title: NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action.
abstract: BACKGROUND
  Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood.
  
  
  METHODS
  Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins.
  
  
  RESULTS
  Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC₅₀ values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC₅₀ of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively.
  
  
  CONCLUSIONS
  These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.
authors.affiliation: Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland. vincent.jaquet@unige.ch
authors.forename: Vincent
authors.initials: V
authors.lastname: Jaquet
authors.forename: Julien
authors.initials: J
authors.lastname: Marcoux
authors.forename: Eric
authors.initials: E
authors.lastname: Forest
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Leidal
authors.forename: Sally
authors.initials: S
authors.lastname: McCormick
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Westermaier
authors.forename: Remo
authors.initials: R
authors.lastname: Perozzo
authors.forename: Olivier
authors.initials: O
authors.lastname: Plastre
authors.forename: Laetitia
authors.initials: L
authors.lastname: Fioraso-Cartier
authors.forename: Becky
authors.initials: B
authors.lastname: Diebold
authors.forename: Leonardo
authors.initials: L
authors.lastname: Scapozza
authors.forename: William M
authors.initials: WM
authors.lastname: Nauseef
authors.forename: Franck
authors.initials: F
authors.lastname: Fieschi
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Krause
authors.forename: Karen
authors.initials: K
authors.lastname: Bedard
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002461:Cell Line, Transformed; D006224:Cricetinae; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D053978:Pentacyclic Triterpenes; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014315:Triterpenes / Q000494:pharmacology*; D018909:src Homology Domains / Q000187:drug effects
chemical_list: D009861:Onium Compounds; D053978:Pentacyclic Triterpenes; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D014315:Triterpenes; D013481:Superoxides; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; C050414:celastrol; D010100:Oxygen