Abstract

Senolysis holds promise for geroprotection but is limited by efficacy and safety; here we show that Celastrol, a pentacyclic triterpenoid, surpasses benchmark agents ABT-263 and fisetin in senolytic potency and elucidate its mechanism and a prodrug strategy to improve safety. Using stress- and replication-induced senescent cells, we demonstrate that Celastrol selectively triggers intrinsic apoptosis—evidenced by viability assays, Annexin V/PI, cleaved caspase-3 and blockade by the pan-caspase inhibitor Z-VAD-FMK—while ferroptosis is excluded by specific inhibitors. Proteomic, co-immunoprecipitation/mass spectrometry, biolayer interferometry, ubiquitination assays and RNAi identify Hsc70 as a binding partner; Celastrol disrupts an Hsc70–Bim–CHIP complex, reduces Bim ubiquitination and stabilizes Bim protein, and Bim knockdown attenuates caspase activation and senolysis. In vivo, Celastrol reduces intestinal senescence and extends Drosophila median and maximum lifespan, and mitigates bleomycin- and CCl₄-induced pulmonary and hepatic fibrosis in mice with increased cleaved caspase-3 in p16⁺ cells. A β-galactosidase-activated prodrug (CeGal) preserves efficacy, preferentially releases Celastrol in β-galactosidase-high cells, and markedly reduces systemic toxicity, supporting clinical translation of this targeted senolytic approach.