Measuring Phosphodiesterase Inhibitor Residence Times with the Transcreener AMP 2 /GMP 2 FP Assay

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1 CASE STUDY Measuring Phosphodiesterase Inhibitor Residence Times with the Transcreener AMP 2 /GMP 2 FP Assay Biology at Work Meera Kumar BellBrook Labs

2 Selective Nucleotide Detection The Transcreener assays rely on antibodies that are able to differentiate between nucleotides on the basis of subtle structural differences. This makes detection of the product nucleotide possible even in the presence of excess substrate nucleotide. AMP vs camp or ATP GMP vs cgmp or GTP ADP vs ATP UDP vs UDP Sugar GDP vs GTP

3 Sensitive Assay for AMP-Producing Enzymes one assay hundreds of targets Ligases & Synthetases Ubiquitin ligases SUMO ligases NAD Synthetase Acyl CoA Synthetase Phosphodiesterases Sialyltransferases (CMP) Direct AMP/GMP Detection Transcreener AMP 2 /GMP 2 Assays are single-step, homogenous, fluorescence assays. Binding of AMP or GMP to antibody displaces a fluorescent tracer to generate the assay signal. UNIVERSAL AMP/GMP DETECTION Detect any AMP/GMP producing enzyme regardless of substrate camp, cgmp, ATP, NAD. SENSITIVE ANTIBODY New monoclonal AMP 2 /GMP 2 Antibody detects AMP and GMP at low nanomolar concentrations so you can use less enzyme. OUTSTANDING DECK & SIGNAL STABILITY Reagents and assay signal are both stable for more than 8 hours, providing flexibility for automated protocols.

4 Protocol Sensitivity % Conversion 0.1 µm 1 µm 10 µm 100 µm Example standard curves show the conversion of ATP to AMP. 4

5 The Enzyme: Phosphodiesterase 7A (PDE7A) PDE7A is a high-affinity camp-specific PDE that is expressed in T cell lines, peripheral blood T lymphocytes, epithelial cell lines, airway and vascular smooth muscle cells, lung fibroblasts, and eosinophils. PDE7 plays a critical role in the regulation of human T cell function and selective PDE7 inhibitors are being examined to treat immunological and inflammatory disorders. PDE7A plays an important role in the regulation of osteoblastic differentiation. PDE7A depletion by RNAi upregulates expression of several osteogenic genes and increases mineralization.

6 PDE7A Titration: 30-Minute Time Point Raw Data Converted Data (Product Formed) mp AMP, µm camp - camp camp - camp PDE7A, pm PDE7A, pm Enzyme Buffer: 50 mm Tris (ph 7.5), 5 mm MgCl 2 [Substrate]: 1 µm camp [Antibody]: 1 µg/ml [Tracer]: 2 nm Optimal [PDE7A]: 5 pm

7 Dose Response Curves AMP, µm Inhibitor + Enzyme (5 pm PDE7A) TC3.6+E BRL50481+E Zardaverine+E Inh, µm IC50 TC3.6+E BRL50481+E Zardaverine+E

8 Protocol for Determining Residence Time Streamlined Protocol Determine EC 80 of target. Determine IC 50 values of the compounds. Preincubate 10 [IC 50 ] compound [EC 80 ] target Perform a 100X Jump Dilution into a reaction mixture of camp and Transcreener detection reagents. Continuously read over time and monitor the change in fluorescence. Analyze data and calculate residence times.

9 Preincubation for [EI] Complex Formation Enzyme for [EI] 100 EC 80 (PDE7A) = 5 pm 100 = 500 pm Inhibitor for [EI] 10 IC 50 (TC3.6) = 637 µm 10 IC 50 (BRL50481) = 169 µm 10 IC 50 (Zardaverine) = 100 µm Preincubation Protocol Preincubation mixes were prepared with PDE7A enzyme at a concentration of 100 EC 80, to provide a robust signal after dilution, and three different inhibitors (TC3.6, BRL50481, Zardaverine) at 10 IC 50, to ensure that the enzyme was saturated with the inhibitor. The mix was incubated for 1 hour at room temperature to insure formation of the EI complex.

10 Jump Dilution Method The dilution of the EI complex keeps the inhibitor concentration is at least 100-fold below its IC 50 value. A 100-fold dilution (0.2 μl of PDE7Al/inhibitor mixture into 19.8 μl of Detection Mixture) was made in an LV-384 well plate. The Detection mixture comprised of 1 μm camp in buffer that also contained 2 nm tracer and 1.4 μg/ml of AMP 2 antibody. The plate was mixed well and read kinetically every 5 minutes for 4 hours in a Tecan Safire plate reader using the fluorescent polarization mode with EXC at 630 nm and EMS at 670 nm.

11 Determining Residence Time TC3.6+E BRL50481+E Zardaverine+E DMSO AMP, µm Time [s] TC3.6+E BRL50481+E Zardaverine+E Koff Tau(min) E E E-07

12 Summary The single-step, homogenous fluorescent Transcreener AMP 2 /GMP 2 FP Assays enable biochemical characterization of a broad range of targets, including PDE7A. Residence time determinations can be made using a simple Jump Dilution protocol of an EI complex into a detection mixture with a plate reader able to continuously monitor kinetic reactions.

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