ROS Detection

ROS Detection Enzymes

Targeting Fibrotic Diseases at their Source: Enzymes

Inflammatory- and fibrotic-related diseases can be difficult to manage, and today’s therapies may only help ease the symptoms and not address factors associated with the root cause. Finding appropriate therapies that target key components implicated in the development and progression of the disease can be a helpful method for improving patient care.

Some of the most common inflammatory and fibrotic diseases include:

  • Acute respiratory disease syndrome
  • Chronic obstructive pulmonary disease
  • Liver fibrosis
  • Idiopathic pulmonary fibrosis
  • Diabetic kidney disease
  • Neurodegenerative diseases
    • Alzheimer disease
    • Parkinson disease
    • Ischemic stroke

Targeting enzymes implicated in these diseases has been hypothesized as an effective approach for reducing disease severity, particularly over current therapies that use steroids and/or various other anti-inflammatory medications.

Nox Enzyme-Generated Reactive Oxygen Species

It is a widely accepted fact that Nox enzyme-generated reactive oxygen species (ROS) plays a noticeable role in fibrotic and inflammatory diseases. Medical therapy with Nox inhibitors is limited, and very few of these inhibitors are isoform-specific. Small molecules are being sought out by researchers to perform as isoform-selective Nox inhibitor agents in inflammatory and fibrotic diseases.

Nox1 to Nox5 and Duox1 and Duox2 belong in the Nox family of enzymes and are major sources of cellular ROS. Mitochondrial electron transport enzymes are also significant sources of cellular ROS. These sources of ROS include the following:

  • Hydrogen peroxide (H2O2)
  • Superoxide radical anion (O2)

Detecting O2⨪ and H2O2 Using Electron Paramagnetic Resonance (EPR)

A reliable method of detection for O2⨪ and H2O2 has been consistently difficult as the commonly used ROS probes lack specificity and reactivity to produce both O2⨪ and H2O2. Zielonka J et al believes that species- and site-specific probes may allow for more effective high-throughput screening in the study and discovery of Nox isoenzyme-specific inhibitors.

Researchers in this study developed a rapid high throughput-compatible assay for determining generation of O2⨪ and H2O2 using two probes: coumarin boronic acid and hydropropidine probes. During the study, researchers found they were able to identify Nox enzyme inhibitors with reliability and accuracy using a medium-throughput plate reader-based oximetry and electron paramagnetic resonance (EPR) spin trapping.

A Bruker X-band EPR spectrometer was used in this experiment to help identify short-lived free radicals via spin trapping. This spectrometer enabled a better monitoring of reaction oxygen species (ROS) activity in the studied samples. Bruker EPR spectrometers include:  

  • EMXnano, a benchtop instrument with best in class sensitivity and stability, microwave and digital technologies and an integrated reference standard, nitrogen variable temperature unit.
  • EMXmicro, a compact spectrometer with multi-frequency capabilities and a helium variable temperature unit
  • EMXplus which provides multi-frequency and multi-resonance options/capabilities and helium variable temperature unit

Additionally, this method, among others used in this study, helped in understanding the mechanisms of action of the discovered selective inhibitors of Nox isoenzymes. Researchers concluded that the rigorous and swift identification of O2⨪ and H2O2 will enable a deeper comprehension of the biology behind H2O2 and O2⨪-producing enzymes, potentially assisting in the discovery of selective Nox isoform inhibitors and the advancement of care for patients with inflammatory- and fibrosis-related diseases.


  • Zielonka J, Cheng G, Zielonka M, et al. High-throughput Assays for Superoxide and Hydrogen Peroxide: Design of a Screening Workflow to Identify Inhibitors of NADPH Oxidases. J Biol Chem. 2014;289(23):16176–16189.
  • Liang S, Kisseleva T, and Brenner DA. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts. Front Physiol. 2016;7:17.
  • Mannam P, Srivastava A, Sugunaraj JP, Lee PJ, and Sauler M. Oxidants in Acute and Chronic Lung Disease. J Blood Lymph. 2014;4:1000128.
  • Cifuentes-Pagano ME, Meijles DN, Pagano PJ. Nox Inhibitors & Therapies: Rational Design of Peptidic and Small Molecule Inhibitors. Curr Pharm Des. 2015;21(41):6023-6035.