Design of Small Molecule Autophagy Modulators: A promising

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Design of Small Molecule Autophagy Modulators: A promising druggable strategy Siyu He, Qi Li, Xueyang Jiang, Xin Lu, Feng Feng, Wei Qu, Yao Chen, and Haopeng Sun J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01019 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Design of Small Molecule Autophagy Modulators: A promising druggable strategy

Siyu He a, Qi Li a, Xueyang Jiang b, Xin Lu a, Feng Feng b, Wei Qu b, Yao Chen c, Haopeng Suna, *

a

Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing,

210009, China b

Key Laboratory of Biomedical Functional Materials, School of Science, China

Pharmaceutical University, Nanjing 211198, China c

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023,

China;

Correspondence: [email protected] (Haopeng Sun).

ACS Paragon Plus Environment

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Abstract: Autophagy is a lysosome-dependent mechanism of intracellular degradation for maintaining cellular homeostasis. Dysregulation of autophagy has been verified to be closely linked to a number of human diseases. Consequently, targeting autophagy has been highlighted as a novel therapeutic strategy for clinical utility. Mounting efforts have been done in recent years to elucidate the mechanisms of autophagy regulation and to identify potential modulators of autophagy. However, most of the compounds target complex and multifaceted pathway and proteins, which may limit the evaluation of therapeutic value and in-depth studies as chemical tools. Therefore, the development of specific and active autophagy modulators becomes most desirable. Here, we briefly review the regulation of autophagy, and then summarize the recent development of small molecules targeting the core autophagic machinery. Finally, we put forward our viewpoints on the current problems, with the aim to provide reference for future drug discovery and potential therapeutic perspectives on novel, potent, selective autophagy modulators.

1.

Introduction Autophagy was discovered more than half a century ago. The 2016 Nobel Prize

in Medicine or Physiology was awarded to professor Yoshiniri Ohsumi for his excellent works on elucidating mechanisms of autophagy regulation. Eukaryotes have two major intracellular protein degradation pathways required for protein homeostasis and cell survival, namely the ubiquitin-proteasome system (UPS) and autophagy.


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These two systems have a coordinated and complementary relationship.1 Under physiological conditions, autophagy plays a fundamental role in maintaining cellular homeostasis by degrading intracellular damaged proteins and organelles for energy maintenance and cytoplasmic quality control.2 Due to different formation processes and related functions, at least three forms of autophagy exist as routes to eliminate accumulation of toxic materials and keep cells healthy, including macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA).3 Macroautophagy (hereafter referred to as autophagy) is characterized by double-membraned autophagosomes which involve engulfment of portions of cytoplasm and proteins. Degradation by autophagy can be either nonselective for bulk intracellular components or selective for damaged organelles (mitophagy, pexophagy, lysophagy and nucleophagy), invasive pathogens (xenophagy), protein aggregates (aggrephagy).4 Autophagy has miscellaneous physiologic roles.5 Recent studies have shown that autophagy can be induced by diverse stimulations, such as reactive oxygen species (ROS),6 hypoxia impairments,7 subcellular organelle damages,8 and protein aggregation.9 Hence, autophagy is closely associated with a wide range of human diseases, including cancer, neurodegeneration, infection, cardiovascular disorders, metabolic diseases, pulmonary diseases and aging.5 Intriguingly, both up- and down-regulation of autophagy have been shown to be protective against cancer, neurodegeneration, infectious diseases and ischaemic insult in the heart;10 thus it shows the intricate relationship between autophagy and diseases, further efforts are necessary to shed light on the precise regulation of autophagy in human disease.



Up to recent decade, autophagy has become an enormously attractive field in biology, current substantial progress has been made in identifying autophagy-related genes (Atgs), proteins, signaling pathways, and related molecular mechanisms that are involved in autophagy regulation. To sum up, there are a lot of demands for diverse small molecule compounds with different mechanisms serving as autophagy regulators. These chemical modulators can be utilized for understanding the pleiotropic functions of autophagy as pharmacological probes, as well as for therapeutic applications. Excellent reviews have described that targeting autophagy provides a promising strategy for drug discovery and therapeutic modulation.11 In this review, we provide an updated overview on advances in the discovery of small molecular compounds selectively targeting the autophagic machinery and the potential therapeutic application of these compounds in human pathogenesis. We select some representative cases, focusing on the molecular design and optimization process, as well as the mechanism and efficacy characteristics, in order to provide a new strategy for discovery and development of more novel, potent, selective autophagy modulators for therapeutics. 2.

The signaling pathways and regulation of autophagy Understanding the cellular and molecular basis of autophagy machinery is

prerequisite for identifying new diagnostic and therapeutic targets. The study of autophagy has accelerated in the last decade because of the discovery of more than 36 Atgs evolutionarily conserved mammalian homologs, which are responsible for the


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core machinery of autophagosome formation as well as required specifically for selective modes of autophagy (Table 1). However, the functions of Atgs are still elusive for the majority. Several Atgs play an indispensable role in the induction of autophagy (Figure. 1), while a subset of them also has autophagy-independent functions.12 Autophagy can be divided into canonical and non-canonical pathways. The canonical pathway requires the hierarchical intervention of all of the Atg proteins for autophagosome construction, while the non-canonical autophagy requires only a subset of Atg proteins. It is important to note that non-canonical autophagy has also been shown to function in other processes.13 Schematically, the execution of autophagy involves several major steps (Figure 1): induction, nucleation, elongation and completion, docking and fusion, degradation and recycling. The pathway is initiated by the formation of isolation membranes. The endoplasmic reticulum (ER) seems to be one of the main components of nascent autophagosomes.14 One of the earliest steps involves the redistribution of the unc-51 like autophagy activating kinase 1 (ULK1) complex to the surface of the ER for recruitment of the vacuolar protein sorting 34 (Vps34) complex. Vps34 complex is responsible for synthesis and deposition of phosphatidylinositol-3-phosphate (PtdIns3P) at autophagosome formation sites. This process will subsequently lead to the recruitment of PtdIns3P-binding proteins and additional factors.15 The ULK1 complex controls the trafficking of Atg9 (the only identified multimembrane-spanning Atg protein in eukaryotes), which involves in the recruitment of membrance to phagophore, at the origin of the autophagic vesicle.16 Two ubiquitin-like conjugation



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systems are necessary for phagophore elongation. The first system involves Atg7 and Atg10 conjugation of Atg12 into the Atg12-Atg5-Atg16L1 complex. The second conjugation system consists of the microtubule-associated protein light chain 3 (LC3), Atg7 and Atg3; in this system, LC3-I is modified into LC3-II by Atg7 and Atg3. The first conjugation system acts as an E3-like enzyme for the second conjugation system, which generates the lipidated phosphatidylethanolamine (PE)-conjugated form of LC3 (LC3-II) downstream of the unconjugated form LC3-I.17 LC3-II locates on the membrane of the autophagosome until it is degraded at the autolysosome. Therefore LC3 can be a common reliable marker to monitor autophagy.18 The completed autophagosome is transported to the lysosome; fusion of the autophagosome vesicle with

either

late

endosomes

or

lysosomes

involves

a

set

of

soluble

N-ethylmaleimide-sensitive fusion attachment protein receptors (SNARES) proteins;19 the ectopic P-granules autophagy protein 5 homolog (EPG5) acts as a tethering factor for this step, allowing the assembly and stabilization of SNAREs and determining the fusion specificity of autophagosomes with late endosomes/lysosomes. Once the fusion is complete, acid hydrolases and the cathepsins in the lysosomal lumen degrade the autophagosomal cargoes to remove harmful materials and simultaneously recoup essential building blocks and adenosine triphosphate (ATP) that are necessary for cell survival.20


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Table 1. Major mammalian Atgs and Atg-related proteins involved in autophagosome formation. ULK1 complex

Vps34 complex

Mammalian protein ULK1

Yeast ortholog Atg1

Features Serine/threonine kinase, phosphorylated by mTORC1 and AMPK, also regulates Vps34 and Beclin 1

Atg13

Atg13

Atg101

-

Interacts with Atg13

FIP200

-

Scaffold for ULK1 and Atg13, phosphorylated by ULK1

Vps34

Vps34

Class III PI3K, core component of Vps34 complexes, alternates between closed cytosolic and open membranous forms

Vps15

Vps15

Serine/threonine kinase, myristoylated, required for the assembly of the catalytic and regulatory subunits

Beclin 1

Atg6

BH3-only protein, positively regulates the catalytic activity of Vps34, phosphorylated by AMPK

Atg14 (L)

Atg14

Autophagy-specific subunit, localizes on the endoplasmic reticulum (ER), core component of Vps34 complex I, increases the phosphorylation of Beclin 1 by AMPK

UVRAG

-

Phosphorylated by mTORC1 and ULK1

Regulator of Vps34 complex II


Function in autophagy regulation Major initiator of autophagy. This complex is negatively regulated by mTORC1 and positively regulated by AMPK, mainly in nutrient- or energy-deprived conditions.

Vps34 complex I participates in autophagy, which is responsible for synthesis and deposition of PtdIns3P. Vps34 complex II takes part in endocytic sorting (as well as autophagy and cytokinesis in mammalian cells).

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Atg12conjugation system

LC3conjugation system

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Atg12

Atg12

Ubiquitin E3-like enzyme, conjugates to Atg5 in Atg8-PE conjugation, assists in autophagosomal elongation

Atg7

Atg7

Acts as an E1-like enzyme for Atg12 conjugation to Atg5

Atg10

Atg10

Ubiquitin E2-like enzyme

Atg5

Atg5

Forms a complex with Atg12, assists in autophagosomal elongation

Atg16L

Atg16

Interacts with Atg5 and is postulated to specify LC3 lipidation sites in autophagic membranes, acts as an E3-like enzyme in Atg8-PE conjugation, assists in autophagosomal elongation

LC3A/B/C

Atg8

Ubiquitin-like modifier, conjugates to associates with autophagosomal membrane

Atg4A-D

Atg4

Cysteine protease, especially Atg4B, converts pro-LC3 to LC3-I, delipidates autophagosomal LC3-II

Atg7

Atg7

E1-like enzyme, facilitates conjugation of LC3 proteins to PE,

Atg3

Atg3

Ubiquitin E2-like enzyme, involved conjugation of Atg8-like proteins to PE


PE,

in

The Atg12-Atg5-Atg16L complex acts as an E3-like enzyme for LC3/Atg8-PE conjugation, which s essential for proper elongation of the isolation membrane.

LC3 is present on both inner and outer membranes of the autophagosome. The formation of LC3-PE conjugates and their deconjugation by Atg4 is important to format the autophagosome membrane.

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Autophagy is stimulated during various physiological and pathological conditions; multiple layers of the regulatory mechanism may exist for organisms to adapt to stress and extracellular cues.21 Multiple stimuli including the presence of growth factors, nutrient and energy levels, amino acid, stress and pathogen infection, promote autophagy through two distinct categories mechanism of action, referring to as the mammalian target of rapamycin (mTOR)-dependent and mTOR-independent pathways.22 Especially, the mTOR-dependent signaling pathway has been well-characterized (Figure 1). mTOR forms two distinct complexes, complex 1 of mTOR (mTORC1) and mTORC2. The kinetics of these two complexes are finely tuned to respond to dynamic changes in cellular metabolism and environmental cues.23 mTORC1 plays a significant role in regulating autophagy, whereas mTORC2 is not a direct autophagy regulator.24 Growth factors such as insulin or insulin-like growth factors activate the class I phosphoinositide 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway.25 Activated Akt directly phosphorylates tuberous sclerosis complex (TSC) and prevents the formation of the TSC1/2 protein complex, resulting in activation of mTORC1.26 Of note, Akt mediates positive crosstalk from mTORC2 to mTORC1. The hydrophobic motif of Akt (Ser473), known as an upstream stimulator of mTORC1, is also a substrate of mTORC2, whose phosphorylation is critical for maximal activity of Akt. Therefore, mTORC2 may indirectly suppress autophagy by activating mTORC1.27 Amino acids are also crucial regulators for mTORC1 activation. In the presence of amino acids, Ras-like small GTPases (Rags) serves as a key mediator of



mTORC1 activation, and recruits mTORC1 to the lysosomal surface.28 In mTOR-dependent signaling pathway, activated mTORC1 down-regulates autophagy by preventing the activity of ULK1 and Vps34 complexes.29 During starvation, inhibited mTORC1 relieves its negative regulation on ULK1 and Vps34 complexes, allowing the nucleation of autophagosomes. AMP-activated protein kinase (AMPK) is sensitive to the level of ATP, increased intracellular ratio of AMP/ATP leads to the activation of AMPK. AMPK activation induces autophagy via both inhibition of mTORC1 and activation of ULK1, forming a negative feedback regulation.30 Activated mTORC1 phosphorylates ULK1 Ser757 to prevent its activation.31 Under glucose starvation, AMPK inhibits mTORC1 to relieve Ser757 phosphorylation, then phosphorylating ULK1 on Ser317 and Ser777.32 The coordinated phosphorylation of ULK1 by mTORC1 and AMPK provides a mechanism for signal integration. AMPK also oppositely regulates non- and pro-autophagy class III PI3K complex by directly phosphorylating Vps34 and Beclin 1 subunits.33 It has been reported that the ULK1 complex can also activate the Vps34 complex, directly phosphorylate Beclin 1.34 The function of Beclin 1 can be suppressed or activated through different binding partners, cell death regulating protein B-cell lymphoma 2 (Bcl-2) and the tumor-suppressing gene UV radiation resistance-associated gene (UVRAG).35


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Figure 1. Concise schematic representation of the mTOR-dependent autophagy pathway. The functional group of Atg proteins involved in autophagosome formation are highlighted. A number of small-molecule modulators can target autophagy at multiple points on regulatory pathways. The process of autophagy is best understood in the context of nutrient starvation. Similarly, deprivation from growth factors and/or amino acids also leads to the inhibition of TORC1, which when active represses conventional autophagy. Under low energy level, AMPK negatively regulates mTORC1 and also phosphorylates ULK1, thereby acting as a positive regulator of autophagy. Activation of ULK1 complex allowing the induction of autophagy machinery. Active ULK1 also phosphorylates the Vps34 complex, thus allowing translocation of Vps34 complex to the site of autophagosome nucleation. The first step is the formation of a double membrane phagophore, then phagophore expands and forms the autophagosome. Two ubiquitin-like conjugation systems are necessary for the elongation. Autophagosome finally fuses with lysosome and the contents are degraded by the lysosomal hydrolases.

3.

Relevance of Autophagy to Human Diseases



Autophagy can play either a protective or destructive role in different diseases, or in different stages of the same disease.10 Here, we concisely summarize part of autophagy-related diseases which attract increasing attentions in recent years. The development of pharmacologic agents that modulate autophagy may prevent the occurrence, delay the progression and decrease the mortality of certain diseases. 3.1 Cancer The association between cancer and autophagy is complex, as the role of autophagy may differ in different stages of this disease. Autophagy is generally considered as a mechanism by which cells conserve and recycle resources, therefore the pathway can tip cell fate and is referred to as type II programmed cell death. It is generally accepted that autophagy acts as a tumor suppressor; the deficiency of autophagy contributes to the development of cancer. It has been proved that multiple autophagy-related genes serve as tumor suppressors. For instance, a study using tissue-specific Atg5 and Atg7 knockout mice shows that autophagy deficiency can lead to tumors in liver.36 Activation of Beclin 1 can induce autophagy, resulting in suppression of tumorigenesis.37 In addition, active epidermal growth factor receptor (EGFR) suppresses autophagy by down-regulating the function of Beclin 1, resulting in the tumor progression in non-small-cell lung cancer (NSCLC).38 Furthermore, autophagy deficiency can cause oxidative stress. Increased oxidative stress activates nuclear factor erythroid-2-like 2 (NRF2), which also stimulates tumor growth.39 The expression of p62/SQSTM1 (p62), a significant substrate of autophagy, promotes oxidative stress and tumor growth. p62 deficiency reduces both the toxicity and


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tumorigenesis caused by defective autophagy.40 Autophagy interacts with cell death pathways via multiple mechanisms; many signals in the context of apoptosis activation induce autophagy, whereas signals that inhibit apoptosis also inhibit autophagy. The cross-talk between autophagy and apoptosis contributes to tumor suppression.41 However, in many cases, cancer cells are more autophagy-dependent than normal cells. Deletion of the essential autophagy-related protein focal adhesion kinase (FAK)

family

kinase-interacting

protein

of

200

kDa

(FIP200)

impairs

oncogene-induced tumorigenesis in a mouse model of breast cancer.42 Human pancreatic cancer cell lines and tumors exhibit increased basal levels of autophagy, inhibiting autophagy in these cells leads to tumor regression in a xenograft mouse model.43 Metabolic stress is a common feature of the tumor microenvironment, characterized by lack of nutrients and oxygen for long periods of time. In these unfavorable circumstances, activation of autophagy has the potential to accommodate the acute energy demands by providing cancer cells with amino acid, free fatty acid and glucose, which are significant nutrients for life maintenance. Treatment with chemotherapeutic agents results in increased cellular stress; autophagy may promote the growth of established tumors and contribute to chemo-resistance. In addition, autophagy is up-regulated in rat sarcoma (RAS)-transformed cancer cells and promotes the growth, survival, invasion, and metastasis of cancer cells. For example, autophagy promotes tumor growth in a mouse model of RAS-driven pancreatic cancer by suppressing p53 activation.44 It has been revealed that chemotherapy and



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radiotherapy can trigger the high level of autophagy, thus leading to multiple drug resistant (MDR) effect in the patients.45 In summary, autophagy is tightly linked to oncogenic signaling. Modulating autophagy indeed plays a therapeutic role in many types of tumors, especially some malignant tumors, such as pancreatic cancer

46

and melanoma.47 However, there still

exist controversy and uncertainty on the mechanism of autophagy in the control of cancers. 3.2 Neurodegeneration Degradation of disease-related mutant proteins to maintain cellular homeostasis is highly dependent on autophagy, thus there is no doubt that autophagy has a beneficial effect of protecting against neurodegeneration, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and neuroinflammation.48 Autophagy induction in response to pathogenic proteins and protein aggregates plays a neuroprotective role at the early stage of these disease.49 Evidence has shown that pharmacologic stimulation of autophagy can alleviate symptoms related to neurodegeneration in mouse models.50 Aggregates (lewy bodies) formation in neurons is one of the characteristics in PD. As the major protein in lewy bodies, mutated α-synuclein causes early-onset PD. It has shown that aggregated α-synuclein is preferentially eliminated by autophagy. Pharmacological enhancement of autophagy may be an attractive strategy to combat α-synuclein aggregation in PD.51 HD associates with the accumulation of mutant proteins with polyglutamine-rich

extensions.

Evidence

also

shows


that

the

extended

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polyglutamine-containing mutant proteins can be degraded through the autophagic pathway in model of HD.52 Surprisingly, autophagy plays a dual role in AD. On the one hand, the accumulation of autophagosomes is accelerated in the brain of AD patients.53 Pharmacologic stimulation of autophagy can alleviate symptoms in AD by providing a selective pathway for the clearance of aggregate-prone proteins. On the other hand, dysfunction in autophagy may enhance γ-secretase activity, which in turn increases the toxic proteolytic product amyloid-β (Aβ) by cleaving Aβ precursor protein (APP).54 These results suggest that accumulation of autophagosomes may process the APP into toxic forms. From this point of view, inhibition of autophagy may benefit the therapy of AD. 3.3 Metabolic Disease Autophagy degrades and recycles a variety of substances such as proteins, lipids, carbohydrates, nucleic acid release amino acids, fatty acids in order to generate the essential metabolites required for the maintenance of cell viability in cardiac, skeletal muscle, adipose tissue and pancreatic β cells. It is not surprise that dysregulation of autophagy has been linked to metabolism diseases such as obesity and diabetes. Previous studies have shed light on the important role of autophagy in regulation of lipid metabolism and insulin sensitivity.55 Autophagy down-regulates in hepatocytes both genetic and dietary models of obesity, thus contributing to insulin secretion and sensitivity. In addition, autophagy is necessary for maintenance of the structure, mass, and function of pancreatic β cells. Autophagy-defective mice in the pancreatic β-cell



show reduced β-cell mass and decreased insulin secretion.56 Metabolic changes incurred by autophagic stimulation can affect the progression of related human disease. 3.4 Infection, inflammation, and immunity Autophagy is a fundamental cell biological pathway contributing to the host defense mechanisms via complex regulatory interactions with immunological signaling. It embodies four principal manifestations: direct elimination of microbes, control of inflammation, antigen presentation, lymphocyte homeostasis, and secretion of immune mediators.57 Autophagy may be the most primordial form of innate immunity against invading microbes including bacteria, virus and parasites. Studies have elucidated the mechanisms by which intracellular microbes are targeted to autophagosomes for degradation.58 However, it must be underlined the de facto mechanisms of evasion from the host defense. For example, the human immunodeficiency virus type 1 (HIV-1) virulence factor Nef suppresses autophagy by binding to Beclin 1;59 certain pathogens such as Porphyromonas gingivalis can utilize autophagosomes as a site of replication for survival, which suggests a bivalent role of autophagy in pathogen defense.60 Understanding the characteristics of the pathogen-autophagy interaction in each of the individual instances is essential. In innate immunity, activation of inflammation results in recruitment of immune cell in pathogen defense. Autophagy has important effects on the anti-inflammatory reaction.61 Production of the pro-inflammatory cytokines interleukin (IL) 1β and


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IL18 is pivotal in antimicrobial host defense; the chronic inflammatory disease of the intestine Crohn’s disease (CD) is associated with increased IL-1β and IL-18 production. Autophagy can target multiple steps of inflammasomes activation and thus can potently suppress the production of IL1β and IL18.62 In addition, autophagy can regulate innate immune responses by digestion of dysfunctional mitochondria; prevent mitochondrial ROS release, and thereby suppressing inflammasomes activation. Apart from the immunological roles in eliminating infected pathogens and alleviating inflammation, autophagy can simultaneously assist the immune system to mount a specialized immune response. In adaptive immunity, it contributes to antigens presentation and the development and function of lymphocytes including T cells and B cells. The relationship between autophagy-associated immune signals and cancer immune responses has been long recognized. The role of crosstalk between autophagy and the immune system in cancer is ambiguous.63 In pro-death mechanism, autophagy enhances anti-tumor immunity in the abnormal tumor microenvironment; CD4+ or CD8+ T lymphocytes and many cytokines contribute to tumor suppression. In pro-survival mechanism, autophagy may impair the anticancer immune response and avail tumor cells to escape from immune surveillance or provide intrinsic resistance against anticancer therapy. Inhibition of autophagy may enhance anticancer immune responses.64 Autophagy significantly contributes to tumorigenesis and efficacy of chemo- and radiotherapy. Understanding the autophagic double-edged role that shapes the interface between cancer and immune



response is of great value to enhance the effects of anticancer approaches. 4.

Detection methods in autophagy Autophagy is a highly dynamic process able to sense intracellular signals and

rapidly mount a response. Accurately monitoring autophagy plays a fundamental role in both basic research and proper understanding of the physiological role of autophagy in disease manifestation. In addition, reliable detection methods are highly significant for discovery of autophagy modulators. Tremendous efforts have been made to develop utility approaches for detection of autophagy.18 Generally, autophagy can be monitored by directing observation of autophagy-related structures and quantification of autophagy/lysosome-dependent degradation of components.65 Transmission electron microscopy (TEM) is the most traditional method to monitor autophagy by analyzing the morphological structures. The autophagosome has a double-membrane structure containing undigested cytoplasmic components, while the autolysosome has a single-membrane structure containing degraded components; both of the autophagy-related structures can be easily distinguished from other cellular membranous compartments by TEM. However, the difficulties of identification of autophagy by TEM can not be ignored, such as time consuming, high technical requirement. It is best used in combination with other methods to ensure the complex and holistic approach.18 The amount of LC3-II reflects the number of autophagosomes. As the most widely used autophagy marker, autophagic flux is often inferred on the basis of LC3-II turnover, which can be measured by immunoblotting. However, it should be


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noted that LC3-II levels cannot quantify actual autophagic activity, since LC3-II is not involved in all cargo sequestration events, and LC3-II can be found on non-autophagosomal membranes. In addition, both autophagy activation and inhibition of autophagosome degradation increase the amount of LC3-II. It is highly recommended to compare the amount of LC3-II with the housekeeping proteins for quantification, such as tubulin or β-actin.66 Green fluorescent protein (GFP)-LC3 is an exogenous construct to increase detection efficiency when the level of the endogenous LC3 is very low. Changes in the number of GFP-LC3 can be monitored using flow cytometry. GFP-LC3 assays are widely used to monitor autophagy through immunofluorescence or fluorescence. However, these assays are time consuming, and require high technical expertise to transfect exogenous gene. It is recommended that results obtained by GFP-LC3 fluorescence microscopy should be verified by additional assays. The fluorescence of GFP can be quenched in the lysosome due to the low pH. To overcome the issue, the GFP can be replaced by the red fluorescent protein (RFP) that exhibits more intense fluorescence in acidic compartments.67 Evaluating changes of canonical autophagy biomarkers such as LC3 and GFP-LC3 has been applied to discovery of autophagy modulators.68 Recently, based on the inverse correlation between cellular lipid droplets (LDs) and autophagy, a novel image-based autophagy assay platform using a fluorescent LD bioprobe as a late-stage marker has been established. LD serves as a late-stage biomarker for monitoring autophagic process and discovering autophagy modulators.69 However,



the presence and potential activation of cytoplasmic lipases that are unrelated to lysosomal degradation must be considered when utilizing this assay. Besides conventional measurements of the autophagic process, some nanostructure-based probes has been developed for in vivo biological imaging, especially an innovative nanostructure-based probe based on an “in vivo self-assembly” strategy.70 These nanoprobes with various controllable physicochemical properties can provide promising application in in vivo autophagy detection owing to their high sensitivity to autophagic enzymes or autophagy-related signal molecules. The “molecule-like” responsive nanoprobe can be cleaved by Atg4B resulting in in situ self-aggregation with enhanced ﬂuorescence. Another probe provides a useful guide-line for real-time monitoring autophagy by detecting the change of lysosomal polarity during the member fusion in autophagy, namely two-photon fluorescent probe. Meaningfully, it is highly sensitive to the change of micro-environmental polarity and can monitor autophagy of a single cell and multiple cells.71 Finding autophagy specific modulators depends on accurately monitoring autophagy. Autophagy is a multi-step process. Small-molecule autophagy modulators with certain specificities can serve as available pharmacological tools for manipulating signaling pathways and evaluating autophagy substrate.72 5.

Autophagy modulators To date, different compounds have been described in literatures and patents as

potential modulators of the autophagy process with therapeutic potentials.73 A recent search of Clinical Trials. Gov. using the search term “autophagy” brings up over 70


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clinical trials. Meanwhile, several the U.S. Food and Drug Administration (FDA)-approved drugs have been shown to stimulate or inhibit autophagy (Table 2).74 Many currently available drugs have been shown to enhance autophagy, which indicates the overlap between diseases that occur in the setting of autophagy deficiency and diseases that respond to these drugs. This autophagy-enhancing action may be repurposed for novel clinical indications.75 However, most of autophagy modulators are discovered from nature products or compounds originally targeting other proteins or pathways, it is still not fully understood how these compounds interact with autophagic targets to induce or inhibit autophagy and whether some clinical agents exert their benefits through autophagy or other pathways. Autophagy is a complicated process, with multiple pathways involved in the signal transduction; poorly selective autophagy modulators may influence multiple targets. So far, understanding the relevance between autophagy and diseases is still limited by lack of specific and potent autophagy modulators. As a result, it is alluring and necessary to discover autophagy-selective modulators targeting the autophagy machinery proteins. Furthermore, selective autophagy modulators is expected to be applied to clinical treatment with maximal clinical benefits and minimal toxicity in the future. To date, several druggable targets that play significant roles in the autophagy process have attracted the attention of medicinal chemists, including two kinase complexes essential for the initiation of autophagy and Atg4 crucial for the formation of autophagosome membrane. Focusing on the discovery, structural modifications,



structure-activity relationship (SAR) and mechanisms of action of these modulators provides a lot of opportunities and challenges for small molecular design.


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Table 2 Several representative FDA-approved drugs that also target autophagy. Name

Molecular Mechanism

Cellular Mechanism

Disease

Ref.

Temozolomide

Inhibits Akt/mTOR pathway

Autophagy inducer

Gliomas

74a

Rapamycin (and rapalogs)

Inhibits mTOR

Autophagy inducer

Cancers

74b

Metformin

Activates AMPK

Autophagy inducer

Type 2 diabetes, Lymphoma

74c

Gefitinib

Activates AMPK

Autophagy inducer

Non-small-cell lung cancer

74d

Bortezomib

Activates AMPK

Autophagy inducer

pancreatic cancer

74e

Sodium phenylbutyrate

Improves cathepsins D and B activities and lysosomal-autophagic function

Autophagy inducer

Sporadic inclusion-body myositis

74f

Carbamazepine

Reduces inositol and Ins(1,4,5)P3 levels

Autophagy inducer

Anticonvulsant, liver diseases

74g

2+

Verapamil

Inhibits Ca channel, lowers intracytosolic Ca2+ levels

Autophagy inducer

Huntington’s disease

74h

Rilmenidine

Reduces cAMP levels

Autophagy inducer

Huntington’s disease

74i

Chloroquine and Hydroxychloroquine

Inhibits lysosome function

Autophagy inhibitor

Malaria, rheumatoid arthritis, lupus erythematosus

74j

Pantoprazole

Proton Pump Inhibitor, increased endosomal pH

Autophagy inhibitor

Solid tumors

74k

Celecoxib

Inhibits lysosome function

Autophagy inhibitor

Acute myeloid leukemia

74l



5.1 Non-specific autophagy modulators 5.1.1 Modulators targeting the PI3K/Akt/mTOR signaling pathway The PI3K/Akt/mTOR signaling pathway primarily affects diverse cellular functions, including proliferation, growth, differentiation, and survival, thus the pathway is densely interconnected with a multitude of important regulatory systems. The negative feedback loop of the PI3K/Akt/mTOR pathway is involved in the switching between activation of pro-survival effectors and induction of constitutive autophagy, suggesting beneficial outcomes can be achieved by modulating the pathway.76 To date, drugs targeting various levels of this pathway have been developed. The PI3K/Akt/mTOR pathway is one of the most frequently disrupted intracellular pathways in human cancer, where it significantly contributes to tumor progression and development of resistance to chemotherapeutic drugs. Small molecules targeting key proteins and simultaneous targeting multiple members within the pathway have shown promise in preclinical and clinical evaluation. 5.1.1.1 Dual PI3K/mTOR inhibitors Since both PI3K and mTOR belong to the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily of kinases, mTOR shares high sequence homology in the hinge-region with PI3K.77 Small molecule inhibitors blocking both PI3K and mTOR would have an advantage of shutting down the PI3K/Akt/mTOR pathway to modulate autophagy (Figure 2). However, it also should be noted that these inhibitors may lead to unwanted effects on different related kinases with similar structures. The first described PI3K/mTOR dual inhibitor PI-103 (1) was a morpholinoquinazoline


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derivative acting on both mTORC1 (IC50 = 20 nM) and mTORC2 (IC50 = 83 nM). It was identified after a high-throughput screening (HTS) aimed to identify p110α inhibitors.78 Compound 1 massively increased the level of LC3-II (LC3B-II) (~10 fold) in HeLa cells and perturbed autophagy flux. After compound 1 treatment, the clearance of p62 (one of the autophagy substrates) was impaired in HeLa cells and compound 1-induced decrease in p62 clearance was maintained at both 24 and 48 hours post drug treatment. Experiments also demonstrated that compound 1-induced cell death was independent of apoptosis and necroptosis; inhibition of autophagic flux might contribute to its-induced cell death.79 Unfortunately, this ATP-competitive inhibitor targeted the active sites of both holoenzymes; the specificity of it for PI3K could elicit off-target effects on related PIKKs, thus causing a higher toxicity profile. PI3K inhibitors with multiple-target abilities could be uncovered using a structure-based approach. Imidazoquinoline derivatives NVP-BGT226 (2) and NVP-BEZ235 (3) inhibited PI3K and mTOR kinase activity by binding to the ATP binding cleft of these enzymes. Especially, compound 2 showed no significant inhibitory activity against other tested kinases, which was available in phase I/II clinical trials for the treatment of advanced solid tumors; this compound represented a promising therapeutic approach in the treatment of hepatocellular carcinoma (HCC), since it was cytotoxic for HCC cell lines in normoxia and in hypoxia condition, meanwhile inhibiting the expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF).80 Combination with chloroquine (CQ, 29) and 2, an increased cytotoxicity in both SNU475 and Mahlavu cells could be detected.



Orally available compound 3 obviously suppressed the PI3K/Akt/mTOR proteins p-AKT, p-mTOR, and p-70S6K. In combination with autophagy inhibitors or autophagy gene knockdown, compound 3 enhanced growth inhibition and apoptosis in MCF-7 cells, providing a therapeutic strategy in breast cancer.81 PF-04691502 (4) was discovered through integration of structure-based drug design (SBDD) and physical properties-based optimization (PPBO). The ATP-competitive PI3K/mTOR dual inhibitor with a promising antitumor activity, which potently inhibited recombinant class I PI3K and mTOR in both biochemical and cell-based selectivity assays. Experiments showed that it induced cell apoptosis, DNA damage and autophagy in NSCLC cells. More interestingly, effects of compound 4 on toxicity and DNA damage were remarkably increased by co-treatment with an autophagy inhibitor.82 Activation of PI3K/Akt/mTOR pathway plays an important role in pancreatic cancer progression and chemo-resistance. PKI-587 (5), an intravenous (IV), ATP-competitive, highly selective and potent dual PI3K/mTOR kinase inhibitor containing the structure of morpholino-triazines, showed an IC50 of 0.4 nM for p110α, 6 nM for p110β, 6 nM for p110γ, 8 nM for p110δ, and 1 nM for mTOR, respectively.83 Recently, it has been demonstrated that the treatment with 5 was able to enhance sensitivity to cetuximab in head and neck squamous cell carcinomas (HNSCC) cells in vitro, and to revert constitutive or acquired resistance. The combination treatment induced autophagy by increase and decrease of the autophagy transducers Beclin 1 and p62, respectively.84 The novel class I PI3K/mTOR kinase inhibitor GDC-0980 (6) inhibited Akt/mTOR activation in pancreatic cancer cells


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(PANC-1 and Capan-1 lines) by blocking Akt-S6K1 phosphorylation. Experiments validated that inhibition of autophagy sensitized 6-induced anti-pancreatic cancer activity, suggesting a novel therapeutic strategy for 6 sensitization.85 Puquitinib mesylate (XC-302, 7) was a new molecular-targeted anticancer drug, which suppressed the activity of PI3K directly (subtype IA, IC50 against PI3K isoforms: p110α = 766.6 nM, p110β = 699.4 nM, p110δ = 2.8 nM and p110γ = 89.7 nM). Exposure to compound 7 exhibited a dose-dependent inhibition of cell viability (the IC50 of 72h was 5.2 µM). Moreover, the compound could induce autophagy in CNE-2 cells (poorly differentiated nasopharyngeal cancer cell line), promoting the program of cell death and inhibiting the PI3K/Akt/mTOR signaling pathway.86

Figure 2. Structure of dual PI3K/mTOR inhibitors.

5.1.1.2 Pan-PI3K inhibitors PI3Ks are divided into class I, class II and class III, and encompass eight isoforms. Besides the autophagy-related class III PI3K (Vps34), the class I PI3K can also modulate autophagy by triggering mTOR signaling pathway, while the



contribution of class II PI3K activity on autophagy is still unclear.87 A group of pan-PI3K inhibitors have been reported to affect autophagy (Figure 3). 3-Metheyladenine (8) is the first discovered and widely used autophagy inhibitor but it is effective only at high concentrations due to the poor solubility.88 Novel 3-MA derivatives have been described with improved solubility and more effective autophagy inhibition, more importantly, without inhibiting class I PI3K.89 Wortmannin (9) was a highly potent pan-PI3K inhibitor with IC50s from 10 to 50 nM for PI3K classes I, II and III.90 It achieved its inhibitory effect via covalent irreversible binding. However, it also inhibited other kinases with poor selectivity. The first synthetic inhibitor Ly294002 (10) inhibited autophagy on cellular assays and was a competitive inhibitor for the ATP binding site of PI3K, very similar to the inhibiting modes of the related protein kinases. The most crucial part of its structure was the morpholine ring which formed a key hydrogen bond with Val882. This kinase-ligand interaction plays a key role in the enzyme inhibition for drug discovery. Compound 10 contributed to understanding the importance of the PI3K pathway and indicated the therapeutic potential of small molecule inhibitors.91 However, the poor potency limited its further therapeutic application (IC50s at the µM level). Besides, compound 9 and 10 had off-target effects on different related lipid and protein kinases. Another inhibitor GSK-2126458 (11) developed for the class I PI3K and mTOR, was also highly active on Vps34 using biochemical assays and recombinant proteins. Unfortunately, no report was available about the regulation of autophagy by these compounds.92 These kinase inhibitors are potential for further study in the future.


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Figure 3. Chemical structures of Pan-PI3K inhibitors.

5.1.1.3 Pan-mTORC inhibitors mTORC1 and mTORC2 have been identified in many diseases. Activation of both mTORC1 and mTORC2 is important for several cancerous behaviors, including cancer cell growth, proliferation, survival, migration and apoptosis resistance.93 Small molecules with an ATP-competitive mechanism inhibiting both mTORC1 and mTORC2 offer an efficient alternative to rapalogs for the treatment of cancer (Figure 4). Based on protein-based screening assay and rational design, the first pan-mTOR inhibitor PP242 (12) and its analogue PP30 (13) containing the adenine-mimetic pyrazolopyrimidine scaffold have been generated, selectively inhibited both mTORC1 and mTORC2 with IC50 values of 8 nM and 80 nM without affecting other kinases.94 Importantly, compound 12 was a more effective mTORC1 inhibitor than Rapamycin (sirolimus, 21), at the molecular level. It inhibited cap-dependent translation under conditions, in which compound 21 had no effect. Torin 1 (14) and Torin 2 (15) were potent mTOR inhibitors. Compound 14 was identified by a biochemical screen for



inhibitors of mTOR kinase activity in a library of heterocyclic chemical compounds and inhibited both mTOR-containing complexes with IC50 values between 2 and 10 nM in vitro kinase assay.95 Now it is widely used as an autophagy inducer. Compound 15 was discovered through a systematic medicinal chemistry effort to improve the pharmacologic and solubility properties of compound 14. This compound inhibited mTORC1-dependent Thr389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM and approximately 800-fold selectivity for cellular mTOR versus PI3K. However, it also exhibited potent biochemical and cellular activity against PIKK family kinases including ataxia-telangiectasia, mutated (ATM, EC50 = 28 nM), ATM and Rad3-related (ATR, EC50 = 35 nM) and DNA-dependent protein kinase (DNA-PK, EC50 = 118 nM).96 Other ATP-competitive inhibitors such as Ku-0063794 (16),97 AZD8055 (17),98 AZD2014 (18)99 and WYE-354 (19)100 have been reported recently. Compound 16 inhibited both mTORC1 and mTORC2 with an IC50 of ~ 10 nM and induced cytoprotective autophagy activation in HepG2 cells. Studies indicated that 16 inhibited HepG2 cell growth in vitro and in vivo, whose activity could be enhanced with autophagy inhibitors.101 The clinical candidates 17 and 18, derivatives of 16, inhibited mTOR kinase with IC50s of 0.8 and 2.8 nM. These two compounds showed excellent selectivity against all class I PI3K isoforms and other members of the PI3K-like kinase family. Compound 17 inhibited the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate Akt and downstream proteins. In H838 and A549 cells, it also induced the formation of acidic vesicles, consistent with a process of autophagy.


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Compound 18 showed consistent exposure in rodents and a low turnover in human hepatocyte incubations. Compound 19 was also a selective mTOR inhibitor with a pyrazolopyrimidine scaffold. It induced autophagy in the colon cancer cells. These results suggest that inhibiting both mTORC1 and mTORC2 may be sufficient for achieving a broad and robust anticancer effect. To discovery novel scaffold mTOR inhibitors, a ligand docking method was used to screen 60,000 compounds from the Natural Product Database, which led to selection 3HOI-BA-01 (20) for further study of antitumor activity in vitro and in vivo. According to the docking results, 20 formed a good interaction with the mTOR inside the ATP-binding pocket. 4 hydrogen bonds were formed with the backbone atoms of the hinge residue Val2240, the side chain atoms of Lys2187, Tyr2225, and Asp2357, respectively. In addition, phenyl ring formed strong π–π stacking interaction with the Tyr2225.102 Compound 20 bound directly to mTOR protein, resulting in biological responses in vivo. Autophagy has been

demonstrated

to

protect

cardiomyocytes

from

ischemia/reperfusion

(I/R)-induced damage. Recent research illustrated the cardioprotective effects of 20 and an AMPK agonist PT1 (27) after I/R injury exposure through enhanced autophagy.103 Both 20 and 27 induced autophagy in cardiomyocytes through inhibiting mTOR signaling and activating AMPK pathway. Furthermore, simultaneous administration of these two compounds profoundly upregulated autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) and significantly promoted survival of cardiomyocytes.



Figure 4. Chemical structures of pan-mTORC inhibitors.

5.1.1.4 mTORC1 inhibitors mTORC1 integrates signals from growth factors, oxygen levels, amino acid, energy status, promotes protein synthesis and participates in other anabolic processes involved in cell growth and metabolism.104 Furthermore, mTORC1 activity is regulated by nutrient cues and can be inhibited by 21, whereas TORC2 is resistant to 21 treatment and its regulation is not well understood.105 These researches make mTORC1 an attractive target to design novel inhibitors. mTORC1 is a target for a natural product, compound 21 and its analogs (rapalogs, Figure 5).74b Compound 21 binds the peptidylprolyl cis-trans isomerase FK506 binding protein 12 (FKBP12) and the complex binds to the FKBP12-rapamycin binding (FRB) domain of mTOR, resulting in mTOR allosteric inhibition, thereby, prevent further phosphorylation of P70S6K, 4E-BP1 even other proteins involved in transcription, translation and cell cycle control, indirectly.106 Compared with pan-mTOR inhibitors, Temsirolimus (CCI-779) and Everolimus (RAD001) are generally cytostatic with less toxicity.107 Therefore, in the treatment of neurodegenerative or metabolic diseases, rapalogs are


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probably more desirable, as they have fewer side effects. Recently, two N-(1-Benzyl-3,5-dimethyl-1H-pyrazol-4-yl) benzamides 22 and 23 showed submicromolar antiproliferative activity and good metabolic stability (Figure 5). Both 22 and 23 disrupted autophagy flux by inhibiting mTORC1 under basal conditions and interfering with the reactivation of mTORC1 under reefed conditions. SAR studies of this class of compounds unclosed the process of discovery the potent and selective mTORC1 inhibitors. The A ring and the D ring were the most important moieties of these compounds. Separately, an amide at the A ring provided an opportunity to serve as both a hydrogen bond donor and a hydrogen bond accepter. Notably, the meta position might be more amenable to structural modifications. At the D ring, incorporation of an aromatic ring showed enhanced anticancer activity. Moreover, the electron-donating group methyl at the ortho or para position enhanced activity. Remarkably, the trifluoromethyl group was preferred substituent at the para position. Although compound 22 and 23 showed submicromolar EC50 values of 0.8 µM and 0.62 µM, respectively, these two compounds represent a new class of autophagy modulators with potent anticancer activity and potentially a novel mechanism of action.108



Figure 5. Chemical structures of mTORC1 inhibitors.

5.1.2 AMPK activators AMPK is a serine/threonine protein kinase that serves as a pleotropic regulator of the whole body energy homoeostasis by down-regulating energy consuming pathways. The role of AMPK in autophagy is complex and highly dependent on both cell type and metabolic conditions.109 Metformin (24, Figure 6), a well-known antidiabetic drug, is one of the AMPK activators widely used as autophagy inducer, also endowed with antineoplastic property.74c Phosphorylation of AMPK through 24 regulated downstream signaling pathways in the neurons during oxidative stress and neurodegeneration.110 However, recent results suggested that 24 inhibited mTOR signaling pathway by processes that did not depend on AMPK.111 A-769662 (25, Figure 6) was a screened and optimized AMPK activator (EC50 = 0.8 µM). It stimulated AMPK via allosteric activation of AMPKα at Thr172 and stabilized the active conformation of AMPK through allosterically binding to the γ-subunit. Compound 25 exerted its cellular effects not as


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a consequence of cellular stress, toxicity, or by altering the AMP/ATP ratio increased.112 A recent literature demonstrated that AMPK was a valuable pro-survival target at least in osteoblasts.113 H2O2 induced AMPK-dependent and cytoprotective autophagy in cultured osteoblasts, compound 25 might be further investigated as a novel anti-osteonecrosis agent by inducing pro-survival effect against H2O2.114 Another specific AMPK agonist GSK621 (26, Figure 6) also activated cytoprotective autophagy in the osteoblasts and ameliorated H2O2-induced osteoblast cell injuries.115 It was previously demonstrated that the specific AMPK agonist 26-induced cytotoxicity by activating autophagy and 26-induced AMPK activation suppressed mTORC1 activity, which could be observed in HEK293 cells. Unexpectedly, a recent study showed that the cytotoxicity induced by 26 indeed required mTORC1 activation that was unique to acute myelocytic leukemia (AML) cells; the eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway was also involved in the synthetic lethality in AML.116 AMPK is a heterotrimer consisting of a catalytic α subunit and two noncatalytic subunits. PT1 (27, Figure 6) was discovered by screening a chemical library (EC50 = 0.3 µM). It interacted with Glu96 and Lys156 residues near the auto-inhibiting domain (AID) in α1 subunit and directly relieved the autoinhibition.117 Recent study elucidated that 27 enhanced AMPK signaling and subsequently augmented autophagy in cardiomyocytes with a protective efficacy on cardiomyocytes after OGD/R in vitro.103 Orally bioavailable AMPK activator OSU-53 (28), directly activated AMPK (EC50 = 0.3 µM) independently of its upstream kinase liver kinase B1 (LKB1). According to the molecular docking based on homology



modeling, it bound to AID through interactions with His152, Lys156, and Tyr277.118 Compound 28 exhibited both in vitro and in vivo antitumor activity against triple negative breast cancer (TNBC) cell lines and in MDA-MB-231 tumor-bearing mice. Daily oral administration of 28 suppressed tumor growth and modulated relevant intratumoral biomarkers of drug activity. However, this compound also induced protective autophagy that attenuated its antiproliferative potency. A recent study demonstrated that 28 also directly inhibited mTOR activity with consequent suppression of mTOR/p70S6K signaling.119 A lot of potent AMPK activators have been reported these years. Here, we only illuminated some representative compounds confirmed to influence autophagy pathways. Additional in-depth studies are required to determine the precise underlying mechanism whether autophagy is involved in the diseases treated by AMPK activators.

Figure 6. Chemical structures of AMPK activators.

5.1.3 Lysosomotropic agents


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Lysosome is a highly membrane-bound organelle which is involved in a sequence of biological functions including protein degradation, cell signal transduction and metabolic homeostasis.120 Lysosomal dysfunctions associate with various autophagy-related diseases such as AD.121 Foci on critical functions of lysosomes may provide cancer therapeutic strategies. However, the ways in which lysosomes cause tumorigenesis and cancer progression are intricate.120 Recent compelling evidences have indicated that lysosomes play a critical role in nutrient sensing and signaling pathways involved in cell metabolism and growth. For instance, the functional activation of lysosome is achieved via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion; suppression of mTORC1 activity by starvation or mTOR catalytic inhibitors leads to activation of lysosomal function.122 Surprisingly, the lysosome not only is required for autophagic degradation but also affects autophagy activation through the Rag/RRAG GTPase pathway. Therefore, lysosome inhibitors own a dual effect in suppressing autophagy degradation and in initiating autophagy.123 The characteristic of lysosome is its highly acidic environment (pH 4.5-5.0) which provides optimal settings for lysosomal hydrolytic enzymes to perform their catalytic function and digest the luminal contents. Lipophilic or amphiphilic lysosomotropic agents cross the lysosomal membrane and then are protonated within the acidic vesicles. They conceivably inhibit the degradation of autophagolysosome by increasing the pH of lysosomes, thereby impairing lysosomal acid hydrolases. Additionally, they indirectly inhibit autophagy, and subsequently prevent cellular



degradation.124 Antimalarial 29 and its derivative hydroxychloroquine (HCQ, 30, Figure 7) are well-known lysosomotropic agents often used as pharmacological tools to evaluate the response to autophagy inhibition. The high concentrations (µM range) of these two compounds required to inhibit autophagy are not consistently achievable in the clinic. As a consequence, a substantial number of active studies have shown the potential of compound 31 and 30 as an adjuvant therapy in various tumors, contributing to the initiation of several clinical trials.74j However, the anticancer effects of 29 might be independent on autophagy inhibition. For instance, in Kirsten rat sarcoma (KRAS) mutant tumors, compound 29 exerted equal anticancer effects in both Atg7-deficient and -proficient cells.125 Recent studies also provided insight into the multifactorial autophagy-independent activities of compound 29, which were related to antineoplastic effects of this compound. Compound 29 reduced tumor growth and restrained tumor invasion and metastasis by autophagy-independent vessel normalization.126 In addition, synergy between mTOR pathway inhibition and 29 caused autophagy-independent cell death in fibroblast growth factor receptor 3 (FGFR3)-mutant cell lines through a suppression of cholesterol metabolism, which proposed a novel approach to future bladder cancer treatment.127 To develop more potent inhibitors of autophagy, dimeric 29 analogs were synthesized. The bisaminoquinoline compound Lys01 (31, Figure 7) was identified as a viable lead compound for development and an autophagy inhibitor for further anticancer therapy. Compound 31 was a >10-fold more potent autophagy inhibitor


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than 29 or 30 at a concentration of 10 µM in LN229 (glioblastoma) cells. Biological studies indicated that the bivalent quinolone structure was critical to enhance the activity. In addition, both the 7-chloro substituent and the bisaminoethyl-methylamine were important. The trihydrochloride salt of 31, named Lys05, was synthesized with enhanced aqueous solubility for in vivo studies. The compound not only blocked autophagy in vitro, but also produced cytotoxicity in multiple cancer cell lines with a ten-fold greater potency than 30. Significantly, Lys05 had single-agent antitumor activity at a lower dose in mice without toxicity.128,129 The combination of Lys05 with v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor was shown to have noticeable activity in vivo.130 The quinoline antimalarial mefloquine (MFQ, 32)131 and quinacrine (QN, 33)132 were also demonstrated to trigger ER stress and exerted cytotoxic effects in tumor cells through blocking autophagosome degradation. Notably, compound 33 contained greater autophagy inhibition properties than 29. Therefore, rational drug design efforts were subsequently centered on the acridine scaffold of 33. Recently, a series of novel acridine and 1,2,3,4-tetrahydroacridine derivatives were identified to inhibit autophagy more potently than 29. SAR studies of the acridine ring led to discovery potent autophagy inhibitors.133 The side chain optimization obviously improved the autophagy inhibition. Compared with compound 34, compounds with short and structurally rigid side chains had improved metabolic instability, high hydrophobicity, and inferior binding affinity. Besides, incorporating methylpiperazine to terminal amino group improved the



inhibitory activity. Further manipulation of the core substitution demonstrated that small groups other than H were required at both R2 and R3 to keep the toxicity under control, compound 37 without substituent at R2 showed enhanced toxicity compared with 36. Apparently, the C ring was critical for the autophagy inhibition and also responsible for the increased toxicity. 1,2,3,4-tetrahydroacridine with a saturated C ring proved effective to attenuate the cytotoxicity. For example, the lethal dose (LD50) of compound 38 and 39 were improved to 27 µM, more than their effective concentrations. The twist-chair conformational change of 38 and 39 could be the reason for their diminishing toxic effects. In addition, furnishing with smaller and more hydrophilic side chain groups improved biophysical properties compared to 33. Based on previous studies, compound 35 and 39 might be used as anticancer agents for adjuvant therapy for their higher potency of autography inhibition than 29. In depth studies on these two potent compounds suggested that both of them acted as autophagy inhibitors with differential effects on cell viability. In addition, these two compounds had the potential to sensitize mutant BRAF melanomas to first-line therapies.134 The druggable circadian nuclear receptor reverse orientation c-erbA gene, variant β (REV-ERBβ) plays an unexpected role in supporting cancer cell viability when autophagy flux is compromised. Accordingly, chemical or genetic inhibition of REV-ERBβ significantly sensitized cancer cells to cytotoxicity induced by 29. Therefore, inhibition of both autophagy and REV-ERBβ revealed potential avenues for the development of new multifunctional agents inducing cytotoxicity in cancer


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cells. ARN5187 (40, Figure 8) was identified from an in silico screen of a diverse and nonredundant small molecules interacting with REV-ERβ with ClogP > 3 and pKa > 7.5. Both of 29 and 40 shared similar lysosomotropic potency and had similar effects on autophagy inhibition in BT-474 cells. Remarkably, compound 40 was significantly more cytotoxic (EC50 values for cytotoxic responses of 29 and 40 were > 1000 µM and 23.5 ± 7.3, respectively).135 Compound 40 provided a novel scaffold for discovering dual inhibitors of REV-ERBβ and autophagy (Figure 8).136 Compound 41 had similar cytotoxic effects to BT-474 cells without cytotoxicity to normal human mammary epithelial cells (HMEC). Remarkably, introduction of the fluorine atom at the meta or para position of the B phenyl ring appeared beneficial for in vitro anticancer activity. Compound 42 significantly increased cytotoxicity in BT-474 without affecting the viability of HMEC. The N-methylpiperazine moiety and the N-methylpiperidine moiety had a good effect on increasing cytotoxicity. On the basis of the structures of 42 and 43, compound 44, the most potent inhibitor was produced. Compound 42, 43 and 44 improved cytotoxicity in BT-474 cell line, related to their enhanced REV-ERBβ inhibitory activity. Indeed, compound 44 was at least 1 order of magnitude more potent than 40 in REV-ERBβ inhibition (IC50 = 1.34 ± 1.18 µM versus IC50 = 17.50 ± 1.08 µM, respectively). It has been verified that compound 44 inhibited both REV-ERB-mediated transcriptional repression and autophagy in SK-BR-3, HEP-G2, and LNCaP cells. Multifunctional inhibitor 40 and its analogs provided an opportunity for development of new anticancer drugs. Further in vivo studies are urgently required for fully evaluation the potential of these compounds.



Figure 7. Chemical structures of quinoline-based lysosome inhibitors.

Figure 8. Chemical structures of dual inhibitors of REV-ERBβ and autophagy.

5.2 Specific autophagy modulators 5.2.1 ULK1 modulators ULK1 was first identified as a key regulator of autophagy via a kinome si-RNA screen.137 As one of the Atg1 homologues, ULK1 consists of an N-terminal serine/threonine protein kinase domain, proline/serine (P/S)-rich domain which


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contains numerous regulatory phosphorylation sites, and a C-terminal interacting domain composed of two microtubule-interacting and transport (MIT) domains, while the ULK1 orthologs do not have the conserved C-terminal sequence and are not thought to be involved in starvation-induced autophagy.138 Notably, between the N and C terminal lobes, a large, positively charged activation loop extends out from the kinase domain from the active sites and stimulates ULK1 activity.139 Recent work has begun to elucidate the function of these domains and their roles of ULK1 in autophagy.140 Serving as the most indispensable autophagy-initiating kinase, ULK1 is regulated by upstream nutrient and energy sensing kinases, and transmits these signals to the core autophagic machinery.31 Aberrant expression of ULK1 is associated with diseases such as poor prognosis in breast cancer and esophageal squamous cell carcinoma.141 ULK1 also represents a potential novel prognostic biomarker for HCC, due to its high expression level.142 ULK1 expression is predominantly localized in hypoxic tumor areas and that high enrichment is associated with a high hypoxic fraction.143 Intriguingly, ULK1 can both suppress and promote tumor growth under different conditions, indicating a novel target for cancer diagnose or therapy.144 Moreover, ULK1 acts as a key mediator of type I interferon receptor (IFNR)-generated signals that control gene transcription and induction of antineoplastic responses, thus ULK1 plays a central role in IFN-dependent immunity.145

Another

study

demonstrated

that

ULK1

contributes

to

the

1-methyl-4-phenylpyridinium ion (MPP+)-induced MN9D death by blocking S6k1 (a major substrate of mTORC1) phosphorylation, providing a new therapeutic strategy



Page 44 of 130

for PD treatment.146 However, recent study identifies that ULK1 can regulate glucose metabolism during nutritional stresses via an autophagy-independent manner. Despite the fundamental role of ULK1 in autophagy regulation, this discovery indicates that ULK1

specific

modulators

may

eventually

show

clinical

relevant

autophagy-independent activities.147 5.2.1.1 ULK1 inhibitors Although ULK1 has been reported as a specific initial kinase of autophagy many years ago, development of its modulators attracts attentions recently. The development of a novel high throughput screening (HTS)-compatible biochemical assay to identify small molecule inhibitors of ULK1 could be used to screen large chemical libraries to discover novel inhibitors; 39 hits (hit rate of 3%) were identified, many of which were known protein kinase inhibitors.148 Another approach to develop ULK1 inhibitors is target-based reverse pharmacology approach. Starting with a FAK inhibitor that showed potent cross-reactivity toward ULK1, the novel ULK1 inhibitor SBI-0206965 (45, Figure 9) was identified by screening a focused library of pyrimidine analogs. Dose-response analysis of 45 revealed an in vitro IC50 of 108 nM for ULK1 kinase activity and 711 nM for the highly related kinase ULK2.149 Notably, rigorous assay confirmed that 45 appeared quite exquisite selectivity for ULK1 in vitro and in vivo without impairing endogenous FAK, AMPK, mTOR, Akt, or extracellular regulated protein kinases (Erk) signaling at 10 µM. Experiments also showed that 45 synergized with starvation and mTOR inhibition to enhance apoptosis in A549 human lung cancer cells, the


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induction of apoptosis cells was dose-dependent. Compound 45 converted the cytostatic response to catalytic mTOR inhibitors into a cytotoxic response due to loss of autophagic maintenance of cell survival. Consequently, ULK1 inhibition may provide an exciting new modality to avoid therapeutic resistance in currently treated with mTOR inhibitors. Compound 45 with high selectivity is a suitable tool to study cell-based mechanism as an autophagy modulator. However, it should be noticed that many tyrosine kinases, such as FAK, Src, Abl, and Jak3, are also affected by 45. Especially, the kinase activity of FAK is significantly inhibited at a nanomolar range. Further efforts are required to explore the interaction between 45 and ULK1, thereby designing highly selective inhibitors against ULK1.

Figure 9. Chemical structure of SBI-0206965.

Shokat et al. first disclosed the structure of ULK1. Two high-resolution crystal structures of the kinase bound to potent inhibitors were further presented in recent years, which facilitates structure-based design of potent and selective ULK1 inhibitors.139 To date, two classes of ULK1 inhibitors have been identified. Unfortunately, most of them display broad inhibitory activity against a panel of many other kinases. Therefore, after the initial reports, these compounds have never been further investigated. Encouragingly, these compounds and their binding modes enrich



our understanding of the structure of kinase domain, resulting in further modification of current potential compounds and development of novel scaffold candidates through a structure-based approach. Compound 46 was selected by screening a collection of 764 compounds against ULK1, with an IC50 of 160 nM inhibiting ULK1. The crystal structure provides important insight of into the binding mode of 46 and ULK1 (Figure 12). Several key structural elements that engaged in the intermolecular interactions are summarized as follows: (1) the heterocyclic moiety amino pyrazole core makes contacts with the ATP binding site participating in a hydrogen bonding network with residues in the hinge region; (2) the cyclopropyl substituent fits into a pocket adjacent to the gatekeeper methionine; (3) the aniline forms a contact with Asp165 of the DFG motif; (4) the quinazoline is hemmed in by the backbone of the kinase and a number of side chains from the crystallographic symmetry-mate. Guided by the crystal structure, a series of pyrazole aminoquinazoline derivatives were synthesized (Figure 10). According to SAR, the quinazoline and amino pyrazole core were maintained because they played a significant role for the inhibitory effect of these compounds. The cyclobutyl ring at the R1 position could maximally take advantage of methionine gatekeeper residue. At the R2 position, 2-amino benzimidazole (47) maintained the potent inhibition of the enzyme while 2-methyl benzimidazole (48) showed no inhibition. Compound 49 and 50 were positional isomers with different ability to inhibit ULK1. Compound 49 containing a benzimidazole structure with an improved IC50 of 8 nM induced conformational


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changes within the kinase domain. The bulkier benzimidazole changed the position of the side chain of Asp165 in the DFG and fitted into the pocket; meanwhile, this core also formed a hydrogen bond with Gln142. Compared with aniline, benzimidazole made strong interaction with the N and C lobes, causing the C-terminal lobe to move closer to the N-terminal lobe. In addition, compound 51 made a potential hydrogen bond with Lys46 through a water molecule. Unfortunately, compound 51 showed no specificity toward ULK1 when profiled in a small panel of protein kinases. The poor selectivity of 51 limited its further usage in cellular assays.

Figure 10. Representative ULK1 inhibitors of pyrazole aminoquinazolines.



Figure 11. Representative ULK1 inhibitors of aminopyrimidine scaffold.

In an attempt to improve the selectivity for ULK1, compounds with aminopyrimidine scaffold were explored. BX-795 (52), with an iodine substituent on the pyrimidine ring, had good potency against ULK1 and showed good selectivity for a few kinases. Further modification by replacing the substituents of the extended diaminopropyl linker, these compounds showed good potency against ULK1 and selectively inhibited autophagy in Hela cells. Compound 53 was more potent than 52. Notably, compound 54 with a less bulky diaminopropyl substituent showed dramatically reduced potency toward 3-phosphoinositide-dependent protein kinase-1 (PDK1) and good selectivity throughout the kinome, though it was not a quite potent ULK1 inhibitor. Compared with previous reported structures, these compounds also bound to the active site of ULK1. The aminopyrimidine core made hinge contacts with the ATP-binding site. A flexible orientation of Ile 22 was required to pack above the aminopropyl group. The pyrrolidine urea moiety provided selectivity to the scaffold, due to taking advantage of unoccupied space in the pocket. Besides, the iodine occupied a similar space with the cyclobutyl substituent of 51; the gatekeeper methionine moved toward the iodine presumably to adopt a favorable dipole-dipole interaction. (Figure 12). MRT67307 (55) and MRT68921 (56) were synthesized as the TANK-binding kinase 1 (TBK1) inhibitors.150 Ganley et al. investigated effects of 55 on kinases with similar catalytic domain; this led to the identification that 55 potently inhibited ULK1 and ULK2 with IC50 of 45 and 38 nM, respectively. Compound 56


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was a more potent inhibitor displaying a ten-fold increased affinity on ULK1 and ULK2 (IC50 of 2.9 and 1.1 nM, respectively).31 Disappointingly, compound 56 inhibited 44 kinases in a panel of 80 protein kinases. Despite of this, 56 acts as a proof-of-principle in developing small molecule inhibitors for the treatment of autophagy related diseases.

Figure 12. Structure of 46 (A, PDB code 4WNO), 51 (B, PDB code 4WNP) and 54 (C, PDB code 5CI7) with ULK1 kinase domain. The ligands are shown in stick model colored by C in blue, N in mazarine, O in red and I in modena. The green dash line indicates a conventional hydrogen bond, the purple dash line indicates alkyl interaction.

5.2.1.2 ULK1 agonist Considering the dual role of autophagy and the important position of ULK1 among an autophagic protein-protein interaction (PPI) network, the design of ULK1 modulator should depend on the expression in the diseases. Recently, a proof-of-principle was applied in discovery ULK1 agonist, aiming to provide a novel therapeutic approach in the treatment of TNBC.151 mAtg13 and FIP200 are required for targeting the ULK1 complex to sites of autophagosome formation and proper



localization, stability and kinase activity of ULK1; they bound to hydrophobic pockets of ULK1, thereby switching the conformation of kinase domain to the on-state.152 Liu et al. put forward an unprecedented approach to design ULK1 agonists, aiming to “lock in” the interaction among ULK1, mAtg13 and FIP200. They selected the nearest pocket of the interaction site and integrated the in silico screening, chemical synthesis, structure modifications and SAR analyses to acquire a series of ULK1 agonists (Figure 13). The leading compound DB01127 was selected to first structural modification, leading to UA1-03 with the best activity. Further structural modifications identified the small molecule LYN-1604 (57) with the best potency. It was the first reported ULK1 agonist designed based upon the X-ray structure of ULK1 with an EC50 of 18.94 nM. Compound 57 formed hydrogen bonds with Lys50 and hydrophobic interaction with Leu53 and Tyr89. On the basis of molecular modeling, the nitrogen atom of the piperazine ring interacted with Lys50 and the naphthyl-based hydrophobic ligand was a functional group for biological potency. Notably, the existence of methylene nearby the carbanyl group as well as the bulky N-substitution played key roles for biological potency. To further explore the autophagic mechanisms induced by 57, a series of biochemical assays indicated that compound 57 induced cell death associated with autophagy and accompanied apoptosis. Conceivably, compound 57 had a good therapeutic potential on TNBC in

vivo, which emphasized the importance of the combination of multidisciplinary in discovering novel potential ULK1 modulators in the future.


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Figure 13. Chemical structure of ULK1 agonist.

5.2.2 Vps34 modulators Vps34 is a member of the PI3K family of lipid kinases, which is divided based on structures and substrate specificities. It is presented in multiple complexes and involves in a variety of cellular functions such as phagocytosis, endocytic traffic and autophagy. Vps34 complex I containing Vps34, Vps15, Beclin 1, and Atg14L functions in autophagy. Vps34 complex II takes part in endocytic sorting (as well as autophagy and cytokinesis in mammalian cells) and contains the same subunits as complex I, except that it has UVRAG instead of Atg14L.153 Vps34 is required for the induction of autophagy during nutrient deprivation, implicated in the regulation of autophagy through the mTOR pathway.154 In addition, Beclin 1 can be phosphorylated by AMPK; simultaneously, Atg14L increases the phosphorylation by AMPK leading to autophagy.33 Recently, it has been identified that nuclear receptor-binding factor 2 (NRBF2) also participates in the induction of starvation-induced autophagy as a specific new member of Vps34 Complex I.155 Accumulating evidences show that inhibition of Vps34 complex successfully inhibits autophagy, thus the complex is an attractive and significant target for developing autophagy inhibitors.33 HTS assay measuring Atg14L and UVRAG levels on the reconstitute pro-autophagy Vps34



complex demonstrated the feasibility of their in vitro assay, providing a reliable tool for the screening of autophagy-specific inhibitors.156 Allosterically impairing Vps34 activity by modulating the PPI of Vps34 complex, such as Beclin 1-Vps34 interaction, is also an effective way to design potent autophagy-specific inhibitors.157 Spautin-1 (58, Figure 14) was a potent and specific small molecule inhibitor of autophagy targeting the Beclin 1 subunit of Vps34 complexes with an effective IC50 of 0.74 µM. This compound did not affect the catalytic activity of Vps34, but promoted the degradation of Vps34 complexes by inhibiting two ubiquitin-specific peptidases (USPs), such as USP10 and USP13.158 Moreover, compound 58 promoted cancer cell death under nutrient deprivation, when autophagy was activated, but not in nutrient-rich conditions. A recent study showed that compound 58 exhibited synergy with imatinib. Co-treatment of 58 and imatinib enhanced cell apoptosis by inhibiting imatinib-induced autophagy, which indicated a potential therapeutic application in chronic myeloid leukemia (CML).159 Compound 58 could also effectively inhibit autophagy flux and reverse the increased level of cytosolic calcium, which ameliorated the pathogenesis of acute pancreatitis.160

Figure 14. Compound targeting Beclin 1-Vps34 complex.

Besides indirectly targeting Vps34 complex activity via modulating the Beclin


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1-Vps34 complex stability and architecture, directly targeting Vps34 catalytic site has attracted more and more attentions. Development of potent and highly selective small molecular modulators of Vps34 is at the forefront of much chemicobiological research. Common to the catalytic subunits of other PI3Ks, Vps34 is composed of an N-terminal C2 domain (residues 1-255), hereafter referred to as helical and catalytic domain (HELCAT), a middle helical domain (residues 293-530), and a C-terminal catalytic kinase domain (residues 533-887) which is required for lipid kinase activity.154 Consequently, some reported pan-PI3K inhibitors also inhibited Vps34 activity, such as 8, 9 and 10. Considering the multiple functions of other PI3Ks, the design of compounds that specifically target Vps34 is of great importance. In recent years, several co-crystal structures have been reported to resolve the Vps34 kinase domain with different types of specific inhibitors (Table 3). These excellent researches facilitate the rational design of selective Vps34 inhibitors.161 Compared with other PI3Ks, the striking features of the Vps34 structure can be briefly summarized: (1) the phosphoinositide-recognition loop which is crucial for the recognizing phosphatidylinositol (PtdIns) and substrate binding is completely ordered, while the loop is largely disordered in other PI3K structures. (2) The ATP-binding site of Vps34 is relatively narrow, hydrophobic compared with other PI3Ks and the active site can best accommodate co-planar aromatic groups. Hence, it is unique in regions critical to inhibitor binding, leading to successful structure-based design of high affinity and highly specific compounds targeting the Vps34. (3) The P-loop curls inward toward the ATP binding pocket. (4) The hinge region between the N- and



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C-lobes is shorter in Vps34 compared with class I PI3Ks, therefore lacking the bulged-out space at the adenine-binding pocket hinge, which is characteristic of class I PI3Ks.162

Table 3. Crystal structures of Vps34-ligand complexes in this review. PDB code

Vps34-ligand complex

reference

4PH4

60

164

5ENN

61

165

4UWF

65

166

5ANL

66

166

4UWH

67

166

4UWL

68

166

4OYS

69

167

Recently reported Vps34 inhibitors can be divided into bis-aminopyrimidine derivatives (Figure 15) and pyrimidinone derivatives (Figure 17), both of which were designed based on the Vps34 structure. The bis-aminopyrimidine compound VPS34-IN1 (59) was discovered to be the first highly selective cell permeable Vps34 inhibitor (IC50 of 25 nM in vitro) and inhibited phosphorylation of PtdIns. This compound did not significantly inhibit the activity of other protein kinases or lipid kinases. While it appeared to be a potent and selective inhibitor; no information was disclosed regarding its in vitro ADME properties and in vivo target engagement.


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Compound 59 could be a useful probe to delineate physiological roles of Vps34. Increasing evidences have shown that a significant number of human tumors that are insensitive to class I PI3K inhibition display elevated levels of serum- and glucocorticoid regulated protein kinase 3 (SGK3). Therefore, SGK3 plays an important role in driving cancer cell survival and proliferation. Treatment with 59 markedly reduced SGK3 activity and phosphorylation. The ability of 59 to suppress SGK3 was dependent on its ability to bind PtdIns3P.163 Another potent and selective Vps34 inhibitor PIK-III (60, IC50 of 0.018 µM) was discovered after medicinal chemistry optimization of a hit screened by the HTS. Biochemical profiling demonstrated that compound 60 was at least 100-fold-selective for Vps34 over related lipid kinases such as PI3Kα, the protein kinase mTOR as well as an additional 44 protein kinases. This compound acutely inhibited de novo lipidation of LC3, leading to the stabilization of autophagy substrates.164 Compound 60 was a highly selective inhibitor of Vps34 catalytic function. The co-structure of human Vps34 in complex with 60 provided an important insight into binding mode (Figure 16), suggesting the ATP-binding site was crucial for selectively targeting Vps34. In detail, the cyclopropyl of 60 could extend in a prominent hydrophobic pocket containing Phe612, Pro618 and Phe684 then interacting with the hinge. This pocket in Vps34 was closer in proximity to the hinge region compared with PI3Kα. Intriguingly, the critical Phe612 was replaced in PI3K with a methionine residue that did not accommodate the cyclopropyl group. Novel bis-aminopyrimidine 61 as a hit compound was identified in a HTS as a



potent inhibitor of Vps34 (IC50 of 110 nM). The binding mode of 61 in the ATP-binding site of the homology model of Vps34 was used as a guide for medicinal efforts. According to the supposed binding mode, chemistry optimization efforts initially centered on replacement of the cyclohexanol portion with a pyridine ring, leading to 62 (IC50 of 37 nM) with enhanced potency and a similar lipid kinase selectivity profile to 61. Compared with other analogues, the incremental enhancement in potency may be due to a possible π-stacking interaction between the aromatic ring and Phe684 as well as the introduction of heteroatoms. The N-demethylation and cyclopropyl oxidation were major pathways of metabolism. Hit compound 61 showed undesirable metabolic stability in mouse (in vitro clearance (CL) = 87 mL min-1 kg-1). In order to improve the metabolic stability for subsequent in vivo studies, further modification concentrated on the N-substituent methyl, compound 63 (IC50 of 15 nM) was then discovered. Incorporation of a hydroxyl group contributed to an additional enhancement in both potency and metabolic stability. Notably, the X-ray co-crystal structure of Vps34 with 63 confirmed the predicted binding mode and provided support for the high selectivity observed (Figure 18). The highly potent and selective inhibitor exhibited nanomolar potency biochemically and in cellular assays, was especially capable of inhibiting autophagy. Compound 63 was an excellent candidate for in vivo pharmacokinetics (PK) evaluation and constituted the first example of a Vps34 inhibitor shown to inhibit autophagy in vivo.165


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H N

N

N

N

N

N

HN

Cl

N

HN

N

N

59 (VPS34-IN1) IC50 = 25 nM

60 (PIK-III) IC50 = 0.018 µ M

interact with the DFG motif

H N

N

H N

N

N

N hinge binder

N

sit in the solvent exposed area of the pocket

OH

N N

HN

HN

H N

N

N

N N

NH2

N

OH

N

N

HN N

N

OH

61 IC 50 = 110 nM

62 IC50 = 37 nM

63 IC 50 = 15 nM

Figure 15. Representative Vps34 inhibitors of bis-aminopyrimidine scaffold.

Figure 16. Structure of Vps34 kinase bound to a small-molecule inhibitor. (A) Overall structure of Vps34 and PIK-III (PDB code 4PH4). (B) Concise docking model of PIK-III with Vps34. Dotted lines indicate hydrogen bonds in green and π-π stacked in pink.

A series of tetra-hydro pyrimido-pyrimidinone derivatives were identified with a



cell-based high-throughput phenotypic screening campaign.166 Compared with the bis-aminopyrimidine derivatives above, these compounds contained a pyrimidinone moiety with a morpholine group as hinge binder; compounds interacted with the hinge region of Vps34 via the oxygen atom of the morpholine moiety, the moiety also made favorable van der Waals interactions with surrounding residues. However, these compounds had cross reactivity against class I PI3Ks. Starting with the hit compound 64, structure-based chemical exploration was based on the binding mode of suitable chemical probes with high Vps34 enzymatic potency compounds (65, 66 and 67). Based on the SAR, the N-substituent pointed toward the entrance of the binding site constituted to improve in vitro ADME properties. Increasing the polarity of the N side chain improved solubility. In addition, modifying the hinge binder moiety led to enhancing selectivity, and replacing the trifluoromethyl moiety gained Vps34 potency and/or lost activity against the other lipid kinases. Intriguingly, compound 67 was at least 60-fold more selective than its corresponding (S)-isomer 64. Compound 68 was selected for in vivo pharmacological studies and displayed an IC50 value of 2 and 82 nM on the Vps34 enzymatic assay and the GFP-FYVE cellular assay, respectively. It exhibited selectivity against mTOR (IC50 > 10 µM), class I PI3Ks (IC50 values of 2.7, 4.5, 2.5, and >10 µM on PI3K α, β, δ, γ isoforms, respectively) and other lipid kinases. In addition, compound 68 exhibited suitable mouse PK and significant pathway modulation in a H1299-GFP-FYVE tumors xenografted mechanistic model in mice. SAR405 (69) had an IC50 of 1 nM in the phosphorylation of a PtdIns substrate by human recombinant Vps34 enzyme, and an on-target IC50 activity on GFP-FYVE


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HeLa cells of 27 nM.167 Notably, no activity on other lipid and protein kinases was determined, demonstrating the exquisite selectivity of 69 for Vps34. Compound 68 and 69 have similar structures and binding modes (Figure 18). The exquisite selectivity could be associated with the presence of methyl-morpholine moiety, which acted as a hinge binder and occupied a hydrophobic pocket. The moiety pointed toward Met682, the orientation provided selectivity against class I PI3Ks. As a consequence, inhibiting the catalytic activity of Vps34 disrupted vesicle trafficking from late endosomes to lysosomes. Compound 69 displayed a 10-fold lower IC50 on autophagy inhibition when induced by an mTOR inhibitor, resulting in a significant synergy on the reduction of cell proliferation using renal tumor cells. These results indicated a potential therapeutic application for Vps34 inhibitors in cancer.168 The binding modes of these novel tetra-hydro pyrimidopyrimidinone derivatives provide a way to improve the selectivity and potency.

Figure 17. Representative Vps34 inhibitors of pyrimidinone scaffold.



Figure 18. Human Vps34 structure and molecular basis for PIK-III selectivity. Binding mode of compound 63 (A, PDB code 5ENN), 68 (B, PDB code 4UWL) and 69 (C, PDB code 4OYS) in complex with Vps34. The ligands are shown in stick model colored by C in green, N in mazarine, and O in red. The green dash line indicates a conventional hydrogen bond, the purple dash line indicates the π-π interaction.

PI3KD/V-IN-01 (70, Figure 19), recent reported highly potent ATP-competitive PI3Kδ/Vps34 dual inhibitor, could efficiently block the PI3Kδ mediated signaling pathway and autophagy. This compound displayed high selectivity over other PI3K isoforms and did not inhibit any other kinases in the kinome. Furthermore, it exhibited better antiproliferative activity against AML, chronic lymphocytic leukemia (CLL) and Burkitt lymphoma (BL) cell lines than known selective PI3Kδ and Vps34 inhibitors. These results suggested that dual inhibition of PI3Kδ and Vps34 may be a preferable approach to improve the PI3Kδ inhibitor’s anti-tumor efficacy.169 However, the dissection of the interaction between Vps34 and this compound was not reported, the high selectivity toward PI3Kδ and Vps34 need further exploration.


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Figure 19. Structure of PI3KD/V-IN-01.

5.2.3 Atg4B modulators The cysteine protease Atg4 plays crucial roles during autophagy by activating LC3 to format the nascent autophagosome membrane. Dysfunctional expression of Atg4 protease in pathologic conditions and potential therapies based on Atg4 modulation have been discussed.170 There are four Atg4 homologs presenting in human cells, Atg4A, Atg4B, Atg4C and Atg4D. Atg4B has the highest catalytic efficiency for cleaving the C terminus of Atg8 family proteins, such as LC3B.171 Phosphorylation of Ser383 and Ser392 at the C-terminus of Atg4B affects the protease activity and correlates with the induction of autophagy, particularly enhancing the ability of Atg4B to delipidate its endogenous substrate LC3.172 In addition, knockdown of Atg4B sensitizes the cancer cells to chemotherapy and radiotherapy.173 So far, Atg4B is the only enzyme in the autophagy cascade shown to convert pro-LC3 to LC3-I in a highly efficient way, indicating its attractiveness as a target for pharmacologic autophagy modulation. Atg4B acts as a role that provides the cell with enough LC3B to amplify autophagy and recycles the lipidated LC3B to sustain autophagy. Recent evidence also demonstrates that Atg4B is endowed with a new role as a potential biomarker of autophagy.173b The crystal structure of Atg4B was first



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reported in 2005.174 Based on the molecular basis for the specificity and catalysis of Atg4B, agonists and antagonists of Atg4B have been development in recent years. 5.2.3.1 Atg4B agonists Only two small molecular agonists have been reported currently. One is an asperphenamate

(As)

derivative

N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D-phenylalaninol (BBP, 71, Figure 20), the first reported Atg4B agonist with increased solubility compared with As; the solubility values of As and 71 were 3.5 and 289.7 µM, respectively. Notably, it was found that human breast tumor MCF-7, T47D and MDA-MB231 cells were more sensitive to the stimulation of 71, suggesting a selective effect of 71 on these human breast tumor cells. Further investigation demonstrated that 71 could produce its growth inhibitory effect through induction of autophagic cell death but not apoptosis in MCF-7 cells. Intriguingly, autophagy induced by 71 in MCF-7 cells was Akt-mTOR signaling-independent, it was confirmed to induce autophagy through both c-Jun N-terminal kinase (JNK) activation and JNK-dependent ROS production. Compound 71 caused autophagy through upregulation of Atg4 protein. However, the mechanism is still not fully understood.175 Besides optimization of nature products to develop Atg4B agonist, current combination of in silico analysis and experimental validation also devotes to discovery

potential

new

Atg4B

targeted

drugs.176

Candidate flubendazole

(ZINC03830847, 72, Figure 20), a FDA-approved small molecule drug, was selected


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for best anti-proliferative effect through virtual screening with molecular docking simulations. Molecular docking displayed 72 bound to Atg4B with the optimal conformations. It formed hydrophobic interactions with residues Lys259 and Glu17. Molecular dynamics (MD) simulations were successfully performed on 72-Atg4B complexes. Treatment with 750 nM 72 for 24h resulted in almost 50% inhibition in the MDA-MB-231 cells. Compound 72 was identified to induce autophagic cell death involved with ROS production; it might regulate the Atg4-LC3 signaling due to binding to the interacted surface of Atg4B and LC3. All these findings would provide a basis for developing Atg4 agonists. The novel Atg4B agonist appeared to have a similar mechanism to 72 but with a better anti-tumor activity especially in TNBC cell lines.

Figure 20. Chemical structures of Atg4B agonists.

5.2.3.2 Atg4B antagonists Based on previous reports on the structure of Atg4B, the Atg4B antagonist NSC185058 (73, Figure 21) was identified by in silico docking of small compounds from the NCI library. The compound made hydrophilic and hydrophobic interactions within the pocket containing His280 and Asp278, which were required for the proteolytic activity of Atg4B.174 Compound 73 inhibited cellular Atg4B activity and



suppressed autophagy without affecting the mTOR or PtdIns3K pathway. Cell-based assay demonstrated its effect in attenuating the growth of osteosarcoma tumors. But the IC50 of the Atg4B inhibition of 73 was only 51 µM. This compound was a weak Atg4B inhibitor with a reversible anticancer effects, other protein targets might exist for this compound to exert its anticancer activity.177

Figure 21. Chemical structure of NSC185058.

To date, the development of specific antagonists of Atg4B is still at a very preliminary stage. The search for antagonists of Atg4B requires efficient assays for enzyme activity in the process of lead identification and characterization. Reed et al. developed a fluorescence-based assay relying on a fusion protein of an LCP (LC3B) and phospholipase A2 (PLA2), then devising an Atg4B HTS assay, resulting in 4 hits against Atg4B with IC50 < 10 µM. However, their effects in cancer therapy were unknown.178 These results provide a proof-of-concept evidence that the HTS is capable of identifying compounds with the potential for selectivity. Young et al. previously developed an assay based on the cleavage of pro-LC3 followed by mass spectrometry (LC-MS assay), while this assay was useful for mechanistic studies, not suitable for HTS.179 To provide an assay more suitable for screening, they reported an efficient assay for monitoring the activity of Atg4B based on a small peptide substrate.


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A series of peptide 7-aminocoumarins showed reasonable rates of cleavage but required high enzyme concentrations. This assay was optimized for HTS, screening over 4500 compounds yielding overall hit rates of about 0.5%. However, none of these hits have yet yielded leads useful for optimization. Several known inhibitors of Atg4B were evaluated using the small peptide substrate and gave IC50 values 3-7 folds higher than previously obtained values using the fluorescence resonance energy transfer (FRET)-LC3 substrate. This assay facilitates efforts to evaluate and screen for inhibitors of Atg4B suitable for clinical evaluation.180 In-depth exploration of the interactions between Atg4B and LC3 is vital to further discovery more specific Atg4B antagonists. Atg4B containing a papain-like fold shows the highest structural similarity to papain and its homologous proteases as well as ubiquitin-specific processing protease (UBP) family. In addition to the papain-like fold, Atg4B has a short finger domain, which is thought to be unique to Atg4 family proteases. The ubiquitin core of LC3 binds to the interface between the two domains of Atg4B. The active catalytic triad site in Atg4B consists of Cys74, Asp278, and His280; mutation of these sites is associated with the complete disappearance of its processing activity.181 Notably, the hydrogen bond network between Atg4B and other cysteine proteases is essentially identic. A region near by the catalytic triad site is important for substrate recognition. The active site cleft of Atg4B is masked by a flexibility inhibitory loop (referred to as regulatory loop, containing residues Lys259, Pro260, Asn261, and Ser262). Another flexible loop consists of Trp142 as the major interacting partner of the inhibitory loop. These two



loops are thought to be a crucial prerequisite for the hydrolysis of substrates by Atg4B (Figure 22). LC3 is comprised of three regions: the N-terminal domain, the ubiquitin-like core and the C-terminal tail. Especially, on complex formation with Atg4B, the only large conformational change is observed in the C-terminal tail; the interaction between the aromatic side chains of Phe119 (LC3) and Trp142 (Atg4B) may be critical for binding and catalysis. In free Atg4B, both the N-terminal tail and the regulatory loop cover the catalytic site, resulting in burying Cys74. During catalysis, a large conformational change is induced in the regulatory loop and the N-terminal tail of Atg4B. The regulatory loop is lifted by LC3 Phe119 to form a groove, along which the LC3 tail enters the active site. Furthermore, the N-terminal tail is detached from the enzyme core and a large flat surface is exposed, enabling the enzyme to access the membrane-bound LC3-PE. As only the open conformation seems to be favorable for the membrane targeting of Atg4B that is required for LC3-PE deconjugation; the conformation of the N-terminal tail could be a regulator for the deconjugation activity.

Figure 22. Overall structure of Atg4B-LC3 complex. (A) The crystal structure of


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human Atg4B- LC3 (1-120) complex (PDB code 2Z0D). Atg4B is colored yellow, and LC3 is colored green. (B) Crystal structure of free Atg4B (PDB code 2CY7). The side chain of catalytic triad residues is shown with stick models, the regulatory loop is colored red, the flexible loop is colored purple and the N-terminal tail is colored blue.

Natural substrates of Atg4B containing peptide sequence SQETFGTA in the C-terminus induce a conformational change in Atg4B, resulting in the formation of several intermolecular contacts with the catalytic pocket.181 Recently, a novel time-resolved TR-FRET assay provided a robust platform to identify specific Atg4B inhibitors. Compounds that could inhibit Atg4B activity showed an enhanced FRET signal in this system. Based on the assay, HTS was employed to screen a library composed of 57,000 chemical compounds. Z-FA-FMK (74) exhibited high inhibitory activity with an IC50 of 14.80 µM. It mimicked the natural substrate peptide sequence (-TFG-) and was expected to form a stable covalent bond with Cys74, thus inactivated Atg4B proteolytic activity. Molecular modeling of compound 74 into the open conformation of Atg4B, using the TFG residues of LC3 as template, suggested a potential steric clash of the additional methyl group in 74 with Trp142, likely explaining the inferior anti-ATG4B activity of this molecule.182 Chemistry modification resulted in an even more potent Atg4B inhibitor with an IC50 of 1.13 µM, named thereafter as Z-FG-FMK (75). Arguably, the fluoromethylketone (FMK) moiety formed a covalent bond with the reactive thiol group of the Cys74 in the catalytic site of Atg4B. Compound 75 was 10-times more potent than 74 with an IC50



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of 1.2 µM in the biochemical assay. Cell-based luciferase reporter assay also demonstrated that compound 75 (IC50 = 1.47 µM) was more potent than 74 (IC50 = 5.09 µM), correlating very well with the values from the TR-FRET assay.183 Subsequent structure-guided optimization of FMK-based peptidomimetics elucidated the mechanism of the autophagy pathway and offered opportunities for drug discovery. In summary, according to the predicted binding mode and SAR, the FMK-based antagonist could be divided into 4 important moieties (Figure 23). The FMK warhead at the P1 position was important for Atg4B inhibition, compared with other Michael acceptors such as aldehyde and nitrile; the FMK was sufficiently fit into the binding site around Cys74. The phenyl group at the P2 position was crucial for anti-Atg4B activity. Intriguingly, halogen substituents could be tolerated at the meta-position of the phenyl group at the P2 position (compound 76). The NH between P2 and P3 functionality made a hydrogen bond with the carbonyl oxygen atom of Tyr143, supported

by

N-methylation

(compound

77

and

78).

The

terminal

N-benzyloxycarbonyl (N-Cbz) group at the P4 position could be replaced by more rigid and smaller amide moieties. Especially, compound 79 showed good anti-Atg4B activity and physicochemical properties. Replacing N-Cbz group with bicyclic aromatic groups at the P4 position, the naphthalene-1-carboxamide analogue compound 80 showed strong inhibition of Atg4B, with IC50 values of 80 and 73 nM in the TR-FRET and cellular-based luciferase refolding assay (LRA). Compared with its β-substituted analogue, compound 81 had the best anti-Atg4B activity with an IC50 of 57 nM and 15 nM in the TR-FRET and cellular LRA assay, respectively. The


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improved activity owed to more efficient stacking of the extended bicyclic π-system with the backbone amide group of Tyr143-Gly144. This should be sensitive to the relative orientation of the naphthyl ring to the plane of the adjacent amide linker. However, the FMK analogues hit a few cysteine proteases with IC50 values < 1 µM.182 Target-based molecular design constructs of Atg4B antagonists revealed key protein-ligand interactions, providing a biochemical basis for compounds design and properties improvement. In response to current deficiencies of reliability, standardized methodologies to quantitate Atg4B activity/inhibition were reported in literatures. Cleenewerck et al. compared three different assay types, a fluorogenic substrate-based assay, an in-gel densitometric quantification assay and a thermal shift protocol. Observably, the in-gel densitometric quantification methodology was shown to combine satisfactory linearity and reproducibility and offered a potential method for Atg4B inhibitor screening. A set of seven Atg4B inhibitors was assessed; these compound classes are known for their broad nonspecific activity against a wide range of biological targets. Structure 2 and 3 (Figure 24) showed modest inhibitory activity. A library of 182 fragment-sized N-7-amino-4-methylcoumarines was subsequently screened. One fragment S069 (Figure 24) was identified as a weak Atg4B inhibitor. Significantly, the active fragment residue 2-methylaminopyrazine substituent could be used as a binding fragment with potential value for fragment-based Atg4B inhibitor discovery.184



Figure 23. Chemical structures of FMK-based peptidomimetics.

Figure 24. Structures of screened Atg4B inhibitors.

6

Perspective Autophagy is an evolutionarily conserved catabolic process that facilitates

nutrient recycling via degradation of unnecessary organelles and proteins through lysosome mediated degradation. Although autophagy was firstly discovered approximately half a century before, the most important advances in this field have been achieved since the last decade. Researchers have made major progress in


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understanding autophagy mechanism and its relationship with other proteins and signaling pathways involved in many diseases. Generally, either inhibition or induction of autophagy plays a significant role in the modulation of the diseases. It is of great importance to carefully consider the strategy of autophagy modulation while designing the therapeutic agents, especially in cancers. On the one hand, induction of autophagy in the early stage of cancers is cytoprotective by suppressing tumorigenesis and maintaining the homeostasis in the tumor cells. It should be noted the crosstalk between autophagy and apoptosis, and other various regulatory pathways. On the other hand, tumor cells can also obtain nutrients and energy through autophagy for survival. Therefore, inhibiting autophagy in late-stage cancers may block the pro-survival mechanism for nutrient-deprived tumor cells by preventing protein recycling and cellular growth.185 Another example is AD. Direct degradation of aggregate-prone proteins by autophagy indeed alleviates symptoms at the early stage of AD. However, impaired autophagic flux characterized by intracellular accumulation of autophagosomes may process the APP into toxic forms, resulting in generation of Aβ which accelerating the progress of AD. Under the circumstance, inhibition of autophagy may benefit the therapy of AD. As autophagy plays a dual role in many diseases, thorough understanding the mechanism of autophagy in different conditions may lead to the identification of new specific targets for both diagnostic and therapeutic approaches. Accurately

detecting/measuring

autophagy

is

critical

to

improve

the

understanding of signaling cascades in various physiological and pathological



conditions. However, autophagy is a highly dynamic and multi-step process that is difficult to measure and quantify. Autophagic activity includes synthesis or lipidation of LC3, process of autophagosome synthesis, delivery of autophagic substrates to the lysosome, and degradation of autophagic substrates inside the lysosome. Static analyses cannot distinguish between autophagy upregulation and degradation inhibition. Autophagic substrates need to be monitored dynamically over time to verify their behaviors.18 Conventional method measuring some autophagy biomarkers/substrates is not always sufficient to quantify autophagy. For example, increased amount of p62 is not necessarily caused by inhibition of autophagy. In addition, lysosomotropic reagent 29 increases modified LC3 proteins and LC3 puncta without inducing autophagy.156 Therefore, considering the dynamic nature of autophagy and complexity of its regulation, as well as strengths and weakness of established method measuring autophagy, it is recommended that simultaneous assessment of several independent assays is required to assess autophagy status, avoiding false interpretations.186 Autophagy modulation may be a suitable therapeutic strategy. However, no small molecule that directly targets the machinery of autophagy has entered the clinical stage. Although many compounds have been reported to exert biological effects on autophagy modulation, they also influence other pathways or targets, which may amplify or limit its therapeutic efficacy. Meanwhile, the poor target selectivity limits the application of these compounds for the mechanism study of autophagy. To date, many autophagy modulators are initially noticed for their bioactivity against


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other targets or phenotypes. Although acting as autophagy modulators, many of these compounds have no detailed information about demonstrating the molecular mechanism on autophagy regulation. Not surprisingly, these compounds can also influence other signaling pathways involved in apoptosis, necrocytosis, immune and inflammation, leading their mechanism of autophagy modulation to be more obscure. In order to avoid these problems, the identification of novel specific autophagy targets as well as the development of selective modulators are challenging and demanding tasks for medicinal chemists. Great efforts have been recently made to measure receptor-ligand affinity on multiple targets in the autophagic pathway. Benefiting from these methods, researchers can efficiently discover more specific modulators to analyze dynamic processes in autophagy for complex diseases. From this point of view, precisely understanding of the intermolecular recognition mechanism between the autophagic targets and their endogenic ligands, especially those peptides with critical binding motifs, will further facilitate the development of efficient screening methodologies for the identification of new autophagy modulators. Therefore, as concluded in this review, diverse strategies can be used to screen selective modulators of autophagy, including HTS, modification of existing structures, fragment-based screening, combining effective fragments together, etc. Classical and modified HTS campaigns are effective screening tools widely used to identify potential autophagy modulators. Combination of basal phenotypic screen assays, cell-based assays, HTS, image-based high-content screen and proteomics



provides a better knowledge in autophagy research.69, 187 In order to avoid pan-assay interference, the approach requires a prior characterization of the proteins involved in autophagy machinery as well as their interactions with other proteins. For example, TR-FRET assay on the basis of the protein-ligand interactions facilitates efforts to evaluate and screen for Atg4B antagonists suitable for clinical evaluation. In addition, a chemical library should be established for HTS with desirable precision to improve the hit rates of promising compounds. Presently, multifunctional or multi-targeted small molecules may be favorable in treatment of some types of cancer, avoiding the complexities of drug-drug interactions and increased risk of toxicity, such as ATP-competitive PI3Kδ/Vps34 dual inhibitors and REV-ERBβ inhibitors. They not only target key proteins of autophagy pathway but also other multiple proteins related to cancer, indeed exerting anticancer activity. However, the mechanism is still not fully understood, whether these compounds will be a boon for preclinical trials still needs to be further evaluated. Up till now, the development of specific modulators of autophagy is still at a very preliminary stage. Driven by intensive studies on autophagy and an abundance of structural information on the essential components of the autophagy machinery such as ULK1, Vps34 and Atg4B, X-ray crystallography, nuclear magnetic resonance (NMR), precision biomarkers and structural bioinformatics-docking techniques provide the direction for the future development of autophagy modulators. Notably, SBDD has also been shown to be critical for discovering new generation of drugs that overcome drug resistance.188 In this review, compounds designed by SBDD successfully target the


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allosteric site of mTORC1, the catalytic domains of kinases as well as the PPI between Atg4B and LC3. However, most compounds targeting the catalytic domains of mTORC1, ULK1 and Vps34 display broad inhibitory activity against other kinases, such as FAK and PI3Ks. Hence the poor selectivity limited their further biological investigations as research tools. Combination of medicinal chemistry with other academic disciplines such as molecular biology, chemical biology, structural biology, cell biology contributes to the in-depth understanding of the structural characteristics of these targets, especially the ligand binding domains. The interdisciplinary research is of great importance to develop novel autophagy modulators. Nowadays, discovery of active and specific autophagy modulators provides an important direction for medicinal chemists. Small molecule compounds able to take great effects in autophagy related diseases with less off-target effects will provide individual treatment options. Moreover, on the basis of crystal structures, along with adequate structural modifications and accurate evaluation strategies, potent and selective autophagy modulators will be definitely obtained and show promise in preclinical and clinical evaluation. However, there are still many issues required future investigation, such as the indistinct autophagy-independent functions of Atgs, the clinical relevant autophagy-independent activities when targeting autophagy machinery,147 possible dose- and time-dependent effects in a clinical setting and the double-edged sword properties depending on concentration give.75

AUTHOR INFORMATION



Corresponding Authors HP. S.: phone, +86-13951934235; e-mail, [email protected]. Address: China pharmaceutical University, Nanjing. Author Contributions Haopeng Sun and Siyu He are responsible for writing the whole passage. Haopeng Sun, Yao Chen, Feng Feng and Wei Qu are in charge of checking and revision. Docking mode pictures are created by Qi Li, and others are made by Xueyang Jiang and Xin Lu. Notes The authors declare no competing financial interest. Biographies Siyu He got Bachelor degree from China Pharmaceutical University in 2016. She is currently a postgraduate student at the Department of Medicinal Chemistry (China Pharmaceutical University) under the supervision of Associate Prof. Haopeng Sun. Her research mainly focuses on the discovery, synthesis and biological evaluation of small molecules targeting autophagy machinery. Qi Li graduated in Pharmacy at China Pharmaceutical University in 2016. She went on advanced study in medicinal chemistry, China Pharmaceutical University, in the research group of associate professor Haopeng Sun. Her research focuses on design and synthesis of small molecules targeting neurodegenerative disease and autophagy modulation. Xueyang Jiang studied pharmacy at Anhui University of Chinese Medicine and


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received his bachelor degree in 2013. He pursues his doctorate of nature medicinal chemistry in China Pharmaceutical University under the supervision of Prof. Feng Feng. His research is focused on the structural optimization and mechanism study of the natural product in the field of anti-Alzheimer disease. Xin Lu graduated in Pharmacy at the China Pharmaceutical University in 2016. In the same year, he followed Associate Professor Haopeng Sun for Master degree. His research focuses on the development of small molecule targeting neurodegenerative diseases. Feng Feng graduated in Chemistry at Shaanxi Normal University in 1991. He received his Ph.D in 2001 in Pharmacy, with a thesis about natural products with anti-tumor activities supervised by Prof. S. X. Zhao. He studied in the University of California, Irvine as visiting scholar in 2005. In 2010, he was promoted to Professor of Natural Medicinal Chemistry at China Pharmaceutical University. So far, he has published more than 70 papers on journals indexed by Science Citation Index. His major research interests include extraction and isolation of chemical constituents from natural medicines, structural modification of active compositions and drug analysis in vivo. In addition, he is focusing on the prevention and treatment of cancer and neurodegenerative agents. Wei Qu graduated in Traditional Chinese Pharmacy at the China Pharmaceutical University in 2004. She received her Ph.D. in 2009 in natural medicinal chemistry, with a thesis about natural products with anti-tumor activities supervised by Prof. J. Y. Liang. In 2016, she was promoted to Associate Professor of Natural Medicinal



Chemistry at China Pharmaceutical University. Her current research interests focus on design, synthesis and biological evaluation of natural compounds, and active chemical constituents from natural medicines, in particular, agents targeting neurodegenerative diseases and cancer. Yao Chen graduated in Pharmacy at the China Pharmaceutical University in 2006. She received her Ph.D. in 2012 in Medicinal Chemistry guided by Prof. Y. H. Zhang, with a thesis about the design, synthesis and bioevaluation of new compounds targeting Alzheimer’s disease. In 2013, she became a Lecturer of Nanjing University of Chinese Medicine, China. So far, she has published thirteen papers on medicinal chemistry journals, including Journal of Medicinal Chemistry. Her major research interests include the design and optimization of small molecules targeting neurodegenerative diseases. In addition, she is interested in the identification of autophagy modulators. Haopeng Sun graduated in Pharmacy at the China Pharmaceutical University in 2006. He received his Ph.D. in 2011 in medicinal chemistry, with a thesis about the structural optimization and mechanism study of the natural product. Advisor Prof. Q. D. You. In 2014, he was promoted to Associate Professor of Medicinal Chemistry at China Pharmaceutical University. So far, he has published more than 70 papers on peer-review journals indexed by Science Citation Index. His major research interests include the design, synthesis and biological evaluation of small molecule bioactive compounds, in particular, agents targeting neurodegenerative diseases. In addition, he is focusing in the field of anti-cancer and anti-inflammatory agents.


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Acknowledgment We gratefully thank the support from the grants 81402851 and 81573281 of National Natural Science Foundation of China, BK20140957 of Natural Science Foundation of Jiangsu Province. We also thank the support from Fundamental Research Funds for the Central Universities (2015ZD009), Jiangsu Qing Lan Project, Top-notch Academic

Programs

Project

of

Jiangsu

Higher

Education

Institutions

(TAPP-PPZY2015A070) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). ABBREVIATIONS USED UPS, ubiquitin-proteasome system; CMA, chaperone-mediated autophagy; Atg, autophagy-related gene; ROS, reactive oxygen species; Atgs, autophagy-related genes; ULK1, unc-51 like autophagy activating kinase 1; Vps34, vacuolar protein sorting 34; PtdIns3K,

phosphatidylinositol

3-kinase;

SNARES,

soluble

N-ethylmaleimide-sensitive fusion attachment protein receptors; EPG5, ectopic P-granules autophagy protein 5 homolog; mTOR, the mammalian target of rapamycin; mTORC1/2, complex 1/2 of mTOR; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PtdIns3P, phosphatidylinositol 3-phosphate; LC3, microtubule-associated protein light chain 3; PE, lipidated phosphatidylethanolamine; TSC, tuberous sclerosis complex; Rags, Ras-like small GTPases; AMPK, AMP-activated protein kinase; UVRAG, UV radiation resistance-associated gene; EGFR, epidermal growth factor receptor; NRF2, nuclear factor erythroid-2-like 2; FAK, focal adhesion kinase; FIP200, FAK family kinase-interacting protein of 200 kDa; RAS, rat sarcoma; MDR,



multiple drug resistant; TEM, Transmission electron microscopy; GFP/RFP, green/red fluorescent protein; SAR, structure-activity relationship; PIKK, phosphatidylinositol 3-kinase-related kinase; HTS, high-throughput screening; HIF-1α, hypoxia-inducible factor 1-α; VEGF, vascular endothelial growth factor; SBDD, structure-based drug design; PPBO, physical properties-based optimization; ATM, ataxia-telangiectasia, mutated; ATR, ATM and Rad3-related; DNA-PK, DNA-dependent protein kinase; OGD/R, oxygen glucose deprivation/reoxygenation; FKBP12, FK506 binding protein 12; FRB, FKBP12-rapamycin-binding; AID, auto-inhibiting domain; ATF4, the eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4; LBK1, liver kinase B1; FGFR3, fibroblast growth factor receptor 3; BRAF, v-Raf murine sarcoma viral oncogene homolog B; REV-ERBβ, reverse orientation c-erbA gene, variant β; MIT, microtubule-interacting and transport; IFNR, interferon receptor; MPP+, 1-methyl-4-phenylpyridinium ion; Erk, extracellular regulated protein kinases; PDK1, 1-phosphoinositide-dependent protein kinase-1; TBK1, TANK-binding kinase 1; PPI, protein-protein interaction; NRBF2, nuclear receptor-binding factor 2; USP, ubiquitin-specific peptidase; HELCAT, helical and catalytic domain; SGK3, serum and glucocorticoid regulated protein kinase 3; JNK, c-Jun N-terminal kinase; UBP, ubiquitin-specific processing protease; FRET, fluorescence resonance energy transfer; NMR, nuclear magnetic resonance. REFERENCES 1. Wang, X. J.; Yu, J.; Wong, S. H.; Cheng, A. S. L.; Chan, F. K. L.; Ng, S. S. M.; Cho, C. H.; Sung, J. J. Y.; Wu, W. K. K. A novel crosstalk between two major protein


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Barreiro, E.; Bartel, B.; Bartolome, A.; Bassham, D. C.; Bassi, M. T.; Bast, R. C., Jr.; Basu, A.; Batista, M. T.; Batoko, H.; Battino, M.; Bauckman, K.; Baumgarner, B. L.; Bayer, K. U.; Beale, R.; Beaulieu, J. F.; Beck, G. R., Jr.; Becker, C.; Beckham, J. D.; Bedard, P. A.; Bednarski, P. J.; Begley, T. J.; Behl, C.; Behrends, C.; Behrens, G. M.; Behrns, K. E.; Bejarano, E.; Belaid, A.; Belleudi, F.; Benard, G.; Berchem, G.; Bergamaschi, D.; Bergami, M.; Berkhout, B.; Berliocchi, L.; Bernard, A.; Bernard, M.; Bernassola, F.; Bertolotti, A.; Bess, A. S.; Besteiro, S.; Bettuzzi, S.; Bhalla, S.; Bhattacharyya, S.; Bhutia, S. K.; Biagosch, C.; Bianchi, M. W.; Biard-Piechaczyk, M.; Billes, V.; Bincoletto, C.; Bingol, B.; Bird, S. W.; Bitoun, M.; Bjedov, I.; Blackstone, C.; Blanc, L.; Blanco, G. A.; Blomhoff, H. K.; Boada-Romero, E.; Bockler, S.; Boes, M.; Boesze-Battaglia, K.; Boise, L. H.; Bolino, A.; Boman, A.; Bonaldo, P.; Bordi, M.; Bosch, J.; Botana, L. M.; Botti, J.; Bou, G.; Bouche, M.; Bouchecareilh, M.; Boucher, M. J.; Boulton, M. E.; Bouret, S. G.; Boya, P.; Boyer-Guittaut, M.; Bozhkov, P. V.; Brady, N.; Braga, V. M.; Brancolini, C.; Braus, G. H.; Bravo-San Pedro, J. M.; Brennan, L. A.; Bresnick, E. H.; Brest, P.; Bridges, D.; Bringer, M. A.; Brini, M.; Brito, G. C.; Brodin, B.; Brookes, P. S.; Brown, E. J.; Brown, K.; Broxmeyer, H. E.; Bruhat, A.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bryson-Richardson, R. J.; Buch, S.; Buchan, A. M.; Budak, H.; Bulavin, D. V.; Bultman, S. J.; Bultynck, G.; Bumbasirevic, V.; Burelle, Y.; Burke, R. E.; Burmeister, M.; Butikofer, P.; Caberlotto, L.; Cadwell, K.; Cahova, M.; Cai, D.; Cai, J.; Cai, Q.; Calatayud, S.; Camougrand, N.; Campanella, M.; Campbell, G. R.; Campbell, M.; Campello, S.; Candau, R.; Caniggia, I.; Cantoni, L.; Cao, L.; Caplan, A. B.; Caraglia, M.; Cardinali, C.; Cardoso, S. M.;


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Carew, J. S.; Carleton, L. A.; Carlin, C. R.; Carloni, S.; Carlsson, S. R.; Carmona-Gutierrez, D.; Carneiro, L. A.; Carnevali, O.; Carra, S.; Carrier, A.; Carroll, B.; Casas, C.; Casas, J.; Cassinelli, G.; Castets, P.; Castro-Obregon, S.; Cavallini, G.; Ceccherini, I.; Cecconi, F.; Cederbaum, A. I.; Cena, V.; Cenci, S.; Cerella, C.; Cervia, D.; Cetrullo, S.; Chaachouay, H.; Chae, H. J.; Chagin, A. S.; Chai, C. Y.; Chakrabarti, G.; Chamilos, G.; Chan, E. Y.; Chan, M. T.; Chandra, D.; Chandra, P.; Chang, C. P.; Chang, R. C.; Chang, T. Y.; Chatham, J. C.; Chatterjee, S.; Chauhan, S.; Che, Y.; Cheetham, M. E.; Cheluvappa, R.; Chen, C. J.; Chen, G.; Chen, G. C.; Chen, G.; Chen, H.; Chen, J. W.; Chen, J. K.; Chen, M.; Chen, M.; Chen, P.; Chen, Q.; Chen, Q.; Chen, S. D.; Chen, S.; Chen, S. S.; Chen, W.; Chen, W. J.; Chen, W. Q.; Chen, W.; Chen, X.; Chen, Y. H.; Chen, Y. G.; Chen, Y.; Chen, Y.; Chen, Y.; Chen, Y. J.; Chen, Y. Q.; Chen, Y.; Chen, Z.; Chen, Z.; Cheng, A.; Cheng, C. H.; Cheng, H.; Cheong, H.; Cherry, S.; Chesney, J.; Cheung, C. H.; Chevet, E.; Chi, H. C.; Chi, S. G.; Chiacchiera, F.; Chiang, H. L.; Chiarelli, R.; Chiariello, M.; Chieppa, M.; Chin, L. S.; Chiong, M.; Chiu, G. N.; Cho, D. H.; Cho, S. G.; Cho, W. C.; Cho, Y. Y.; Cho, Y. S.; Choi, A. M.; Choi, E. J.; Choi, E. K.; Choi, J.; Choi, M. E.; Choi, S. I.; Chou, T. F.; Chouaib, S.; Choubey, D.; Choubey, V.; Chow, K. C.; Chowdhury, K.; Chu, C. T.; Chuang, T. H.; Chun, T.; Chung, H.; Chung, T.; Chung, Y. L.; Chwae, Y. J.; Cianfanelli, V.; Ciarcia, R.; Ciechomska, I. A.; Ciriolo, M. R.; Cirone, M.; Claerhout, S.; Clague, M. J.; Claria, J.; Clarke, P. G.; Clarke, R.; Clementi, E.; Cleyrat, C.; Cnop, M.; Coccia, E. M.; Cocco, T.; Codogno, P.; Coers, J.; Cohen, E. E.; Colecchia, D.; Coletto, L.; Coll, N. S.; Colucci-Guyon, E.; Comincini, S.; Condello, M.; Cook, K. L.; Coombs, G. H.;



Cooper, C. D.; Cooper, J. M.; Coppens, I.; Corasaniti, M. T.; Corazzari, M.; Corbalan, R.; Corcelle-Termeau, E.; Cordero, M. D.; Corral-Ramos, C.; Corti, O.; Cossarizza, A.; Costelli, P.; Costes, S.; Cotman, S. L.; Coto-Montes, A.; Cottet, S.; Couve, E.; Covey, L. R.; Cowart, L. A.; Cox, J. S.; Coxon, F. P.; Coyne, C. B.; Cragg, M. S.; Craven, R. J.; Crepaldi, T.; Crespo, J. L.; Criollo, A.; Crippa, V.; Cruz, M. T.; Cuervo, A. M.; Cuezva, J. M.; Cui, T.; Cutillas, P. R.; Czaja, M. J.; Czyzyk-Krzeska, M. F.; Dagda, R. K.; Dahmen, U.; Dai, C.; Dai, W.; Dai, Y.; Dalby, K. N.; Dalla Valle, L.; Dalmasso, G.; D'Amelio, M.; Damme, M.; Darfeuille-Michaud, A.; Dargemont, C.; Darley-Usmar, V. M.; Dasarathy, S.; Dasgupta, B.; Dash, S.; Dass, C. R.; Davey, H. M.; Davids, L. M.; Davila, D.; Davis, R. J.; Dawson, T. M.; Dawson, V. L.; Daza, P.; de Belleroche, J.; de Figueiredo, P.; de Figueiredo, R. C.; de la Fuente, J.; De Martino, L.; De Matteis, A.; De Meyer, G. R.; De Milito, A.; De Santi, M.; de Souza, W.; De Tata, V.; De Zio, D.; Debnath, J.; Dechant, R.; Decuypere, J. P.; Deegan, S.; Dehay, B.; Del Bello, B.; Del Re, D. P.; Delage-Mourroux, R.; Delbridge, L. M.; Deldicque, L.; Delorme-Axford, E.; Deng, Y.; Dengjel, J.; Denizot, M.; Dent, P.; Der, C. J.; Deretic, V.; Derrien, B.; Deutsch, E.; Devarenne, T. P.; Devenish, R. J.; Di Bartolomeo, S.; Di Daniele, N.; Di Domenico, F.; Di Nardo, A.; Di Paola, S.; Di Pietro, A.; Di Renzo, L.; DiAntonio, A.; Diaz-Araya, G.; Diaz-Laviada, I.; Diaz-Meco, M. T.; Diaz-Nido, J.; Dickey, C. A.; Dickson, R. C.; Diederich, M.; Digard, P.; Dikic, I.; Dinesh-Kumar, S. P.; Ding, C.; Ding, W. X.; Ding, Z.; Dini, L.; Distler, J. H.; Diwan, A.; Djavaheri-Mergny, M.; Dmytruk, K.; Dobson, R. C.; Doetsch, V.; Dokladny, K.; Dokudovskaya, S.; Donadelli, M.; Dong, X. C.; Dong, X.; Dong, Z.; Donohue, T. M.,


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Jr.; Doran, K. S.; D'Orazi, G.; Dorn, G. W., 2nd; Dosenko, V.; Dridi, S.; Drucker, L.; Du, J.; Du, L. L.; Du, L.; du Toit, A.; Dua, P.; Duan, L.; Duann, P.; Dubey, V. K.; Duchen, M. R.; Duchosal, M. A.; Duez, H.; Dugail, I.; Dumit, V. I.; Duncan, M. C.; Dunlop, E. A.; Dunn, W. A., Jr.; Dupont, N.; Dupuis, L.; Duran, R. V.; Durcan, T. M.; Duvezin-Caubet, S.; Duvvuri, U.; Eapen, V.; Ebrahimi-Fakhari, D.; Echard, A.; Eckhart, L.; Edelstein, C. L.; Edinger, A. L.; Eichinger, L.; Eisenberg, T.; Eisenberg-Lerner, A.; Eissa, N. T.; El-Deiry, W. S.; El-Khoury, V.; Elazar, Z.; Eldar-Finkelman, H.; Elliott, C. J.; Emanuele, E.; Emmenegger, U.; Engedal, N.; Engelbrecht, A. M.; Engelender, S.; Enserink, J. M.; Erdmann, R.; Erenpreisa, J.; Eri, R.; Eriksen, J. L.; Erman, A.; Escalante, R.; Eskelinen, E. L.; Espert, L.; Esteban-Martinez, L.; Evans, T. J.; Fabri, M.; Fabrias, G.; Fabrizi, C.; Facchiano, A.; Faergeman, N. J.; Faggioni, A.; Fairlie, W. D.; Fan, C.; Fan, D.; Fan, J.; Fang, S.; Fanto, M.; Fanzani, A.; Farkas, T.; Faure, M.; Favier, F. B.; Fearnhead, H.; Federici, M.; Fei, E.; Felizardo, T. C.; Feng, H.; Feng, Y.; Feng, Y.; Ferguson, T. A.; Fernandez, A. F.; Fernandez-Barrena, M. G.; Fernandez-Checa, J. C.; Fernandez-Lopez, A.; Fernandez-Zapico, M. E.; Feron, O.; Ferraro, E.; Ferreira-Halder, C. V.; Fesus, L.; Feuer, R.; Fiesel, F. C.; Filippi-Chiela, E. C.; Filomeni, G.; Fimia, G. M.; Fingert, J. H.; Finkbeiner, S.; Finkel, T.; Fiorito, F.; Fisher, P. B.; Flajolet, M.; Flamigni, F.; Florey, O.; Florio, S.; Floto, R. A.; Folini, M.; Follo, C.; Fon, E. A.; Fornai, F.; Fortunato, F.; Fraldi, A.; Franco, R.; Francois, A.; Francois, A.; Frankel, L. B.; Fraser, I. D.; Frey, N.; Freyssenet, D. G.; Frezza, C.; Friedman, S. L.; Frigo, D. E.; Fu, D.; Fuentes, J. M.; Fueyo, J.; Fujitani, Y.; Fujiwara, Y.; Fujiya, M.; Fukuda, M.; Fulda, S.;



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Fusco, C.; Gabryel, B.; Gaestel, M.; Gailly, P.; Gajewska, M.; Galadari, S.; Galili, G.; Galindo, I.; Galindo, M. F.; Galliciotti, G.; Galluzzi, L.; Galluzzi, L.; Galy, V.; Gammoh, N.; Gandy, S.; Ganesan, A. K.; Ganesan, S.; Ganley, I. G.; Gannage, M.; Gao, F. B.; Gao, F.; Gao, J. X.; Garcia Nannig, L.; Garcia Vescovi, E.; Garcia-Macia, M.; Garcia-Ruiz, C.; Garg, A. D.; Garg, P. K.; Gargini, R.; Gassen, N. C.; Gatica, D.; Gatti, E.; Gavard, J.; Gavathiotis, E.; Ge, L.; Ge, P.; Ge, S.; Gean, P. W.; Gelmetti, V.; Genazzani, A. A.; Geng, J.; Genschik, P.; Gerner, L.; Gestwicki, J. E.; Gewirtz, D. A.; Ghavami, S.; Ghigo, E.; Ghosh, D.; Giammarioli, A. M.; Giampieri, F.; Giampietri, C.; Giatromanolaki, A.; Gibbings, D. J.; Gibellini, L.; Gibson, S. B.; Ginet, V.; Giordano, A.; Giorgini, F.; Giovannetti, E.; Girardin, S. E.; Gispert, S.; Giuliano, S.; Gladson, C. L.; Glavic, A.; Gleave, M.; Godefroy, N.; Gogal, R. M., Jr.; Gokulan, K.; Goldman, G. H.; Goletti, D.; Goligorsky, M. S.; Gomes, A. V.; Gomes, L. C.; Gomez, H.; Gomez-Manzano, C.; Gomez-Sanchez, R.; Goncalves, D. A.; Goncu, E.; Gong, Q.; Gongora,

C.; Gonzalez,

C.

B.; Gonzalez-Alegre,

P.;

Gonzalez-Cabo,

P.;

Gonzalez-Polo, R. A.; Goping, I. S.; Gorbea, C.; Gorbunov, N. V.; Goring, D. R.; Gorman, A. M.; Gorski, S. M.; Goruppi, S.; Goto-Yamada, S.; Gotor, C.; Gottlieb, R. A.; Gozes, I.; Gozuacik, D.; Graba, Y.; Graef, M.; Granato, G. E.; Grant, G. D.; Grant, S.; Gravina, G. L.; Green, D. R.; Greenhough, A.; Greenwood, M. T.; Grimaldi, B.; Gros, F.; Grose, C.; Groulx, J. F.; Gruber, F.; Grumati, P.; Grune, T.; Guan, J. L.; Guan, K. L.; Guerra, B.; Guillen, C.; Gulshan, K.; Gunst, J.; Guo, C.; Guo, L.; Guo, M.; Guo, W.; Guo, X. G.; Gust, A. A.; Gustafsson, A. B.; Gutierrez, E.; Gutierrez, M. G.; Gwak, H. S.; Haas, A.; Haber, J. E.; Hadano, S.; Hagedorn, M.; Hahn, D. R.; Halayko, A. J.;


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Hamacher-Brady, A.; Hamada, K.; Hamai, A.; Hamann, A.; Hamasaki, M.; Hamer, I.; Hamid, Q.; Hammond, E. M.; Han, F.; Han, W.; Handa, J. T.; Hanover, J. A.; Hansen, M.; Harada, M.; Harhaji-Trajkovic, L.; Harper, J. W.; Harrath, A. H.; Harris, A. L.; Harris, J.; Hasler, U.; Hasselblatt, P.; Hasui, K.; Hawley, R. G.; Hawley, T. S.; He, C.; He, C. Y.; He, F.; He, G.; He, R. R.; He, X. H.; He, Y. W.; He, Y. Y.; Heath, J. K.; Hebert, M. J.; Heinzen, R. A.; Helgason, G. V.; Hensel, M.; Henske, E. P.; Her, C.; Herman, P. K.; Hernandez, A.; Hernandez, C.; Hernandez-Tiedra, S.; Hetz, C.; Hiesinger, P. R.; Higaki, K.; Hilfiker, S.; Hill, B. G.; Hill, J. A.; Hill, W. D.; Hino, K.; Hofius, D.; Hofman, P.; Hoglinger, G. U.; Hohfeld, J.; Holz, M. K.; Hong, Y.; Hood, D. A.; Hoozemans, J. J.; Hoppe, T.; Hsu, C.; Hsu, C. Y.; Hsu, L. C.; Hu, D.; Hu, G.; Hu, H. M.; Hu, H.; Hu, M. C.; Hu, Y. C.; Hu, Z. W.; Hua, F.; Hua, Y.; Huang, C.; Huang, H. L.; Huang, K. H.; Huang, K. Y.; Huang, S.; Huang, S.; Huang, W. P.; Huang, Y. R.; Huang, Y.; Huang, Y.; Huber, T. B.; Huebbe, P.; Huh, W. K.; Hulmi, J. J.; Hur, G. M.; Hurley, J. H.; Husak, Z.; Hussain, S. N.; Hussain, S.; Hwang, J. J.; Hwang, S.; Hwang, T. I.; Ichihara, A.; Imai, Y.; Imbriano, C.; Inomata, M.; Into, T.; Iovane, V.; Iovanna, J. L.; Iozzo, R. V.; Ip, N. Y.; Irazoqui, J. E.; Iribarren, P.; Isaka, Y.; Isakovic, A. J.; Ischiropoulos, H.; Isenberg, J. S.; Ishaq, M.; Ishida, H.; Ishii, I.; Ishmael, J. E.; Isidoro, C.; Isobe, K.; Isono, E.; Issazadeh-Navikas, S.; Itahana, K.; Itakura, E.; Ivanov, A. I.; Iyer, A. K.; Izquierdo, J. M.; Izumi, Y.; Izzo, V.; Jaattela, M.; Jaber, N.; Jackson, D. J.; Jackson, W. T.; Jacob, T. G.; Jacques, T. S.; Jagannath, C.; Jain, A.; Jana, N. R.; Jang, B. K.; Jani, A.; Janji, B.; Jannig, P. R.; Jansson, P. J.; Jean, S.; Jendrach, M.; Jeon, J. H.; Jessen, N.; Jeung, E. B.; Jia, K.; Jia, L.; Jiang, H.; Jiang,



H.; Jiang, L.; Jiang, T.; Jiang, X.; Jiang, X.; Jiang, X.; Jiang, Y.; Jiang, Y.; Jimenez, A.; Jin, C.; Jin, H.; Jin, L.; Jin, M.; Jin, S.; Jinwal, U. K.; Jo, E. K.; Johansen, T.; Johnson, D. E.; Johnson, G. V.; Johnson, J. D.; Jonasch, E.; Jones, C.; Joosten, L. A.; Jordan, J.; Joseph, A. M.; Joseph, B.; Joubert, A. M.; Ju, D.; Ju, J.; Juan, H. F.; Juenemann, K.; Juhasz, G.; Jung, H. S.; Jung, J. U.; Jung, Y. K.; Jungbluth, H.; Justice, M. J.; Jutten, B.; Kaakoush, N. O.; Kaarniranta, K.; Kaasik, A.; Kabuta, T.; Kaeffer, B.; Kagedal, K.; Kahana, A.; Kajimura, S.; Kakhlon, O.; Kalia, M.; Kalvakolanu, D. V.; Kamada, Y.; Kambas, K.; Kaminskyy, V. O.; Kampinga, H. H.; Kandouz, M.; Kang, C.; Kang, R.; Kang, T. C.; Kanki, T.; Kanneganti, T. D.; Kanno, H.; Kanthasamy, A. G.; Kantorow, M.; Kaparakis-Liaskos, M.; Kapuy, O.; Karantza, V.; Karim, M. R.; Karmakar, P.; Kaser, A.; Kaushik, S.; Kawula, T.; Kaynar, A. M.; Ke, P. Y.; Ke, Z. J.; Kehrl, J. H.; Keller, K. E.; Kemper, J. K.; Kenworthy, A. K.; Kepp, O.; Kern, A.; Kesari, S.; Kessel, D.; Ketteler, R.; Kettelhut Ido, C.; Khambu, B.; Khan, M. M.; Khandelwal, V. K.; Khare, S.; Kiang, J. G.; Kiger, A. A.; Kihara, A.; Kim, A. L.; Kim, C. H.; Kim, D. R.; Kim, D. H.; Kim, E. K.; Kim, H. Y.; Kim, H. R.; Kim, J. S.; Kim, J. H.; Kim, J. C.; Kim, J. H.; Kim, K. W.; Kim, M. D.; Kim, M. M.; Kim, P. K.; Kim, S. W.; Kim, S. Y.; Kim, Y. S.; Kim, Y.; Kimchi, A.; Kimmelman, A. C.; Kimura, T.; King, J. S.; Kirkegaard, K.; Kirkin, V.; Kirshenbaum, L. A.; Kishi, S.; Kitajima, Y.; Kitamoto, K.; Kitaoka, Y.; Kitazato, K.; Kley, R. A.; Klimecki, W. T.; Klinkenberg, M.; Klucken, J.; Knaevelsrud, H.; Knecht, E.; Knuppertz, L.; Ko, J. L.; Kobayashi, S.; Koch, J. C.; Koechlin-Ramonatxo, C.; Koenig, U.; Koh, Y. H.; Kohler, K.; Kohlwein, S. D.; Koike, M.; Komatsu, M.; Kominami, E.; Kong, D.; Kong, H. J.; Konstantakou, E. G.; Kopp,


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B. T.; Korcsmaros, T.; Korhonen, L.; Korolchuk, V. I.; Koshkina, N. V.; Kou, Y.; Koukourakis, M. I.; Koumenis, C.; Kovacs, A. L.; Kovacs, T.; Kovacs, W. J.; Koya, D.; Kraft, C.; Krainc, D.; Kramer, H.; Kravic-Stevovic, T.; Krek, W.; Kretz-Remy, C.; Krick, R.; Krishnamurthy, M.; Kriston-Vizi, J.; Kroemer, G.; Kruer, M. C.; Kruger, R.; Ktistakis, N. T.; Kuchitsu, K.; Kuhn, C.; Kumar, A. P.; Kumar, A.; Kumar, A.; Kumar, D.; Kumar, D.; Kumar, R.; Kumar, S.; Kundu, M.; Kung, H. J.; Kuno, A.; Kuo, S. H.; Kuret, J.; Kurz, T.; Kwok, T.; Kwon, T. K.; Kwon, Y. T.; Kyrmizi, I.; La Spada, A. R.; Lafont, F.; Lahm, T.; Lakkaraju, A.; Lam, T.; Lamark, T.; Lancel, S.; Landowski, T. H.; Lane, D. J.; Lane, J. D.; Lanzi, C.; Lapaquette, P.; Lapierre, L. R.; Laporte, J.; Laukkarinen, J.; Laurie, G. W.; Lavandero, S.; Lavie, L.; LaVoie, M. J.; Law, B. Y.; Law, H. K.; Law, K. B.; Layfield, R.; Lazo, P. A.; Le Cam, L.; Le Roch, K. G.; Le Stunff, H.; Leardkamolkarn, V.; Lecuit, M.; Lee, B. H.; Lee, C. H.; Lee, E. F.; Lee, G. M.; Lee, H. J.; Lee, H.; Lee, J. K.; Lee, J.; Lee, J. H.; Lee, J. H.; Lee, M.; Lee, M. S.; Lee, P. J.; Lee, S. W.; Lee, S. J.; Lee, S. J.; Lee, S. Y.; Lee, S. H.; Lee, S. S.; Lee, S. J.; Lee, S.; Lee, Y. R.; Lee, Y. J.; Lee, Y. H.; Leeuwenburgh, C.; Lefort, S.; Legouis, R.; Lei, J.; Lei, Q. Y.; Leib, D. A.; Leibowitz, G.; Lekli, I.; Lemaire, S. D.; Lemasters, J. J.; Lemberg, M. K.; Lemoine, A.; Leng, S.; Lenz, G.; Lenzi, P.; Lerman, L. O.; Lettieri Barbato, D.; Leu, J. I.; Leung, H. Y.; Levine, B.; Lewis, P. A.; Lezoualc'h, F.; Li, C.; Li, F.; Li, F. J.; Li, J.; Li, K.; Li, L.; Li, M.; Li, M.; Li, Q.; Li, R.; Li, S.; Li, W.; Li, W.; Li, X.; Li, Y.; Lian, J.; Liang, C.; Liang, Q.; Liao, Y.; Liberal, J.; Liberski, P. P.; Lie, P.; Lieberman, A. P.; Lim, H. J.; Lim, K. L.; Lim, K.; Lima, R. T.; Lin, C. S.; Lin, C. F.; Lin, F.; Lin, F.; Lin, F. C.; Lin, K.; Lin, K. H.; Lin, P. H.; Lin, T.; Lin, W. W.;



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Lin, Y. S.; Lin, Y.; Linden, R.; Lindholm, D.; Lindqvist, L. M.; Lingor, P.; Linkermann, A.; Liotta, L. A.; Lipinski, M. M.; Lira, V. A.; Lisanti, M. P.; Liton, P. B.; Liu, B.; Liu, C.; Liu, C. F.; Liu, F.; Liu, H. J.; Liu, J.; Liu, J. J.; Liu, J. L.; Liu, K.; Liu, L.; Liu, L.; Liu, Q.; Liu, R. Y.; Liu, S.; Liu, S.; Liu, W.; Liu, X. D.; Liu, X.; Liu, X. H.; Liu, X.; Liu, X.; Liu, X.; Liu, Y.; Liu, Y.; Liu, Z.; Liu, Z.; Liuzzi, J. P.; Lizard, G.; Ljujic, M.; Lodhi, I. J.; Logue, S. E.; Lokeshwar, B. L.; Long, Y. C.; Lonial, S.; Loos, B.; Lopez-Otin, C.; Lopez-Vicario, C.; Lorente, M.; Lorenzi, P. L.; Lorincz, P.; Los, M.; Lotze, M. T.; Lovat, P. E.; Lu, B.; Lu, B.; Lu, J.; Lu, Q.; Lu, S. M.; Lu, S.; Lu, Y.; Luciano, F.; Luckhart, S.; Lucocq, J. M.; Ludovico, P.; Lugea, A.; Lukacs, N. W.; Lum, J. J.; Lund, A. H.; Luo, H.; Luo, J.; Luo, S.; Luparello, C.; Lyons, T.; Ma, J.; Ma, Y.; Ma, Y.; Ma, Z.; Machado, J.; Machado-Santelli, G. M.; Macian, F.; MacIntosh, G. C.; MacKeigan, J. P.; Macleod, K. F.; MacMicking, J. D.; MacMillan-Crow, L. A.; Madeo, F.; Madesh, M.; Madrigal-Matute, J.; Maeda, A.; Maeda, T.; Maegawa, G.; Maellaro, E.; Maes, H.; Magarinos, M.; Maiese, K.; Maiti, T. K.; Maiuri, L.; Maiuri, M. C.; Maki, C. G.; Malli, R.; Malorni, W.; Maloyan, A.; Mami-Chouaib, F.; Man, N.; Mancias, J. D.; Mandelkow, E. M.; Mandell, M. A.; Manfredi, A. A.; Manie, S. N.; Manzoni, C.; Mao, K.; Mao, Z.; Mao, Z. W.; Marambaud, P.; Marconi, A. M.; Marelja, Z.; Marfe, G.; Margeta, M.; Margittai, E.; Mari, M.; Mariani, F. V.; Marin, C.; Marinelli, S.; Marino, G.; Markovic, I.; Marquez, R.; Martelli, A. M.; Martens, S.; Martin, K. R.; Martin, S. J.; Martin, S.; Martin-Acebes, M. A.; Martin-Sanz, P.; Martinand-Mari,

C.;

Martinet,

W.;

Martinez,

J.;

Martinez-Lopez,

N.;

Martinez-Outschoorn, U.; Martinez-Velazquez, M.; Martinez-Vicente, M.; Martins, W.


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Design of Small Molecule Autophagy Modulators: A promising

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