Simple, Sensitive Detection of Protein Phosphodiesterase Activity Measure Real-Time Signal Differences in the Picomole Range with the Phosphate Sensor Method

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APPLICATION NOTE Phosphodiesterase Activity: A Phophate Sensor Application

Simple, Sensitive Detection of Protein Phosphodiesterase Activity Measure real-time signal differences in the picomole range with the Phosphate Sensor method

Described in this application is a method utilizing the Life Technologies Phosphate Sensor, a simple tool to interrogate the activity of phosphate-releasing enzymes. The assays described detect the increase of fluorescence intensity when free inorganic phosphate binds to a bacterially derived phosphate-binding protein modified with a fluorophore.To evaluate the Phosphate Sensor methods, we compared detection by coupling to an alkaline phosphatase against a commonly used coupled luciferase assay, examined responses for human phosphodiesterase A (PTE5A) in a titration, in real-time kinetic mode, and with a sildenafil citrate inhibitor. Phosphate Sensor is orders of magnitude more sensitive than the coupled luciferase method, faster and simpler to use than other competitor methods, and uniquely qualified for determining enzymatic rates.

Second messenger systems that involve an increase or decrease of cyclic nucleotides (cAMP or cGMP) mediate intracellular signal trans- duction. Nucleotide degradation regulates this process and is mediated through the action of phosphodiesterase (PDE) enzymes, which also play an integral role in a number of disorders including erectile dysfunction [1], asthma [2], and chronic obstructive pulmonary disease [2], as well as schizophrenia, bipolar disorder, and major depression [3]. As research identifies additional potential drug targets, methodologies for measur- ing activity become vital. Figure 1. Phosphate Sensor assay principle. The protein ribbon diagram illustrates the modified We describe here the use and optimization of a simple, flexible reagent phosphate-binding protein with the MDCC fluoro- for measurement of phosphodiesterase activity through coupling it with phore (shown in blue). Upon binding inorganic a phosphatase. Phosphate Sensor is a phosphate-binding protein modi- phosphate, fluorescence of Phosphate Sensor increases approximately 6- to 8-fold and can be fied with a fluorophore [4]. As the sensor binds free inorganic phosphate measured in real time. (Pi), fluorescence intensity increases (Figures 1 and 2). This simple direct measurement of released phosphate is not dependent on a specific substrate or enzyme, making it amenable to almost any target of interest.

www.lifetechnologies.com/phosphatesensor Note: Phosphate is common in biological materials, buffers, and reagents, as well as on plastics and glassware. Care should be taken to minimize contamination from these sources when using the Phosphate Sensor product. Using the phosphate mop (Box 1) in control samples can rule out phosphate contamination problems or help identify sources. Care also should be used when selecting a microplate as some surface-coated plates contain significant amounts of phosphate. We recommend using Corning 384-well uncoated plates (P/N 3677).

Box 1. The phosphate mop. The phosphate mop, comprised of 7-methyl guanosine (7-MEG) and purine nucleoside phosphorylase (PNPase), can be used to sequester potentially contaminating inorganic phosphate that may be present in experimental solutions or materials in the form of ribose-1-phosphate [7]. For typical applications, 200 µM 7-MEG and 0.1 to 1.0 U/mL PNPase are used. Water is used to dissolve 7-MEG (Sigma P/N M0627) to a 30 mM stock solution (stored at –80°C) and PNPase (Sigma P/N N8264) to 500 U/mL (dispensed into small aliquots to avoid freeze/thaw cycles and stored at –80°C).

Materials and methods Phosphodiesterase 5A (PDE5A; SignalChem P/N P93-31G), cGMP (Sigma P/N G6129), GMP (Sigma P/N G8377), alkaline phosphatase (Calbiochem P/N 524545), sildenafil citrate (Tocris P/N 3784), PDE-Glo™ Phosphodiesterase Assay (Promega P/N V1361), 3-isobutyl-1Simple,-methylxanthine Sensitive (IBMX, Sigma Detection-Aldrich P/N I5879)of Phosphodiesterase, and Phosphate Sensor Activity (Invitrogen P/N PV4406), were used in these experiments. The PDE enzymatic reaction buffer consisted of 50 mM Tris Note:pH 7.6, Phosphate 100 mM isNaCl, common 10 mM in MgCl biological2, 0.01% materi Triton- XExperimental-100, and 0.5 mM Procedures DTT. The 1X termination buffer for the PDE- als,Glo™ buffers, reaction and consisted reagents, of as 100 well mM as IBMX on plastics in 100% DMSO. The Phosphate Sensor detection buffer consisted of 20 mM Tris pH 7.6 and 0.05% Triton X-100. All assays were performed in black 384-well low-volume, round-bottom, non-treated and glassware. Care should be taken to minimize plates (Corning P/N 3677), except for the “CompetitorMaterials A” assays and, which methods were performed in white 384-well low volume, contaminationround bottom, from non these-treated sources plates when (Corning using Phos P/N- 3674)Phosphodiesterase. 5A (PDE5A; SignalChem P/N P93-31G), cGMP (Sig- phate Sensor. Using the phosphate mop (Box 1) in ma P/N G6129), GMP (Sigma P/N G8377), alkaline phosphatase (Cal- controlOptimization samples of can alkaline rule out phosphatase phosphate contami concentration- biochem P/N 524545), sildenafil citrate (Tocris P/N 3784), PDE-Glo™ nationThe first problems step of or the help comparison identify sources. process Care was also optimization of the alkaline phosphatase read-out reaction. A two-fold dilution series of alkaline phosphatase ranging from 250Phosphodiesterase mU/mL to 0.00012 Assay mU/mL (Promega was run P/Nin a finalV1361), 10 µL 3-isobutyl-1-methylx reaction - should be used when selecting a microplate as some volume and incubated for 60 minutes with no nucleotideanthine, 5 µM (IBMX, GMP, Sigma-Aldrichor 5 µM cGMP. AfterP/N I5879),the reaction, and Phosphate 10 µL of 2X Sensor (1 (Invit- surface-coatedµM) Phosphate plates Sensor contain Protein significant in Phosphate amounts S ofensor detection buffer was added to a final volume of 20 µL and rogen P/N PV4406), were used in2 theseTM experiments. The PDE enzymatic phosphate.concentration We recommendof 0.5 µM. Theusing plate Corning was 384-wellmixed and read immediately on a Tecan Safire at excitation 430 nm (10) and uncoatedemission plates 450 nm(P/N (10 3677).) (Figure 3). reaction buffer consisted of 50 mM Tris pH 7.6, 100 mM NaCl, 10 mM ® MgCl2, 0.01% Triton X-100, and 0.5 mM DTT. The 1X termination buffer 60,000 for the PDE-Glo™ reaction consisted of 100 mM IBMX in 100% DMSO. 50,000 The Phosphate Sensor detection buffer consisted of 20 mM Tris pH 7.6 40,000 and 0.05% Triton® X-100. All assays were performed in black 384-well 30,000 low-volume, round-bottom, non-treated plates (Corning P/N 3677), except for the “Competitor A” assays, which were performed in white 384- 20,000 well low volume, round bottom, non-treated plates (Corning P/N 3674). 10,000

(Em 450) Fluorescence 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 Optimization of alkaline phosphatase concentration

[Alkaline PPase] (mU/mL) The first step of the comparison process was optimization of the alka-

nono nucleotide nucleotide ▲ 5 5µMµM GMP ▼ 5 µM5 µ McGMP cGMP ■ line phosphatase read-out reaction. A two-fold dilution series of alka-

Figure 3. Alkaline phosphatase titration. A two- line phosphatase ranging from 250 mU/mL to 0.00012 mU/mL was run Figure 3. Alkaline phosphatase titration. A two-fold serial dilution of alkaline phosphatase was incubated for 60 fold serial dilution of alkaline phosphatase was in a final 10 µL reaction volume and incubated for 60 minutes with no minutes with no nucleotide, 5 µM GMP, or 5 µM cGMP. While alkaline phosphatase had no affect on cGMP, signal above incubatedbackground for was60 minutes observed with when no nucleotide, GMP hydrolyzed 5 μM intonucleotide, guanosine 5and µM inorganic GMP, or phosphate. 5 µM cGMP. After the reaction, 10 µL of 2X GMP, or 5 μM cGMP. While alkaline phosphatase (1 µM) Phosphate Sensor in Phosphate Sensor detection buffer was add- hadHuman no affect PDE5A on cGMP,titration signal above background ed to a final volume of 20 µL and concentration of 0.5 µM. The plate was was observed when GMP hydrolyzed into guanos- In the second step of the evaluation, a direct comparisonmixed of theand Phosphate read immediately Sensor to on a competitora Tecan Safire2™ technology microplate was reader at inedetermined and inorganic via a phosphate. human phosphodiesterase 5A (PDE5A) titration. For the Phosphate Sensor, a two-fold serial dilution of PDE5A was made in PDE enzymatic reaction buffer andexcitation incubate 430d fornm 60 (10) minutes and emission in the presence 450 nm or (10) absence (Figure of 3). 5 µM cGMP and 10 mU/mL alkaline phosphatase. The PDE reaction was performed in 10 µL and inorganic phosphate was detected by adding 10 µL of 2X (1 µM) Phosphate Sensor Protein to a final volume of 20 µL and concentration of 0.5 µM. The plate was mixed and read as previously described. For the PDE-Glo™ technology, which relies on a multiple enzymaticA. Coupled cascade Luciferase before readout Method, the PDE5A reaction was run accordingB. Phosphate to the user Sensor guide supplied Method with the kit. In short, a two-fold serial dilution of PDE5A in PDE-Glo™ reaction buffer was made, incubated for 60 minutes with 5 µM cGMP in 5 µL, and followed byphophorylated the addition of 2.5 µL of a 1X termination buffer with the IBMXAlkaline inhibitor and incubatedPhosphate for 10 minutes. Next, 2.5 µL of proteinPKA substratekinase a (PKA) was added in a 1X detectionPDE buffer andPPase incubated for 20 minutesSensor. Finally, 10 µL of Kinase-Glo® reagent was added and incubated for 10 minutes in a final reaction volume of 20 µL before —P reading luminescence in the plate on a—P BMG LabTech PHERAStar Plus microplate reader (Figure 4). The enzymatic ADP cNMP cNMP R R cNMP NMP N-osine + Pi

R R ATP C C C C O2 + luciferin PKA substrate PKA PKA holoenzyme KEY: Alkaline PPase = Alkaline Phosphatase luciferase (active) (inactive) cNMP = cAMP or cGMP NMP = AMP or GMP N-osine = Adenosine or Guanosine PDE = Phosphodiesterase oxyluciferin + AMP + light Pi = Inorganic Phosphate

Figure 2. Comparison of a coupled luciferase assay to the Phosphate Sensor method. (A) The Promega PDE-Glo™ assay involves coupled reactions that measure phosphodiesterase activity by following the activation of protein kinase A (PKA) from holoenzyme and assessing the amount of ATP depleted with Kinase-Glo® Reagent in a luciferase reaction. (B) By coupling Phosphate Sensor with a hydro- lase enzyme like alkaline phosphatase (alkaline PPase), the Phosphate Sensor method detects phosphodiesterase activity in fewer steps.

2 steps and assay setups for the two methods compared are shown in Figure 2 and Table 1 shows the comparison of these two assay setups. steps and assay setups for the two methods compared are shown in Figure 2 and Table 1 shows the comparison of these two assay setups. Phosphate Sensor Method PDE-Glo™ Method detection Phosphatefluorophore Sensor Method PDEmultiple-Glo™ enzymatic Method cascade

detectionassay format fluorophore384-well multiple384-well enzymatic cascade

assayfinal assay format volume 38420 -µLwell 38420- wellµL

finalassay assay time volume 2060 µL minutes 2010 µL0 minutes

assay inhibitable time steps 602 (PminutesDE5A and alkaline PPase reactions) 1030 ( minutesPDE5A, PKA, and luciferase reactions)

stepsinhibitable steps 23 (PDE5A and alkaline PPase reactions) 3 5(PDE5A, PKA, and luciferase reactions) steps 3 5 protocol 1. Add 5 µL of 2X PDE5A 1. Add 2.5 µL of 2X PDE5A protocol 1.2. AddAdd 5 5 µL µL of of 2X 2X P cGMPDE5A and 2X alkaline 1.2. Add Add 2. 52 .µL5 µL of of 2X 2X PDE5A cGMP , incubate 60 2.PPase Add ,5 incubateµL of 2X cGMP60 minutes and 2X at alkalineRT 2.minutes Add 2.5 atµL RTof 2X cGMP, incubate 60 PPase3. Add, incubate10 µL of 2X60 minutesPhosphate at RTSensor minutes3. Add at2.5 RT µL IBMX in 1X termination 3.Protein, Add 10 read µL of plate 2X Phosphate immediately Sensor 3.buffer Add 2.5 µL IBMX in 1X termination Protein, read plate immediately buffer4. Add 2.5 µL PKA in 1X detection buffer 4.5. Add Add 2.5 10 µL µL PKA Kinase in 1X-Glo® detection buffer 5. Add 10 µL Kinase-Glo®

Table 1. Comparison of PDE5A assays between the Phosphate Sensor and competitor methods. The Phosphate TableSensor 1. Assay Comparison requires of fewer PDE5A steps assays (also betweensee Figure the 5) Phosphate in a shorter Sensor total assay and timecompetitor than the methods. multiple enzymatic The Phosphate cascade Sensortechnology. Assay The requires Phosphate fewer Sensorsteps (also assay see also Figure has 5)fewer in a “inhibitable”shorter total steps.assay time than the multiple enzymatic cascade technology. The Phosphate Sensor assay also has fewerSimple, “inhibitable” Sensitive steps. Detection of Phosphodiesterase Activity

A Human PDE5A titration A 55,000 55,00050,000 In the second step of the evaluation, a direct comparison of Phos- 50,00045,000 phate Sensor to a competitor technology was determined via a hu- 45,00040,000 40,00035,000 man phosphodiesterase 5A (PDE5A) titration. For Phosphate Sensor, 35,00030,000

30,00025,000 a two-fold serial dilution of PDE5A was made in PDE enzymatic reac-

25,00020,000 tion buffer and incubated for 60 minutes in the presence or absence of 20,00015,000 15,00010,000 5 µM cGMP and 10 mU/mL alkaline phosphatase. The PDE reaction was 10,000 (Em 450) Fluorescence 5,000 performed in 10 µL and inorganic phosphate was detected by adding

(Em 450) Fluorescence 5,0000 010 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 µL of 2X (1 µM) Phosphate Sensor to a final volume of 20 µL and -3 -2 -1 0 1 2 3 4 5 10 10 10 [PDE5A]10 10 (pg/well)10 10 10 10 concentration of 0.5 µM. The plate was mixed and read as previously de- [PDE5A] (pg/well) nono cGMP, cGMP, no Alk. no PPaseAlk. PPase ▲ 5 µMno cGMP,cGMP, no 10 Alk. mU/mL PPase Alk. PPase scribed. For the PDE-Glo™ technology, which relies on a multiple enzy- ■ no cGMP, no Alk. PPase no cGMP, 10 mU/mL Alk. PPase ▼ no5 cGMP,µM cGMP, 10 mU/mL no Alk. Alk. PPase PPase ♦ 5 µM5 cGMP,µM cGMP, 10 mU/mL 10 mU/mL Alk. PPase Alk. PPasematic cascade before readout, the PDE5A reaction was run according to 5 µM cGMP, no Alk. PPase 5 µM cGMP, 10 mU/mL Alk. PPase B the user guide supplied with the kit. In short, a two-fold serial dilution of B B 110 110 110 110 PDE5A in PDE-Glo™ reaction buffer was made, incubated for 60 minutes

90 90 with 5 µM cGMP in 5 µL, and followed by the addition of 2.5 µL of a 1X Normalized

90 90 signalRLU Normalized

signalRLU 70 70 termination buffer with the IBMX inhibitor and incubated for 10 minutes. 70 70

50 50 Next, 2.5 µL of protein kinase a (PKA) was added in a 1X detection buffer 50 50 and incubated for 20 minutes. Finally, 10 µL of Kinase-Glo® reagent was

RFU RFU signal 30 30 Normalized

RFU RFU signal 30 30 Normalized added and incubated for 10 minutes in a final reaction volume of 20 µL 10 10 10 10 before reading luminescence in the plate on a BMG LabTech PHERAStar -10 -10 -10 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10-105 -2 -1 0 1 2 3 4 5 10 10 10 10 10 10 10 10 Plus microplate reader (Figure 4). The enzymatic steps and assay setups [PDE5A] (pg/well) [PDE5A] (pg/well) for the two methods compared are shown in Figure 2 and Table 1 shows Phosphate Sensor "Competitor A" Assay ■ PhosphatePhosphate Sensor Sensor ▲"Competitor"Competitor A" assay A" Assay the comparison of these two assay setups.

Figure 4. Human PDE5A titrations. (A) Human Kinetic enzyme reaction PDE5A titration using the Phosphate Sensor assay. The PDE5A generates a sigmoidal curve with In the third portion of this comparison, Phosphate Sensor was utilized to analyze a PDE5A reaction in real time kinetic-mode. The PDE5A as- an EC50 of 290 pg/well. As expected, PDE5A activity was only observed in the presence of both cGMP say was run as described previously except that after addition of cGMP and alkaline phosphatase. Background signals and alkaline phosphatase, there was no incubation period. Rather, the from the alkaline phosphatase or cGMP are neg- 2X Phosphate Sensor was added immediately and the plate was read ligible. (B) The Phosphate Sensor assay as com- in kinetic mode, collecting data every minute for a total of 60 minutes pared to “Competitor A” assay. (Figure 5).

Phosphate Sensor Method PDE-Glo™ Method detection fluorophore multiple enzymatic cascade assay format 384-well 384-well final assay volume 20 µL 20 µL assay time 60 minutes 100 minutes inhibitable steps 2 (PDE5A and alkaline PPase reactions) 3 (PDE5A, PKA, and luciferase reactions) steps 3 5 protocol 1. Add 5 µL of 2X PDE5A 1. Add 2.5 µL of 2X PDE5A 2. Add 5 µL of 2X cGMP and 2X alkaline PPase, incubate 2. Add 2.5 µL of 2X cGMP, incubate 60 minutes at RT 60 minutes at RT 3. Add 2.5 µL IBMX in 1X termination buffer 3. Add 10 µL of 2X Phosphate Sensor, read plate 4. Add 2.5 µL PKA in 1X detection buffer immediately 5. Add 10 µL Kinase-Glo® reagent

Table 1. Comparison of PDE5A Assays between Phosphate Sensor and a competitor method. The Phosphate Sensor Assay requires fewer steps (also see Figure 5) in a shorter total assay time than the multiple enzymatic cascade technology. The Phosphate Sensor assay also has fewer “inhibitable” steps.

3 Figure 4. Human PDE5A titrations.(A) Human PDE5A titration using the Phosphate Sensor assay. The PDE5A generates a sigmoidal curve with an EC50 of 290 pg/well. As expected, PDE5A activity was only observed in the presence of both cGMP and alkaline phosphatase. Background signals from the alkaline phosphatase or cGMP are negligible. (B) The Phosphate Sensor assay as compared to “Competitor A” assay. The Phosphate Sensor with an EC50 of 240 pg/well is about 10-fold more sensitive than the “Competitor A” assay with an EC50 of 2.6 ng/well.

Kinetic enzyme reaction In the third portion of this comparison, the Phosphate Sensor Protein was utilized to analyze a PDE5A reaction in real Figuretime 4 .kinetic Human-mode. PDE5A The titrations PDE5A.(A) assayHuman was PDE5A run titration as described using the previously Phosphate Sensorexcept assay. that after The PDE5Aaddition of cGMP and alkaline phosphatase, there was no incubation period. Rather, the 2X Phosphate Sensor Protein was added immediately and the generatesFigure 4. Humana sigmoidal PDE5A curve titrations with an .(ECA) 50Human of 290 PDE5A pg/well .titration As expected, using thePDE5A Phosphate activity Sensor was only assay. observed The PDE5A in the presencegeneratesplate wasof a bothsigmoidal read cGMP in curve kineticand alkalinewith mode, an ECphosphatase. 50collecting of 290 pg/well Backgrounddata. Asevery expected, signals minute PDE5Afrom for thea activitytotal alkaline of was 60phosphatase onlyminutes observed (Figureor cGMP in the 5) are. negligible.presence of (B both) The cGMP Phosphate and alkaline Sensor phosphatase. assay as compared Background to “Competitor signals from A” assay.the alkaline The Phosphate phosphatase Sensor or cGMP with anare EC 50 of 240 pg/well is about 10-fold more sensitive than the “Competitor A” assay with an EC50 of 2.6 ng/well. negligible. (B) The Phosphate30000 Sensor assay as compared to “Competitor A” assay.4 ng PDE5The Phosphate Sensor with an EC50 of 240 pg/well is about 10-fold more sensitive than the “Competitor A” assay with2 ng an PDE5 EC50 of 2.6 ng/well. Kinetic enzyme reaction25000

InKinetic the third enzyme portion reaction of this comparison , the Phosphate Sensor Protein was utilized1 ng to PDE5 analyze a PDE5A reaction in real timeIn the kinetic third -portionmode. ofThe this20000 PDE5A comparison assay, wasthe Phosphaterun as described Sensor previously Protein was except utilized that0.5 to ngafter analyze PDE5 addition a PDE5A of cGMP reaction and alkaline in real phosphatase,time kinetic-mode. there The was PDE5A no incubation assay wasperiod. run Rather,as described the 2X previously Phosphate except Sensor that Protein after addition was added of cGMP immediate and alkalinely and the 15000 0.25 ng PDE5 platephosphatase, was read there in kinetic was nomode, incubation collecting period. data Rather,every minute the 2X for Phosphate a total of Sensor60 minutes Protein (Figure was 5) added. immediately and the 0.125 ng PDE5 plate was read in kinetic10000 mode, collecting data every minute for a total of 60 minutes (Figure 5). 30000 PDE5A4 ng (ng) PDE5 Inhibition0.063 ng of PDE5 PDE5A activity using sildenafil citrate 42 ng PDE5 2500030000 5000 4 ng PDE5 In the0.031 final ng PDE5 step of this comparison, Phosphate Sensor was used to ana- 1 ng PDE5 (Em 450) Fluorescence 22 ng PDE5 0.016 ng PDE5 2000025000 0 10.51 ng ng PDE5 PDE5lyze inhibition of PDE5A activity by sildenafil citrate. The assay was run 0 1000 2000 3000 4000 1500020000 0.50.250.5 ng ng PDE5 PDE5as previously described, except that the order of addition for the PDE5A

Time (sec) 0.250.1250.25 ng ng PDE5 PDE5 1000015000 reaction was modified. First, a 3-fold serial dilution of sildenafil citrate 0.1250.0630.125 ng PDE5

Figure 5. PDE5A10000 titration in kinetic mode using the0.0630.031 Phosphate ng PDE5(2.5 µL Sensor of 4X concentrate) Protein. The was ability made of the and Phosphate incubated Sensor with 5 µL of 2X (1.6 5000 0.063 ng PDE5 to directly (Em 450) Fluorescence measure the amount of inorganic phosphate0.0310.0160.031 present ng PDE5 allows for real-time detection of its release. Because 50000 nM) PDE5A (total of 7.5 µL) for 10 minutes. Then, 2.5 µL of 4X (20 µM)

other competitor (Em 450) Fluorescence technologies cannot do this, determining enzymatic rates is a unique feature of the Phosphate Sensor 0 1000 2000 3000 4000 0.160.016 ng PDE5cGMP and 4X (40 mU/mL) alkaline phosphatase was added to start the technology. 0 0 1000 Time2000 (sec) 3000 4000 reaction and incubated for 60 minutes. Last, 10 µL of 2X (1 µM) Phos- Time (sec) Figure Inhibition 5. PDE5AFigure of titration PDE5A 5. PDE5A in activity kinetic titration mode using in using kineticsildenafil the mode Phosphate citrate using Sensor phate Protein. Sensor The ability was of added the Phosphate and read Sensor as described earlier (Figure 6). The toFigure directlyIn the 5. PDE5Ameasurefinal step titration the of amount this in comparisonkinetic of inorganic. modeThe , phosphate using abilitythe Phosphate the of Phosphatepresent Phosphate Sensor allows Sensor for protein real Protein.-time was detection The used ability toof analyzeitsof therelease Phosphate inhibition. Because Sensor of PDE5A activity by other competitorPhosphate technologies Sensor cannot do this, determining enzymatic rates finalis a unique concentration feature of the of Phosphate PDE5A Sensorwas 0.8 nM per reaction. to directlysildenafil measureSensor citrate. the to The amountdirectly assay of measure inorganic was run the phosphate as amount previously presentof inorganic described, allows for real except-time detectionthat the orderof its release of addition. Because for the PDE5A reaction technology.other competitor technologies cannot do this, determining enzymatic rates is a unique feature of the Phosphate Sensor technology.was modified. phosphate First, allowsa 3-fold for serial real-time dilution detection of sildenafil of its citrate Conclusions(2.5 µL of 4X concentrate) was made and incubated with 5 InhibitionµL of 2X of release.PDE5A(1.6 nM) activity Because PDE5A using (total other sildenafil of competitor 7.5 citrateµL) for technologies 10 minutes . Then, 2.5 µL of 4X (20 µM) cGMP and 4X (40 mU/mL) alkaline InInhibition thephosphatase final ofstep cannotPDE5A of thiswas doactivity comparison added this, using determining to, startthe sildenafil Phosphate the enzymaticreaction citrate Sensor and rates protein incubated is was a used for to60 analyze minutes. inhibition Last, of 10 PDE5A µL of activity2X (1 byµM) Phosphate Sensor sildenafil citrate. The assay was run as previously described, except that thePhosphate order of addition Sensor for theis aPDE5A simple reaction assay system used to directly measure In thewas final added stepunique of and this feature read comparison as of describedthe, Phosphatethe Phosphate earlier Sensor Sensor (Figure technology. protein 6). The was final used concentration to analyze inhibition of PDE5A of PDE5A was activity 0.8 nM by per reaction. wassildenafil modified. citrate. First, The a assay3-fold wasserial run dilution as previously of sildenafil described, citrate (except2.5 µL ofthat 4X the theconcentrat order amount of eaddition) was of inorganic made for the and PDE5A incubatedphosphate reaction with generated 5 in an enzymatic reaction. µLwas of modified. 2X (1.6 nM) First, PDE5A a 3-fold (total serial of dilution7.5 µL) forof sildenafil 10 minutes citrate. Then (2.5, 2.5 µL µL of of4X 4X concentrat (20 µM) ecGMP) was madeand 4X and (40 incubated mU/mL) alkaline with 5 50,000 This tool can also be configured to perform kinetic reads, which allows phosphataseµL of 2X (1.6 wasnM) PDE5Aadded to (total start of the 7.5 reaction µL) for and10 min incubatedutes. Then for ,60 2.5 minutes. µL of 4X Last, (20 µM)10 µL cGMP of 2X and (1 µM) 4X (40Phospha mU/mL)te Sensor alkaline wasphosphatase added and was read added as described to start the earlier reaction (Figure and 6)incubated. The final for concentration 60 minutes.users Last,of PDE5A 10the µL uniquewas of 2X 0.8 (1 nMability µM) per Phospha reaction.to measurete Sensor enzymatic rates. was added and read40,000 as described earlier (Figure 6). The final concentration of PDE5A was 0.8 nM per reaction. 50,000 Phosphate Sensor is very sensitive, detecting picomole quantities of in- 50,00030,000 40,000 organic phosphate in phosphodiesterase (PDE), protein phosphatase,

40,00020,000 ATPase, GTPase, and other activity assays. 30,000 30,000 20,00010,000 References 20,000 (Em 450) Fluorescence 10,000 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 1. Boolell M, Allen MJ, et al (1996) Int J Impot Res 8: 47-52. Fluorescence (Em 450) Fluorescence 10,000 0 -5 -4 -3 -2 [Sildenafil-1 0 1 2Citrate]3 4 (nM)5 (Em 450) Fluorescence 10 10 10 10 10 10 10 10 10 10 10 2. Nials AT, Tralau-Stewart CJ, et al. (2011) J Pharmacol Exp Ther 337: 137-44. 0 -5 -4 [Sildenafil-3 -2 -1 Citrate]0 1 (nM)2 3 4 5 Figure10 10 6. 10Inhibition10 10 10 of10 human10 10 10 PDE5A10 activity 3. Fatemi SH, Folsom TD, et al. (2010) Schizophr Res 119: 266-267. Figure 6. Inhibition of human PDE5A activity with sildenafil citrate using the Phosphate Sensor Protein. The IC50 with sildenafil[Sildenafil citrate Citrate] using (nM) Phosphate Sensor. Figure value 6. Inhibitionof 2.3 nM of obtained human PDE5A agrees activity with the with reported sildenafil value citrate of using 3.74. nMBrune the [ 5Phosphate]. M, Hunter Sensor JL, et Protein.al (1994) The Biochemistry IC50 33: 8262-8271. value of 2.3 nMThe obtained IC50 value agrees of 2.3with nM the obtainedreported valueagrees of 3.7with nM the [5 ]. Figure 6. Inhibition of human PDE5A activity with sildenafil citrate using the Phosphate Sensor Protein. The IC50 valueConclusions of 2.3 nMreported obtained value agrees of 3.7 with nM the [5]. reported value of 3.7 nM [5]. 5. Blount MA, Beasley A, et al. Mol Pharmacology 66: 144-152. Conclusions The Phosphate Sensor is a simple assay system used to directly measure the amount of inorganic phosphate generated The Phosphate Sensor is a simple assay system used to directly measure the amount of inorganic phosphate generated Conclusionsin an enzymatic reaction. This tool can also be configured to perform kinetic reads, which allows users the unique ability in an enzymaticBox reaction. 1. The This phosphatetool can also be mop. configured to perform kinetic reads, which allows users the unique ability Theto Phosphate measure Sensor enzymatic is a simple rates. assay system used to directly measureLearn the amount more of inorganic phosphatewww.lifetechnologies.com/phosphatesensor generated toin anmeasure enzymatic enzymatic reaction. rates. This tool can also be configured to perform kinetic reads, which allows users the unique ability to measure enzymaticThe phosphate rates. mop, comprised of 7-methyl TheThe Phosphate Phosphate Sensor Sensor is very sensitive,is very sensitive, detecting picomoledetecting quantities picomole of inorganicquantitiesFor Technical phosphate of inorganic Support in phosphodiesterase phosphate for this or in other phosphodiesterase Invitrogen Discovery Sciences guanosine (7-MEG) and purine nucleoside phos- (PDE),The(PDE), Phosphate protein protein phosphatase, Sensor phosphatase, is very ATPase, sensitive, ATPase, GTPase, detecting andGTPase, picomole other activity and quantities other assays. ofactivity inorganic Products, assays. phosphate dial 760 in 603phosphodiesterase 7200, select option 3, extension 40266. (PDE), protein phosphatase,phorylase (PNPase), ATPase, canGTPase, be used and toother sequester activity assays. potentially contaminating inorganic phosphate Learn more Learn morethat may be present in experimental solutions or Learn more materials in the form of ribose-1-phosphate [7]. For typical applications, 200 μM 7-MEG and 0.1 to 1.0 U/mL PNPase are used. Water is used to dissolve 7-MEG (Sigma P/N M0627) to a 30 mM stock solution (stored at –80°C) and PNPase Product Description Catalog Number Size (Sigma P/N N8264) to 500 U/mL (dispensed into Phosphate Sensor PV4406 10 nmol small aliquots to avoid freeze/thaw cycles and Phosphate Sensor PV4407 100 nmol stored at –80°C). DTT, 1M P2325 1 ml

The Life Technologies products discussed are For Research Use Only, and are not intended for any animal or human therapeutic or diagnostic use. © 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respec- tive owners. Kinase-Glo® is a registered trademark and PDE-Glo™ a trademark of Promega Corporation. Safire2™ is a trademark of Tecan Group Ltd. Triton® is a registered trademark of Union Carbide Corporation. [1211]

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