qHTS for Antagonist of cAMP-regulated guanine nucleotide exchange factor 3 (EPAC1): primary screen
In multi-cellular eukaryotic organisms, the effects of cAMP are transduced by two ubiquitously-expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) [1, 2]. Both PKA and EPAC are ubiquitously more ..
BioActive Compounds: 517
In multi-cellular eukaryotic organisms, the effects of cAMP are transduced by two ubiquitously-expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) [1, 2]. Both PKA and EPAC are ubiquitously expressed in all tissues, and an increase in intracellular cAMP levels will lead to the activation of both PKA and EPAC. Therefore, careful dissections of the individual role and relative contribution of EPAC and PKA within the overall cAMP signaling in various model systems are critical for further elucidating the mechanism of cAMP signaling. To date no EPAC-specific antagonists and agonist, as well as isoform-specific agonists have been reported, and developing EPAC-specific pharmacological probes to determine the physiological functions that EPAC play in the overall cAMP-mediated signaling remains a major challenge within the research field. Furthermore, identified regulators of EPAC may lead to new mechanism-based therapeutic strategies specifically targeting the EPAC / cAMP-signaling components as EPAC has been implicated to play important roles in major human pathological conditions such as diabetes, heart disease, and cancer.
This qHTS will be used to screen the Molecular Libraries Small Molecule Repository (MLSMR) as a concentration-titration series using the NCGC unique quantitative screening (qHTS) platform to find inhibitors for EPAC1. In addition, this assay will also be used as a counterscreen for the EPAC2 qHTS. This fluorescent based assay is developed and validated for EPAC1 that measures exchange using EPAC effector Rap1 complex with BODIPY-GDP, a GDP analog with a tethered fluorophores. Inhibitors are identified by monitoring the change in fluorescence signal as the exchanged free BODIPY-GDP has a reduced fluorescence compared to Rap1-bound BODIPY-GDP. Decrease in signal (inhibition) is the desired outcome for the EPAC1 antagonist while no activity in this assay is desired for the EPAC2 antagonist counterscreen.
 de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J.L. (1998) Nature 396, 474-477
 Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Science 282, 2275-2279
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: NS066510
Assay Submitter (PI): Xiaodong Cheng, The University of Texas, Medical Branch
Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control ATA and DMSO.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)