Abstract 303: Discovery of autophagy-inhibiting compounds as a novel class of senolytics

Abstract Introduction: Senolytics are compounds capable of selectively eliminating senescent cells, holding promise for therapies targeting age-related diseases and cancer. To identify novel senolytic agents, we screened a chemical library using cellular models of replicative senescence induced by replication stress. This screen yielded several hits, including MCOPPB, a known nociceptin receptor agonist. Subsequent in vivo studies in a murine model confirmed the potent senolytic activity of MCOPPB. Surprisingly, further investigations revealed that this activity was independent of nociceptin receptor signaling, suggesting an off-target mechanism. Methods: To explore the underlying mechanism, we performed a cell-painting assay, which phenotypically links compounds based on their cellular effects. MCOPPB clustered closely with another senolytic hit from the screen, Daurisolin, a natural autophagy inhibitor. Autophagy inhibition by MCOPPB was validated through assays measuring LC3 accumulation and other markers. Additional studies with known autophagy inhibitors, including Dauricin and Chloroquine, demonstrated both senolytic activity and clustering with MCOPPB in the cell-painting assay. Results: Our findings establish autophagy inhibition as the common mechanism of senolytic action for this newly identified class of compounds. MCOPPB, Daurisolin, and related inhibitors displayed consistent phenotypes in cell-painting assays and induced selective clearance of senescent cells. The clustering of these compounds in phenotypic assays provides a powerful approach for identifying senolytics with similar mechanisms of action. Conclusion: This study highlights autophagy inhibition as a novel senolytic mechanism and identifies a unique chemical cluster of autophagy-inhibiting senolytic compounds. These insights could drive the development of targeted therapies for conditions associated with cellular senescence. Acknowledgements: This work was supported by the Technology agency of the Czech Republic project PERMED: T2BA (TN02000109) and the National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102). Infrastructure was supported by projects CZ-OPENSCREEN (LM2023052), EATRIS-CZ (LM2023053) and Czech Bioimaging (LM2023050) Citation Format: Martin Mistrik, Matthew Lacey, Lucie Beresova, Alzbeta Srovnalova, Josef Srovnal, Petr Dzubak, Marian Hajduch. Discovery of autophagy-inhibiting compounds as a novel class of senolytics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 303.