title: A cell-based quantitative high-throughput image screening identified novel autophagy modulators.
abstract: Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: McGreal
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Zhao
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, 200030 China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, 200030 China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhong
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA. Electronic address: wxding@kumc.edu.
authors.forename: Wen-Xing
authors.initials: WX
authors.lastname: Ding
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D001681:Biological Assay; D002746:Chlorpromazine / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005476:Fluphenazine / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007189:Indans / Q000494:pharmacology; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011954:Receptors, Dopamine / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D018492:Dopamine Antagonists; D007189:Indans; D008744:Methylamines; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D011954:Receptors, Dopamine; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C418119:indatraline; D058570:TOR Serine-Threonine Kinases; D005476:Fluphenazine; D002746:Chlorpromazine
keywords: Autophagy; Dopamine receptor; GFP-LC3; High-throughput screening; mTOR