Targeting fibrotic diseases at their source: enzymes

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^⨪)

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.