Covalently targeting HSP90 with a natural small-molecule costunolide to inhibit necroptosis and ulcerative colitis

Necroptosis exacerbates ulcerative colitis (UC) by disrupting intestinal barriers and promoting necroinflammation. Heat shock protein 90 (HSP90) regulates receptor-interacting protein kinase 1/3 (RIPK1/3) activity through its chaperone function, thereby participating in necroptosis and emerging as a potential therapeutic target for its inhibition in UC therapy. Costunolide (CTL), a principal sesquiterpene lactone from Aucklandia lappa, has poorly defined mechanisms in UC treatment. This study aimed to elucidate CTL's role in necroptosis inhibition and UC therapy by targeting HSP90.

Multiple cellular models were employed to assess CTL's inhibitory effects on necroptosis and necroinflammation. Subsequently, western blotting and co-immunoprecipitation (Co-IP) were used to analyze the impact of CTL on necroptosis signaling. Functional targets and binding sites of CTL were identified through drug affinity responsive target stability (DARTS), mass spectrometry, and cellular thermal shift assay (CETSA). Molecular dynamics simulation and tryptophan quenching were then conducted to elucidate CTL-mediated allosteric regulatory mechanisms. In vivo, dextran sulfate sodium (DSS)-induced colitis mice were used to evaluate the therapeutic efficacy of CTL.

CTL is identified as a novel necroptosis inhibitor that suppresses RIPK1/3-mixed lineage kinase domain-like protein (MLKL) signaling and mitigates necroinflammation in vitro. Mechanistically, CTL covalently binds to the conserved cysteine (Cys) 572/564 in the middle domain of HSP90, inhibiting its dimerization and chaperone function via an allosteric effect without significantly compromising ATPase activity or triggering substantial heat shock response. This covalent interaction further disrupts HSP90-RIPK1 association, consequently blocking RIPK1/3-MLKL necroptosis signaling. In vivo, CTL is capable of effectively alleviating DSS-induced colitis via blocking RIPK1/3-MLKL axis activation.

Our findings highlight Cys572/564 as a druggable site for allosteric HSP90 inhibition and propose that CTL acts as a promising chemical scaffold for developing therapeutics targeting necroptosis- and HSP90-driven pathologies, such as UC.