Macroautophagy, herein referred to as autophagy, is an intracellular degradation process essential for ensuring cellular homeostasis. This well-conserved catabolic process mediates the targeted degradation of unwanted or excess cytoplasmic materials, such as aggregation-prone proteins, pathogens and damaged organelles like mitochondria, amongst others (Ravikumar et al., 2010). This process is also involved in the bulk degradation of cytoplasmic macromolecules and recycling of the breakdown products especially during nutrient deprivation to provide energy homeostasis, thereby forming a crucial connection between anabolism and catabolism (Boya et al., 2013; Kaur and Debnath, 2015). Due to its vital function as a homeostatic regulator, impairment of the autophagy is implicated in several human pathologies including certain cancer, metabolic syndromes, infectious diseases, liver diseases, myopathies, aging and neurodegenerative disorders (Mizushima et al., 2008). Therefore, therapeutic modulation of autophagy holds great potential in the development of treatment strategies for these diseases (Rubinsztein et al., 2012).

Autophagy is evolutionarily-conserved from yeast to humans. The de novo formation of phagophores, the double-membrane structures that expand to form double-membrane vesicles called autophagosomes, require multiple autophagy-related (Atg) genes in the autophagic machinery, such as the Atg5-Atg12-Atg16 complex and the phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3 (LC3-II) (Kabeya et al., 2000; Mizushima et al., 2011; Ktistakis and Tooze, 2016). Maturation of autophagosomes into the degradative autolysosomes occurs either via the multi-step route involving the fusion of autophagosomes with late endosomes to form amphisomes which subsequently fuse with the lysosomes, or via the direct route involving the fusion between autophagosomes and the lysosomes (Nakamura and Yoshimori, 2017). The autophagic cargo engulfed by the autophagosomes are ultimately degraded in the acidic autolysosomes by the lysosomal hydrolases, which are only active at the low pH maintained by the vacuolar-type H⁺-ATPase (V-ATPase) on the lysosomal membrane (Saftig and Klumperman, 2009). Finally, the breakdown products are recycled and utilized as inputs to cellular metabolism for energy generation (Rabinowitz and White, 2010). The rate at which this dynamic turnover of cellular contents occurs through the process of autophagy is referred to as autophagic flux. Autophagic flux encompasses all stages of autophagy which includes autophagosome formation, fusion with the lysosomes and cargo degradation in the autolysosomes (Figure 1).

Key upstream modulators of autophagy include the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which promotes cellular biosynthesis and inhibits autophagy (Saxton and Sabatini, 2017). Regulation of autophagosome formation by mTORC1 is mediated via the ULK1–Atg13–FIP200 complex; mTORC1 suppresses autophagy under nutrient-rich conditions by phosphorylation-dependent inactivation of ULK1 and Atg13 (Mizushima, 2010; Zachari and Ganley, 2017). Various signals such as growth factors and nutrients impinge on mTORC1 to negatively influence autophagy (Kim and Guan, 2015). Conversely, during nutrient starvation, autophagy is promoted by inhibition of the mTORC1 activity (Carroll et al., 2014; Russell et al., 2014). Furthermore, ULK1 can be directly phosphorylated and activated by the energy sensor AMPK to stimulate autophagy (Egan et al., 2011; Kim et al., 2011). In addition, several mTORC1-independent pathways have been described where autophagy is negatively regulated by the elevation in intracellular inositol, Ca²⁺ and nitric oxide levels, amongst others (Sarkar, 2013b). Molecular mediators of the late stage of autophagy involving autophagosome maturation include Rab7, SNAREs (N-ethylmaleimide-sensitive factor-attachment protein receptors), GABARAPs, BRUCE and Beclin1-interacting partners such as Atg14L, UVRAG and Ambra1 (He and Levine, 2010; Nguyen et al., 2016; Wang et al., 2016; Reggiori and Ungermann, 2017; Ebner et al., 2018). At a transcriptional level, autophagy is governed by the transcription factor EB (TFEB) (Settembre et al., 2011), which in itself is activated by lysosomal Ca²⁺ (Medina et al., 2015).

Chemical modulation of autophagy by targeting the mTOR-dependent and mTOR-independent pathways has proven to be of potential biomedical relevance due to therapeutic advantages, especially in neurodegenerative disorders as well as in diverse human pathological conditions such as in certain liver diseases, myopathies, infectious diseases, metabolic diseases, cancer and aging (Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015). Hence, the discovery of potent small molecules regulating autophagy is of great interest. Here we review the chemical screening strategies for autophagy drug discovery, and highlight the potential benefits of autophagy modulators in human diseases.

A number of in vitro screening methods have been designed for identifying compounds (Sarkar, 2013a; Joachim et al., 2015; Seranova et al., 2019). The assays are primarily based on the perturbations of autophagy reporters or autophagy cargoes as readouts (Figure 1), which can be measured via fluorescence or high-content imaging, immunoblotting, flow cytometry and microplate reader (Mizushima et al., 2010; Klionsky et al., 2016; Figure 2 and Table 1). Some of these screening methods can be subjected to high-throughput applications. Below are descriptions of the common screening approaches in mammalian cells, and the identification and therapeutic benefits of key autophagy modulators.

Screening methods based on autophagy reporters are the most commonly used approaches to detect changes in the numbers of autophagosomes and autolysosomes (Table 1). The protein reporter that is widely used to study autophagy is microtubule-associated protein 1 (MAP1) light chain 3 (LC3). The nascent LC3 is cleaved at its C-terminal arginine residue by Atg4 to form the cytoplasmic LC3-I, which is then post-translationally conjugated with phosphatidylethanolamine at its C-terminal glycine residue by Atg7 to form the autophagosome-associated LC3-II (Kabeya et al., 2000). The lipidated LC3-II remains associated to the autophagosomes throughout their lifespan, and is present on both the outer and inner membranes. Following the maturation of autophagosomes with lysosomes to form autolysosomes, the LC3-II on the inner surface is degraded whereas the LC3-II on the outer surface is delipidated and removed by Atg4B for recycling (Tanida et al., 2004). A number of fluorescent-tagged reporters of LC3, such as GFP-LC3 (Kabeya et al., 2000), mRFP-GFP-LC3 (Kimura et al., 2007) and GFP-LC3-RFP-LC3ΔG (Kaizuka et al., 2016), have been used to study autophagy and undertake chemical screening.

In addition to the screening approaches based on LC3 reporters, autophagy substrate clearance has also been utilized as a primary screening assay for identifying autophagy modulators (Table 1). This method measures the autophagic cargo flux, which together with LC3-based secondary assays for autophagosome flux can indicate the overall autophagic flux.

Autophagy plays an essential role for tissue homeostasis and cellular survival by removing unwanted materials like malfunctioning aggregated proteins and damaged organelles from the cells; however, deregulation of this process could contribute to cytotoxicity (Mizushima et al., 2008). Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases (Levine and Kroemer, 2008; Jiang and Mizushima, 2014), and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance (Figure 3). A number of independent studies and chemical screens have identified several autophagy modulators, which have been shown to impart beneficial effects in various transgenic disease models (Table 2; Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015). Some of the key studies in specific disease contexts are highlighted below.

Basal autophagy in the brain is critical for maintaining cellular homeostasis in post-mitotic cells like neurons, which is evident from the genetic studies in mice where brain-specific deletion of essential autophagy genes resulted in neurodegenerative phenotypes (Hara et al., 2006; Komatsu et al., 2006). Particularly, autophagy is the primary degradation pathway for several aggregation-prone proteins associated with neurodegeneration (Rubinsztein, 2006; Nixon, 2013). However, defective autophagy has been reported in several neurodegenerative diseases, including neurodegenerative lysosomal storage disorders, and is considered a major causative factor for neurodegeneration (Nixon, 2013; Sarkar, 2013b; Menzies et al., 2017; Seranova et al., 2017). Therefore, induction of autophagy for enhancing the clearance of mutant aggregation-prone proteins is considered a potential treatment strategy. The therapeutic benefits of autophagy inducers have been robustly demonstrated in the context of neurodegeneration where upregulation of autophagy was protective in several in vitro and in vivo transgenic models of neurodegenerative diseases (Rubinsztein et al., 2012; Sarkar, 2013b; Levine et al., 2015; Seranova et al., 2017). Stimulating autophagy with mTOR inhibitors like rapamycin or its analogs had beneficial effects in fly and mouse models of Huntington’s disease, Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD), spinocerebellar ataxia type 3 (SCA3) and prion disease (Ravikumar et al., 2004; Berger et al., 2006; Sarkar et al., 2008; Menzies et al., 2010; Spilman et al., 2010; Cortes et al., 2012; Wang et al., 2012; Ozcelik et al., 2013; Jiang et al., 2014). Likewise, several mTOR-independent autophagy inducers such as, but not limited to, lithium, carbamazepine (inositol lowering agents), rilmenidine (cAMP reducing agent), trehalose (AMPK activator), SMERs and BRD5631 have been shown to be protective in fly, Zebrafish, mouse or induced pluripotent stem cell (iPSC) models of AD, FTD, HD, amyotrophic lateral sclerosis (ALS) and Niemann-Pick type C1 (NPC1) disease (Sarkar et al., 2005, 2007a,b; Fornai et al., 2008; Williams et al., 2008; Rose et al., 2010; Zhang et al., 2011, 2018; Shimada et al., 2012; Wang et al., 2012; Li et al., 2013; Maetzel et al., 2014; Kuo et al., 2015). The most widely used mTOR-independent autophagy inducer in vivo is trehalose (Sarkar et al., 2007a), a disaccharide that stimulates autophagy by inhibiting SLC2A family of glucose transporters and activating AMPK (DeBosch et al., 2016), which in turn can directly influence the phosphorylation of the autophagy-initiating kinase ULK1 (Egan et al., 2011; Kim et al., 2011). Remarkably, trehalose had beneficial effects in mouse models of AD, PD, HD, FTD, SCA17, ALS, as well as cellular and iPSC-derived neuronal models of prion and NPC1 disease, respectively (Tanaka et al., 2004; Aguib et al., 2009; Rodriguez-Navarro et al., 2010; Schaeffer et al., 2012; Castillo et al., 2013; Du et al., 2013; Zhang et al., 2014; Chen et al., 2015; Tanji et al., 2015). Additional autophagy-inducing agents reported to be cytoprotective in neurodegenerative models such as HD, PD, ALS, FTD and Lafora disease include Tat-Beclin 1 peptide, calpastatin, verapamil, metformin, AUTEN-67, AUTEN-99, 6-Bio and fluphenazine (Ma et al., 2007; Williams et al., 2008; Shoji-Kawata et al., 2013; Barmada et al., 2014; Berthier et al., 2016; Billes et al., 2016; Papp et al., 2016; Kovacs et al., 2017; Suresh et al., 2017). A combinatorial approach in enhancing autophagy has been shown with rapamycin and mTOR-independent autophagy inducers such as lithium, trehalose or SMERs. Higher efficacy was achieved via the additive effects of dual treatment on autophagy induction and cytoprotection in cell and fly models of HD than the effects of single compounds (Sarkar et al., 2007a,b, 2008).

The ability of autophagy in the maintenance of metabolic homeostasis has drawn considerable attention as a potential target for cancer therapy via its pro-survival and pro-death mechanisms (Rabinowitz and White, 2010; Levy et al., 2017). Autophagy plays tumor suppressive role by mitigating oxidative stress, removing superfluous mitochondria and preventing DNA damage and genome instability; and on the other hand, shows pro-tumor activity by preventing the induction of tumor suppressors, increasing resistance to apoptosis and maintaining tumor metabolism through recycling of nutrients (Mathew et al., 2007; Galluzzi et al., 2015; Kimmelman and White, 2017). Depending on the cancer context and the opposing effects of autophagy, either inhibitors or inducers of autophagy could be exploited for cancer therapy (Galluzzi et al., 2017; Levy et al., 2017). Since autophagy promotes tumorigenesis in most contexts, inhibition of autophagy has gathered considerable interest for cancer therapy. Accumulating evidence demonstrate that autophagy inhibitors/blockers exerted therapeutic benefits in cancer models. The clinically- approved autophagy inhibitors chloroquine or hydroxychloroquine (HCQ), which impair lysosomal acidification and block autophagic flux (Murakami et al., 1998; Boya et al., 2005), caused tumor shrinkage in preclinical studies; and thus HCQ being more potent with lesser side-effects is used in ongoing clinical trials either alone or in combination with other treatments (Briceno et al., 2003; Amaravadi et al., 2007; Cook et al., 2014; Chude and Amaravadi, 2017; Levy et al., 2017; Onorati et al., 2018). Autophagy inhibitory compounds, such as Lys05 and ROC-325, which exhibited anti-tumor activity in mice have been suggested to be more potent than HCQ (McAfee et al., 2012; Carew et al., 2017). In addition, autophagy inhibitors preventing autophagosome formation such as ATG4B antagonists (compounds NSC185058 and UAMC-2526), Vps34 (vacuolar protein sorting protein 34) inhibitor (compound SAR405), ULK1 (Unc-51-like kinase 1) inhibitor (compound SBI-0206965), USP10/USP13 (ubiquitin-specific peptidases) inhibitor (Spautin-1) and agents causing transcriptional inhibition of autophagy genes (pyrvinium pamoate), also exerted anti-proliferative and anti-tumor effects in cellular and in vivo models of cancer (Liu et al., 2011; Deng et al., 2013; Akin et al., 2014; Ronan et al., 2014; Shao et al., 2014; Egan et al., 2015; Kurdi et al., 2017). On the contrary, various chemical agents or natural products exerting antiproliferative or anti-tumor activity either alone or in combination with chemotherapeutic agents could induce autophagy or autophagic cell death, which include Torin 1, AC-73, MC-4, metformin, silibinin, Abrus agglutinin, curcumin, liensinine, spermidine, vitamin D3, and imatinib (Buzzai et al., 2007; Ertmer et al., 2007; Wang et al., 2008; Thoreen et al., 2009; Qian et al., 2011; Francipane and Lagasse, 2013; Law et al., 2014; Jiang et al., 2016; Pietrocola et al., 2016; Panda et al., 2017; Son et al., 2018; Spinello et al., 2018).

Autophagy plays an important role in innate defense mechanism by removing intracellular pathogens; a process termed xenophagy (Levine et al., 2011; Deretic et al., 2013). The role of autophagy in regulating intracellular infections initially emerged through studies on Mycobacterium tuberculosis (Mtb) (Gutierrez et al., 2004; Singh et al., 2006). Subsequently, several other bacterial pathogens like Salmonella and Listeria, and viral pathogens like HIV and Dengue were shown to utilize host autophagy pathways for their own advantage (Jia et al., 2009; Kyei et al., 2009; Yoshikawa et al., 2009; Heaton and Randall, 2010). A genome-wide siRNA screen to identify host factors required for intracellular Mtb survival within macrophages revealed that a large number of host factors acted via regulation of autophagy to help the bacteria (Kumar et al., 2010). Induction of autophagy with rapamycin, carbamazepine, SMER28, and vitamin D3 were shown to prevent bacterial survival or HIV replication in macrophages (Gutierrez et al., 2004; Floto et al., 2007; Yuk et al., 2009; Kumar et al., 2010; Campbell and Spector, 2011, 2012; Schiebler et al., 2015). Notably, carbamazepine reduced bacterial burden, improved lung pathology and stimulated adaptive immunity in mice infected with multidrug-resistant Mtb (Schiebler et al., 2015). Rapamycin also controlled viral and bacterial pathogens both in vitro and in vivo (Donia et al., 2010). In an integrated chemical and RNAi screening for modulators of intracellular mycobacteria, one of the top three compounds was nortriptyline which significantly suppressed Mtb survival within macrophages and induced autophagy (Sundaramurthy et al., 2013). Other compounds limiting bacterial or HIV infections through activation of autophagic flux were nitazoxanide (anti-protozoan drug) and flubendazole (antihelminthic drug) (Lam et al., 2012; Chauhan et al., 2015). Similarly, the naturally occurring disaccharide trehalose, a potent mTOR-independent enhancer of autophagy in diverse cell-types (Sarkar et al., 2007a), can also induce autophagy and xenophagy in Mtb-infected macrophages that resulted in the killing of bacteria (Sharma et al., 2017). In this study, trehalose was found to act as a PI(3,5)P₂ (phosphatidylinositol 3,5-bisphosphate) agonist for activating the lysosomal Ca²⁺ channel TRPML1 (Sharma et al., 2017), which in turn released lysosomal Ca²⁺ that caused nuclear translocation of TFEB to induce autophagy (Medina et al., 2015). Excitingly, trehalose also seemed to be effective during HIV-Mtb co-infection and limits Mtb survival by reversing the HIV-mediated block in autophagy flux (Sharma et al., 2017). Similarly, vitamin D3 could also kill Mtb during HIV co-infection by inducing autophagy (Campbell and Spector, 2012). Several host factors currently being tested for anti-Mtb therapeutics function by regulating host autophagy and xenophagy. For example, inhibition of host Src kinase by the compound AZD0530 induced autophagy and lysosomal maturation to clear Mtb (Chandra et al., 2016). A pioneering anti-infective, autophagy-inducing agent is Tat-Beclin 1, which is a peptide representing a region of the autophagy regulator Beclin 1 that interacts with the HIV-1 accessory protein NEF, and this domain is linked with the HIV-1 Tat transduction domain to make it cell permeable (Shoji-Kawata et al., 2013). Tat-Beclin 1 prevented the replication of a number of viral and bacterial pathogens in vitro in autophagy-dependent manner, as well as induced autophagy and anti-viral activity in mice infected with chikungunya or West Nile virus (Shoji-Kawata et al., 2013). Thus, it is evident that regulators of autophagy and xenophagy have tremendous potential for novel therapeutics against various infectious diseases. It is now clear that within an infected host cell, there is a possibility of uncoupling between homeostatic autophagy and anti-bacterial xenophagy (Chandra et al., 2015; Sharma et al., 2018). Therefore, it is desirable to perform chemical screening pertaining to infection-specific xenophagy flux for identifying novel regulators of bacterial/viral survival within the host cells through the autophagy pathway.

Liver autophagy is essential for various hepatic functions and is implicated in various liver conditions including α1-antitrypsin (AAT) deficiency, non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma and viral hepatitis (Rautou et al., 2010; Ueno and Komatsu, 2017). Chemical modulation of autophagy has been shown to have beneficial effects in some of these diseases. Carbamazepine, an mTOR independent autophagy inducer acting by reducing inositol levels (Sarkar et al., 2005), reduced hepatic load of mutant α1-antitrypsin Z and hepatic fibrosis in a mouse model of AAT deficiency (Hidvegi et al., 2010), as well as decreased hepatocellular aggregate-related toxicity in patients suffering from fibrinogen storage disease (Puls et al., 2013). A high-throughput drug screen in hepatocyte-like cells derived from iPSC lines of patients with AAT deficiency also revealed inositol-lowering autophagy-inducing agents, such as carbamazepine, lithium, and valproic acid, in facilitating the clearance mutant AAT (Choi et al., 2013). Carbamazepine as well as the mTOR inhibitor rapamycin also rescued dysfunctional autophagic flux and improved cell viability in hepatic-like cells differentiated from patient-derived iPSC lines of Niemann-Pick type C1 (NPC1) disease (Maetzel et al., 2014). In addition, autophagy induction with trehalose, carbamazepine, rapamycin or hydrogen sulfide reduced steatosis, lipid accumulation and liver injury in high-fat diet-induced NAFLD in mice (Lin et al., 2013; Sun et al., 2015; DeBosch et al., 2016). Furthermore, the anti-diabetic drug metformin, which indirectly inhibits mTOR, induced SIRT1-mediated autophagy in primary hepatocytes and ameliorated hepatic steatosis in vivo (Song et al., 2015). Overall, these studies indicate that activation of autophagy via inhibition of mTOR, lowering inositol levels or with trehalose are effective modes of inducing autophagy in the liver.

Basal autophagy is required for maintaining muscle mass and myofiber integrity (Masiero et al., 2009), and thus deregulation of muscle autophagy is implicated in myopathies and muscular dystrophies (Sandri et al., 2013). Sustained activation of mTORC1 in skeletal muscle of TSC1-deficient mice could cause late-onset myopathy related to suppression of autophagy (Castets et al., 2013). Upregulation of autophagy, primarily by inhibiting the mTORC1 pathway, has been reported to have beneficial effects in certain transgenic disease models. Autophagy induction by rapamycin or low-protein diet increased myofiber survival and attenuated dystrophic phenotype in a mouse model of collagen type VI muscular dystrophy (Grumati et al., 2010). Likewise, activation of autophagy by dietary changes or with the AMP-activated protein kinase (AMPK) agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), improved dystrophic phenotypes in mouse models of Duchenne muscular dystrophy (DMD) (De Palma et al., 2012; Pauly et al., 2012). A potential role of simvastatin, which has been reported to induce autophagy by inhibiting the Rac1-mTOR pathway (Wei et al., 2013), has been suggested in improving the physiological function of skeletal muscle in DMD transgenic mice (Whitehead et al., 2015). In addition, rapamycin or its analog, temsirolimus, ameliorated cardiomyopathy and improved skeletal and cardiac muscle function in mouse models of LMNA (lamin A/C gene) cardiomyopathy that recapitulate Emery-Dreifuss muscular dystrophy (EDMD) (Choi et al., 2012; Ramos et al., 2012).

The functionality of autophagy declines with aging (Rubinsztein et al., 2011), and thus restoring adequate autophagy is considered a possible anti-aging strategy for lifespan extension. There are a number of lifespan expanding strategies, and in many of such approaches, autophagy acts as a common denominator for promoting longevity (Madeo et al., 2010; Hansen et al., 2018). Pharmacological treatment with autophagy inducers has been linked to increasing longevity in transgenic in vivo models (Madeo et al., 2015). Lifespan extension via induction of autophagy with naturally- occurring polyamines such as spermidine, which is an acetyltransferase inhibitor, was shown in yeast, flies, worms and mice (Eisenberg et al., 2009, 2016); and likewise also reported with the natural phenol resveratrol, which is a deacetylase activator, in yeast, flies, worms as well as in mice on high-fat diet (Howitz et al., 2003; Wood et al., 2004; Baur et al., 2006; Morselli et al., 2010). Although both spermidine and resveratrol impacts on the acetylproteome, stimulation of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) whereas the effect of spermidine was SIRT1 independent (Morselli et al., 2010, 2011). Inhibition of mTOR by rapamycin also extended lifespan in yeast, flies and mice (Alvers et al., 2009; Harrison et al., 2009; Bjedov et al., 2010; Lamming et al., 2013). In addition, lifespan extension in multiple organisms including mice and apes could be achieved by caloric restriction, which is a physiological inducer of autophagy via AMPK activation, mTORC1 inhibition and SIRT1 activation (Mair and Dillin, 2008; Colman et al., 2009; Mercken et al., 2014; Mattison et al., 2017). In some of these studies reporting lifespan extension by autophagy activation, the role of autophagy has been specifically determined by abolishing the anti-aging effects via knockdown of essential autophagy genes (Madeo et al., 2015; Nakamura and Yoshimori, 2018).

The methodologies for measuring autophagy have evolved over the past decade and it is now feasible to undertake high-throughput chemical screens for identifying modulators of autophagic flux. A number of pharmacological modulators of autophagy have been identified via screening approaches or individual studies; some of which have been demonstrated to exert therapeutic benefits in diverse human diseases. Most of the key autophagy modulators have been identified either by the GFP-LC3 screening method in HeLa cells or via assessing the clearance of aggregation-prone proteins in inducible PC12 cell lines. While analysis of changes in autophagosome number with GFP-LC3 reporter requires shorter treatment period (such as 8–24 h), analysis of clearance of aggregation-prone proteins requires longer treatment duration (such as 24–72 h) depending on the nature of the transgene product. Following the primary screen, it is pertinent to characterize the high-confidence screen hits with secondary autophagy assays because there are no single assays to determine autophagic flux. These normally include analysis of autophagosome formation with bafilomycin A₁ via immunoblotting with anti-LC3 antibody, analysis of autophagosome maturation with mRFP-GFP-LC3 reporter, and analysis of autophagy substrate (p62) clearance via immunoblotting with anti-p62 antibody (Mizushima et al., 2010; Klionsky et al., 2016).

Although the methods described in this review are those that have been generally used in the field, alternative autophagy assays could also be employed for chemical screening. One potential approach is the use of Keima, a fluorescent acid-stable protein that exhibits bimodal excitation spectra in neutral and acidic pH, such as in autophagosomes and autolysosomes, respectively (Katayama et al., 2011). The cumulative fluorescence readout can be used to measure bulk autophagic flux. This protein can also be utilized for selective autophagic flux, such as with mitochondria-targeted Keima to measure mitophagy (Katayama et al., 2011; Sun et al., 2017). However, Keima-based assays solely depend upon the lysosomal acidity and thus cannot be performed in fixed cells where the pH gradient across lysosomal membranes is lost. In addition, other screening approaches could be based on fluorescent-tagged early markers of autophagy initiation, such as with WIPI-1 (Proikas-Cezanne and Pfisterer, 2009) and DFCP1 (Axe et al., 2008); however, these methods will not capture the late events of autophagy pathway involving autophagosome maturation and cargo degradation.

For the therapeutic exploitation of autophagy modulators, mTOR-independent autophagy inducers are generally favorable and considered to have lesser side-effects than the mTOR inhibitors like rapamycin. This is because mTOR controls vital cellular functions like cell growth and translation and thus its inhibition can lead to undesirable side-effects unrelated to autophagy induction. For clinical translation to patients, it is important to determine the efficacy and penetrance of the autophagy modulators in the target organs. Future directions could include identifying specific inducers of autophagy acting at the level of autophagic machinery rather than the upstream signaling pathways.

PP, AF, SV, DK, and SS wrote the manuscript. ES and SS made the figures. PP and SS made the tables. PP, AF, SV, ES, VS, MC, PD, JT, TR, DK, and SS reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.