Category: Assay Development and Screening
The cyclic nucleotide cGMP is an important second messenger in signal transduction, mediating many important processes including vision, the sense of smell, and cardiovascular tone. Phosphodiesterase (PDE) inhibitors and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions, so these enzymes are important drug targets. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP. We present here the first live cell assay to test the specificity of drugs targeting PDEs in any cell type.
Montana Molecular has developed a new genetically-encoded fluorescent biosensor to monitor cGMP levels in response to phosphodiesterase inhibitors or sGC activators/stimulators. The assay produces robust changes in fluorescence intensity that is readily detected on standard fluorescence plate readers and indicates changes in cGMP by either activators of guanylate cyclase or inhibitors of PDEs. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP. HEK 293 cells expressing either the cGMP biosensor or the cAMP biosensor (cADDis), produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.
Jake Quinn– Sales and Customer support, Montana Molecular, Bozeman, Montana