qHTS Assay for Inhibitors of PDE-IV
The cyclic nucleotide phosphodiesterases (PDEs) are proteins 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 an important role in controlling cellular concentrations of cyclic nucleotides and have a central role in a variety of intracellular signaling events. cAMP PDEs are emerging as a promising class of drug targets in asthma and cardiovascular disease therapeutic areas. ..more
BioActive Compounds: 34
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
NCGC Assay Overview:
The cyclic nucleotide phosphodiesterases (PDEs) are proteins 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 an important role in controlling cellular concentrations of cyclic nucleotides and have a central role in a variety of intracellular signaling events. cAMP PDEs are emerging as a promising class of drug targets in asthma and cardiovascular disease therapeutic areas.
There are no commercially available cell-based screening assays for PDE inhibitors: currently biochemical assays are done using pure substrates (cAMP or cGMP) and purified recombinant PDE enzymes. As with other biochemical screens on intracellular targets, false positives and false negatives may result from issues involving cell membrane permeability, cellular metabolism, and the like.
Here we report the development of a cell-based screening assay for cAMP phosphodiesterase (PDE) inhibitors. The assay is based on the BD ACTOne# technology (BD Biosciences) which utilizes a modified cyclic nucleotide-gated channel (CNG) to monitor intracellular cAMP changes in live cells. The CNG channel is localized on the plasma membrane, and opens when the intracellular cAMP level increases, resulting in ion flux and cell membrane depolarization. The influx of cations through the CNG channel can be quantified using membrane potential (MP) dyes. We modified the BD ACTOne technology to allow the detection of PDE inhibitors, and performed an inhibitor screen for PDE-IV, which is expressed endogenously in HEK293 cells. We optimized this assay in homogeneous 1536-well plate assay format for high throughput screening.
In order to help eliminate false positives, a counterscreen of the entire library using the parental cell line was conducted and compounds that showed signaling in both the parental and PDE assay cell line were labeled as 'Inconclusive'. Compounds which were active only in the PDE assay cell line were labeled as 'active' and compounds which signaled only in the parental cell line were labeled 'inactive'.
NCGC Assay Protocol Summary:
ACTOneTM cells (HEK293 cells stably expressing a modified cyclic nucleotide-gated channel and optimized for screening PDE inhibitors) were maintained in DMEM (Invitrogen, Carlsbad, CA) at 37 degrees Celsius under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS (Invitrogen), 2 mM L-glutamine, and 250 ug/ml G418 (Cellgro) and 1 ug/ml puromycin (Clontech). ACTOneTM parental cells (BD Biosciences) were maintained in the same medium without puromycin.
The activity of the CNG channel was measured by BD ACTOneTM membrane potential dye kit (BD Biosciences). Forskolin, a positive control for cAMP activation and RO 20-1724, a PDE inhibitor, were both purchased from Sigma and solublized in DMSO.
1,Reagent,3 uL,1000 cells/well 1536 TC treated Black plate
2,Time,12 hours,37 C 5% CO2
3,Compound,0.5 uL,Kalypsys dispense RO 20-1724 100 uM final [A]
4,Reagent,3 uL,BD membrane potential dye
5,Time,1 hour,Room temp
6,Compound,23 nL,Final concentration was 0.7nM to 22.9uM
7,Time,1 hour,Room temp
8,Detector,EX 535 / EM 590 [B],Envision
Footnotes: [A] The stock 2 mM RO 20-1724 PDE inhibitor was made in 10 % DMSO. Parental cell line received 10 uM forskolin + 100 uM RO. [B] Envsion settings: Bottom Excitation 535/20 nm top emission 590/20, gain of 150 and 5 flashes per well.
Keywords: PDE, PDE-IV, cytotoxicity, flourescence, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
1. Compounds are first classified as being probable activators (compounds causing an increase in membrane potential dye flourescence, presumably through inhibition of PDE), inhibitors (compounds causing a decrease in membrane porential dye flourescence), inactive, or inconclusive based on flourescence readout in ACTOne(TM) (pde) and parental cells.
2. Within the activators, compounds were ranked by efficacy and potency. The most potent and highest efficacy compounds were ranked highest, with a score of 99 to 40. Inhibitors were ranked with a score of 20, inconclusive with a rank of 10, and inactive with a rank of 0.
Categorized Comment - additional comments and annotations
Data Table (Concise)