A team of researchers based at West Virginia University has devised an innovative way to potentially monitor enzyme activity in vivo using electron paramagnetic resonance imaging. The method could provide new insights into the molecular underpinnings of many types of disease.
The team specifically focused on tracking enzymatic dephosphorylation. Abnormalities in dephosphorylation have been linked to disorders ranging from cancer to Alzheimer disease. Monitoring such malfunction in vivo can provide crucial details into disease state and progression, but direct measurement of enzyme activity within a living organism remains extremely challenging. Many imaging approaches that might be used for this purpose are hampered by concerns such as low sensitivity and penetration depth.
Such limitations prompted the researchers to turn to EPRI – a method with high intrinsic sensitivity and specificity. But despite these advantages, using EPRI for in vivo monitoring of enzyme activity comes with its own set of complications. Most notably, there’s a limited abundance of endogenous EPR-active enzymes, making it necessary to use an exogenous paramagnetic substrate to get a good read.
To generate an appropriate substrate, the team synthesized a phosphorylated nitroxide radical that can be modified by the action of alkaline phosphatase – an enzyme that catalyzes numerous dephosphorylation reactions. Because of its far-reaching effects, alkaline phosphatase is of critical physiological importance. When the enzyme cleaves the phosphate moiety on the substrate, it causes a spectral shift that can be picked up with EPRI.
Using the novel substrate, the team successfully imaged a time course of alkaline phosphatase activity with EPRI. They also showed that the reaction was highly specific to this enzyme, potentially reducing the chances for off-target reads. Perhaps most importantly, cells treated with the substrate showed no significant drop in viability even at concentrations ten times that needed for in vivo imaging, suggesting the method is safe.
Overall, the work lays a foundation for monitoring alkaline phosphatase reactions in a natural environment. Although more work is needed to get to the point of imaging a living organism, the team’s findings take the field one step closer to non-invasive imaging of enzyme activity