Caspase Recruitment Domain 6 (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.