key: cord-0900576-vweqjnlt authors: Rein, Theo title: Harnessing autophagy to fight SARS‐CoV‐2: An update in view of recent drug development efforts date: 2021-10-20 journal: J Cell Biochem DOI: 10.1002/jcb.30166 sha: c8555d2ce6ed13197622a69009755ad306bd5832 doc_id: 900576 cord_uid: vweqjnlt Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid‐19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid‐19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life‐threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS‐CoV‐2 life cycle. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has gained notoriety for causing the currently raging coronavirus disease 2019 (Covid-19) pandemic. 1 Humongous efforts are ongoing worldwide to cope with the impact on health and society. Although vaccines could be developed and marketed with unprecedented swiftness, drug development will take significantly longer. In light of the obvious exigency, drug repurposing is a promising strategy that is being followed by many scientists in preclinical and clinical research. 2 For example, the World Health Organization (WHO) launched the research program "Solidarity" in 2020 to test four compounds as options for antiviral treatment, namely remdesivir (originally developed as an inhibitor of viral RNA polymerase to treat hepatitis C, Ebola, or Marburg virus infection), interferon β1a (boosting the host response to viral infection), hydroxychloroquine (a malaria drug), and a combination of lopinavir and ritonavir (both HIV drugs). Unfortunately, an interim report of the study, including 11 330 in-patients with Covid-19 at 405 hospitals in 30 countries, revealed little or no effect. 3 A more recent initiative in the Solidarity program evaluates three established immune-modulatory drugs for Covid-19 treatment. 4 The selection of these drugs was based on a different rationale, that is, instead of trying to fight the virus directly, the aim is to confine the damage of an exaggerated immune response to the own body. A previous study showed that limiting the host defense can have beneficial effects in critically ill patients with Covid-19. 5 The selected drugs are infliximab, imatinib, and artesunate. The aim of this short review is to point to a potential involvement of autophagy in the action of these drugs, which may play a more prominent role than generally acknowledged. The review additionally includes the drug ivermectin, which received media attention as it promising results were reported in clinical trials, 6 and also covers antidepressants. In general, autophagy is an evolutionary conserved intracellular degradation process pivotal for cellular protein, energy, and organelle homeostasis. 7 It is active under the basic condition at a low level ensuring continuous turnover and can be activated under certain stress conditions such as proteotoxicity or starvation. 8 Material destined for degradation or recycling is engulfed by or transported into a double-membrane structure called "autophagosome." Through additional membrane remodeling processes, these autophagosomes fuse with lysosomes producing autolysosomes, with prior fusion with late endosomes as a potential intermediate step. 9 As detailed in excellent reviews, this process is tightly controlled and executed by a vast array of proteins, ATG proteins in particular, but also EPG proteins required for the more complex autophagy in multicellular organisms. [7] [8] [9] [10] [11] Autophagic flux refers to the activity through all consecutive steps of autophagy and typically is defined as a measure of autophagic degradation activity. 12 Analytical tools assessing autophagic flux need to be chosen with great care to avoid erroneous conclusions. 13 Several compounds currently are being developed, targeting different proteins in the autophagic cascade, given its involvement in various physiological and pathophysiological conditions, including viral infection. 14,15 The link between autophagy and invading pathogens is anything but new and both pro-and antiviral roles of autophagy were identified. 16 For example, evidence suggests that double-membrane structures derived from the endoplasmic reticulum both are required for the initial steps of autophagy and serve as replication sites for coronaviruses [17] [18] [19] [20] [21] [22] (see also Figure 1 ). Later on, several coronavirus proteins were shown to induce the formation of double-membrane structures, such as the nonstructural proteins 2, 3, 4, and 6. 23 The broad activity against coronavirus replication of compounds that interfere with the generation of these structures further corroborates their importance. In contrast to this appearing congruence of viral mechanisms and early steps of autophagy, there is also firm evidence that coronaviruses interfere with late steps of autophagy to evade degradation. Very recently, for example, ORF3a (the protein derived from open reading frame 3a) of SARS-CoV-2 has been demonstrated to inhibit the fusion of autophagosomes with lysosomes, thereby increasing the number of autophagosomes but decreasing autophagic flux, 24, 25 in line with the effect of coronavirus infection. 26, 27 However, the exact details of how coronaviruses in general, and SARS-CoV-2 in particular, are intertwined with autophagy await further elucidation. 23, 28, 29 Nevertheless, it appears plausible that coronaviruses may benefit from earlier steps of the autophagic pathway, but are vulnerable to the increased autophagic flux that clears out viral particles. This is supported by reports showing that induction of autophagy has the potential to fight coronavirus infection. 26, 27 F I G U R E 1 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication and endosomal/autophagic pathways, simplified scheme. Current knowledge supports both beneficial and detrimental effects of the autophagic pathway for SARS-CoV-2 replication. A major entry route for the virus is endocytic uptake, which requires lysosomal acidification for viral RNA release. The autophagic pathway is a multifactorial and multistep pathway with a vast range of possibilities for pharmacological targeting. In the more initial phases, phagophores, and double-membrane vesicles (DMVs) are formed, most likely from the endoplasmic reticulum, possibly also promoted by some coronavirus proteins. SARS-CoV-2 replication takes place at the endoplasmic reticulum as well, at very similar, if not identical, membrane structures. SARS-CoV-2 inhibits the last step of autophagy leading to viral degradation, that is, the fusion of autophagosomes with lysosomes to form autolysosomes, thus inhibiting autophagic flux. Accordingly, compounds impacting autophagy are expected to be efficient in fighting SARS-CoV-2 only if they enhance autophagic flux Chloroquine inhibits autophagy by interfering with autophagosome-lysosome fusion. 30 However, chloroquine and hydroxychloroquine exert additional effects like disorganizing the endo-lysosomal system that might have been the basis for the initial hope put on this drug for Covid-19 treatment. 31, 32 However, with more studies coming up, no overall beneficial effect of this drug on Covid-19 was apparent, [33] [34] [35] and the drug now is abandoned in the WHO Solidarity program. Therefore, it appears likely that autophagy-targeting drugs need to promote autophagy flux rather than other aspects of autophagy. Although COVID-19 primarily is a respiratory disease, multiple organs are affected, either through cytokines or directly upon invasion of SARS-CoV-2. [36] [37] [38] As autophagy is a conserved mechanism operative in most cells, pharmacological induction of autophagy has the potential to fight SARS-CoV-2 in all organs that are reached by the compound. However, the effect on overall health may depend on existing comorbidities, such as cancer, for example, where the effect of autophagy depends on the circumstances. 39, 40 4 | ARTESUNATE Like the other two drugs infliximab and imatinib, artesunate was added to the WHO Solidarity program because of its effects on the immune system. 4 Artesunate is a derivate of artemisinin with established antimalaria features, but also potent anticancer effects. For considering its potential effects on autophagy, it is important to differentiate general effects on autophagy from effects on autophagy flux, given the complex interaction of SARS-CoV-2 with autophagy. The vast majority of publications assessing artesunate for its effects on autophagy report induction 41-44 not all publications, however, assess autophagic flux following the established guidelines. 13 Nevertheless, some flux assays have been performed such as the use of the late autophagy blockers chloroquine or bafilomycin A, where artesunate still enhances the autophagy marker LC3BII/I. [45] [46] [47] Although all these studies support autophagy promoting function of artesunate, an inhibitory effect of artesunate has been observed using the tandem fluorescence tagged LC3B stably transfected into HeLa cells, 48 which is recognized as a valid method to determine autophagic flux. 13 The reason for this seeming discrepancy is not known, which makes further studies mandatory. Infliximab is a chimeric antibody targeting TNF-α used in clinical practice to treat autoimmune diseases such as Crohn's disease. Recently, it has been put forward that a range of drugs, including infliximab, that are either approved or in a clinical trial with great promise to treat Crohn's disease induces autophagy as a relevant mechanism at least contributing to their effect. 49, 50 At least for infliximab, however, there is a scarcity of studies investigating the effect on autophagy directly, 51, 52 and no reports were found presenting autophagic flux assays for infliximab. Imatinib is an ABL tyrosine kinase inhibitor used to treat chronic myeloid leukemia. Like chloroquine, it is a cationic amphiphilic drug and thus should have the potential to inhibit autophagy by accumulating in lysosomes and disturbing their function. 53 However, several studies report an autophagy-inducing effect of imatinib, including the assessment of autophagic flux using chloroquine as an inhibitor. [54] [55] [56] [57] Nevertheless, more studies are needed applying a broader range of autophagic flux assessments to solidify the conclusion that imatinib induces autophagy. Furthermore, it has been argued that compounds prone to induce phospholipidosis such as cationic amphiphilic drugs should be excluded from drug repurposing for SARS-CoV-2 treatment. 58 Nevertheless, it should be noted, that several other cationic amphiphilic drugs such as some antidepressants also induce autophagic flux. 59 Thus, this compound class may as well elicit more specific effects. Evidence is accumulating that patients with Covid-19 benefit from antidepressant treatment: A multicentric observational retrospective study with 7230 adults hospitalized for Covid-19 reported that those receiving antidepressant treatment had a reduced risk of intubation or death. 60 Similarly, a study with 3238 Covid-19 patients revealed a beneficial effect of the antidepressant fluvoxamine, reducing the need for emergency room observation or hospitalization. 61 A small randomized clinical trial with 152 COVID-19 outpatients revealed a lower likelihood of clinical deterioration for patients receiving fluvoxamine. 62 Furthermore, a recent preclinical study found the antidepressant fluoxetine as an inhibitor of SARS-CoV-2 in human lung tissue. 63 The beneficial effects of antidepressants frequently are conceptualized as cytokine effects 64 thus reducing the risk of a fatal cytokine storm. 65 However, antidepressants are known to induce autophagy as well. 59, 66 Thus, their effect on autophagy might not only be important for treating depression but also to fight SARS-CoV-2. In fact, tricyclic antidepressants inhibit lysosomal acidic sphingomyelinase, thereby not only enhancing autophagy but also reducing SARS-CoV-2 entry into epithelial cells. 67, 68 Ivermectin is an antihelmintic macrolide of the avermectin group. 69 It is investigated as a potential anti-SARS-CoV-2 treatment with promising initial results, but also very recent dispute. 6, 71 Several mechanisms are discussed for its apparent antiviral activity 70, 71 and this viewpoint argues for adding autophagy to this panel. A number of publications report an autophagy-inducing effect of ivermectin, 72-74 including a study carefully determining autophagic flux. 75 Therefore, autophagy should be considered as a mediator of the manifold effects of ivermectin in general, 70 and of its antiviral activity in particular. Of note, another antihelmintic drug, niclosamide, not only is known for its autophagyinducing action but also has been demonstrated to reduce replication of the Middle East Respiratory Syndrome Coronavirus 26 as well as of SARS-CoV-2. 27 The point of this article is to draw attention to autophagy as a potential contributing mechanism of selected drugs currently under investigation for repurposing to Covid-19 treatment. In other words, it is possible that the three drugs recently added to the WHO Solidarity program may not just prevent a life-threatening overreaction of the body during a SARS-CoV-2 infection, but also actually limit SARS-CoV-2 replication through activating autophagy. The interaction of SARS-CoV-2 with the autophagic pathway is complex (Figure 1) , with evidence for both the virus taking advantage of the autophagic pathway and trying to tame the full activity of this pathway to prevent its degradation. It is obvious from this scenario that it will be essential to learn how exactly the SARS-CoV-2 life cycle is intertwined with the autophagic pathway. Future research should include all known forms of autophagy, such as macroautophagy, microautophagy, chaperone-mediated autophagy, secretory autophagy, and so forth. This also applies to better understanding the action of to be repositioned or new drugs at each level of the autophagic pathway. Open Access funding enabled and organized by Projekt DEAL. https://orcid.org/0000-0003-2850-4289 A pneumonia outbreak associated with a new coronavirus of probable bat origin Therapeutic effectiveness and safety of repurposing drugs for the treatment of COVID-19: position standing in 2021 Repurposed antiviral drugs for Covid-19-Interim WHO Solidarity Trial Results International COVID-19 trial to restart with focus on immune responses Published online Interleukin-6 receptor antagonists in critically ill patients with Covid-19 Ivermectin and mortality in patients with COVID-19: a systematic review, meta-analysis, and meta-regression of randomized controlled trials Mechanisms governing autophagosome biogenesis Mechanism and medical implications of mammalian autophagy Machinery, regulation and pathophysiological implications of autophagosome maturation Molecular definitions of autophagy and related processes The role of Atg proteins in autophagosome formation Defining and measuring autophagosome flux-concept and reality Guidelines for the use and interpretation of assays for monitoring autophagy Biological functions of autophagy genes: a disease perspective Autophagy in human diseases Autophagy and viruses: adversaries or allies? SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum Coronavirus replication complex formation utilizes components of cellular autophagy Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate Coronavirus interactions with the cellular autophagy machinery ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers hosttargeting antivirals Highlights in the fight against COVID-19: does autophagy play a role in SARS-CoV-2 infection? Taming the autophagy as a strategy for treating COVID-19 Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Effect of hydroxychloroquine in hospitalized patients with Covid-19 A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19 Hydroxychloroquine in patients with COVID-19: a systematic review and metaanalysis SARS-CoV-2 cell tropism and multiorgan infection Neuroinvasion of SARS-CoV-2 in human and mouse brain Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series Autophagy Takes center stage as a possible cancer hallmark Paradoxical roles of autophagy in different stages of tumorigenesis: protector for normal or cancer cells Artesunateinduced ATG5-related autophagy enhances the cytotoxicity of NK92 cells on endometrial cancer cells via interactions between CD155 and CD226/TIGIT Artesunate alleviates diabetic retinopathy by activating autophagy via the regulation of AMPK/SIRT1 pathway Artemisinin-type drugs for the treatment of hematological malignancies Targeting autophagy enhances the anticancer effect of artemisinin and its derivatives Artesunate alleviates liver fibrosis by regulating ferroptosis signaling pathway Ferroptotic agentinduced endoplasmic reticulum stress response plays a pivotal role in the autophagic process outcome Artesunate impairs growth in cisplatin-resistant bladder cancer cells by cell cycle arrest, apoptosis and autophagy induction Artesunate induces necrotic cell death in schwannoma cells Crohn's disease: potential drugs for modulation of autophagy Autophagy: a new target or an old strategy for the treatment of Crohn's disease? Autophagy contributes to the induction of anti-TNF induced macrophages Tumor necrosis factor-alpha regulates photoreceptor cell autophagy after retinal detachment Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein The anticancer drug imatinib induces cellular autophagy A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications Imatinib induces autophagy via upregulating XIAP in GIST882 cells Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2 Is autophagy involved in the diverse effects of antidepressants? Cells Association between antidepressant use and reduced risk of intubation or death in hospitalized patients with COVID-19: results from an observational study. Mol Psychiatry Effect of early treatment with fluvoxamine on risk of emergency care and hospitalization among patients with COVID-19: the TOGETHER randomized platform clinical trial Fluvoxamine vs placebo and clinical deterioration in outpatients with symptomatic COVID-19: a randomized clinical trial The serotonin reuptake inhibitor Fluoxetine inhibits SARS-CoV-2 in human lung tissue Peripheral alterations in cytokine and chemokine levels after antidepressant drug treatment for major depressive disorder: systematic review and meta-analysis The pathogenesis and treatment of the 'Cytokine Storm' in COVID-19 Antidepressants encounter autophagy in neural cells Antidepressants act by inducing autophagy controlled by sphingomyelinceramide Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells wonder drug' from Japan: the human use perspective Ivermectin: an anthelmintic, an insecticide, and much more The mechanisms of action of ivermectin against SARS-CoV-2: an evidence-based clinical review article Progress in understanding the molecular mechanisms underlying the antitumour effects of ivermectin Ivermectin confers its cytotoxic effects by inducing AMPK/mTOR-mediated autophagy and DNA damage ROS accumulation contributes to abamectin-induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells Ivermectin induces cytostatic autophagy by blocking the PAK1/Akt axis in breast cancer How to cite this article: Rein T. Harnessing autophagy to fight SARS-CoV-2: An update in view of recent drug development efforts