Affinity‐Based Protein Profiling Reveals IDH2 as a Mitochondrial Target of Cannabinol in Receptor‐Independent Neuroprotection

Phytocannabinoids are attracting growing attention because of their potential for treatment of neurodegenerative diseases. Among them, the "minor" cannabinoid, cannabinol (CBN), has emerged as a promising neuroprotective agent, acting independently of classical cannabinoid receptors through as-yet unidentified mitochondrial targets. To uncover the molecular basis of its neuroprotective effects, we designed and synthesized a chemical probe based on CBN, incorporating a minimalist diazirine linker. Functional assays confirmed that the probe retains CBN's mitochondrial activity and exhibits strong mitochondrial enrichment, as demonstrated by fluorescence microscopy and click-correlative light and electron microscopy (click-CLEM). By affinity-based protein profiling (AfBPP), we identified isocitrate dehydrogenase 2 (IDH2) as a key mitochondrial target of CBN. This finding was further substantiated by siRNA knockdown studies, which revealed that the absence of IDH2 partially phenocopies CBN's effects, validating its role as a critical mediator of CBN's neuroprotective activity.