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.
Copyright © 2015 Elsevier B.V. All rights reserved.
Publication Date: 2015-08-04 PubMed ID: 26452955DOI: 10.1016/j.talanta.2015.08.007Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The researchers developed a technique called EquiNox2 for measuring the activity of an enzyme, NADPH oxidase, and for studying the interaction between potential inhibitors and the enzyme. The technique was applied to understand the effect of curcumin, a medicinal compound, and its water-soluble derivative, NDS27, on the enzyme.
EquiNox2: A New Technique for NADPH Oxidase Research
- The paper revolves around the development of a new method called the EquiNox2 to measure the activity of the neutrophil NADPH oxidase enzyme (NOX).
- NOX, which plays a significant role in producing superoxide anion, is a derivative of reactive oxygen species (ROS); an excess of which is implicated in various human and horse diseases.
- Due to the therapeutic potential of modulating NOX, the EquiNox2 method is designed to measure direct interactions between potential inhibitors and NOX.
- The method avoids the risk of interference between the inhibitors and the probe used to measure activity, which is a common issue with other cell-free systems used for the same purpose.
- EquiNox2 was validated using diphenyliodonium chloride and Gp91ds-tat, two known inhibitors for human NOX.
EquiNox2 Application: Curcumin and NDS27
- Beyond method development, the paper also applies the technique to understand the effect of curcumin and its water-soluble derivative, NDS27, on the assembly and activity of NOX.
- The researchers found that both curcumin and NDS27 bind strongly to the enzyme, preventing its assembly.
- As curcumin and NDS27 prevent the assembly of the enzyme, they are thus identified as good candidates for the treatment of various diseases in humans and horses that are associated with an excessive activation of neutrophils.
Significance and Future Implications
- The research sheds light on the therapeutic potential of curcumin and NDS27 in mitigating the issues arising from the excessive stimulation of neutrophils.
- It also presents a technique for further investigation into NOX and its inhibitors, providing a foundation for designing more effective treatment methods for related pathologies.
Cite This Article
APA
Derochette S, Serteyn D, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Neven P, Franck T.
(2015).
EquiNox2: A new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme.
Talanta, 144, 1252-1259.
https://doi.org/10.1016/j.talanta.2015.08.007 Publication
Researcher Affiliations
- 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.
- 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.
- Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
- Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
- Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée du 6 Août 13, B-4000 Liège, Belgium.
- Faculty of Pharmacy, Laboratory of Medicinal Chemistry, B36, University of Liège, Avenue de l'Hôpital,1, B-4000 Liège, Belgium.
- 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.
MeSH Terms
- Animals
- Cell Membrane / metabolism
- Curcumin / chemistry
- Curcumin / pharmacology
- Cytochrome b Group / antagonists & inhibitors
- Cytochrome b Group / chemistry
- Cytochrome b Group / metabolism
- Enzyme Activation / drug effects
- Enzyme Assays / methods
- Horses
- Luminescent Agents / chemistry
- Membrane Proteins / metabolism
- NADPH Oxidases / antagonists & inhibitors
- NADPH Oxidases / chemistry
- NADPH Oxidases / metabolism
- Neutrophils / cytology
- Neutrophils / enzymology
- Protein Binding
Citations
This article has been cited 3 times.- Franck T, Ceusters J, Graide H, Niesten A, Duysens J, Mickalad AM, Serteyn D. Equine Muscle Derived Mesenchymal Stem Cells Loaded with Water-Soluble Curcumin: Modulation of Neutrophil Activation and Enhanced Protection against Intracellular Oxidative Attack. Int J Mol Sci 2023 Jan 5;24(2).
- Saleem N, Goswami SK. Activation of adrenergic receptor in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived ROS mediate the downstream responses. Mol Cell Biochem 2017 Dec;436(1-2):167-178.
- Stanić Z. Curcumin, a Compound from Natural Sources, a True Scientific Challenge - A Review. Plant Foods Hum Nutr 2017 Mar;72(1):1-12.
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