Optimization of a Phosphodiesterase (PDE) Assay to Determine the Effects of Vindeburnol on cAMP Levels
Session Number
Q21
Advisor(s)
David Braun, University of Illinois at Chicago Douglas Feinstein, University of Illinois at Chicago
Location
B-131 Grainger
Start Date
28-4-2016 9:15 AM
End Date
28-4-2016 9:40 AM
Abstract
Derived from the vincamine plant, vindeburnol is a drug that has been shown to enhance release of noradrenaline, a key chemical in the regulation of neuroinflammation, in locus coeruleus neurons. Evidence shows that vincamine modulates cAMP levels in neurons, suggesting that vindeburnol is able to as well. Since the enzyme phosphodiesterase (PDE) breaks down cAMP, we use the PDE- Glo™ Assay to explore the idea that vindeburnol is a direct inhibitor of PDE, increasing cAMP levels. The assay is comprised of two separate reactions. In the first, PDE converts cAMP into AMP. In the second, AMP is converted into ATP, which is quantified as light, and is proportional to the amount of cAMP converted. Completed optimization of the second reaction shows that consistent peak light levels can be obtained for up to 20 uM AMP using a pH 7.8 buffer with 5mM Mg after 30 minutes of incubation at room temperature. We are currently optimizing the first reaction with respect to the concentration of cAMP, incubation time, and temperature. Following optimization, we will use similar conditions to thoroughly determine the effects of vindeburnol on PDE, and how its derivatives may work in animal models of Alzheimer’s disease and multiple sclerosis.
Optimization of a Phosphodiesterase (PDE) Assay to Determine the Effects of Vindeburnol on cAMP Levels
B-131 Grainger
Derived from the vincamine plant, vindeburnol is a drug that has been shown to enhance release of noradrenaline, a key chemical in the regulation of neuroinflammation, in locus coeruleus neurons. Evidence shows that vincamine modulates cAMP levels in neurons, suggesting that vindeburnol is able to as well. Since the enzyme phosphodiesterase (PDE) breaks down cAMP, we use the PDE- Glo™ Assay to explore the idea that vindeburnol is a direct inhibitor of PDE, increasing cAMP levels. The assay is comprised of two separate reactions. In the first, PDE converts cAMP into AMP. In the second, AMP is converted into ATP, which is quantified as light, and is proportional to the amount of cAMP converted. Completed optimization of the second reaction shows that consistent peak light levels can be obtained for up to 20 uM AMP using a pH 7.8 buffer with 5mM Mg after 30 minutes of incubation at room temperature. We are currently optimizing the first reaction with respect to the concentration of cAMP, incubation time, and temperature. Following optimization, we will use similar conditions to thoroughly determine the effects of vindeburnol on PDE, and how its derivatives may work in animal models of Alzheimer’s disease and multiple sclerosis.