title: EquiNox2: A new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme.
abstract: Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacological tool, to determine the direct interaction of potential inhibitors with equine phagocytic NOX and their effect on the enzyme activity or assembly. This method consists in binding the membrane fractions of neutrophils containing flavocytochrome b558 or the entire complex, reconstituted in vitro from membrane and cytosolic fractions of PMNs, onto the wells of a microplate followed by incubation with potential inhibitors or drugs. After incubation, the excess of the drug is simply eliminated or washed prior measuring the activity of the reconstituted complex. This latter step avoid the risk of interference between the inhibitor and the revelation solution and can distinguish if inhibitors, strongly bound or not, could interfere with the assembly of the enzymatic complex or with its activity. The EquiNox2 was validated using diphenyliodonium chloride and Gp91ds-tat, two well-known inhibitors largely described for human NADPH oxidase. The present technique was used to study and understand better the effect of curcumin and its water-soluble derivative, NDS27, on the assembly and activity of NOX. We demonstrated that curcumin and NDS27 can strongly bind to the enzyme and prevents its assembly making these molecules good candidates for the treatment of horse or human pathologies implying an excessive activation of neutrophils.
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium. Electronic address: sderochette@ulg.ac.be.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Derochette
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium; Faculty of Veterinary Medicine, Equine Clinic, B41, University of Liège, Boulevard de Colonster 20, B-4000 Liège, Belgium.
authors.forename: Didier
authors.initials: D
authors.lastname: Serteyn
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
authors.forename: Ange
authors.initials: A
authors.lastname: Mouithys-Mickalad
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
authors.forename: Justine
authors.initials: J
authors.lastname: Ceusters
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
authors.forename: Ginette
authors.initials: G
authors.lastname: Deby-Dupont
authors.affiliation: Faculty of Pharmacy, Laboratory of Medicinal Chemistry, B36, University of Liège, Avenue de l'Hôpital,1, B-4000 Liège, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Neven
authors.affiliation: Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium; Faculty of Veterinary Medicine, Equine Clinic, B41, University of Liège, Boulevard de Colonster 20, B-4000 Liège, Belgium.
authors.forename: Thierry
authors.initials: T
authors.lastname: Franck
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003474:Curcumin / Q000737:chemistry / Q000494:pharmacology; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D006736:Horses; D049408:Luminescent Agents / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology; D011485:Protein Binding
chemical_list: D003573:Cytochrome b Group; D049408:Luminescent Agents; D008565:Membrane Proteins; C036888:cytochrome b558; D019255:NADPH Oxidases; D003474:Curcumin
keywords: Cell-free system; Curcumin; Cyclodextrin; Diphenyliodonium chloride; Gp91-ds-tat; NDS27