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Application: Study enzyme kinetics and screen small molecule inhibitors for drug discovery in high throughput screening (HTS) applications.
Background: Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) signaling, by hydrolyzing them. The PDE superfamily is composed of 11 families, with PDE4, 7 and 8 being cAMP-specific hydrolases, and thus regulating positive and negative responses to it. PDE4 is the second most abundant PDE in cardiovascular tissues. PDE4, is a cAMP-specific PDE and the largest PDE subfamily with over 35 different isoforms and as such, the most widely characterized PDE isoenzyme. PDE4 proteins are involved in cancer, depression, autism, and inflammation. Inhibitors of PDE4 are being used for the treatment of psoriasis, COPD (chronic obstructive pulmonary disease) and atopic dermatitis. PDE4D plays multiple roles, contributing to cell adhesion, migration, proliferation and angiogenesis. PDE4D is found in the brain and thought to contribute to the formation of long-term memory by increasing the levels of cAMP, which promotes the activation of PKA (protein kinase A) and activate CREB (cAMP response element binding protein). This protein has been linked to cancer, and in lung adenocarcinoma (LUAD) the PDE4D/IL-23/IL-9 axis seems to be required for CTL (cytotoxic T lymphocytes) action on the cancer cells. Targeting PDE4D may thus be a therapeutic strategy in Alzheimer’s disease (AD). A deeper understanding of PDE4D functions is still required and the development of PDE4D inhibitors may prove beneficial for treatment of PDE4D related disorders. PDE4 inhibitor roflumilast has been approved for the treatment of chronic obstructive lung disease.
Contraindications: The final concentration of DMSO in the assay should not exceed 1%. Fluorescent compounds that have λex=470 nm and detection at λem=528 nm can interfere with the readouts.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.
Description: The PDE4D7 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE4D7 (Phosphodiesterase 4D7) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant human PDE4D7, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE4D7 Assay Kit principle. The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE4D7), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP). PDE4D7 catalyzes the hydrolysis of the phosphodiester bond in the cyclic nucleotide and frees the phosphate group. In a second step the free phosphate group is recognized by a specific phosphate-binding nanobead (Binding Agent) leading to the formation of a large complex, with restricted movement (high FP). FP is proportional to PDE4D7 activity.This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note: FP, assay principles and applications. Interested in having us perform the screening and profiling of inhibitors? Visit our Phosphodiesterase Screening & Profiling Services.
Format: Catalog #NameAmount Storage 60047PDE4D7, GST-Tag*˃1 µg-80°C60200FAM-Cyclic-3′, 5′-AMP**1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete)25 ml-20°C60390PDE Binding Agent100 µl4°C60391Binding Agent Diluent (cAMP)10 ml4°C827350.5 M DTT200 µl-20°C79685Low binding, black 96-well plate1Room Temp. * The concentration of protein is lot-specific and will be indicated on the tube containing the protein.** FAM-Cyclic-3′, 5′-AMP is provided as a powder. Vial will need to be resuspended in 600 µl of Complete PDE Assay Buffer before use.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Target: PDE4D7
Uniprot: Q08499
Warnings: Avoid freeze/thaw cycles.This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note: FP, assay principles and applications.
Biosafety Level: Not applicable (BSL-1)
References: Lai S., et al., 2020 Oncogene 39(19):3791-3801. Tibbo A. and Baillie G., 2020 Cells 9(5):1254.
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