ACTOne technology is the key platform used for development of cell-based assays for GPCR and PDE.
It is based on a modified cyclic nucleotide-gated (CNG) ion channel as a biosensor of cAMP activity in live cells. This channel responds in real time to increases or decreases in intracellular cAMP levels by coordinately altering cation flux (e.g., calcium, potassium, or sodium), which can be measured by calcium-sensitive fluorescent indicators, or on broadly available plate readers or single-cell imagers. By juxtapositioning this biosensor adjacent to adenylyl cyclase, the enzyme responsible for synthesis of cAMP, in the membrane of cells that also express the GPCR target of interest, the biosensor provides ultrasensitive responses to cAMP fluctuations (Rich et. al., J. Gen. Physiol. 2000;116:147–161). The technology platform enables live-cell GPCR screening or deorphanization of agonists, antagonists, and allosteric modulators of Gs, Gi or Gq-coupled receptors in 1536-well formats. Because the technology directly measures cAMP through CNG channels, it detects GPCR activity through its nature pathway. No engineering or modifications to the G protein is required. The assay is highly sensitive to intracellular cAMP change; it has competitive signal to noise ratio and z’ value. This assay is also economical and easy to use. Preliminary evidence demonstrates the ability of this assay to freeze live cell lines in ready-to-screen format, and to enable receptor profiling for specificity assessment.
The cyclic nucleotide phosphodiesterases (PDEs) are enzymes that catalyze hydrolysis of 3′, 5′-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5′-nucleotide monophosphates. These enzymes play important roles in controlling cellular concentrations of cyclic nucleotides and have central roles in a variety of intracellular signaling events. As such, phosphodiesterases are emerging as a promising class of drug targets, particularly in asthma, cardiovascular disease, and CNS diseases. Currently, PDE inhibitors are identified in screens employing biochemical assays using pure substrates (cAMP or cGMP) and purified recombinant PDE enzymes. We have leveraged the CNG channels in the ACTOne Technology to detect intracellular cAMP/cGMP changes and developed the first commercially available live-cell PDE inhibitor assays.
To measure cAMP specific PDE activity, we have over-expressed a constitutively active Gs-coupled GPCR in the cells containing CNG channels to stimulate adenylyl cyclases, leading to cAMP synthesis. The produced cAMP is hydrolyzed by intracellular PDE, so the steady cAMP is kept at low level. Upon the PDE activity inhibition, the cellular cAMP level rises quickly and is measured by the CNG biosensor. This assay has been used to identify inhibitors of PDE4 in a 1536 well format (Titus et al, J Biomol Screen 2008; 13: 609-618).
To assess cGMP specific PDE activity, we transfected soluble guanylate cyclase (sGC) into the cells containing CNG channels. Cellular cGMP can be increased by the sGC stimulator, BAY 41-2272. In the presence of cGMP specific PDE, the produced cGMP is hydrolyzed quickly by PDE so the steady level of cGMP is low. With the added cGMP specific PDE inhibitor, the cGMP can accumulate quickly and activate CNG channels.