Scientists at the National Institutes of Health’s National Chemical Genomics Center and BD Biosciences have developed and validated a high-throughput assay that does not require adding exogenous cAMP stimuli such as forskolin or GPCR agonists, and that can be used as a primary screen in phosphodiesterase 4 inhibition studies, an NCGC official told CBA News this week.
This PDE4 assay uses a HEK293 cell line coexpressing a constitutively active G protein-coupled receptor as a driving force for cAMP production together with a cyclic nucleotide-gated channel as a cAMP sensor in a 1,536-well plate format.
It can forgo exogenous cAMP stimuli because the constitutive activity of a cotransfected thyroid-stimulating hormone receptor produces enough cAMP to generate a measurable signal in the presence of a PDE inhibitor, according to the findings, which appear online in the June 30 issue of the Journal of Biomolecular Screening.
Other potential advantages over other assays are that it does not require the addition of exogenous cAMP stimuli; the assay protocol is simplified and standard fluorescent plate readers can be used; and its ability to detect cAMP accumulation and subsequent cation influx with a membrane potential dye enables researchers to homogenously measure PDE activity.
This compares favorably to other methods, such as those using Fura-2 dye or voltage-clamp methods, which have lower throughput and require a cell-wash step.
Researchers traditionally use cell-free enzyme assays to perform compound screening in drug discovery, said Wei Zheng, group leader for cellular signaling assay technologies at the NCGC. However, the difficulty of using PDE4 and other intracellular enzymes is that compounds must penetrate the cell surface membrane to become active.
In addition, these compounds often lose activity in cell-based assays, even though they are very active in the cell-free enzyme assay. In drug discovery, investigators who use PDE4 enzyme assays to screen compounds must eventually test the compound in cell-based assays.
“Traditionally, those cell-based assays are low-throughput and a little difficult to perform,” said Zheng.
“This assay that we developed has increased throughput and is more robust for the primary screen.”
He said the NCGC and BD scientists wanted to develop a cell-based assay as a secondary screen or as a primary screen to identify the compound that can penetrate the cell membrane; and use that compound as a lead compound to quickly move to the hit-to-lead process and accelerate the lead-discovery process.
“This assay that we developed has increased throughput and is more robust for the primary screen,” Zheng said. “Previous assays were only used for a few compounds in the confirmation stage, and were relatively complicated.”
He also pointed out that this is the first time that the CNG reporter has been used to measure the enzyme activity of PDE4; it has previously been used for GPCR screening.
The assay was developed based on BD’s ACTOne biosensor technology, said Xiao Li, a manager and principal scientist at BD Biosciences. Li said that the company developed the ACTOne biosensor technology in-house “to measure intracellular cAMP in live cells.” The cell line was also used for GPCR screening.
Several years ago, the company wanted to expand the GPCR platform to other assays that involve cAMP change, Li said.
“One [assay] that we thought about is the PDE assay,” she said. Since PDE hydrolyzes cAMP, the company thought that its activity can be measured by this type of technology, too.
So, said Li, “We went ahead and ran some experiments, and we developed a system that can be used to measure the intracellular PDE activity.”
Li said that the company has already filed a US patent application to protect the assay. However, “in order to test a lot more compounds, we decided to collaborate with the NIH, because they have a nice compound library and they have the capacity to run a big screening campaign.”
In terms of this screen, the project is done, Li said. “It looks like we have accomplished our goal, which was to demonstrate that the assay works in a ‘real-world’ screening environment.”
She added that BD intends to commercialize the cell line.
The assay uses a stably transfected CNG cation channel as a biosensor whose signal can be detected by a change in membrane potential. The PDE substrate, cAMP, was produced by adenylate cyclase, which is stimulated by the constitutive activity of stably transfected TSH receptors coupling through the GαS G-protein subtype.
The constitutive activity of transfected TSH receptors in the PDE4 cell line maintained a moderate level of cAMP production that provided the basis for the measurement of PDE activity without addition of a TSH receptor agonist.
In the absence of a PDE inhibitor, the moderate level of cAMP in this PDE4 cell line was rapidly hydrolyzed by endogenous PDEs, predominantly PDE4 in the host HEK293 cells, and is counted as a baseline. In the presence of a PDE4 inhibitor, cAMP accumulates, and the increased levels of cAMP bind to and open the CNG cation channels, resulting in an influx of cations, including Na+ and Ca2+.
This cation influx triggers a cell membrane depolarization that can be quantified using a fluorescent membrane potential dye that does not require cell wash or medium change steps. The inhibition of PDE activity can therefore be monitored homogeneously in live cells using a standard fluorescence plate reader.
Cells were dispensed at a density of 1,000 cell per well in black, clear-bottom, tissue culture-treated, 1,536-well plates in 3 µL of assay medium and were incubated for 24 hours; 3 µL per well of 1x membrane potential dye was added and incubated for 1 hour at room temperature.
The library compound or the positive control, RO-20-1724, were added at 23 nL per well and were incubated for 1 hour at room temperature. The assay plate was then read in PerkinElmer’s EnVision plate reader.
The NCGC has an internal program to develop PDE4 inhibitors that looks at the effect of PDE4 inhibition on learning and memory, Zheng said.
He added that the assay is already being used as a secondary screen for those initial hits found in the enzyme-free PDE4 assay. “We are also thinking about using it to screen an entire compound library to see if we can find different effects of the compounds compared to the enzyme assay.”
Zheng said that the assay can also be extended to other PDEs. ”The reason that we deal with PDE4 is that it is a very important target, and the HEK293 cell line we used predominantly expresses PDE4.”