Caspase Recruitment Domain 6 <b>(</b> CARD6) is the therapeutic target of dihydroartemisinin for lupus nephritis based on Proteolysis Targeting Chimera (PROTAC) technology

Background and Purpose Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) that symbolizes a primary contributor of morbidity and mortality. Dihydroartemisinin (DHA; artenimol) shows good therapeutic effects on LN. Its pharmacological target remains unclear. Proteolysis targeting chimeras (PROTAC) technology was used to identify the targets of DHA in LN treatment. Experimental Approach The activity of a novel compound termed A1 was assessed through cytotoxicity and anti‐inflammatory assays in vitro . The target of DHA was screened by the A1 integrating with proteomics and then validated via western blotting, co‐immunoprecipitation (Co‐IP), immunofluorescence, molecular docking, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) in vitro. Twenty‐four‐hour proteinuria, inflammatory cytokines, serum biochemical indicators, immune complex deposition and renal pathological injury were detected. Adeno‐associated virus‐mediated RNA interference in silencing CARD6 was conducted in vivo to comprehensively evaluate the effect and the target of DHA on MRL/lpr mice. Key Results CARD6 was the most notably degraded protein affected by A1 that identified as the target of DHA. Specifically, DHA interacted with CORE‐CARD6 at the 481–949aa region. DHA blocked CARD6 to inhibit RIP2/NF‐κB pathway, which manifested anti‐inflammatory properties in vivo and in vitro . Silencing CARD6 ameliorated the inflammatory response of MRL/lpr mice. Notably, the inhibition effects of DHA on the MRL/lpr mice were abrogated when CARD6 was depleted. Conclusion and Implications CARD6 was a therapeutic target of DHA for LN management. This finding highlights the great potential of using PROTAC technology to effectively identify therapeutic targets of natural products.