title: Phenolic 1,3-diketones attenuate lipopolysaccharide-induced inflammatory response by an alternative magnesium-mediated mechanism.
abstract: Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4-myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined.
  
  
  
  Binding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4-MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells.
  
  
  
  Both curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of pro-inflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4-MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9.
  
  
  
  These results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPS-mediated TLR4-MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Morena
authors.initials: M
authors.lastname: Zusso
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Mercanti
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Bologna, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Belluti
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Bologna, Italy.
authors.forename: Rita Maria Concetta
authors.initials: RMC
authors.lastname: Di Martino
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pagetta
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Marinelli
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Brun
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Ragazzi
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
authors.forename: Rita
authors.initials: R
authors.lastname: Lo
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
authors.forename: Stefano
authors.initials: S
authors.lastname: Stifani
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Giusti
authors.affiliation: Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Moro
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D007249:Inflammation / Q000188:drug therapy* / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000037:antagonists & inhibitors / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008297:Male; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D016207:Cytokines; D007659:Ketones; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D008274:Magnesium