Blockage of Autophagy for Cancer Therapy: A Comprehensive Review.

The incidence and mortality of cancer are increasing, making it a leading cause of death worldwide. Conventional treatments such as surgery, radiotherapy, and chemotherapy face significant limitations due to therapeutic resistance. Autophagy, a cellular self-degradation mechanism, plays a crucial role in cancer development, drug resistance, and treatment. This review investigates the potential of autophagy inhibition as a therapeutic strategy for cancer. A systematic search was conducted on Embase, PubMed, and Google Scholar databases from 1967 to 2024 to identify studies on autophagy inhibitors and their mechanisms in cancer therapy. The review includes original articles utilizing in vitro and in vivo experimental methods, literature reviews, and clinical trials. Key terms used were "Autophagy", "Inhibitors", "Molecular mechanism", "Cancer therapy", and "Clinical trials". Autophagy inhibitors such as chloroquine (CQ) and hydroxychloroquine (HCQ) have shown promise in preclinical studies by inhibiting lysosomal acidification and preventing autophagosome degradation. Other inhibitors like wortmannin and SAR405 target specific components of the autophagy pathway. Combining these inhibitors with chemotherapy has demonstrated enhanced efficacy, making cancer cells more susceptible to cytotoxic agents. Clinical trials involving CQ and HCQ have shown encouraging results, although further investigation is needed to optimize their use in cancer therapy. Autophagy exhibits a dual role in cancer, functioning as both a survival mechanism and a cell death pathway. Targeting autophagy presents a viable strategy for cancer therapy, particularly when integrated with existing treatments. However, the complexity of autophagy regulation and the potential side effects necessitate further research to develop precise and context-specific therapeutic approaches.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Novel combinatorial autophagy inhibition therapy for triple negative breast cancers.

BACKGROUND: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. METHODS: Immunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. RESULTS: Metabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. CONCLUSION: In this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Inhibition of autophagy initiation: A novel strategy for oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer and is known to have poor prognostic outcomes. Autophagy is known to be associated with aggressive tumor biology of OSCC. Hence, this study aimed to develop a novel therapeutic strategy against OSCC by targeting the autophagic pathway. METHODS: Immunoblotting, and confocal microscopy were used to examine the effect of tumor microenvironmental stressors on the autophagy activity. Cellular proliferation and migration assays were performed to assess the anti-cancer activity of standard chemotherapy and autophagy initiation inhibitors, either alone or in combination. High resolution mass-spectrometry based proteomic analysis was utilized to understand the mechanisms behind chemoresistance in OSCC models. Finally, immunohistochemistry was performed to determine associations between autophagy markers and clinicopathological characteristics. RESULTS: Tumor microenvironmental stressors were shown to induce autophagy activity in OSCC cell lines. Novel combinations of chemotherapy and autophagy inhibitors as well as different classes of autophagy inhibitors were identified. Combination of MRT68921 and SAR405 demonstrated marked synergy in their anti-proliferative activity and also showed synergy with chemotherapy in chemoresistant OSCC cell models. Autophagy was identified as one of the key pathways involved in mediating chemoresistance in OSCC. Furthermore, TGM2 was identified as a key upstream regulator of chemoresistance in OSCC models. Finally, positive staining for autophagosome marker LC3 was shown to be associated with low histological grade OSCC. CONCLUSION: In conclusion, this study identified a combination of novel autophagy inhibitors which can potently inhibit proliferation of both chemosensitive as well as chemoresistant OSCC cells and could be developed as a novel therapy against advanced OSCC tumors.

Unraveling the Janus-Faced Role of Autophagy in Hepatocellular Carcinoma: Implications for Therapeutic Interventions.

This review aims to provide a comprehensive understanding of the molecular mechanisms underlying autophagy and mitophagy in hepatocellular carcinoma (HCC). Autophagy is an essential cellular process in maintaining cell homeostasis. Still, its dysregulation is associated with the development of liver diseases, including HCC, which is one of leading causes of cancer-related death worldwide. We focus on elucidating the dual role of autophagy in HCC, both in tumor initiation and progression, and highlighting the complex nature involved in the disease. In addition, we present a detailed analysis of a small subset of autophagy- and mitophagy-related molecules, revealing their specific functions during tumorigenesis and the progression of HCC cells. By understanding these mechanisms, we aim to provide valuable insights into potential therapeutic strategies to manipulate autophagy effectively. The goal is to improve the therapeutic response of liver cancer cells and overcome drug resistance, providing new avenues for improved treatment options for HCC patients. Overall, this review serves as a valuable resource for researchers and clinicians interested in the complex role of autophagy in HCC and its potential as a target for innovative therapies aimed to combat this devastating disease.

Pharmacotherapy developments in autophagy inhibitors for bladder cancer.

INTRODUCTION: Autophagy is an intracellular process that plays a key role in the cellular homeostasis. Recently, it has been described as a potential therapeutic target in oncology, whether by activating or inhibiting its different cascades. Autophagy inhibitors interact with different molecular processes of the hallmarks of cancer. AREAS COVERED: Multiple proteins of the autophagy cascade could be aimed by specific inhibitors in many tumors, notably bladder cancer. In fact, bladder cancer has been increasing in prevalence over the last decade, and resistance to conventional treatment has been extensively reported in the literature. Autophagy inhibitors in bladder cancer have been described in preclinical studies to increase the sensitivity of the tumor to chemotherapy and radiotherapy. This paper is a review of the literature, which selected randomized trials, cohort studies, and case-control studies documenting the relationship between autophagy inhibitors and bladder cancer treatment. EXPERT OPINION: Autophagy is a promising pathway for cancer cell targeting that opens the horizons for a potential new therapeutic area in particular the multidisciplinary management of bladder cancer.

Autophagy up-regulation upon FeHV-1 infection on permissive cells.

FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.

Autophagy inhibitors for cancer therapy: Small molecules and nanomedicines.

Autophagy is a conserved process in which the cytosolic materials are degraded and eventually recycled for cellular metabolism to maintain homeostasis. The dichotomous role of autophagy in pathogenesis is complicated. Accumulating reports have suggested that cytoprotective autophagy is responsible for tumor growth and progression. Autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), are promising for treating malignancies or overcoming drug resistance in chemotherapy. With the rapid development of nanotechnology, nanomaterials also show autophagy-inhibitory effects or are reported as the carriers delivering autophagy inhibitors. In this review, we summarize the small-molecule compounds and nanomaterials inhibiting autophagic flux as well as the mechanisms involved. The nanocarrier-based drug delivery systems for autophagy inhibitors and their distinct advantages are also described. The progress of autophagy inhibitors for clinical applications is finally introduced, and their future perspectives are discussed.

Autophagy and Breast Cancer: Connected in Growth, Progression, and Therapy.

Despite an increase in the incidence of breast cancer worldwide, overall prognosis has been consistently improving owing to the development of multiple targeted therapies and novel combination regimens including endocrine therapies, aromatase inhibitors, Her2-targeted therapies, and cdk4/6 inhibitors. Immunotherapy is also being actively examined for some breast cancer subtypes. This overall positive outlook is marred by the development of resistance or reduced efficacy of the drug combinations, but the underlying mechanisms are somewhat unclear. It is interesting to note that cancer cells quickly adapt and evade most therapies by activating autophagy, a catabolic process designed to recycle damaged cellular components and provide energy. In this review, we discuss the role of autophagy and autophagy-associated proteins in breast cancer growth, drug sensitivity, tumor dormancy, stemness, and recurrence. We further explore how autophagy intersects and reduces the efficacy of endocrine therapies, targeted therapies, radiotherapy, chemotherapies as well as immunotherapy via modulating various intermediate proteins, miRs, and lncRNAs. Lastly, the potential application of autophagy inhibitors and bioactive molecules to improve the anticancer effects of drugs by circumventing the cytoprotective autophagy is discussed.

Natural Compounds Targeting the Autophagy Pathway in the Treatment of Colorectal Cancer.

Autophagy is a highly conserved intracellular degradation pathway by which misfolded proteins or damaged organelles are delivered in a double-membrane vacuolar vesicle and finally degraded by lysosomes. The risk of colorectal cancer (CRC) is high, and there is growing evidence that autophagy plays a critical role in regulating the initiation and metastasis of CRC; however, whether autophagy promotes or suppresses tumor progression is still controversial. Many natural compounds have been reported to exert anticancer effects or enhance current clinical therapies by modulating autophagy. Here, we discuss recent advancements in the molecular mechanisms of autophagy in regulating CRC. We also highlight the research on natural compounds that are particularly promising autophagy modulators for CRC treatment with clinical evidence. Overall, this review illustrates the importance of autophagy in CRC and provides perspectives for these natural autophagy regulators as new therapeutic candidates for CRC drug development.

Modifications of the PI3K/Akt/mTOR axis during FeHV-1 infection in permissive cells.

FeHV-1 is the causative agent of infectious rhinotracheitis in cats. The relationship between viral infection and the PI3K/Akt/mTOR pathway, as well as its function in crucial physiological processes like as autophagy, apoptosis or the IFN induction cascade is known for other varicelloviruses. However, there is no information on whether autophagy is activated during FeHV-1 infection nor on how this infection modifies PI3K/Akt/mTOR pathway. In this work, we aim to elucidate the involvement of this pathway during cytolytic infection by FeHV-1 in permissive cell lines. Using a phenotypic approach, the expression of proteins involved in the PI3K/Akt/mTOR pathway was examined by Western blot analysis. The findings demonstrated the lack of modifications in relation to viral dose (except for phospho-mTOR), whereas there were changes in the expression of several markers in relation to time as well as a mismatch in the time of activation of this axis. These results suggest that FeHV-1 may interact independently with different autophagic signaling pathways. In addition, we found an early phosphorylation of Akt, approximately 3 h after infection, without a concomitant decrease in constitutive Akt. This result suggests a possible role for this axis in viral entry. In a second phase, the use of early autophagy inhibitors was examined for viral yield, cytotoxic effects, viral glycoprotein expression, and autophagy markers and resulted in inefficient inhibition of viral replication (12 h post-infection for LY294002 and 48 h post-infection for 3-methyladenine). The same markers were examined during Akt knockdown, and we observed no differences in viral replication. This result could be explained by the presence of a protein kinase in the FeHV-1 genome (encoded by the Us3 gene) that can phosphorylate various Akt substrates as an Akt surrogate, as has already been demonstrated in genetically related viruses (HSV-1, PRV, etc.). For the same reasons, the use of LY294002 at the beginning of infection did not affect FeHV-1-mediated Akt phosphorylation. Our findings highlight changes in the PI3K/Akt/mTOR pathway during FeHV-1 infection, although further research is needed to understand the importance of these changes and how they affect cellular processes and viral propagation.

Recent Update and Drug Target in Molecular and Pharmacological Insights into Autophagy Modulation in Cancer Treatment and Future Progress.

Recent evidence suggests that autophagy is a governed catabolic framework enabling the recycling of nutrients from injured organelles and other cellular constituents via a lysosomal breakdown. This mechanism has been associated with the development of various pathologic conditions, including cancer and neurological disorders; however, recently updated studies have indicated that autophagy plays a dual role in cancer, acting as a cytoprotective or cytotoxic mechanism. Numerous preclinical and clinical investigations have shown that inhibiting autophagy enhances an anticancer medicine's effectiveness in various malignancies. Autophagy antagonists, including chloroquine and hydroxychloroquine, have previously been authorized in clinical trials, encouraging the development of medication-combination therapies targeting the autophagic processes for cancer. In this review, we provide an update on the recent research examining the anticancer efficacy of combining drugs that activate cytoprotective autophagy with autophagy inhibitors. Additionally, we highlight the difficulties and progress toward using cytoprotective autophagy targeting as a cancer treatment strategy. Importantly, we must enable the use of suitable autophagy inhibitors and coadministration delivery systems in conjunction with anticancer agents. Therefore, this review briefly summarizes the general molecular process behind autophagy and its bifunctional role that is important in cancer suppression and in encouraging tumor growth and resistance to chemotherapy and metastasis regulation. We then emphasize how autophagy and cancer cells interacting with one another is a promising therapeutic target in cancer treatment.

Unique guanidine-conjugated catechins from the leaves of Alchornea rugosa and their autophagy modulating activity.

Natural guanidines, molecules that contain the guanidine moiety, are structurally unique and often exhibit potent biological activities. A phytochemical investigation of the leaves of Alchornea rugosa (Lour.) Müll.Arg. by MS/MS-based molecular networking revealed eight undescribed guanidine-flavanol conjugates named rugonines A-H. The chemical structures of the isolated compounds were comprehensively elucidated by NMR spectroscopy, HRESIMS, and circular dichroism (CD) analysis. All isolated compounds were tested for autophagosome formation in HEK293 cells stably expressing GFP-LC3. The results revealed that compounds rugonines D-G showed potential autophagy inhibitory activity.

The SAR and action mechanisms of autophagy inhibitors that eliminate drug resistance.

Autophagy is an essential homeostatic and catabolic process crucial for the degradation or recycling of proteins and cellular components. Drug resistance has been demonstrated to be closely implicated in increased autophagy. Autophagy inhibition to reverse drug resistance involves in the five stages of autophagy, including phagophore initiation, vesicle nucleation, vesicle elongation, vesicle fusion and cargo degradation. Herein, emphases were placed on discussions on the targets responsible for the upstream phagophore initiation and nucleation of autophagosome, as well as the ones mediating the downstream autophagosome and lysosome fusion and cargo degradation. The structure-activity relationships (SARs) and action mechanisms of the corresponding target-based small molecule autophagy inhibitors were analyzed and delineated. This review will provide a promising guidance for the design and optimization of drug-like scaffolds in the discovery of autophagy inhibitors able to eliminate drug resistance.

Inhibition of Autophagy Negates Radiofrequency-Induced Adaptive Response in SH-SY5Y Neuroblastoma Cells.

In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.

An Autophagy-Related Gene Signature can Better Predict Prognosis and Resistance in Diffuse Large B-Cell Lymphoma.

Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous disease, and about 30%-40% of patients will develop relapsed/refractory DLBCL. In this study, we aimed to develop a gene signature to predict survival outcomes of DLBCL patients based on the autophagy-related genes (ARGs). Methods: We sequentially used the univariate, least absolute shrinkage and selector operation (LASSO), and multivariate Cox regression analyses to build a gene signature. The Kaplan-Meier curve and the area under the receiver operating characteristic curve (AUC) were performed to estimate the prognostic capability of the gene signature. GSEA analysis, ESTIMATE and ssGSEA algorithms, and one-class logistic regression were performed to analyze differences in pathways, immune response, and tumor stemness between the high- and low-risk groups. Results: Both in the training cohort and validation cohorts, high-risk patients had inferior overall survival compared with low-risk patients. The nomogram consisted of the autophagy-related gene signature, and clinical factors had better discrimination of survival outcomes, and it also had a favorable consistency between the predicted and actual survival. GSEA analysis found that patients in the high-risk group were associated with the activation of doxorubicin resistance, NF-κB, cell cycle, and DNA replication pathways. The results of ESTIMATE, ssGSEA, and mRNAsi showed that the high-risk group exhibited lower immune cell infiltration and immune activation responses and had higher similarity to cancer stem cells. Conclusion: We proposed a novel and reliable autophagy-related gene signature that was capable of predicting the survival and resistance of patients with DLBCL and could guide individualized treatment in future.

Autophagy: A Key Player in Pancreatic Cancer Progression and a Potential Drug Target.

Pancreatic cancer is known to have the lowest survival outcomes among all major cancers, and unfortunately, this has only been marginally improved over last four decades. The innate characteristics of pancreatic cancer include an aggressive and fast-growing nature from powerful driver mutations, a highly defensive tumor microenvironment and the upregulation of advantageous survival pathways such as autophagy. Autophagy involves targeted degradation of proteins and organelles to provide a secondary source of cellular supplies to maintain cell growth. Elevated autophagic activity in pancreatic cancer is recognized as a major survival pathway as it provides a plethora of support for tumors by supplying vital resources, maintaining tumour survival under the stressful microenvironment and promoting other pathways involved in tumour progression and metastasis. The combination of these features is unique to pancreatic cancer and present significant resistance to chemotherapeutic strategies, thus, indicating a need for further investigation into therapies targeting this crucial pathway. This review will outline the autophagy pathway and its regulation, in addition to the genetic landscape and tumor microenvironment that contribute to pancreatic cancer severity. Moreover, this review will also discuss the mechanisms of novel therapeutic strategies that inhibit autophagy and how they could be used to suppress tumor progression.

Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy.

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.

Autophagy: A promising target for triple negative breast cancers.

Triple negative breast cancer (TNBC) is the most aggressive type of breast cancers which constitutes about 15% of all breast cancer cases and characterized by negative expression of hormonal receptors and human epidermal growth factor receptor 2 (HER2). Thus, endocrine and HER2 targeted therapies are not effective toward TNBCs, and they mainly rely on chemotherapy and surgery for treatment. Despite recent advances in chemotherapy, 40% of TNBC patients develop a metastatic relapse and recurrence. Therefore, understanding the molecular profile of TNBC is warranted to identify targets that can be selected for the development of a new and effective therapeutic approach. Autophagy is an internal defensive mechanism that allows the cells to survive under different stressors. It has been well known that autophagy exerts a crucial role in cancer progression. The critical role of autophagy in TNBC progression is emerging in recent years. This review will discuss autophagic pathway, how autophagy affects TNBC progression and recent therapeutic approaches that can target autophagy as a new treatment modality.

Targeting autophagy in disease: established and new strategies.

Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible.Abbreviations: ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.