New Case Study: Determination of Inhibitor Residence Times for a Phosphodiesterase using the Transcreener® AMP2/GMP2 FP Assay
Analysis of inhibitor residence times is increasingly being incorporated into lead development efforts because longer engagement with the target can result in improved efficacy, increased therapeutic window and reduced side effects. BellBrook Labs scientist, Meera Kumar, recently used a jump-dilution protocol with the Transcreener® AMP2/GMP2 FP Assay to determine the residence times of three phosphodiesterase (PDE) inhibitors.
The PDE7A target used for this study is a high-affinity cAMP-specific PDE expressed in T cell lines, peripheral blood T lymphocytes, epithelial cell lines, airway and vascular smooth muscle cells, lung fibroblasts, and eosinophils. The enzyme plays a critical role in the regulation of human T cell function, where selective PDE7 inhibitors may have therapeutic potential for immunological and inflammatory disorders. In an earlier webinar, we described a jump dilution approach for measuring residence time for protein kinase inhibitor drugs using the Transcreener® ADP2 Kinase Assay to continuously monitor the recovery of enzyme activity as the inhibitor compound dissociates from the kinase. Here we applied a similar methodology to assess the residence time of PDE7A inhibitors, including:
- Titrating PDE7A in reactions with Transcreener® AMP2/GMP2 FP Assay reagents determine the optimal enzyme concentration (EC80) for initial velocity detection.
- Running inhibitor dose-response curves to determine IC50 values for inhibitors TC3.6 (637 µM), BRL50481 (169 µM), and Zardaverine (100 µM) with PDE7A.
- Allowing enzyme/inhibitor (EI) complex formation in preincubation mixtures containing 100-fold excess of PDE7A and saturating amounts of each inhibitor.
- Diluting EI complexes 100-fold into Transcreener® AMP2/GMP2 FP Assays to monitor PDE7A rate increases as the complexes dissociates.
Use of the jump dilution method for measuring inhibitor dissociation rates obviates the need for ligand immobilization for SPR, or the requirement for a labeled active site probe for competitive binding assays. Homogenous, direct detection of reaction products with Transcreener®assays enables the critical step in the process, by allowing continuous monitoring of enzyme activities.
See the data: Download a copy of the Case Study.
BellBrook Labs Transcreener® AMP2/GMP2 Assays are homogenous, direct AMP or GMP detection assays with far red fluorescent readouts for detecting the activity of any enzyme class that produces either AMP or GMP (including ubiquitin, SUMO, nucleic acid and protein ligases, phosphodiesterases (PDEs), and synthetases) using any precursor substrate (including cAMP, cGMP, ATP, or NAD). Learn more about Transcreener® AMP2/GMP2 FP Assays.