Celastrol Targets Hsc70-Bim Interaction as a Novel Senolytic to Extend Lifespan and Mitigate Organ Fibrosis.

Celastrol demonstrates superior senolytic potency compared to benchmark agents ABT-263 and fisetin in senescent cells.

• Celastrol was tested against both stress-induced and replication-induced senescent cells.
• Senolytic activity was assessed using viability assays comparing Celastrol directly to ABT-263 and fisetin.
• Celastrol was described as surpassing benchmark agents ABT-263 and fisetin in senolytic potency.

Celastrol selectively triggers intrinsic apoptosis in senescent cells while ferroptosis is excluded as a mechanism.

• Apoptosis was evidenced by viability assays, Annexin V/PI staining, and cleaved caspase-3 detection.
• Apoptosis was blocked by the pan-caspase inhibitor Z-VAD-FMK, confirming caspase-dependent intrinsic apoptosis.
• Ferroptosis was excluded as a mechanism using specific ferroptosis inhibitors.
• The apoptotic pathway was characterized as intrinsic (mitochondrial) rather than extrinsic.

Celastrol disrupts an Hsc70-Bim-CHIP complex, reduces Bim ubiquitination, and stabilizes Bim protein in senescent cells.

• Hsc70 was identified as a binding partner of Celastrol using proteomic analysis, co-immunoprecipitation/mass spectrometry, and biolayer interferometry.
• Ubiquitination assays demonstrated that Celastrol reduces Bim ubiquitination.
• Celastrol was shown to disrupt the Hsc70-Bim-CHIP complex, leading to Bim protein stabilization.
• RNAi-mediated Bim knockdown attenuated caspase activation and senolysis, confirming Bim's mechanistic role.

Celastrol reduces intestinal senescence and extends both median and maximum lifespan in Drosophila.

• In vivo studies were conducted in Drosophila melanogaster.
• Celastrol treatment reduced intestinal senescence in Drosophila.
• Both median and maximum lifespan were extended with Celastrol treatment.
• These findings support geroprotective activity of Celastrol in a whole-organism model.

Celastrol mitigates bleomycin-induced pulmonary fibrosis and CCl4-induced hepatic fibrosis in mice with increased cleaved caspase-3 in p16+ cells.

• Mouse models of organ fibrosis were used: bleomycin-induced pulmonary fibrosis and CCl4-induced hepatic fibrosis.
• Celastrol treatment resulted in mitigation of fibrosis in both pulmonary and hepatic models.
• Increased cleaved caspase-3 was observed specifically in p16+ (senescent) cells in treated animals, indicating selective senolysis in vivo.
• These results support the therapeutic relevance of senolysis for fibrotic diseases.

A β-galactosidase-activated prodrug of Celastrol (CeGal) preserves senolytic efficacy while markedly reducing systemic toxicity.

• CeGal was designed as a β-galactosidase-activated prodrug strategy to improve the safety profile of Celastrol.
• CeGal preferentially releases Celastrol in β-galactosidase-high cells, which are characteristic of senescent cells.
• CeGal was shown to preserve the senolytic efficacy of the parent compound.
• Systemic toxicity was markedly reduced compared to Celastrol, supporting clinical translation.