High-Throughput Screening of NOX Inhibitors. Methods Mol Biol 2019;1982:429-446
Date
06/07/2019Pubmed ID
31172487Pubmed Central ID
PMC6953630DOI
10.1007/978-1-4939-9424-3_25Scopus ID
2-s2.0-85067079084 (requires institutional sign-in at Scopus site) 10 CitationsAbstract
Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.
Author List
Zielonka J, Zielonka M, Cheng G, Hardy M, Kalyanaraman BAuthors
Gang Cheng PhD Assistant Professor in the Biophysics department at Medical College of WisconsinMicael Joel Hardy PhD Visiting Assistant Professor in the Biophysics department at Medical College of Wisconsin
Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Adenosine TriphosphateBiomarkers
Cell Culture Techniques
Cell Line
Chromatography, High Pressure Liquid
Data Interpretation, Statistical
Drug Discovery
Drug Evaluation, Preclinical
Enzyme Inhibitors
High-Throughput Screening Assays
Humans
Isoenzymes
Molecular Structure
NADPH Oxidases
Oxidation-Reduction
Phenanthridines
Quaternary Ammonium Compounds
Reactive Oxygen Species
Structure-Activity Relationship
Superoxides
