the subject.
Expert opinion: The self-catabolic intracellular process autophagy occurs in organisms throughout their
lifetime, supporting its critical role in organismal health across life stages. Because of the detrimental
influence of dysfunctional cells to an organism and their etiology in numerous diseases, maintaining cellular
quality control by recycling components through autophagy is essential to prevent health decline.
ARTICLE HISTORY
Received 9 December 2019
Accepted 20 May 2020
KEYWORDS
Autophagy; autophagy-
related diseases; autophagy-
targeting strategy; cancer;
proteopathies; vascular
diseases
1. Introduction
‘Autophagy’ indicates a ‘self-eating’ process wherein a cell
degrades its cytoplasmic material by lysosomal digestion [1–3].
This self-digestion removes damaged organic substrates and
exogenous microorganisms as a cellular defense strategy.
Based on these homeostatic roles that maintain vital cellular
functions, many recent studies have highlighted links between
autophagy modulation and various phenotypic diseases such as
cancer and vascular, neurodegenerative, and metabolic diseases
[4–7]. Therefore, discovering autophagy-regulating drugs has
been emphasized to provide potential disease-alleviating
approaches. However, the therapeutic application of autophagy
modulators without an understanding of their mechanism of
action could lead to undesirable adverse effects, since these
drugs trigger different responses based on the specific autop-
hagy pathways involved in each disease [4,8]. Therefore, eluci-
dating the molecular mechanisms of autophagy-regulating small
molecules in a disease-specific manner is crucial for their use in
treating autophagy-related diseases.
This review covers major autophagy-related diseases
along with emerging protein factors in autophagy pathways
and the small molecules that target them. To highlight
these diseases, 11 of the most deadly autophagy-related
maladies and symptoms are ranked based on the number
of cases reported during 2000–2009 and 2010–2019. During
both periods, cancer ranked highest, followed by aging,
proteopathies, diabetes, vascular disease, obesity, viral infec-
tion, microbial infection, myopathy, lysosomal storage dis-
orders (LSDs), and sepsis. Among them, aging, diabetes, and
obesity were excluded, as they are considered ‘certain
symptoms or metabolic statuses’ of the human body rather
than ‘diseases’. Thus, in this review, we primarily focus on
the three highest-ranking diseases (cancer, proteopathies,
and vascular disease), followed by other infectious and
lysosomal diseases. Because the autophagic process is
linked to diverse signaling networks in the cell, disease
pathogenesis is differentially affected by autophagy, based
on the signaling pathways involved. Therefore, it is critical
to clearly understand the implications of targeting autop-
hagy-regulating pathways and their roles in disease before
building a therapeutic approach. To support this, we review
small molecules that target autophagy-related pathways
and/or newly emerging factors and their roles in each dis-
ease. In addition, we cover therapeutic drugs that are cur-
rently used or undergoing clinical trials. This exploration
into the pharmacological intervention of autophagy-related
diseases and regulatory factors offers promising opportu-
nities for the strategic development of therapeutic
approaches against autophagy-related diseases.
2. Regulatory factors in the autophagy process
2.1. Regulatory factors in autophagy: from phagophore
to autolysosome
When autophagy is induced upon certain stress stimuli, cells
promote the dynamic interaction of several proteins that play
a part in the autophagy process. This comprises multiple steps,
including initiation/nucleation, elongation/expansion, and
CONTACT Ho Jeong Kwon kwonhj@yonsei.ac.kr Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science and
Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea
*These authors contributed equally to this work
EXPERT OPINION ON DRUG DISCOVERY
https://doi.org/10.1080/17460441.2020.1773429
© 2020 Informa UK Limited, trading as Taylor & Francis Group
maturation/degradation [9]. During autophagic ‘flux’ or turnover,
the first two steps, considered early-stage, relate to the formation
of the autophagosome, while the last step, considered late-stage,
relates to autophagosomal degradation [10].
At the beginning of initiation/nucleation, autophagy-related
gene (ATG) proteins assemble at the phagophore assembly site
(PAS) to form a small cup-shaped membrane precursor. These
ATGs include E1-like ligase ATG7, ATG6 homolog Beclin-1,
cysteine protease ATG4A-D, and microtubule-associated protein
1 light-chain 3 (LC3) [11]. The precursor phagophores eventually
close to form double- or multi-membraned vacuoles called
autophagosomes in the later elongation/expansion step [12].
During this step, LC3 undergoes lipidation through covalent
binding with phosphatidylethanolamine (PE), thereby resulting
in the conversion of LC3-I to LC3-II. This has been used as
a quantitative biomarker in the assessment of autophagic
activity.
In the final maturation/degradation step, autophagosomes
traffic toward the microtubule-organizing center (MTOC) to
fuse with lysosomes and degrade cargo through lysosomal
hydrolases. Two essential processes are pivotal for proper
autophagy turnover: (1) autophagosome-lysosome fusion,
and (2) lysosomal hydrolase activation. Although the mechan-
ism underlying autophagosome-lysosome fusion is currently
unclear, it is under intense investigation. Factors involved in
fusion include the endosomal sorting complexes required for
transport (ESCRTs), soluble N-ethylmaleimide-sensitive factor
attachment protein receptors (SNARE), ultraviolent radiation
resistance-associated gene (UVRAG), Rubicon (RUBCN), small
GTPase of the Ras-related protein 7 (RAB7), lysosomal asso-
ciated membrane protein 2 (LAMP2), ion channels, and other
tethering factors [13–17]. Because lysosomal enzymes are acid
hydrolases (including proteases, glycosidases, nucleases,
phosphatases, and lipases) that are active at acidic pH (~5)
but not neutral pH, maintaining low pH is essential for ‘active
lysosomes’ to induce sequential autophagic turnover.
Accordingly, lysosomal ion channel proteins such as vacuolar-
type H
+
-ATPase (V-ATPase) and transient receptor potential
mucolipin 1 (TRPML1) play essential roles to maintain lysoso-
mal ionic homeostasis and membrane potential, resulting in
lysosome activation by hydrogen cation influx [18].
2.2. Regulatory factors in the expression of autophagy
genes
Several transcription factors act as master regulators during
signal transfer in the autophagic process [19]. Under nutrient-
rich conditions, these are sequestered in the cytoplasm in the
inactive state. However, upon certain stimuli to the cells, they
are modified by cytoplasmic enzymes and translocate into the
nucleus to participate in autophagy regulation at the tran-
scriptional level [19]. The primary master transcription factors
are portrayed below with a description of the mechanism by
which they are regulated to facilitate the expression of genes
encoding autophagy-related proteins (Figure 1).
2.2.1. Microphthalmia/transcription factor e (mit/tfe)
MiT/TFE family members (MITF, TFEB, TFE3, and TFEC) are
the most recognized master genes of autophagy, as they
play a pivotal role in autophagosomal and lysosomal bio-
genesis [20]. They share a common structure in the basic
helix-loop-helix (bHLH) leucine zipper (LZ) dimerization
motif, except for TFEC (transcription inhibition rather than
activation) [21]. MITF, TFEB, and TFE3 directly bind to the
coordinated lysosomal expression and regulation (CLEAR)
element [22,23], which is a common feature in the promoter
regions of lysosome genes. These proteins are primarily
phosphorylated by kinases such as mTORC1 and bound to
14-3-3 protein, which masks their nuclear localization signal
(NLS) to maintain them in the cytoplasm [24]. Upon autop-
hagy initiation, the repressive phosphorylation is removed
by phosphatases such as calcineurin, resulting in their
nuclear translocation for transcription of autophagy/lysoso-
mal genes. Indeed, several studies have revealed that mod-
ulation of Mit/TFE family members is a pivotal step in
a variety of pathologic phenotypes [25].
2.2.2. Nuclear factor erythroid-derived 2-like 2 (nfe2l2/
nrf2)
NFE2L2/NRF2 is considered a master regulator of cellular
homeostasis, as it controls the expression of protective
genes [26]. The protective genes, including antioxidant
genes, share a common cis-acting antioxidant response ele-
ment (ARE) in their upstream promoter region [26,27]. Under
benign conditions, KEAP1 directly binds to NRF2, leading to its
ubiquitination and degradation by the ubiquitin-proteasome
system (UPS) [28]. Upon oxidative stress, cysteine thiols on
KEAP1 are modified, releasing NRF2 to translocate into the
nucleus to control target gene expression [29]. Furthermore,
NRF2 is reported to regulate autophagy and vice versa. NRF2
modulates the expression of autophagy genes, including
SQSTM/p62 and LAMP2A [26,27]. However, increased SQSTM/
Article highlights
●Autophagy is a fundamental catabolic process to maintain cellular
homeostasis in response to cell proliferation, hypoxia, stress gener-
ated by reactive oxygen species, various pathological invasions,
nutrient starvation, and aggregation of cytoplasmic components
leading to human pathological conditions. Recently, many reports
have highlighted autophagy-targeting strategies for therapeutic
purposes.
●Many small molecule modulators perturbing autophagy have been
used to elucidate the interplay between specific autophagy pathways
and autophagy-related diseases.
●In cancer, there have been significant disputes over autophagy-
targeting therapies due to their complex functions. Targeting autop-
hagy in cancer depends either on its tumor-suppressive or tumori-
genic actions. Targeting strategies using autophagy modulators can
be classified into autophagy activation and autophagy inhibition.
●Maintaining protein homeostasis through autophagy degradation in
proteopathies can restore or remove toxic cellular protein aggregates
to restore normal physiological conditions. Targeting strategies with
pharmacological interventions will be emphasized through the reg-
ulation of various autophagy pathways in diverse protein conforma-
tional disorders such as amyloidosis, tauopathies, synucleinopathies,
and prion diseases.
●Pharmacological perturbation through the cannibalizing effects of
autophagy can be considered a robust targeting strategy due to its
fundamental roles in mediating physiological processes across
diverse vascular diseases such as atherosclerosis, restenosis, intrao-
cular neovascularization, and ischemia-reperfusion injury.
2D. KIM ET AL.
p62 sequesters KEAP1 and promotes its degradation as
SQSTM/p62 itself is an autophagy substrate, thereby creating
a positive feedback loop for active NRF2 signaling [29]. This
master autophagy regulator is highlighted due to its preven-
tive role against human diseases.
2.2.3. Forkhead box o (foxo)
FoxO transcription factor family members (FoxO1, FoxO3,
FoxO4, and FoxO6) have established roles in diverse cellular
functions such as aging, longevity, and development [30].
Similar to the MiT/TFE family, the FoxO transcription factor
family is mostly controlled by phosphorylation [31]. AKT/JNK
kinase signaling is a major upstream regulator for FoxO1,
FoxO3, and FoxO4, but less for FoxO6 due to its lack of
a C-terminal AKT-dependent site [30,32]. Among these,
FoxO3 [33] and FoxO1 [34] were reported to be autophagy
gene transcription factors. A recent study exhibited the rele-
vance of FoxO4 to autophagy gene expression through
a triple genetic deletion of FoxO1, FoxO3, and FoxO4 in mus-
cle [35]. However, it is currently unclear whether FoxO4 binds
directly to the promoter region of autophagy genes. The FoxO
family is regarded as another pivotal modulator for cellular
homeostasis through autophagy, as these members have cen-
tral roles in diverse diseases.
2.2.4. CCAAT/enhancer-binding protein (c/ebp)
C/EBP family members (C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε,
and C/EBPζ) are characterized by a conserved b-Zip domain at
the C-terminus that mediates dimerization and DNA binding
[36]. C/EBP family members are known to regulate cell prolifera-
tion and differentiation at the transcriptional level [37]. Recent
studies have revealed that they also have a role in autophagic
regulation. Among them, C/EBPβ was reported to control the
expression of autophagy genes, including Beclin-1, ATG5, and
ATG4 [38,39]. More recently, C/EBPδ (CEBPD) was found to
activate transcription of the autophagy genes LC3B and ATG3
in hepatocellular carcinoma (HCC) [40]. Another transcription
factor, C/EBPζ (C/EBP-homologous protein/CHOP), alone or in
combination with ATF4, was revealed to bind the promoter
regions of a set of autophagy genes through eIF2α/ATF4 path-
way activation under ER stress or starvation conditions, thereby
regulating autophagy [41]. Thus, the C/EBP family can regulate
cellular homeostasis as autophagy master genes, supporting
their utility as molecular targets for related diseases.
2.2.5. GATA transcription factor
Transcription factors in the GATA family (GATA1, GATA2, GATA3,
GATA4, GATA5, and GATA6) are characterized by their ability to
bind the (A/T)GATA(A/G) sequence in DNA [42]. Although GATA
Figure 1. Master genes in the transcriptional regulation of autophagy. (i) MiT/TFE family members are attenuated by phosphorylation in the cytoplasm. When they
are modified by phosphatases such as calcineurin, they translocate into the nucleus and bind the CLEAR domain in DNA to promote transcription of autophagy-
related genes. (ii) NFE2 L2/NRF2 are inhibited by KEAP1 binding in the cytoplasm. Upon oxidative stress, a disulfide bond forms in KEAP1, thereby releasing NFE2L2/
NRF2 for nuclear translocation. The transcription factors bind to ARE domains in DNA and promote target gene expression. (iii) FoxO family members remain inactive
in the cytoplasm by phosphorylation. Upon signaling stimuli, phosphatase PP2A dephosphorylates them, leading to their nuclear translocation to enhance target
gene expression. (iv) C/EBP family members undergo posttranslational modifications in the cytoplasm. Cellular kinases such as ERK1/2 modify them by
phosphorylation to promote their nuclear translocation and transcriptional activity. (V) GATA family members are cell type-specifically regulated. GATA1 is mainly
known as a transcription factor for autophagy genes. GATA1 is activated upon acetyl modification by the acetyltransferase CBP, which promotes autophagy gene
transcription.
EXPERT OPINION ON DRUG DISCOVERY 3
family members were originally considered to play roles in hema-
topoietic cells and cardiac tissue [43], it was found that GATA1
directly activates the transcription of pivotal autophagy genes
such as LC3B and its homologs, and genes involved in lysosomal
biogenesis and function [44]. Because GATA1 is a cell type-specific
master regulator in hematopoietic development, it can be lever-
aged due to its specificity and suggests a new strategy for target-
ing related diseases.
3. Autophagy and disease targeting
Numerous studies have revealed an interplay between autop-
hagy and a variety of human diseases. In this section, major
diseases such as cancer, proteopathies, vascular disease, and
others are discussed (Figure 2), followed by recent chemother-
apeutic strategies that target autophagy (Table 1).
3.1. Cancer
Autophagy-targeting strategies for cancer treatment have been
considered promising therapeutic approaches but are controver-
sial due to duals roles for autophagic function in cancer therapy.
The potential complexity of autophagy in cancer is likely depen-
dent on cancer type, location, genetic variation, stage, and tumor
microenvironment [46,47]. Some investigations present autop-
hagy activation as tumor suppressive function, while the others
suggest its tumorigenic characteristics according to specific autop-
hagy pathways. Hence, understanding these differences and the
exact biological roles autophagy plays in cancer is critical for
therapeutic importance. Notably, recent studies have identified
numerous modulators of autophagy as cancer targets.
3.1.1. Autophagy activation in cancer
Autophagy was originally considered a tumor inhibitory
mechanism to reduce cancer progression or increase cell
ProteopathiesCancer
Other diseases
Microbial
infection
Viral
infection
Targeting
Autophagy
Vascular diseases
Restenosis
Atherosclerosis
BBB disruption
Aβ
plaque
α-syn
body
Tau
NFT
Prion
Aggregates
degradation
PI3K-Akt-mTOR
pathway
Tubulin
AMPK-mTOR
pathway
Rhes
Autophagy
cycle
Abnormal
growth
Malignant
transformation
Stress
UVRAG
SNARE
PRDX1
ROS
Lipophagy
TFEB/Nrf2
Neovascularization
Akt-mTOR
pathway
Autophagy
VEGF
Dysfunctional
lysosome
Lysosomal
hydrolysis
AMPK
pathway
mTOR-NLRP3
pathway
Autophagy
Inflammation
Replication
Figure 2. Autophagy-targeting strategies are established in various diseases including cancer, proteopathies, vascular disease, and other human diseases. (i)
Applying autophagy for cancer treatment must be carefully considered based on its complex role in cancer. Characteristics of cancer such as protein abnormalities,
morphology, and progressive growth can be modulated by the autophagy pathways depicted in Section 3.1. In brief, targeting autophagy in cancer can be divided
into two strategies: autophagy activation and inhibition. In the autophagy activation approach, the purpose is to prevent cells from malignant transformation by
maintaining cellular protection. The autophagy inhibition approach includes targeting autophagy initiation and its ability to ultimately degrade its contents. The
goal of this approach is to inhibit cancer cells from exploiting autophagy for their propagation. (ii) Proteopathies are protein conformational disorders caused by the
accumulation of abnormal protein aggregates such as amyloid β plaques, tau neurofibrillary tangles (NFTs), α-synuclein aggregates, and prions. The strategy for
‘autophagy activation’ could ameliorate pathological conditions derived from these conformational abnormality through promoting degradation of the pathological
substrates as depicted in Section 3.2. (iii) In vascular disease including atherosclerosis, restenosis, intraocular neovascularization, and ischemia-reperfusion injury,
autophagy plays a fundamental role in maintaining physiological homeostasis as depicted in Section 3.3. Targeting autophagy signaling such as mTOR-S6 K could
improve atherosclerosis and restenosis by attenuating abnormal endothelial cell proliferation and protecting the blood brain barrier (BBB) against ROS-induced
injury. ROS could negatively regulate autophagic flux, implying its role as a therapeutic target for regulating autophagy in vascular diseases. In addition, despite few
clarified mechanisms, autophagy inhibition reduces VEGF protein levels, leading to an anti-intraocular neovascularization effect. (iv) Therapeutic approaches for
targeting other human diseases including viral or bacterial infectious diseases and lysosomal storage diseases. Targeting strategies over autophagy pathways using
specific modulators or combinational approaches with other medications are depicted in Section 3.4. In lysosomal storage disorders and bacterial infectious diseases,
molecular regulators elevating autophagy turnover are promising therapeutics. In viral infections, however, this approach should be carefully determined based on
how autophagy positively or negatively affects the host-virus relationship.
4D. KIM ET AL.
mortality. The dual roles of autophagy can be partially attrib-
uted to different actions from the same factors. Although
increasing some biological elements can be beneficial to
tumor progression in the initial steps of tumorigenesis, such
as ROS generation, accumulation of p62/SQSTM1-mediated
misfolded aggregates, damaged mitochondria, and inflamma-
tion, their accumulation during later stages of tumor growth
can cause deleterious effects such as metabolism dysfunction,
genetic impairment, and eventual tumor suppression [48,49].
For autophagy activation strategies to target cancer progres-
sion, ATG family members like ATG2, ATG5, ATG7, ATG9, and
ATG12, and Beclin-1 and VPS34 are upstream factors necessary
for phagophore formation [50]. The impact of depleting or
mutating these factors has been studied in cancer cell lines
and mouse models, revealing that reduction of autophagy
increases cellular proliferation, supporting a role for autopha-
gic activation against tumorigenesis. In other studies, various
signaling players, such as AKT (curcumin, AZD7328) [51,52],
mTOR (rapamycin, resveratrol, torin 2) [53,54], AMPK (metfor-
min, kaempferol, aspirin, nilotinib) [3,55–57], and MAPK (bor-
tezomib, p38-MAPK-JUNK pathway activator) [46], have been
shown as valuable targets both for inhibition of cancer
Table 1. Representative autophagy-targeting agents in autophagy-related diseases.
Agents Structure Targeting autophagy
Autophagy
pathways Ref.
Cancer Vinblastine Autophagy inhibition and
cancer cell death
Disrupts polymerization of
both non-acetylated or
acetylated microtubules,
leading to inhibition of
fusion between
autophagosomes
and lysosomes
[76]
Berbamine Autophagy inhibition and
cancer cell death
Inhibits SNARE complex
formation, leading to
autophagy inhibition by
disrupting the interaction
between BNIP3 and SNAP29
[77]
Proteopathies Alborixin Autophagy activation and amyloid β
(Aβ) plaque clearance in
amyloidosis
Induces autophagy by
targeting the PTEN/PI3-K/
AKT signaling axis
[94]
Lonafarnib Autophagy activation and tau
aggregate clearance in tauopathies
Induces autophagy by
suppressing Rhes
farnesylation followed by
increased lysosomal activity
[122]
Trehalose and rapamycin Additive autophagy activation and
protection of dopaminergic
neurons in Parkinson’s disease
Induces autophagy to improve
dopaminergic deficits
through the AMPK-mTOR
signaling axis
[134]
AR-12 Autophagy activation and
combinatorial effects with cellulose
esters, a prion conversion inhibitor,
in prion diseases
Induces pro-autophagic
activity that significantly
extends the survival of
prion-infected mice by
targeting the mTOR
pathway
[145]
(Continued )
EXPERT OPINION ON DRUG DISCOVERY 5
progression and autophagy activation in various cancer cell
lines and mouse models. Some of these autophagy activators
have been applied as clinical FDA-approved treatments such
as rapamycin derivatives (renal cell carcinoma, pancreatic or
breast cancer), nilotinib (chronic myelogenous leukemia), and
bortezomib (myeloma/lymphoma) after verifying their effec-
tiveness. However, recent studies demonstrated that autop-
hagy can promote both cell death or cell survival in cancer
according to the stage of progression. For example, elevating
autophagy through an acidic pH microenvironment contri-
butes to cancer progression or survival during initial stages
[58]. It is responsible for the poor response to autophagy
activation in clinical uses, such as with the mTOR inhibitor
rapamycin, and should be considered in detail when targeting
autophagy activation in cancer therapy.
3.1.2. Autophagy suppression in cancer
Autophagy inhibition, resulting in the inability to remove
targeted protein cargo, is also considered a suppressive
approach against tumorigenesis. For example, key biomarkers
such as LC3 and p62 are used to monitor autophagy levels, as
they are associated with the formation of autophagosomal
membranes that envelop protein cargo for subsequent degra-
dation. Autophagic machinery activated by these factors is
correlated with cancer phenotypes such as cancer survival
and metastasis, vessel invasion, and tube formation at the
early stages of progression [59]. When autophagy cannot
occur after phagophore formation, which means blocking
the initiation of forming autophagosomes by inhibiting targets
involved in these pathways, it may support tumor growth in
multiple cells and tumor types, as well as trigger resistance to
Table 1. (Continued).
Agents Structure Targeting autophagy
Autophagy
pathways Ref.
Vascular
diseases
PRDX1 mimetics
(ebselen, gliotoxin)
Maintaining lipophagic flux and
cholesterol efflux in atherosclerosis
Rescues impaired lipophagy
under pathologic conditions
by reducing produced ROS
[154]
Indatraline Autophagy activation and inhibition
of the rapid proliferation of
smooth muscle cells in restenosis
Induces autophagy without
apoptotic cellular toxicity
by targeting the AMPK-
mTOR-S6 K signaling axis
[45]
3-methyladenine or
chloroquine
Autophagy inhibition in initiation or
degradation steps, respectively,
and suppressed neo-
vascularization in ocular vascular
diseases
Inhibits autophagy leading to
regulation of the
angiogenic factors VEGF
and PEDF
[157]
Sirolimus
(rapamycin)
Autophagy activation leading to
maintaining the endothelial barrier
in ischemia-reperfusion injury
Induces autophagy by
targeting mTOR and
regulates the localization of
tight junction protein Cldn5
[159]
Viral infection 3-methyladenine
or
wortmannin
Autophagy inhibition at initiation and
suppressed viral infection and
replication in cells
Inhibits autophagy by
targeting the PI3-K/AKT
signaling axis
[171]
Microbiome-
mediated
diseases
Alpinetin Autophagy activation to ameliorate
the intestinal barrier and colitis
Induces autophagy by
targeting the TSC2/mTOR
signaling axis and
suppressing apoptotic cell
death signaling
[187]
6D. KIM ET AL.
various therapies. Autophagy initiation can also be suppressed
by inhibitors upstream of the phagophore. For instance, phos-
phatidylinositol 3-kinase (PI3-K) is a protein kinase that acti-
vates autophagy through PI3-K-AKT-mTOR, and through ERK
signaling crosstalk is involved in cancer progression and sur-
vival. In several cancers, including glioblastoma, cutaneous
melanoma, and HCC, this signaling cascade is highly activated,
so their inhibition is considered a therapeutic strategy to
suppress cancer [60]. Inhibitors of PI3-K (idelalisib: the class
I inhibitor, 3-methyladenine: the class I and class III dual
inhibitor, wortmannin: the class I inhibitor) [61–63] or MEK/
ERK (PD98059) [64] suppress autophagy, increasing cancer cell
death. This early-stage inhibitory strategy has potential for
clinical applications, as idelalisib has already been developed
as a treatment for chronic lymphocytic leukemia, follicular
B-cell non-Hodgkin’s lymphoma, and small lymphocytic lym-
phoma (FDA approved).
Another emerging cancer target is the UVRAG protein,
which regulates Beclin-1-class III phosphatidylinositol 3-kinase
(PI3-KC3)-mediated autophagic and tumor-suppressive activ-
ity. Beclin-1 is mono-allelically deleted or mutated in several
cancers, including breast, colon, and ovarian cancers [65].
UVRAG positively acts in the Beclin-1-PI3-KC3-dependent acti-
vation of autophagy and therefore inhibits tumorigenesis [66],
making it an interesting target for further investigation. As
a case study for developing small molecules that specifically
target autophagy, autophagy-activating rapamycin and its
derivatives showed antitumor effects in HCC patients by inhi-
biting the mTOR pathway and cancer progression in clinical
trials [67,68]. Although some did not show an antitumor effect
in clinical trials [69], introducing rapamycin as autophagy
activator opened a gateway to take advantage of mTOR-
related autophagy and its practical application to cancer. To
date, combinatorial approaches using different autophagic
pathway modulators with mTOR inhibitors have been
a promising therapeutic strategy in HCC treatment [70].
During autophagosome formation and recycling, LC3
undergoes processing by some ATG proteins (ATG4, ATG7,
ATG3, and the 12-5-16L1 complex, sequentially). Among
them, ATG4 plays roles in priming pro-LC3 into LC3-I and
cleaving LC3-II into LC3-I to maintain the autophagic process
[71]. Recent articles reviewed small molecules targeting
ATG4B, such as FMK-9a, tioconazole, and Z-FG-FMK, suggest-
ing that ATG4B could be a molecular target in cancer through
an autophagy inhibition approach [71]. Inhibiting autophagic
flux in the later stages of the autophagic process has been
increasingly considered a key strategy for promoting cancer
cell death through apoptosis, necrosis, and ferroptosis [72–74].
Many cancers, including glioblastoma, breast cancer, and non-
small cell lung cancer, have the ability to sustain or intensify
autophagic degradation following radiation, playing a key role
in cancer cell resistance to radiotherapy and antitumor drugs
[75]. Therefore, the development of autophagic degradation
inhibitors in combination with other agents has been promis-
ing. The accumulation of cellular cargo proteins in autophago-
somes can be induced by either interrupting their fusion with
lysosomes or inhibiting lysosomal proteolysis. Fusion between
autophagosomes and lysosomes to form autolysosomes can
be suppressed by interrupting a variety of factors such as
microtubules and SNARE complexes. Microtubules are
required for trafficking and fusion of mature autophagosomes
with lysosomes. During this process, microtubule acetylation is
required. Vinblastine disrupts microtubule polymerization
both in non-acetylated and acetylated microtubules and
impairs LC3-associated fusion between autophagosomes and
lysosomes [76]. SNARE complexes comprise cytoplasmic
SNAP29, autophagosomal syntaxin 17, and lysosomal VAMP8,
all of which interact with each other and are required for
autolysosome fusion [77]. Berbamine is an autophagic flux
inhibitor that potently increases BCL2 interacting protein 3
(BNIP3) expression and the interaction between BNIP3 and
cytoplasmic SNAP29, blocking the interaction between
SNAP29 and lysosomal VAMP8 [77]. Berbamine-mediated inhi-
bition of SNARE complex formation and autophagic flux has
shown chemotherapeutic effects in cancer.
As another strategy for inhibiting autophagic flux in cancer,
lysosomotropic reagents such as chloroquine (CQ, under clin-
ical trial) inhibit lysosomal uptake, resulting in increased lyso-
somal pH and impaired autophagy degradation [78]. Either
direct or indirect inhibition of lysosomal proteins can be
a method for disrupting autophagic flux and cancer, as in
the following examples: (1) targeting lysosomal enzymes
with the combinatorial treatment of pepstatin A and E64d
completely blocks lysosomal functionality by inactivating
aspartic/thiol proteases and cathepsin B, D, H, and L in lyso-
somes [79]; (2) targeting the lysosomal membrane protein
V-ATPase, which acidifies lysosomes as an electrogenic H
+
pump, using bafilomycin A1 to inhibit V-ATPase, elevate
vacuolar pH, and inhibit lysosomal uptake [2]; and (3) target-
ing cytoplasmic interactors such as Hsp70 chaperone and
ubiquitin-like protein 4A (UBL4A). Hsp70 stabilizes lysosomes
through a direct interaction with the endolysosomal anionic
phospholipid bis(monoacylglycerol)phosphate (BMP), which
leads to direct binding between lysosomal sphingomyelin
and acid sphingomyelinase (ASM) to maintain lysosomal
homeostasis [80,81]. Pifithrin-μ (PES) directly inhibits Hsp70
activity to suppress tumorigenesis [82]. Alternatively, UBL4A
maintains protein metabolism and cellular homeostasis.
UBL4A causes dysfunction in lysosomal degradation through
direct binding with lysosomal-associated membrane protein 1
(LAMP1), causing an antitumor effect [83]. Therefore, UBL4A
can be a promising target for disrupting autophagy degrada-
tion in cancer therapy.
3.1.3. Crosstalk between autophagy and mechanism of
cell death in cancer
Although autophagy is a cell survival process by recycling
deleterious organic substrates, overactive autophagy can
lead to cellular suicide. Crosstalk between autophagy and
mechanisms of cell death such as apoptosis has been investi-
gated for their crucial roles in cellular homeostasis. Because
autophagy can act as both a tumor-suppressive mechanism by
degrading factors that contribute to cancer as well as an
apoptotic driver for cancer cell death, its impairment can
lead to favorable environments for cancer. However, autop-
hagy also facilitates the survival of cancer cells under stressful
EXPERT OPINION ON DRUG DISCOVERY 7
conditions such as the acidic tumor microenvironment or
hypoxia. Recently, targeting interactions between autophagic
and apoptotic factors has been implicated in cancer therapeu-
tics. Autophagy and apoptosis are interconnected through
several factors, including a Beclin-1-BCL2 interaction, caspase-
and calpain-mediated Beclin-1 cleavage, increased B-cell lym-
phoma 2-interacting mediator of cell death (BIM) expression
by mTOR inhibition, interactions between UVRAG and BCL2-
associated X protein (BAX), and interactions between FAS-
associated protein with death domain (FADD) and ATG5
[43,84]. Overall, the interconnectivity between autophagy
and apoptosis requires an integrative approach for developing
autophagy-targeting small molecules against specific diseases.
3.1.4. Brief discussion about autophagy in cancer
Due to the dual role of autophagy in the pathophysiological
phenotypes of malignant cancers, it is important to determine
whether autophagy activators or inhibitors are required to treat
a specific cancer type based on tumorigenic characteristics and
autophagy-related pathways. To date, multi-dimensional knowl-
edge of autophagy-modulating small molecules in preclinical stu-
dies have given valuable results in cancer research [85]. Therefore,
making use of autophagic modulators without genetic modifica-
tions will contribute to the discovery and control of autophagic
signaling molecules that play key roles in specific autophagy-
related cancer pathways.
3.2. Proteopathies
Proteopathies, also known as proteinopathies or protein con-
formational disorders (PCDs), refer to a class of diseases in
which certain proteins can aggregate in non-native, structu-
rally abnormal conformations, or become more susceptible to
aggregation even in their native conformation. These aggre-
gates become toxic and disrupt the function of cells, tissues,
and organs [86]. Recent studies have reported that inducing
autophagy flux significantly ameliorates these pathologies by
directly degrading the abnormal aggregates [87]. Therefore,
pharmacologically modulating autophagy is regarded as
a promising strategy against these diseases.
3.2.1. Amyloidopathies
Amyloidopathies (also called as β-amyloidopathies) consist of
amyloid β (Aβ) plaque deposition in the brain, which is a key
feature in neurodegenerative disorders including Alzheimer’s dis-
ease (AD) [88], Parkinson’s disease dementia (PDD) [89], and
dementia with Lewy bodies (DLB) [90]. The formation of Aβ aggre-
gates has been well established. The amyloid precursor protein
(APP, a transmembrane protein) undergoes two different cleavage
processes via three secretase enzymes: α-secretase, β-secretase
(BACE1), and γ-secretase [91]. In non-amyloidogenic processes,
APP is cleaved by α-secretase to leave the C-terminal fragment
83 (αCTF) in the cell membrane, which is then cleaved by γ-
secretase and released as the soluble P3 peptide fragment. In the
amyloidogenic process, APP is cleaved by β-secretase that leaves
the C-terminal fragment 99 (βCTF) in the cell membrane, which is
then cleaved by γ-secretase and released as Aβ
1-40
or the less
soluble Aβ
1-42
. These Aβ peptides are prone to aggregate into
oligomers, fibrils, and plaques, hallmarks of neurodegenerative
disorders and AD in particular [92]. Autophagy is a key regulator
of Aβ metabolic activity such as clearance and secretion [93],
implying that balanced regulation of autophagy is critical for
neuronal homeostasis.
As described in Section 3.1, the PI3-K-AKT-mTOR pathway is
a conventional signal for autophagy induction. Recent studies
revealed that several modulators promoted Aβ clearance
through autophagy induction by targeting this pathway,
including alborixin [94], rapamycin [95], AR12 (an analog of
the COX-2 inhibitor celecoxib) [96], and ginsenoside com-
pound K [97]. AMPK is also known to induce autophagy by
directly phosphorylating ULK1/Beclin-1 to form the phago-
phore or through attenuation of the autophagy inhibitory
factor mTOR [98]. Hence, AMPK activators such as resveratrol
[99] and its analogs (RSVA314, RSVA405) [100] and ginseno-
side Rg2 [101] exhibited reduced Aβ levels by activating
autophagy. However, another indirect AMPK activator, metfor-
min, is currently controversial for its effect on amyloid pathol-
ogy, as it exhibited gender-specific responses in an APP mouse
model [102] and showed contradictory results for the regula-
tion of Aβ formation [103]. This indicates that delicate mod-
ulation is required for AMPK targeting approaches.
Additionally, regulation of autophagy by master transcrip-
tion factors has also been considered for amyloid-maladies.
For instance, resveratrol exhibited fAβ degradation by promot-
ing TFEB deacetylation through Sirtuin 1 (SIRT1) [104].
Tacrolimus (FK506) also reduced amyloid plaques resulting in
neuroprotection [105], in which TFEB activation could occur
through interactions with another protein target [2].
3.2.2. Tauopathies
Tauopathies are a group of neurodegenerative disorders
including Alzheimer’s disease (AD), chronic traumatic ence-
phalopathy (CTE), and corticobasal degeneration (CBD) [106].
It is well established that tauopathies feature the pathological
accumulation of abnormal forms of the microtubule-
associated protein tau (MAPT) in neurons and glia, which are
susceptible to forming insoluble aggregates through misfold-
ing [107] or hyper-phosphorylation [108]. These aggregates
lead to neuronal cell death by overcoming the entire neuron
and forming neurofibrillary tangles (NFTs), a major hallmark of
neurodegenerative diseases together with Aβ plaques [109].
Several studies have revealed that autophagy dysregulation
occurs in tauopathies [110]. It is therefore pivotal to rescue
and augment autophagy in order to control pathogenic tau in
tau-mediated neuronal disorders. Currently, studies have
focused on the AKT/mTOR axis to regulate autophagy. For
example, autophagy enhancement with AKT/mTOR axis regu-
lators such as rapamycin [111], selenomethionine [112], and
PP242 [113] reduced tau aggregates or mitigated cognitive
deficits in tauopathy models.
Moreover, the indirect AMPK activator metformin amelio-
rated tauopathy through autophagy induction [114] and also
reduced tau phosphorylation through AMPK-mediated activa-
tion of protein phosphatase 2A (PP2A) [115]. However, the
relevance of PP2A and AMPK is currently controversial [116],
since AMPK is known to induce autophagy through phosphor-
ylation and reportedly elevates tau phosphorylation toward
tau-mediated toxicity [117]. Consequently, several studies
8D. KIM ET AL.
have focused on an alternative approach of promoting autop-
hagy gene master transcription factors or autophagic flux
machinery. According to these reports, tau pathology was
reduced by activating TFEB with trehalose [118], fisetin [119],
flubendazole [120], and activating NRF2 with fisetin [119] and
sulforaphane [121]. This drove the development of trehalose,
fisetin, and sulforaphane dietary supplements for neuronal
health. Furthermore, a recent study has indicated the farnesyl-
transferase inhibitor lonafarnib as a new therapeutic agent
against tau-maladies by reducing Rhes farnesylation and
increasing lysosomal activity and tau protein turnover [122].
These results support the therapeutic potential of targeting
key autophagic pathway components to treat tauopathies.
3.2.3. Synucleinopathies
Synucleinopathies (also called as α-synucleinopathies) are neu-
rodegenerative disorders characterized by abnormal deposits of
α-synuclein (α-Syn) aggregates in the brain [123]. Α-Syn aggre-
gates, which are mainly triggered by genetic mutations promot-
ing misfolding [124] or overexpression [125], disrupt the
dopamine transporter in dopaminergic neurons, leading to
synaptic dysfunction, cell death, and the progression of neuro-
degenerative diseases [126]. The three major types of synuclei-
nopathies are Parkinson’s disease (PD), dementia with Lewy
bodies (DLB), and multiple system atrophy (MSA) [123]. Since
homeostatic autophagy is impaired in synucleinopathies, thera-
peutic approaches to restore autophagy have emerged as clin-
ical strategies against them [127]. Studies have revealed the
amelioration of pathological synuclein features in vitro and
in vivo through pharmacological treatment with autophagy
modulators targeting mTOR such as rapamycin [128], and those
targeting AMPK such as metformin, 5-aminoimidazole-4-carbox-
amide ribonucleotide (AICAR) [129,130], resveratrol [131], and
trehalose [132,133]. This supports the benefit of autophagy path-
way regulation in synuclein-related disorders. Furthermore,
a recent combinatorial approach revealed significant additive
improvements to dopaminergic deficits through dual targeting
of AMPK and mTOR by trehalose and rapamycin, respectively
[134]. Several studies also pursued the pharmacological activa-
tion of autophagy through other pathways. Lithium, which is
known to inhibit both inositol monophosphatase (IMPase) and
glycogen synthase kinase-3β (GSK3β), induced autophagy by
reducing intracellular 1,4,5-inositol trisphosphate (IP3) levels
[135,136] and prevented α-Syn accumulation and neurotoxicity
[137]. Additionally, tyrosine kinase inhibitors, including nilotinib
[138], saracatinib (under clinical trials), and masitinib [139], also
promoted α-Syn clearance and prevented its accumulation
through activation of the AKT/mTOR pathway [140]. In conclu-
sion, numerous studies have reported new autophagic factors
and their modulators as relevant to α-synucleinopathies, provid-
ing beneficial tools for understanding the pathologic process
and offering opportunities for therapeutic development against
to α-Syn-related proteopathies.
3.2.4. Prion diseases
Prion diseases (also known as transmissible spongiform ence-
phalopathies (TSEs)) are a group of progressive neurodegen-
erative disorders caused by the conversion of normal prion
proteins (PrP
C
, cellular form, α-helical structure) to misfolded
prions (PrP
Sc
, scrapie form, β-pleated sheet structure) with
abnormal structure, which have the ability to transmit their
misfolded shape to normal prions [141]. Although the
mechanism of how prion aggregates kill nerve cells remains
unclear, it is reported that these abnormal prions are toxic to
neurons [142], leading to neuronal cell death that leaves
microscopic fluid-filled spaces behind and turns brains into
sponge-like shapes that causes progressive neuronal dete-
rioration [143]. The ‘gain of toxicity’ in prion proteins occurs
as sporadic (spontaneous), genetic (familial), and acquired
(infectious/transmissible), leading to human prion diseases
such as Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann-
Sträussler-Scheinker syndrome (GSS), and familial insomnia
(FI) [144]. Several studies have suggested a role for autophagy
in the pathogenesis of prion diseases. A recent study utilized
the mTOR regulators rapamycin and AR12 to examine their
combinatorial effect with cellulose ethers (CEs, prion conver-
sion inhibitor) in prion diseases [145]. Although there was
only a slight improvement without additive effects in vivo,
the trial itself is significant and implies the application of
mTOR-mediated autophagy in prion disease. In addition to
factors related to autophagosome formation, others con-
nected with autophagic turnover have received considerable
attention. Spermine facilitates autolysosome fusion and
autophagic degradation by inducing the acetylation of struc-
tural tubulin machinery [146]. Tacrolimus exhibited prion
degradation in an autophagy-dependent manner by enhan-
cing autophagy-related gene expression [147], which might
have relevance with autophagy master transcription fac-
tors [2].
3.2.5. Brief discussion about autophagy in proteopathies
Various proteopathies in which certain proteins become struc-
turally abnormal are introduced in Section 3.2. Autophagic
turnover becomes defective by protein aggregates which
aggravates these conditions, suggesting that reactivating
autophagy is required to ameliorate these diseases.
Targeting mTOR signaling has been implicated as an impor-
tant therapeutic approach for conventionally elevating autop-
hagy. However, due to its roles in cellular metabolism,
especially during neuronal development [148], chronic regula-
tion of mTOR should be carefully regulated. Therefore, instead
of targeting essential pathways related to broad energy meta-
bolism, other factors involved in autophagy should be regu-
lated such as autolysosome fusion and lysosomal activity.
3.3. Vascular diseases
Increasing studies have revealed that balanced autophagy
plays an essential role in mediating vascular physiological
processes, including: (1) as a protective cellular mechanism
in vessel walls; (2) in smooth muscle cell survival and prolif-
eration; (3) in the preservation of endothelial function; (4) in
the degradation of abnormal plaques in vessel walls; and (5) in
effective efferocytosis [149–151]. Therefore, pharmacological
intervention with modulators that stimulate the pro-survival
effects of autophagy in the vasculature have been implicated
to target diverse vascular diseases.
EXPERT OPINION ON DRUG DISCOVERY 9
3.3.1. Atherosclerosis
Atherosclerosis is the blockage of blood vessels caused by
plaque deposits. It is classified as an inflammatory pathology
occurring in blood vessels with several risk factors including
sustained diabetes, aging, obesity, arterial hypertension, and
the accumulation of oxidized low-density lipoprotein (oxLDL)
cholesterol. Recent reports have focused on acquired defects
in autophagy that aggravate atherosclerosis. Lipophagy, or the
autophagy-mediated degradation of lipids, has been under
investigation as a targeting strategy for atherosclerosis.
Oxidative stress triggered by excess ROS generation is
known as a main cause of atherosclerosis. The abnormal
occurrence of such stress results in vascular cell-induced
damage and excessive immune responses via direct oxidation
of molecular components and the accumulation of cell death-
induced debris, which is responsible for the occurrence and
aggravation of atherosclerosis [152]. To target autophagy in
atherosclerosis, a modulator of antioxidant proteins can be
used to scavenge ROS in macrophages because uncontrolled
ROS production results in lipophagic flux inhibition [153].
Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that main-
tains lipophagic flux by reducing the produced ROS. Therefore,
the delivery of PRDX1-mimetics (such as ebselen and glio-
toxin) or modulator excavation could rescue impaired lipo-
phagy and maintain cholesterol efflux [154].
3.3.2. Intraocular neovascularization
Intraocular neovascularization is a cause of ocular vascular
diseases and affects transparency in the eye. It results in
hemorrhage, multiple disorders of the eye, and contributes
to loss of eyesight and other serious complications associated
with these diseases, such as branch retinal vein occlusion, age-
related macular degeneration, and diabetic retinopathy [155].
In recent studies, autophagy has been considered a plausible
strategy to target vascular endothelial growth factor (VEGF)
and neovascularization. VEGF and pigment epithelium-derived
factor (PEDF) are intimately related to intraocular neovascular-
ization in retinal pigment epithelium (RPE) cells. A baseline
level of autophagy under normal conditions changes along
with age and pathological processes that can accompany
hypoxia, oxidative stress, and inflammation. Accordingly,
autophagy can be activated in RPE cells [156]. Activation of
autophagy upon hypoxia exposure promotes VEGF expression
and inhibits PEDF expression in RPE cells [157]. When pre-
treated with 3-MA (a PI3-K inhibitor) or CQ (a lysosomal inhi-
bitor), inhibitors of autophagic initiation and lysosomal
degradation, respectively, VEGF expression was reduced and
the expression of PEDF was increased, resulting in neovascular
disruption by inhibiting cell proliferation, tube formation, and
vascular endothelial cell migration [157]. Although the
detailed mechanisms of how autophagy regulates angiogenic
factor expression are yet to be elucidated, autophagy inhibi-
tors could be developed as effective therapeutic agents for
retinal neovascularization.
3.3.3. Ischemia-reperfusion injury
Ischemia-reperfusion injury (IRI) in brain microvascular
endothelial cells is the main cause of blood brain barrier
(BBB) integrity disruption, which can result in fatal ischemic
stroke in patients [158]. Maintaining the integrity of the BBB is
pivotal for homeostasis by exchanging essential nutrients and
ions in the brain. Damage to the BBB is primarily caused by
stress-induced ROS generation, dysfunction of endothelial
tight junctions, and elevated permeability [159]. Therefore,
protecting the structural integrity of the BBB has been
a focus for targeting various central nervous system diseases.
Autophagy is highly induced in ischemic brain injury with
therapeutic activity, including protection of endothelial cells
against stress or stimuli and recovery from abnormal BBB
permeability. In recent studies, autophagy stimulated by
serum starvation or AKT-mTOR-S6 K modulators such as rapa-
mycin and torin-1 was found to protect endothelial brain
barrier integrity by scavenging ROS, inhibiting the dysfunction
of tight junction proteins such as claudin-5 (Cldn5) and zonula
occludens-1 (ZO-1), and reducing elevated permeability in the
BBB [158–160].
3.3.4. Brief discussion about autophagy in vascular
diseases
There is mounting evidence demonstrating that autophagy
plays an essential role in vascular disease through the mod-
ulation of autophagy-specific genes. Yet, how to correctly
induce beneficial autophagy without triggering the detrimen-
tal effects of abnormal immune responses and cell death
remains a challenge. It is therefore vital to identify pharmaco-
logical agents that modulate autophagy with greater specifi-
city toward select molecular factors in order to be a favorable
target for therapeutic development in vascular diseases.
3.4. Other therapeutic applications in human diseases
Autophagy principally serves a protective role to maintain
homeostasis against diverse pathologies, even beyond the
diseases mentioned above. In this section, additional diseases
are discussed in which autophagy has critical relevance but is
less reported, including viral and microbial diseases and lyso-
somal storage diseases (LSDs). In this context, pharmacological
modulation of autophagy in combination with other medica-
tions could be a beneficial strategy for their treatment.
3.4.1. Viral infection & microbiome diseases
Pathogenic viruses cause infectious diseases. Viral infections are
caused by the invasion of viruses and virions (viral particles) into
the host body, where they enter into cells for propagation [161].
Many antiviral medications act directly on virus-specific compo-
nents; however, these result in unfortunate side effects such as
drug-resistance by promoting mutations within the viruses them-
selves [162]. Numerous reports have implicated host cell autop-
hagy in viral diseases by protecting the intracellular machinery
from hijacking by the virus or restricting their growth [163]
(Table 2). Therefore, pharmacological agents targeting autophagy
factors in the host could be beneficial for treating viral infections.
A recent study reported that CALCOCO2/NDP52 and SQSTM1/p62,
receptors of virophagy (viral xenophagy), positively and negatively
regulated the production of coxsackievirus B3 (CVB3), respectively,
supporting their use as host targets for antiviral treatment [164].
Another report revealed that combined treatment with oseltamivir
10 D. KIM ET AL.
(Tamiflu) and rapamycin protected mice against lethal pH1N1
infection by not only blocking viral replication, but also suppres-
sing mTOR-NLRP3-IL-1β axis-mediated immune damage and indu-
cing autophagy [165], portraying autophagy targeting as
a beneficial strategy for viral drug development. In contrast,
other studies highlight that several viruses exploit autophagy to
stimulate their replication (Table 2). For instance, it was established
that after viruses enter into cells, they require ‘replication compart-
ments’ (RCs) for genome replication and final virion assembly [166].
Among cellular vesicular structures, autophagosomes can serve as
viral RCs [167], which could explain the interplay between autop-
hagy and viral prosperity. Hence, blocking autophagosome forma-
tion by inhibiting the PI3-K pathway with 3-MA or wortmannin can
be a strategy to reduce viral infections such as adenovirus, Zika,
Dengue, and Influenza A virus [168–171]. Furthermore, recent
studies have reported reduced viral infections upon treatment
with autophagy flux inhibitors that target lysosomal activity or
autolysosomal fusion, such as CQ, (H
+
ion quencher) [171], bafilo-
mycin A1 (lysosomal V-ATPase/ER-calcium ATPase inhibitor) [172],
and U18666A (Niemann-Pick type C protein inhibitor) [173].
Alternatively, microbiome-mediated diseases are caused by
gut dysbiosis in which microbial balances collapse, altering
symbiotic relationships and perturbing host health [184]. The
symbiotic relationship between the gut microbiome and its
host is regulated through crosstalk using signaling molecules
such as microbial products and metabolites. However, when
this relationship is disrupted, deregulated molecular signaling
may be harmful to the host and can cause diverse disorders
such as immune-mediated/autoimmune diseases, metabolic/
cardiovascular diseases, infectious diseases, neuropsychiatric
disorders, and even cancer [185]. Recent reports have revealed
that autophagy regulation in host intestinal or colonic epithe-
lial cells changes with microbiome composition, and vice versa
[186]. These results indicate that host autophagy has func-
tional relevance with microbiome-mediated disorders, paving
the way for autophagy modulation to be a potential clinical
strategy for treatment. Using pharmacological approaches,
a recent study revealed alpinetin as a new therapeutic agent
for protecting the intestinal barrier by promoting autophagy
through regulation of the AhR/suv39h1/TSC2/mTOR axis [187],
indicating new bioactive molecules for targeting autophagy
upstream of mTOR. In one study, JWH-133 rescued autophagy
dysregulation in cultured inflammatory bowel disease (IBD)
biopsies by acting as an agonist of cannabinoid receptor 2
(CB2) [188], ligands of which are reported to induce autop-
hagy in an AMPK-dependent manner [189]. These results cor-
relate with another study portraying that AMPK activity is
pivotal for inducing host autophagy and clearing invading
bacteria through xenophagy (antibacterial autophagy), sug-
gesting a new approach for antibacterial medication develop-
ment [190].
3.4.2. Lysosomal storage disorders
LSDs belong to a group of rare inherited metabolic diseases
characterized by defects in lysosomal function, which occur
through mutations in lysosomal genes encoding hydrolases,
Table 2. Viruses that regulate autophagy for their propagation.
Virus Virus classification
Autophagy
regulation Targeting factors Ref.
Coxsackievirus B3 (CVB3) (+) ssRNA Naked Evasion of autophagic
degradation
SQSTM1/p62 (through expression of viral
proteinase 2A that cleaves SQSTM1)
[164]
Herpes simplex virus 1 (HSV-1) dsDNA
Enveloped
Evasion of autophagic
degradation
Beclin1 and TANK-binding kinase1 (TBK1)
(through expression of ICP34.5 that
inhibits beclin1 and TBK1)
[174]
Murine γ-herpesvirus 68 (MHV68)
Kaposi’s sarcoma-associated herpesvirus (KSHV)
dsDNA
Enveloped
Evasion of autophagic
degradation
Beclin1 (through expression of viral
homologue of BCL-2 that inhibit beclin1)
Rubicon-Beclin1 interaction and VPS34
(through expression of K7 protein that
promotes the interaction and inhibits
VPS34)
[175,176]
Human immunodeficiency virus type 1 (HIV-1) Dimer RNA
Enveloped
Evasion of autophagic
degradation
Beclin 1 and TFEB (through expression of
Nef that inhibit beclin1 and beclin1
mediated TFEB dephosphorylation)
[177]
Middle East respiratory syndrome coronavirus (MERS-CoV) (+) ssRNA
Enveloped
Evasion of autophagic
degradation
Akt1-SKP2-Beclin1 (through enhancing Akt1
phosphorylation, resulting in beclin1
degradation)
[178]
Sindbis virus (SINV) ssRNA
Enveloped
Vulnerable to autophagic
degradation
p62 (viral capsid protein binds to p62
resulting in virophagy)
[179]
Hepatitis C virus (HCV) ssRNA
Enveloped
Vulnerable to autophagic
degradation
SHISA5 (viral NS5A protein undergoes
transport to autophagosome by SHISA5)
[180]
Enhancing autophagy for
viral replication
Beclin1 (through expression of p7 ion
channel protein that directly binds to
beclin1)
[181]
Adenovirus dsDNA
Naked
Enhancing autophagy for
viral replication
Through induction of Atg12-Atg5 complex
formation
[168]
Zika virus (ZIKV) (+) ssRNA
Enveloped
Enhancing autophagy for
viral replication
Akt-mTOR pathway (through perturbing the
pathway by viral NS4A and NS4B)
[171]
Dengue virus (DV) (+) ssRNA
Enveloped
Enhancing autophagy for
viral replication
LC3 (through inducing LC3 cleavage by viral
NS4A to form autophagosome)
[169]
Influenza A virus (IAV) (-) ssRNA
Enveloped
Enhancing autophagy for
viral replication
Akt-TSC2-mTOR pathway or LC3 (through
modulation of the pathway or
relocalization of LC3 by viral M2 protein
[182]
Vesicular stomatitis virus (VSV) (-) ssRNA
Enveloped
Enhancing autophagy for
viral replication
Dependent on NLRX1
[183]
EXPERT OPINION ON DRUG DISCOVERY 11
enzyme cofactors, membrane proteins, and transporters [191].
The progressive accumulation of macromolecules within lyso-
somes due to defective degradation capacity leads to cellular
damage and affects various organs throughout the body [192].
Several studies have revealed possible therapeutic interven-
tions in some LSDs through pharmacological treatment with
autophagy inducers. At first, the mTOR inhibitor rapamycin
exhibited beneficial effects in a Gaucher disease (GD) droso-
phila model [193], and increased cellular viability in Niemann-
Pick disease type C1 disease model cells deficient in the NPC
intracellular cholesterol transporter 1 (NPC1) gene [194].
However, another study reported contradictory results about
the effects of rapamycin on NPC mice, in that lengthening life
span by rapamycin was dependent on the genetic background
[195]. Due to these controversial mTOR-targeting results,
several studies examined mTOR-independent autophagy indu-
cers to avoid perturbing the critical functions of mTOR [196].
The IMPase inhibitors lithium and L690,330 rescued cerebellar
cells from juvenile neuronal ceroid lipofuscinosis (JNCL)
pathology, in which the lysosomal transmembrane protein
CNL3 gene is defective, through reducing intracellular IP3
levels and enhancing autophagy [135,136]. One of the LSDs,
Vici syndrome, is caused by recessive mutations in the EPG5
gene, which encodes a crucial protein facilitating SNARE
assembly in the autolysosome fusion process. A recent report
revealed that under loss of EPG5 function, SNAP25 interferes
with the SNARE complex instead of SNAP29, resulting in
autophagy dysregulation. This suggests that SNAP25 could
be a potential therapeutic target for restoring autophagy in
LSDs [197].
Table 3. Autophagy-targeting therapeutic agents developed into pharmaceuticals/dietary supplements or under clinical trials.
Application state Agents Structure Targeting diseases Targeting factors/pathways Ref.
FDA approved Rapamycin derivatives Cancer mTOR
(Autophagy activation)
[67,68]
Nilotinib Cancer AMPK
(Autophagy activation)
[57]
Bortezomib Cancer 38-MAPK-JUNK pathway
(Autophagy activation)
[46]
Idelalisib Cancer PI3-K
(Autophagy inhibition)
[61]
Under clinical trial Chloroquine
/Hydroxy-chloroquine
Cancer
Viral infection
Lysosomal hydrogen
(Autophagy inhibition)
[78]
Saracatinib (AZD0530) Neurodegenerative disorders
- Alzheimer’s Disease
- Parkinson’s Disease
Tyrosine kinase Fyn
(Autophagy activation)
[139]
(Continued )
12 D. KIM ET AL.
4. Conclusion
Through a comprehensive understanding of autophagic
machinery, its fundamental importance to cellular health
emerges as an auspicious target for curing various phenotypic
diseases in humans. Pharmacological approaches have been
considered a major priority because of advantages such as
brief pharmacokinetics, easy treatment and delivery into
cells, and minimized immune responses. The development
and application of pharmacological agents are therefore pivo-
tal to providing new insight and exploration into autophagy-
related diseases and their underlying mechanisms. Careful
consideration is also required during this process, since autop-
hagy activation or suppression can yield defensive or destruc-
tive outcomes according to disease type, even at different
stages of the same disease. For instance, autophagy may be
utilized as a resistance mechanism for chemotherapy-treated
tumor cell survival, or alternatively, may induce unintended
death in important cells required for pathological recovery,
such as in proteopathies, vasculature abnormalities, metabolic
disorders, and aging. We have described both conventional
and recent concepts connecting specific autophagic path-
ways, targeting strategies using bioactive compounds, and
autophagy-related diseases. Collectively, the considerations
and recent advances highlighted in this review contributes
to a comprehensive understanding of autophagy signaling
control in human diseases.
5. Expert opinion
The self-catabolic intracellular process autophagy occurs in
organisms throughout their lifetime, supporting its critical
role in organismal health across life stages. Because of the
detrimental influence of dysfunctional cells to an organism
and their etiology in numerous diseases, maintaining cellular
quality control by recycling components through autophagy is
essential to defend against health decline. There is a history of
successful cases of small molecule development targeting
autophagy-related diseases (Table 3).
Because variations in signaling pathways or protein expres-
sion levels specific to each target diseases could change drug
efficacy and cause side effects, understanding autophagic
modulators and their molecular interactors in the cell are
critical components for therapeutic success. To utilize autop-
hagy-modulating compounds without serious side effects,
identification of their target proteins should be a focus during
the process. By clarifying these specific interactions, research-
ers can more comprehensively characterize a compound’s
effect and efficiently define their ideal spatial and temporal
Table 3. (Continued).
Application state Agents Structure Targeting diseases Targeting factors/pathways Ref.
Dietary supplement Resveratrol Neuronal health AMPK pathway
SIRT1-TFEB pathway
(Autophagy activation)
[99,104]
Trehalose Neuronal health Autophagy master gene (TFEB)
(Autophagy activation)
[118]
Fisetin Neuronal health Autophagy master gene (NRF2)
(Autophagy activation)
[119]
Sulforaphane Neuronal health Autophagy master gene (NRF2)
(Autophagy activation)
[121]
Lithium Neuronal health IMPase
GSK3β
(Autophagy activation)
[135,136]
Ginsenoside Rg3 Neuronal health PI3-K pathway
(Autophagy activation)
[198]
EXPERT OPINION ON DRUG DISCOVERY 13
chemotherapeutic characteristics. As outlined in this review,
targeting strategies for autophagic pathways across several
diseases show strong potential as therapeutic strategies.
Several agents are introduced that have been developed
into clinical drugs, are under clinical trials, or are being used
as dietary supplements. For instance, rapamycin has been
developed into clinical anticancer medications, raising its
value in the mTOR inhibitor market which is expected to
significantly grow during 2019–2023 with CAGR over 3%,
size by 455 USD.85 million (Technavio, 2019). Other drugs
such as bortezomib, nilotinib (AMPK/mTOR/MAPK pathway
modulators), and idelalisib (PI3-K inhibitor) are also FDA
approved as anticancer therapeutics, supporting their efficacy
and safety. Other autophagy regulators like chloroquine, sar-
acatinib, and latrepirdine are also undergoing clinical trials,
and natural small molecules that induce autophagy have
been developed into dietary supplements for neuronal health
(resveratrol, trehalose, fisetin, sulforaphane, lithium, and gin-
senoside Rg3). These applications verify the utility and value of
autophagy-targeting strategies for drug development, with
further promise in treating currently undruggable pathologies
such as proteopathies. Instead of the current drugs on the
market for Alzheimer’s disease that merely delay patient
pathologies without removing the pathologic cause, autop-
hagy-targeting strategies offer promising therapeutic options
and new paradigms for future treatments.
Funding
This work was supported by grants from the National Research
Foundation of Korea, funded by the Korean government (MSIP;
2015K1A1A2028365, 2015M3A9B6027818, 2016K2A9A1A03904900,
2018M3A9C4076477), the Brain Korea 21Plus Project in the Republic of
Korea and ICONS (the Institute of Convergence Science), Yonsei University.
Declaration of interest
The authors have no other relevant affiliations or financial involvement
with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript
apart from those disclosed.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
ORCID
Dasol Kim http://orcid.org/0000-0003-1314-2940
Hui-Yun Hwang http://orcid.org/0000-0001-9223-1428
Ho Jeong Kwon http://orcid.org/0000-0002-6919-833X
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