| Assay Method Information | |
| | QPCTL Enzymatic Activity Assay |
| Description: | Compound IC50 values for the inhibition of QPCTL activity were determined using a biochemical fluorescent-based assay. QPCTL activity was measured in a coupled-enzyme assay using the QPCTL substrate, glutamine-7-amido-4-methylcoumarin (H-Gln-AMC, Bachem), which is converted to pyroglutamyl-AMC by QPCTL. Pyroglutamyl-AMC is then converted to the fluorescent molecule, AMC, by incubation with the human enzyme pyroglutamyl peptidase-1. Test compounds or controls (SEN177 and DMSO) diluted in 100% DMSO were preincubated with 10-12 nM QPCTL in multi-well plates in assay buffer (25 mM HEPES pH 7.0) for 30 minutes at 37° C. Final concentrations of DMSO and test compounds were 2% and 30-100 μM, respectively. Following the incubation, H-Gln-AMC substrate was diluted in assay buffer and added to each well for a final concentration of 10 μM. The reaction was incubated for 60 min at 37° C., before stopping it by boiling at 100° C. for 5 minutes and cooling the plate at 4° C. for 3 minutes. An equal volume of 38 nM recombinant, human PGPEP-1 (rhPGPEP-1, R&D Systems) in 100 mM Tris pH 8.0, 5 mM dithiothreitol was added to the reaction for a final concentration of 19 nM and incubated for 25 minutes at room temperature. Following incubation, the fluorescence was measured with a plate reader using excitation at 380 nm and emission at 460 nm. Percent inhibition of enzymatic activity by test compounds was calculated by normalizing the data to the 2% DMSO and 30-100 μM SEN177 control values, which represent 0% and 100% inhibition of enzymatic activity, respectively. |
| Affinity data for this assay | |
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