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 diseases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula britannica 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-κB transcription 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 α-methylene-γ-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 covalent target of 1 and 2 against neuroinflammation.

1,10-Seco-Eudesmane sesquiterpenoids as a new type of anti-neuroinflammatory agents by suppressing TLR4/NF-κB/MAPK pathways.

Dysregulation of neuroinflammation is a key pathological factor in the progressive neuronal damage of neurodegenerative diseases. An in-house natural products library of 1407 compounds were screened against neuroinflammation in lipopolysaccharide (LPS)-activated microglia cells to identify a novel hit 1,6-O,O-diacetylbritannilactone (OABL) with anti-neuroinflammatory activity. Furthermore, a 1,10-seco-eudesmane sesquiterpenoid library containing 33 compounds was constructed by semisynthesis of a major component 1-O-acetylbritannilactone (ABL) from the traditional Chinese medicinal herb Inula Britannica L. Compound 15 was identified as a promising anti-neuroinflammatory agent by nitrite oxide (NO) production screening. 15 could attenuate tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) productions, and inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at a submicromolar level. Mechanistic study revealed that 15 significantly modulated TLR4/NF-kB and p38 MAPK pathways, and upregulated the anti-oxidant response HO-1. Besides, 15 promoted the conversion of the microglia from M1 to M2 phenotype by increasing levels of arginase-1 and IL-10. The structure-activity relationships (SARs) analysis indicated that the α-methylene-γ-lactone motifs, epoxidation of C5=C10 bond and bromination of C14 were important to the activity. Parallel artificial membrane permeation assay (PAMPA) also demonstrated that 15 and OABL can overcome the blood-brain barrier (BBB). In all, compound 15 is a promising anti-neuroinflammatory lead with potent anti-inflammatory effects via the blockage of TLR4/NF-κB/MAPK pathways, favorable BBB penetration property, and low cytotoxicity.