Emission Ratiometric Indicators of Intracellular Cyclic AMP and Epac Activation

Unmet Need: Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. Various methods exist for detecting and measuring intracellular cAMP, but none are ideally suited for monitoring spatial and temporal distributions of cAMP in living cells. Here, inventors developed a series of emission ratiometric reporters allowing measuring the production and degradation of cAMP in living cells after transfection with plasmid constructs encoding the reporter protein, using a Fluorescent Resonance Energy Transfer (FRET) assay. Advantages include:

• Reporter molecules allowing measuring free cellular cAMP in living cells
• Provide high spatial and temporal resolution
• Reporters can be targeted to different subcellular sites to monitor specific pools of cAMP
• Unlike existing methods, does not require destroying large areas of cells or tissue
• Cellular assays negate the need to determine all of the appropriate cofactors

 
Technical Details: The classic second messenger, cAMP, coordinates many cellular functions via its effectors such as Epac (exchange proteins directly activated by cAMP) and PKA (cAMP-dependent protein kinase). The herein-disclosed reporter molecule comprise a pair of fluorescent proteins linked by a region containing Epac. Binding of cAMP results in a conformational change in the reporter and a change in the FRET emission ratio, between yellow and cyan, in a manner dependent on local cAMP concentration. The reporter can also be targeted to different subcellular sites to monitor specific pools of cAMP. These reporters have some unique advantages over previous methods for assessing cAMP dynamics inside cells. They do not require destroying large amounts of cells or tissue, provide high spatial and temporal resolution, and measure free rather than total cAMP. Cellular assays negate the need to determine all of the appropriate cofactors for the intended targets prior to high throughput screening and ensure that the active compounds already possess adequate solubility, membrane permeability, stability and the ability to act within the biological complexity of the cell.

Looking for Partners: These reporters are suitable for use in high throughput assays in single living cells for analyzing:

• the evaluation of the effect of a variety of known agonists, antagonists and siRNA that are known to effect cAMP signaling in one experiment;
• drug screening of a variety of small molecules to determine their effect on cAMP production and degradation as well as possibly define unknown mechanisms for previously established drugs;
• drug screening of new candidates that target G-protein coupled receptors, G proteins (both Gs and Gi), phosphodiesterases and adenylyl cyclase.
• Newer versions of these emission ratiometric reporters are completely reversible and therefore can be used to screen for drugs that block cAMP production or have effects on receptor desensitization. Furthermore, these reporters provide a novel in situ assay for Epac activation that provides high spatial and temporal resolution.

 
Patent Status: Granted US Patent 8,236,523

Associated Publications: Mol Biosyst. 2009 Aug;5(8):832-7.

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