Pde glo phosphodiesterase assay

Manufactured by Promega

Sourced in United States

The PDE-Glo Phosphodiesterase Assay is a luminescent-based assay used to measure phosphodiesterase enzyme activity. The assay quantitates the amount of cyclic nucleotide, such as cAMP or cGMP, remaining after a phosphodiesterase reaction.

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Lab products found in correlation

384 well plate wells, by Thermo Fisher Scientific (1 mentions) Polarstar omega, by BMG Labtech (1 mentions) Pde4b, by BPS Biosciences (1 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions) Microplate luminometer, by PerkinElmer (1 mentions) Winnonlin v 7, by Phoenix Pharmaceuticals (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Phosstop, by Roche (1 mentions)

8 protocols using pde glo phosphodiesterase assay

Phosphodiesterase Inhibitory Assay for PTX, R-(-)-LSF and S-(+)-LSF

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The PDE inhibitory activity of PTX, R-(-)-LSF and S-(+)-LSF was evaluated using the PDE-Glo Phosphodiesterase Assay according to the manufacturer’s instruction (Promega Corporation, Madison, WI, USA). Briefly, 1,5 μl of 1× PDE-Glo reaction buffer containing 10 mU of purified hrPDE4B or hrPDE7A (SignalChem, Richmond, Canada) was pipetted into 384-well plate wells (Thermo Scientific, USA). The tested compounds were dissolved in DMSO, and a serial dilution of the inhibitors was performed using 1× PDE-Glo reaction buffer. Then, 1 μL of diluted inhibitors and 2.5 μL of cAMP solution were added to each well. After 10 min of incubation in 30 °C, 2.5 μL of PDE-Glo™ Termination Buffer and 2.5 μL of PDE-Glo™ Detection Solution were added and the plate was incubated for 20 min at room temperature. Finally, 10 μL of Kinase-Glo® Reagent was pipetted to each well and after 10 min of incubation, the luminescence was measured using a microplate luminometer (POLARstar Omega, BMG LABTECH, Ortenberg, Germany). All data points are the average of two determinations.

Świerczek A., Wyska E., Baś S., Woyciechowska M, & Mlynarski J. (2017). PK/PD studies on non-selective PDE inhibitors in rats using cAMP as a marker of pharmacological response. Naunyn-Schmiedeberg's Archives of Pharmacology, 390(10), 1047-1059.

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Inhibitory Activity of Brilacidin on PDE4

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Example 11

To examine the inhibitory activity of brilacidin on PDE4 phosphodiesterase, inhibition assays of PDE4 were performed using brilacidin. The PDE-Glo phosphodiesterase assay (Promega, Madison, Wis.) was performed using 8 ng of PDE4B3, 1 μM cAMP substrate and brilacidin. The compounds and PDE4B (BPS Biosciences, San Diego, Calif.) were mixed and pre-incubated at room temperature for 15 minutes. Substrate was added and the reaction was incubated for 10 minutes at room temperature. Data are presented as luminescence units (RLU). Brilacidin inhibited PDE4 in a dose dependent manner with an IC50 in the 3 μM range (FIG. 10, Panels A and B, semi-logarithmic and linear axes respectively). This is the first demonstration of a HDP mimetic inhibiting a PDE.

US11771694B2. Arylamide compounds for treatment and prevention of viral infections (2023-10-03). Innovation Pharmaceuticals Inc. [US]. Inventors: Jane A. Harness [US], Leo Ehrlich [US], Warren Kyle Weston [US].

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PDE Enzyme Inhibition Assay

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A PDE activity assay was carried out using the PDE-Glo Phosphodiesterase Assay per manufacturer’s instruction (Promega). Briefly, 5 ul of 1X PDE-Glo Reaction buffer containing 10 mU of purified human recombinant PDE4A4 (Millipore) was added to 96 well plate. All test compounds were dissolved in DMSO and a serial dilution (110) of the inhibitors were performed using 1X PDE-Glo Reaction buffer. 7.5 ul of test compounds were added to the PDE enzyme with the final concentrations in each assay of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 µM, and incubated at room temperature for 10 min. To initiate the PDE reaction, 12.5 ul of 2 µM cAMP was added to to each reaction, mixed well and incubate at room temperature for 10 min. The reactions were terminated using 12.5 ul of PDE-Glo Termination Buffer. 12.5 ul of PDE-Glo Detection Solution containing PDE-Glo Detection Buffer and Protein Kinase A was added to each reaction. The reactions were carried at room temperature for 20 min. 50 ul of Kinase-Glo Reagent was then added to each reaction, mixed well, and incubate for 10 minutes at room temperature. The chemical luminescence signals were measured, quantified and graphed. Compounds 6, 7, 9 and 13 were further tested similarly using 25 mU purified PDE3, 5, 7, 10 (Millipore) enzyme.

Coon T.A., McKelvey A.C., Weathington N.M., Birru R.L., Lear T., Leikauf G.D, & Chen B.B. (2014). Novel PDE4 Inhibitors Derived from Chinese Medicine Forsythia. PLoS ONE, 9(12), e115937.

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PDE3 Inhibition by Brilacidin

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Example 12

Inhibition of phosphodiesterase type 3 was also performed for brilacidin. The PDE-Glo phosphodiesterase assay (Promega, Madison, Wis.) was performed using 2.75 ng of PDE3A, 1 μM cAMP substrate, and brilacidin. The compounds and PDE3A were mixed and pre-incubated at room temperature for 15 minutes. Substrate was added and the reaction was incubated for 12 minutes at room temperature. Data are presented as luminescence units (RLU). Brilacidin inhibited PDE3 in a dose dependent manner with an IC50 of approximately 1.8 μM (FIG. 11, Panels A and B, semi-logarithmic and linear axes respectively). Based upon these data, it is hypothesized that inhibition of PDE3 by brilacidin in vivo causes increased intracellular concentration of cAMP and thereby functions through the cyclic AMP pathway in suppression of the inflammatory response.

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Evaluating Recombinant PDE5 Activity

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The activity of recombinant expressed PDE5 (Enzo Biochem, Madison, USA) was evaluated using the PDE-Glo™ Phosphodiesterase assay (Promega Corporation, Madison, WI, USA). Aliquots of PDE-Glo™ reaction buffer containing appropriate amounts of purified human recombinants PDE5A were added to a 96-well plate. After the addition of diluted compounds to each well, cGMP™ solution was added to initiate the reaction. After an appropriate incubation, Kinase-Glo^® reagent was pipetted into each well and 10 min later luminescence was measured using a plate reader (Thermo Scientific Varioskan^® Flash).

Wisanwattana W., Wongkrajang K., Cao D.Y., Shi X.K., Zhang Z.H., Zhou Z.Y., Li F., Mei Q.G., Wang C., Suksamrarn A., Zhang G.L, & Wang F. (2021). Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway. Frontiers in Endocrinology, 12, 636784.

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Evaluation of PDE5 Activity by PDE-Glo Assay

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The PDE5 activity after incubation with L-ARG and L-CIT was evaluated using the PDE-Glo Phosphodiesterase Assay according to the manufacturer’s instruction (Promega Corporation, Madison, WI, USA). Briefly, 5 μl of 1× PDE-Glo reaction buffer containing 10 mU of purified Bovine PDE5 (Sigma-Aldrich, St Louis, MO) was pipetted into 96- white opaque well plate wells (Thermo Scientific, Waltham MA). The tested compounds were dissolved in water, and serial dilutions of L-ARG and L- CIT were created using 1× PDE-Glo reaction buffer. Then, 10 μL of diluted inhibitors and 12.5 μL of cGMP solution were added to each well. After 90 min of incubation in 25 °C, 12.5 μL of PDE-Glo™ Termination Solution and 12.5 μL of PDE-Glo™ Detection solution were added and the plate was incubated for 20 min at RT. Finally, 50 μL of Kinase-Glo® Reagent was pipetted to each well and after 10 min of incubation, the luminescence was measured using a microplate luminometer (Perkin Elmer, Waltham MA,). All data points were the average of two experiments done by triplicates, and expressed as count per second (CPS).

Ferrini M.G., Abraham A., Nguyen S., Luna R., Flores M., Artaza J.N., Graciano L, & Rajfer J. (2019). EXOGENOUS L-ARGININE DOES NOT STIMULATE PRODUCTION OF NO OR cGMP WITHIN THE RAT CORPORAL SMOOTH MUSCLE CELLS IN CULTURE. Nitric oxide : biology and chemistry, 89, 64-70.

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PDE Inhibition Assay Protocol

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Inhibitory activity of the investigated and reference compounds against various subtypes of PDEs was analyzed using the PDE-Glo Phosphodiesterase Assay following the manufacturer’s instruction (Promega Corp., USA). A brief description of this procedure was presented in the supplementary materials. The values of IC₅₀, I_max, and γ parameters of the investigated and reference compounds were estimated using non-linear regression in Phoenix WinNonlin v. 7.0. Each sample was performed in quadruplicate and the relative activity of each sample was calculated as

E = \frac{sample activity}{control activity} \cdot 100 %

. Subsequently, the following equation was fitted to the effect-concentration data:

E = E_{0} - I_{\max} \cdot (\frac{C^{γ}}{C^{γ} + {IC}_{50}^{γ}})

where E₀ is a baseline relative activity of each enzyme in the absence of a studied compound (fixed to 100%). I_max is the maximal inhibitory activity of a compound, C is the concentration of a compound, IC₅₀ is the concentration of a compound that produces 50% of I_max, and γ is the Hill coefficient.

Świerczek A., Pociecha K., Ślusarczyk M., Chłoń-Rzepa G., Baś S., Mlynarski J., Więckowski K., Zadrożna M., Nowak B, & Wyska E. (2020). Comparative Assessment of the New PDE7 Inhibitor – GRMS-55 and Lisofylline in Animal Models of Immune-Related Disorders: A PK/PD Modeling Approach. Pharmaceutical Research, 37(2), 19.

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PDE4 Activity Assay in Brain Regions

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PDE4 activity was assayed by PDE-Glo™ Phosphodiesterase Assay (Promega, Madison, WI) according to the manufacturer’s protocol. Briefly, the NAc, PFC, and VTA were homogenized in KHEM buffer ([in mM] 50 KCl, 10 EGTA, 1.92 MgCl₂, 1 DTT, and 50 HEPES, pH 7.2) containing protease inhibitor cocktail (nacali tesque, Kyoto, Japan) and PhosSTOP (Roche, Mannheim, Germany), centrifuged, and the supernatant fractions were pre-incubated with 10 μM rolipram or DMSO. cAMP at a final concentration of 2 μM was used as a substrate. PDE-Glo termination buffer containing PDE inhibitor IBMX (Sigma-Aldrich, St. Louis, MO) was added to inhibit most PDEs. After the addition of PDE-Glo detection solution and kinase-Glo reagent, luminescence was measured using a plate-reader. PDE4 activity was designated as the remainder after subtracting the value of PDE activity in the presence of rolipram from that in the presence of DMSO.

Hikida T., Morita M., Kuroiwa M., Macpherson T., Shuto T., Sotogaku N., Niwa M., Sawa A, & Nishi A. (2019). Adolescent psychosocial stress enhances sensitization to cocaine exposure in genetically vulnerable mice. Neuroscience research.

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