PDE5A1 Assay Kit1

• 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. PDE5 is expressed in several tissues, such as the lung, brain, kidney, cardiomyocytes, platelets, and penile corpus cavernosum. It is activated by phosphorylation and targeted for degradation by nitrosylation. It plays a role in vascular tone, vision, renal and intestinal function, peristalsis, cell growth and apoptosis, cancer, amongst others. Its functions make it a prime candidate for pharmacological intervention. The development of inhibitors, mostly competitive inhibitors for the catalytic domain, has resulted in the commercialization of sildenafil, tadalafil, vardenafil and avanafil, for the treatment of erectile dysfunction, pulmonary arterial hypertension, and lower urinary tract symptoms (LUTS) . PDE5 inhibitors have also shown potential as cancer therapy, with a reduced risk of metastasis in colorectal cancer (CRC) when PDE5 inhibitors were used post-operatively. Continuing to understand PDE5 roles and the development of newer specific therapeutic tools will open new treatment avenues in PDE5-related disorders.

• Description:

  The PDE5A1 Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE5A1 (Phosphodiesterase 5A1) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE5A1, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE5A1 Assay Kit principle.The assay uses a fluorescein-labeled cyclic guanosine monophosphate (cGMP-FAM for PDE5A1), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE5A1 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 PDE5A1 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.Note:As of July 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.Need us to run inhibitor screens or profile your compounds against PDE5A1? Check out ourPhosphodiesterase Screening Services.This product has beencited 14 times.

• UniProt:

  O76074

• Applications:

  Study enzyme kinetics and screen small molecule inhibitors for drug discovery in high throughput screening (HTS) applications.

• Format:

  Catalog #NameAmountStorage60050PDE5A1, GST-Tag*>1 µg-80°C60201FAM-Cyclic-3′, 5′-GMP**2 x 1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent100 µl4°C60392Binding Agent Diluent (cGMP) 10 ml4°C0.5 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′-GMP is provided as a powder. Each vial will need to be resuspended in 400 µl of Complete PDE Assay Buffer before use.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  This assay kit will perform optimally for up to6 months from date of receipt when the materials are stored as directed.

• Notes:

  Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For further questions, email[email protected]

• 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.