Proteasome-dependent degradation of DAPK1 by wogonoside blocks PARthanatos and protects against ischemic stroke.

Ischemic stroke remains a leading cause of death and disability, yet effective pharmacological neuroprotection remains elusive. ROS-induced PARthanatos-driven by PARP1 hyperactivation and the mitochondrial-apoptosis-inducing factor (AIF) death axis-is a principal executor of ischemic neuronal death. However, direct PARP1 inhibition inevitably compromises essential DNA repair. Here, we identify a downstream intervention node that uncouples lethal signaling from genomic maintenance. Through phenotype-to-target deconvolution combining reverse pharmacophore mapping, bioinformatic intersection with programmed cell death genes, and integrated biophysical validation (docking, 100-ns molecular dynamics, cellular thermal shift assay, and surface plasmon resonance), we demonstrate that wogonoside (WS), a flavonoid glycoside from Scutellaria baicalensis, directly binds death-associated protein kinase 1 (DAPK1; KD = 6.07 μM) and acts as a ligand-induced degrader that triggers proteasome-dependent DAPK1 clearance. CRISPR/Cas9-mediated DAPK1 knockout fully phenocopied WS-mediated protection, whereas ectopic DAPK1 overexpression abolished it. WS-induced degradation intercepted the JNK-mitochondrial depolarization-AIF nuclear translocation cascade while leaving PARP1 catalytic activity, PAR polymer formation, and γH2AX resolution fully intact-in stark contrast to the clinical PARP1 inhibitor olaparib. In a mouse permanent distal middle cerebral artery occlusion model, a single dose of WS reduced cortical infarct volume by ∼50%, preserved neurons, suppressed microglial activation, and maintained microvascular integrity, accompanied by significant sensorimotor recovery. Single-nucleus RNA sequencing revealed Dapk1 enrichment in activated microglia and co-variation with neuronal MAPK pro-death signaling, suggesting a dual-compartment mechanism. These findings define proteasome-dependent DAPK1 degradation as a mechanistically distinct antioxidant strategy for acute ischemic stroke.