autophagy protease tag-compound addition and cellular supernatant harvest for drug screening

Hochdurchsatz-Screening-Analyse für 4B-Cystein-Peptidase-Autophagie-Inhibitoren

Autophagy is a conserved metabolic process that allows cells to keep intracellular homeostasis and respond to stress by degrading aged, defective, or unnecessary cellular contents via the lysosomes1,2,3. Under some pathophysiologic conditions, this process acts as a crucial cellular response to nutrient and oxygen deprivation, resulting in recycled nutrients and lipids, allowing the cells to adapt to their metabolic needs2,3,4. Autophagy has also been identified as a cellular stress response related to several diseases, such as neurodegenerative disorders, pathogen infection, and various types of cancer. The function of autophagy in cancer is complex and dependent on the type, stage, and status of the tumor. It can suppress tumorigenesis through autophagic degradation of damaged cells, but can also promote the survival of advanced tumors by improving cell survival during stressful conditions, such as hypoxia, nutrient deprivation, and cytotoxic damage2,4,5,6.
Several studies have shown that autophagy inhibition provides a benefit as an anticancer strategy. Thus, the inhibition of critical steps, such as autophagosome formation or its fusion with the lysosome, could be an effective method for cancer control2,4,5,6. Growing evidence has shown that ATG4B is involved in certain pathological conditions, and it has gained attention as a potential anticancer target2,3,4. For instance, it was observed that colorectal cancer cells and human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells had significantly higher ATG4B expression levels than adjacent normal cells2,4. In prostate cancer cells, inhibition of ATG4B resulted in a cell line-specific susceptibility to chemotherapy and radiotherapy7. Recently, strong evidence has emerged that pancreatic ductal adenocarcinoma (PDAC) is particularly vulnerable to ATG4B inhibition. For instance, in a genetically engineered mouse model, it was shown that intermittent loss of ATG4B function reduces PDAC tumor growth and increases survival3,4. Overall, ATG4B is highly overexpressed in some cancer types, is related to the progression of tumor, and is linked to cancer therapy resistance2,4,8.
The ATG4 cysteine proteases in mammals have four family members, ATG4A-ATG4D. These proteins exhibit some target selectivity toward the LC3/GABARAP (ATG8) family of proteins9,10,11 and may have additional functions not linked to their protease activity12,13. Furthermore, ATG4 functions in regulating a novel type of post-translational modification, the ATG8-ylation of proteins11,12. While ATG4B and its main substrate LC3B are the most widely studied, a picture is emerging that suggests a complex role for each subfamily member in the regulation of autophagic and non-autophagic processes. This is further corroborated by a complex network of post-translational modifications that regulate ATG4B activity via phosphorylation, acetylation, glycosylation, and nitrosylation9,10,11,12,13.
Several known ATG4B inhibitors have been published2,4,14,15. While these are suitable as research tools, their pharmacodynamic profile, selectivity, or potency have yet precluded them from development as preclinical candidates4,16. Overall, there is an urgent need to identify more potent and selective compounds. Often, the compounds are good biochemical inhibitors of protein function, yet their efficacy in cell-based assays is poor. There are multiple assays to monitor ATG4B activity, including biochemical methods and cell-based assays4. We have previously developed a simple, luminescence-based, high-throughput assay for monitoring ATG4B activity in cells8,17. This assay utilizes a luciferase protein from Gaussia princeps (GLUC) that is stable and active in the extracellular milieu and can be inducibly released from cells in response to ATG4B proteolytic activity18,19.
In this reporter construct, dNGLUC is linked to the actin cytoskeleton of cells. A protease-specific linker can be introduced between the &#946;-actin anchor and dNGLUC, turning the secretion dependent on cleavage of the linker. We used the full-length open reading frame of LC3B between &#946;-actin and dNGLUC, to be able to monitor LC3B cleavage17,18,19. Although the secretion mechanism of dNGLUC is poorly understood, it is specific for monitoring ATG4B activity, does not depend on overall autophagy as it occurs in ATG5 knockout cells, and is mediated by non-conventional mechanisms that do not require a classical signal peptide4,18,19. We have successfully used this reporter to screen small molecules and siRNA libraries, and have identified novel regulators of ATG4B activity, such as the Akt protein kinases8. This paper describes a detailed protocol for the use of this luciferase reporter in a semi-automated, high-throughput screening format.

Autor im Rampenlicht: Ein selektiver Luciferase-basierter Assay zur Überwachung der ATG4B 27-Aktivität in Zellen 

Zellbasiertes Wirkstoff-Screening auf Inhibitoren der Autophagie-verwandten 4B-Cystein-Peptidase

Watch this Scientific Journal Video about Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening at JoVE.com

Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening  

Autophagie-Protease-Tag-Compound-Addition und zelluläre Überstandsgewinnung für das Wirkstoff-Screening

Beginnen Sie mit dem Auftauen der Selleckchem Small Molecule Compound Library-Platten bei Raumtemperatur. Öffnen Sie die Software und klicken Sie auf Neues Protokoll. Klicken Sie auf der Registerkarte Protokoll auf Probenplattenformat, gefolgt von 384PP.Wählen Sie dann unter Musterplattentyp die Option 384PP_DMSO2 aus. Wählen Sie als Nächstes Cell Carrier 384 Ultra PN als Zielplattentyp aus. Klicken Sie auf Auswahlliste und wählen Sie die Importoptionen aus, um die Tabelle mit dem Dosierprogramm zu importieren.Klicken Sie auf die Option Protokoll ausführen, um die Richtigkeit der angezeigten Informationen zu überprüfen, und klicken Sie dann auf Ausführen. Klicken Sie anschließend im neuen Fenster mit dem Titel Ausführungsstatus auf Start, und führen Sie dann die Schritte aus, die in den Eingabeaufforderungsfenstern angezeigt werden. Verwenden Sie einen akustischen Flüssigkeitsspender im Nanomaßstab und geben Sie 50 Nanoliter der Small Molecule Compound Library pro Well in eine 384-Well-Assay-Platte.Um mit der Ernte von zellulären Überständen zu beginnen, konfigurieren Sie zunächst das Deck der Laborautomatisierungs-Workstation. Platzieren Sie ein Einweg-Rack mit 96 Spitzen auf der Position P1 und platzieren Sie die Assay-Platten auf den Positionen P2 und P4. Zum Schluss platzieren Sie die leeren, massiven schwarzen 384-Well-Platten an den Positionen P3 und P5. Mit den Spitzen von P1 werden 10 Mikroliter des Überstandes von der Platte bei P2 abgesaugt und dann in die leere feste schwarze Platte auf P3 überführt. Werfen Sie die Spitzen an Position P6 ab. Wiederholen Sie den Transfer des Überstandes von P4 auf P5.

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Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening

93 Aufrufe. Beginnen Sie mit dem Auftauen der Selleckchem Small Molecule Compound Library-Platten bei Raumtemperatur. Öffnen Sie die Software und klicken Sie auf Neues Protokoll. Klicken Sie auf der Registerkarte Protokoll auf Probenplattenformat, gefolgt von 384PP.Wählen Sie dann unter Musterplattentyp die Option 384PP_DMSO2 aus. Wählen Sie als Nächstes Cell Carrier 384 Ultra PN als Zielplattentyp aus. Klicken Sie auf Auswahlliste und wählen Sie die Importoptionen aus, um die Tabelle mit dem...

Serie: Autophagy Protease Tag-Compound Addition and Cellular Supernatant Harvest for Drug Screening  

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...