Identification of NLRP3 as a covalent target of 1,6-O, O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP)

Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative dis-eases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula bri-tannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/ NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear. Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-icB tran-scription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the alpha-methylene-gamma-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new co-valent target of 1 and 2 against neuroinflammation.

Automated summary

Neuroinflammation is a key factor in neurodegenerative diseases, and this study discovers that 1,6-O,O-diacetylbritannilactone (OABL) has the potential to reduce neuroinflammation and penetrate the blood-brain barrier. Through quantitative thiol reactivity profiling (QTRP), NLRP3 was identified as a novel covalent target of OABL, regulating cellular response to oxidative stress. Treatment with OABL was shown to reduce inflammation in the brains of 5XFAD mice.