EquiNox2: A new method to measure XXXX oxidase activity and to study effect of inhibitors and their interactions with the enzyme.

@entity1 @entity2233 @entity1641 @entity26 @entity6108 @entity1110 @entity224
Excessive neutrophil stimulation and reactive @entity26 species (ROS) production are involved in numerous @entity1 or @entity1641 pathologies. The modulation of the neutrophil @entity1110 oxidase (NOX) has a great therapeutic potential since this enzyme produces @entity224 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 @entity1641 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 @entity2233 and Gp91ds-tat, two well-known inhibitors largely described for @entity1 @entity1110 oxidase. The present technique was used to study and understand better the effect of @entity6108 and its water-soluble derivative, NDS27, on the assembly and activity of NOX. We demonstrated that @entity6108 and NDS27 can strongly bind to the enzyme and prevents its assembly making these molecules good candidates for the treatment of @entity1641 or @entity1 pathologies implying an excessive activation of neutrophils.
