Tesi etd-03162023-150902
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Tipo di tesi
Dottorato
Autore
AMATO, RACHELE
URN
etd-03162023-150902
Titolo
Delivery of a reliable cell-based assay for autophagy-related drug discovery study
Settore scientifico disciplinare
BIO/11
Corso di studi
Istituto di Scienze della Vita - PHD IN MEDICINA TRASLAZIONALE
Commissione
relatore GRIMALDI, BENEDETTO
Parole chiave
- assay
- autophagy
- Drug discovery
- high content imaging
- primary screening
Data inizio appello
24/10/2023;
Disponibilità
parziale
Riassunto analitico
Autophagy is a complex cellular physiological process involved in the regulation of metabolism and cellular homeostasis. This process is implicated in several pathologies, including cancer, lysosomal disorders, muscle dystrophies, neurodegeneration, inflammatory and heart diseases. Therefore, the discovery of new compounds that modulate autophagy could be useful in treating those diseases in which autophagy is the cause or consequence of pathological condition.
In this work, we developed a suitable assay for rapid screening of autophagy-affecting compounds in mammalian cells that allows the measure of cytotoxicity and EC50 of positive candidates. The autophagy reporter used in this screening platform is based on the autophagy marker LC3. LC3-containing autophagosomes accumulate in cells during induction or blockade of autophagy, allowing to quantify autophagy flux. The Operetta high-content imager and Harmony software is used to set the count conditions of GFP-LC3 spots after CQ treatment, well-known autophagy inhibitor. We measured the drug potency of CQ (2 µM) by evaluating dose-response curves and toxic effects according to the number of nuclei. From our analysis, we defined a cut-off, ≥75% of the number of nuclei, to exclude from the EC50 investigation those doses that affect autophagy but reduce the number of cells by 25%. The validation of the assay is confirmed by treating A375 GFP-LC3 cells with Lys05, a dimeric CQ, and by a compound synthesized in our laboratory, ARN1987, an autophagy blocker that shares the same potency of CQ. Assay validation is achieved using a secondary approach: western blot analysis of the expression of endogenous LC3-II, LC3 bound to GFP and p62 protein, confirmed the Operetta assay system. Toxic effects are supported by the expression of cleaved-Parp, an apoptotic marker.
Using our assay, a high content screen of 336 pure natural compounds was performed to identify compound affecting autophagy. From this screen, we identified 13 positive hits but focused on one compound, ARN1967. This compound is not toxic to A375 GFP-LC3 cells up to a concentration of 10 μM and showed an EC50 of 2.8 μM against the autophagy process. Expression analysis of GFP-LC3, LC3-II endogenous and p62 markers showed that the compound blocks autophagy by inhibiting the late-stage of autophagic flux. All data collected so far support the identification of a novel autophagy blocker, ARN1967.
In this work, we developed a suitable assay for rapid screening of autophagy-affecting compounds in mammalian cells that allows the measure of cytotoxicity and EC50 of positive candidates. The autophagy reporter used in this screening platform is based on the autophagy marker LC3. LC3-containing autophagosomes accumulate in cells during induction or blockade of autophagy, allowing to quantify autophagy flux. The Operetta high-content imager and Harmony software is used to set the count conditions of GFP-LC3 spots after CQ treatment, well-known autophagy inhibitor. We measured the drug potency of CQ (2 µM) by evaluating dose-response curves and toxic effects according to the number of nuclei. From our analysis, we defined a cut-off, ≥75% of the number of nuclei, to exclude from the EC50 investigation those doses that affect autophagy but reduce the number of cells by 25%. The validation of the assay is confirmed by treating A375 GFP-LC3 cells with Lys05, a dimeric CQ, and by a compound synthesized in our laboratory, ARN1987, an autophagy blocker that shares the same potency of CQ. Assay validation is achieved using a secondary approach: western blot analysis of the expression of endogenous LC3-II, LC3 bound to GFP and p62 protein, confirmed the Operetta assay system. Toxic effects are supported by the expression of cleaved-Parp, an apoptotic marker.
Using our assay, a high content screen of 336 pure natural compounds was performed to identify compound affecting autophagy. From this screen, we identified 13 positive hits but focused on one compound, ARN1967. This compound is not toxic to A375 GFP-LC3 cells up to a concentration of 10 μM and showed an EC50 of 2.8 μM against the autophagy process. Expression analysis of GFP-LC3, LC3-II endogenous and p62 markers showed that the compound blocks autophagy by inhibiting the late-stage of autophagic flux. All data collected so far support the identification of a novel autophagy blocker, ARN1967.
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