Analytical Biochemistry 449 (2014) 132–138

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Development of phosphocellulose paper-based screening of inhibitorsof lipid kinases: Case study with PI3Kb

0003-2697/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ab.2013.12.029

⇑ Corresponding author. Fax: +91 040 66929900.E-mail address: sayan.mitra@gvkbio.com (S. Mitra).

Mahesh Yanamandra a,c, Labanyamoy Kole b, Archana Giri c, Sayan Mitra a,⇑a Biology Division, GVK Biosciences Pvt. Ltd., Hyderabad 500076, Andhra Pradesh, Indiab VINS BIO, Kothur Mandal, Mahaboobnagar District 509325, Andhra Pradesh, Indiac Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, 500085 Hyderabad, Andhra Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 October 2013Received in revised form 18 December 2013Accepted 20 December 2013Available online 29 December 2013

Keywords:Phosphatidyl inositol 3-kinasePhosphocelluloseLipidPtdInsPtdSerIC50

The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transductionpathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of thesepathways are common in oncogenesis, and they are known to be altered in other metabolic disorders aswell. Despite its huge potential as an attractive target in these diseases, there is an unmet need for thedevelopment of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhib-itors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinasescreening platform using a phosphocellulose plate that involves transfer of radiolabeled [c-32P]ATP tophosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phos-phocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format usingstandard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies towardPI3K isoforms. ATP and lipid apparent Km for both were determined and IC50 values generated thatmatched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay(PI3Kb as the target) as an excellent platform for the identification of novel chemical entities in PI3K drugdiscovery.

� 2013 Elsevier Inc. All rights reserved.

A multitude of kinases involved in signal transductionpathways comprise a major target class in therapeutic areas ofcancer and inflammation, among others. The phosphatidylinositol3-kinase (PI3K) pathway is one of the most important signal trans-duction pathways implicated in cell growth, survival, proliferation,and motility [1,17]. In addition to its functions in cancer andinflammation, the PI3K signaling pathway promotes the activationof platelets [2,3,15]. PI3Ks belong to the structurally andfunctionally different lipid kinase family that phosphorylates theD3-hydroxyl position of the membrane phospholipid phosphor-4,5-biphosphate (PIP2) to phosphor-3,4,5-triphosphate (PIP3).PI3Ks are subclassified into classes I, II, and III depending onsubstrate specificity and protein sequence. Class I has been widelystudied and comprises four isoforms, viz., p110a, p110b, p110c,and p110d.

The pharmacological significance of p110a lies in cancer cellgrowth; p110b in thrombosis and insulin signaling; p110c ininflammation, leukocyte chemotaxis, and neutrophil activation;and finally p110d in immune cell function, including B and T cellsignaling. Since PI3Ks are involved in a large spectrum of cellular

activities [17], there lies a huge potential and need for the develop-ment of isoform-specific PI3K inhibitors targeting distinct thera-peutic areas.

Lipid kinase assay methods such as homogeneous time-resolved fluorescence, phospholipid flash plate assay, and biolumi-nescent ADP detection assay have been reported in 96- and384-well formats [5–7]. However, they have certain limitationsin terms of cost, accuracy, and sensitivity. Radioactive methodsusing c-32P-labeled adenosine triphosphate (ATP) are the goldstandard for the study of kinase reactions and their phosphoryla-tion, avoiding the false positives and negatives associated withfluorescence assay formats. Typically, in a radiometric kinase as-say, the phosphorylated substrate (usually a peptide) is capturedon phosphocellulose paper. The complexity associated with thedevelopment of a lipid kinase assay arises from the difficulty incapturing lipid substrate/product (PIP2/PIP3) on the negativelycharged phosphocellulose plate for estimating transferred radioac-tive phosphate. In contrast, protein kinase assays convenientlycapture positively charged peptide substrate/product on thenegatively charged phosphocellulose plate. Conventional methodssuch as organic solvent extraction of lipids and separation onthin-layer chromatography are not suitable for high-throughputscreening. Despite their limitations, fluorescence (and other

Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138 133

format) assays are commonly used for inhibitor identification [11–13,16] because of the difficulties related to radioactive lipid prod-uct capture in the filter plate format. Recently, Knight et al. [4] re-ported usage of a nitrocellulose membrane of screening for lipidkinase.

Here we report, for the first time, the development of a radioac-tive method for the detection of [c-32P]ATP transferred by PI3K ona lipid substrate that was captured on phosphocellulose platesmanufactured by Millipore or Whatman. The assay parameterswere set with PI3Kb as the model enzyme and later extended toother PI3K isoforms. This is a simple and extensively validatedin vitro kinase assay in 96-well format, which can also be extendedto a 384-well layout. The IC50 for known PI3K inhibitors such asLY294002, TGX-221, and wortmannin subjected to this assay for-mat matched very well to the reported values. This assay was fur-ther used for the screening and profiling of novel chemicalstructures that inhibit pan-PI3Ks or show high selectivity.

Materials and methods

Materials

Recombinant human phosphatidylinositol 3-kinase b, a, c, andd (full-length enzymes) were purchased from Upstate (Millipore,UK) expressed in Spodoptera frugiperda (SF-21) cells. Phosphatidyl-inositol (PtdIns) and phosphatidylserine (PtdSer) were purchasedfrom Sigma Chemicals. Two types of phosphocellulose filter plateswere purchased: MultiScreenHTS-PH plates from Millipore andUnifilter P81 plates from Whatman Corp. Unlabeled ATP was pro-cured from Sigma Chemicals and [c-32P]ATP was from the Centerfor Cellular and Molecular Biology (Jonaki lab, Hyderabad, India).Dimethyl sulfoxide (Me2SO), Mops, EGTA, and MgCl2 were fromSigma Chemicals. Microscint ‘‘O’’ was obtained from PerkinElmerand polystyrene 96-well assay plates were from Greinerbio.

Lipid micelle preparation

PIP2 powder was dissolved in the manufacturer’s prescribedsolution containing 1 N HCl with methanol and chloroform. Dis-solved PIP2 was transferred into glass vials that were purged withnitrogen gas and immediately sealed with paraffin paper andplugged with caps. These aliquots were stored at �80 �C for furtheruse. PtdSer was procured from the manufacturer as a solution. Inthe final assay conditions, 1 mg of PIP2 was mixed with 1 mg ofPtdSer (ratio 1:1) in 1 ml sonication buffer (25 mM Mops, pH 7.0,1.0 mM EGTA) resulting in 1 lg/ll concentration of each of the lip-ids in the micelles. Sonication was carried out in a water bath soni-cator at room temperature for three cycles of 5 min each. Themultilamellar liposomes generated were stored at �20 �C. Thawedliposome vials were sonicated again to ensure micelle formation.These micelles were stable for 1 week when stored at 4 �C andfor 2 to 3 months when stored at �20 �C.

Processing the phosphocellulose plate

Phosphocellulose plates were equilibrated with 100 ll of 70%Ethyl alcohol followed by washing with 100 ll of Milli-Q waterusing a vacuum manifold. The pressure in the system was adjustedto 15 psi and maintained throughout the experiment. The com-plete sample volume (from each well) was transferred to the phos-phocellulose plate and filtered through the same, following which200 ll of 0.8% phosphoric acid was added for washing. Threewashes were done to make sure that the unbound radioactivitywas completely removed from the plate. Following this, the platewas completely dried and 100 ll of Microscint ‘‘O’’ was added to

the wells and mixed gently for 2 min on a lab MixMate. The platewas sealed and read in a Top Count radioactivity plate reader.

Assay setup in 96-well format

PI3K assays were performed in 96-well Greinerbio polystyreneplates. The enzyme was mixed with 5� reaction buffer (125 mMMops, pH 7.0, 25 mM MgCl2, 5 mM EGTA) and added into the plate.After brief shaking, lipid substrate (sonicated mixture of PtdIns(PIP2) and PtdSer in a 1:1 ratio) was added followed by additionof 3 lCi [c-32P]ATP mixed with 25 lM ATP. The reaction volumewas adjusted to 50 ll. The reactants were briefly mixed and incu-bated for the desired time at 30 �C. The reaction was terminated byadding 90 ll stop buffer (1:1 methanol:1 N HCl) to each well andincubating at room temperature for 10 min. The reaction mixwas transferred to phosphocellulose plates and processed as de-scribed above.

Compound screening and IC50 generation

A master mix of enzyme, 5� buffer, and water was added toeach well, followed by addition of presonicated lipid mix (1:1 mix-ture of PIP2 and PtdSer). A mixture of cold ATP including [c-32-

P]ATP was added to each well such that the final concentrationof the mix was 50 ng of enzyme, 1 lg of lipid, 3 lCi of [c-32P]ATP,with 25 lM ATP. Primary screening for compounds was performedat three concentrations in duplicate. Serially diluted compoundstocks were prepared in 50� concentration in 100% Me2SO in 96-well plates. One microliter of compound was added in duplicate(final concentration of Me2SO was 2%), mixed, and kept on ice.After the reaction was initiated with ATP, the plate was incubatedat 30 �C with gentle shaking. Reaction continued for 2 h and wasterminated as previously described. The dose–response curveswere generated by serially diluting the stock threefold up to 10concentrations and adding 1 ll to each well. Final concentrationswere 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.13, 0.04, 0.015, and0.005 lM. Concentrations of reference compounds were chosendepending on the compound potency, e.g., TGX-221 and wortman-nin ranged from 10 to 0.0005 lM. Percentage of inhibition ob-tained from primary screening was used to determine startingconcentration for compound IC50. The assay schematic flow is illus-trated in Fig. 1.

Determination of assay parameters

Tris and Mops buffers at various pH were used to obtain thebest signal over background. The assessment of various buffers atdifferent pH was carried on phosphocellulose paper with spottingof the reaction mix and subsequent washings followed by scintilla-tion counting. Various pH conditions, 6.0, 7.0, and 8.0, were testedfor both Tris–HCl and Mops buffers. Velocity of the reaction wasestimated at various time points (10, 20, 30, 60, 120, and180 min). Reactions were set up with 100 ng enzyme, 10 lg/wellPIP2:PtdSer (1:1), 1 lM ATP, and 3 lCi [c-32P]ATP. Reactions wereterminated at each time point by adding stop solution. The samevolume for each reaction was spotted on the phosphocellulose pa-per followed by washes for scintillation reading.

Enzyme titration was performed with various amounts of en-zyme ranging from 1 to 200 ng, 1 lM ATP, 3 lCi [c-32P]ATP, andPIP2:PtdIns = 10 lg/well (1:1 ratio). Lipid mixtures (10 lg/reac-tion) of PIP2 and PtdSer at different ratios were tested for optimalsignal:background. Ratios of PIP2:PtdSer ranged from 0:100, 20:80,40:60, 50:50, 60:40, 80:20, and 100:0. Mixes of 0.001 to 5 lg PIP2and PtdSer (1:1) were titrated with 50 ng of enzyme and a fixedATP concentration and incubated at 30 �C up to 2 h. With 1 lgPIP2 and PtdSer (1:1) mix and 50 ng of enzyme, ATP concentrations

Step-1

PI3K β kinaseLipid mix ( PIP2+PS)

ATP mix( cold + [γ -32P]ATP )

Me2SO orcompound

Reaction is carried for 2 hours at 30oC

Termination of reaction by addition of (1:1 Methanol: 1(N) HCl)

Step-2Complete reaction volume is transferred on pre equilibrated Phosphocellulose plates

Wash phosphocellulose plates to remove unbound radioactivity

PIP2 , PIP3, γphosphate PIP3 will be bound to phosphocellulose plate,unbound [γ -32P] ATP will be washed

Dry the plate to evaporate residual aqueous solution

Add microscint and read in Top Count plate reader

Fig.1. Assay setup. The enzyme reaction setup details and schematic of the assayare shown. PS, phosphatidylserine.

pH Tolerance

5 6 7 8 90

100002000030000400005000060000700008000090000

MOPS, pH 6.0

MOPS, pH 7.0MOPS, pH 8.0

TRIS, pH 8.0TRIS, pH 7.0

TRIS, pH 6.0

pH variants

CPM

Assay linearity

0 25 50 75 100 125 150 175 20025000

50000

75000

100000

125000

Time in Minutes

CPM

Protein optimization

0 50 100 150 200 2500

102030405060708090

100110

Protein concentration (ng/well)

Fold

cha

nge

(S/B

)

A

B

C

Fig.2. (A) Buffer composition optimization. ATP, 1 lM; [c-32P]ATP, 5 lCi; PIP2:Pt-dIns, 10 lg/well (1:1 ratio); temperature, 30 �C; enzyme concentration, 100 ng/well/reaction. (B) Assay linearity. ATP, 1 lM; [c-32P]ATP, 5 lCi; PIP2:PtdIns, 10 lg/well (1:1 ratio); temperature, 30 �C; enzyme concentration, 100 ng/well/reaction.(C) Enzyme concentration optimization. ATP, 1 lM; [c-32P]ATP, 5 lCi; PIP2:PtdIns,10 lg/well (1:1 ratio); temperature, 30 �C; enzyme concentration, 1 to 250 ng per/well/reaction.

134 Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138

were varied from 0.9 to 1000 lM. All assays were performed in 96-well plates. Substrate Km for lipid and ATP were determined byMichaelis–Menten plot in GraphPad Prism.

Various concentrations of Me2SO ranging from 0.1 to 10% weretested for their interference in the enzyme assay. IC50 values weredetermined for various inhibitors by using end-point reactions. Thereaction was carried out with 50 ng of enzyme at a 1:1 ratio ofPIP2:PtdSer, 25 lM ATP, and 3 lCi [c-32P]ATP for 2 h at 30 �C. Ref-erence and test compounds were dissolved in 100% Me2SO andserially diluted threefold in 100% Me2SO. The final concentrationof Me2SO was made to 2% in 50 ll of reaction mix. Concentrationsof LY294002 ranged from 100 to 0.005 lM, TGX-221 from 10 to0.0005 lM, and wortmannin from 10 to 0.0005 lM. The percent-age of inhibition was calculated by subtracting the blank fromthe compound treated activity and total enzyme activity. The totalenzyme activity was taken as 100% activity and percentage of inhi-bition was calculated with a defined formula: 100 � (test com-pound counts per minute (cpm) � 100/total activity cpm). Thesedata were plotted in a nonlinear regression analysis sigmoidaldose–response curve using variable slope for the determinationof IC50. The regression coefficient (r2) was kept above 0.9 for allthe IC50. The raw data were fitted into GraphPad Prism version 5for data analysis. Percentage coefficients of variation (%CVs) werecalculated from the standard deviations and mean counts betweenthe duplicates of the experiment. For each plate the Z factor wasdetermined according to Zhang et al. [8] to evaluate the qualityof the experiment. Various isoforms of PI3K (a, b, c, and d, fromMillipore) were assayed with the same reference compounds toestimate isoform selectivity if any.

Results and discussion

Phosphorylated forms of PtdIns (PIP, PIP2, PIP3) phosphoinosi-tides are lipid signaling molecules in the PI3K pathway that are

implicated in cell growth, proliferation, and survival. PIP isphosphorylated to PIP2 and PIP3 by PI3K-mediated subsequentphosphorylation. An imbalance in the bioactive lipids often leadsto diseases such as cancer, inflammation, diabetes, andatherosclerosis.

These biologically active lipid moieties are present in hydropho-bic membrane areas. Because of their chemical nature, they areinsoluble in aqueous solutions. They tend to aggregate, leading tomicelle formation. This makes it very cumbersome to develop anin vitro PI3K assay for a small-molecule screening campaign.

A very good fold change generated from the ratio of signal tobackground (S:B) in an assay system helps to rank order the

Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138 135

compounds tested, identify the structure–activity relationship(SAR), and prioritize pharmacophores that exhibit isoform selec-tivity. We developed a unique method for the screening ofsmall-molecule lipid kinase inhibitors using phosphocellulose pa-per. This paper-based method was translated onto phosphocellu-lose plates that are extensively used for the screening of proteinkinase inhibitors. Phosphocellulose papers/plates are gold stan-dard methods for the capture of phosphorylated products of pro-tein kinase reactions. Phosphocellulose plates from Millipore(MSPH) or Whatman have been widely used for kinase reactionsin the 96-well format that capture the c-32P-labeled positivelycharged peptide substrate on the negatively charged phosphocel-lulose paper. The peptide substrates, having basic amino acids,increase the net positive charge thereby facilitating the bindingof the peptide.

Phosphatidylinositol 4,5-diphosphate gets phosphorylated andbecomes phosphatidylinositol 3,4,5-triphosphate in an inositolring in a reaction catalyzed by PI3K. We used l-a-phosphatidylin-ositol 4,5-diphosphate sodium salt (PtdIns) and 1,2-diacyl-sn-gly-cero-3-phospho-l-serine (PtdSer) in the reaction mix that plays amajor role in interfacial enzyme kinetics by assisting the associa-tion of the enzyme with the surface-attached substrate [10]. BothPtdIns and PtdSer were sonicated as a mixture for the micelle

Effect of percentage of PtdIns ( PIP2) in the reaction mix

0 10 20 30 40 50 60 70 80 90 1000

10

20

30

40

% of PtdIns ( PIP2)

Fold

cha

nge

(S/B

)

Percentage of γ phosphate to PIP2(PIP2 to PIP3 conversion)

10 µM 25 µM 50 µM 100 µM 200 µM 1000µM-10

01020304050607080

0.01 µg

0.1 µg

0.5 µg

1 µg

5 µg

ATP Concentrations

% r

adio

activ

e in

corp

orat

ion

A

B D

C

Vel

ocity

(CPM

min

-1)

Vel

ocity

( CPM

min

-1)

Fig.3. (A) Optimizing PIP2/PtdSer composition. ATP, 1 lM; [c-32P]ATP, 5 lCi; PIP2:PtdIn50 ng/well/reaction; time of the reaction, 2 h; buffer, Mops. (B) PIP2 to PIP3 conversion. ATratio); temperature, 30 �C; enzyme concentration, 50 ng/well/reaction; time for the reactiMichaelis–Menten template in GraphPad Prism software. ATP, 1 to 1000 lM; [c-32P]ATP,time, up to 2 h; temperature, 30 �C. Buffer, Mops. (D) Lipid Km determination. ATP, 25 lenzyme concentration, 50 ng/well/reaction; time, up to 2 h; buffer, Mops. PS, PtdSer.

formation. These lipid micelles presumably facilitate PI3K inphosphorylating PtdIns at their respective positions.

Upon completion, the reaction was terminated by methanol:1 Nhydrochloric acid. Adding HCl to the PtdIns protonates thephosphate groups [14]. Net positive charge increases in the pres-ence of strong acid owing to protonation of phosphate groupsand carbon atoms in the fatty acid chains. PtdSer also gets a posi-tive charge owing to the presence of strong acid. This makes themolecules acquire a high positive charge facilitating the bindingof the lipid substrate to the negatively charged phosphocellulosepaper by electrostatic interactions, which is captured for the read-ing. The residual unincorporated radioactivity is washed out withphosphoric acid washing.

Optimizing assay parameters

The initial set of experiments was carried out on precut papersfrom Millipore, and the experiments were performed in 0.5-mlEppendorf tubes. After the reaction, half the reaction volume wasspotted on phosphocellulose paper followed by washing and read-ing in the Top Count using Microscint.

The optimal pH for PI3K activity was examined with Mops andTris buffers generally used for kinase assays. We limited ourselves

ATP Km

0 250 500 750 1000 12500

25

50

75

100

125

150

175

Michaelis-MentenBest-fit values VMAX KM

160.523.42

ATP Concentration (µM)

Lipid Km

0 1 2 3 4 5 60

60

120

180

240

Michaelis-MentenBest-fit values VMAX KM

243.60.9220

1:1 ratio PIP2/PS (conc in µg)

s, 10 lg/well in different compositions; temperature, 30 �C; enzyme concentration,P, 10 to 1000 lM; [c-32P]ATP, 5 lCi; PIP2:PtdIns composition, 0.01 to 5 lg/well (1:1

on, 2 h; buffer, Mops. (C) ATP Km determination. Km was derived by calculating in the5 lCi; PIP2:PtdIns, 1 lg/well (1:1 ratio); enzyme concentration, 50 ng/well/reaction;M; [c-32P]ATP, 5 lCi; PIP2:PtdIns, 0.01 to 5 lg/well (1:1 ratio); temperature, 30 �C;

PI3K beta inhibition

-10 -9 -8 -7 -6 -5 -4 -30

102030405060708090

100110120

WortmanninTGX-221LY-294002

EC50Wortmannin9.572e-009

TGX-2214.771e-009

LY-2940029.057e-007

Log drug Conc [M]

%in

hibi

tion

Effect of Me2SO

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8

0100002000030000400005000060000700008000090000

100000110000120000

% of Me2SO

CPM

PI3K delta inhibition

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

102030405060708090

100110120

LY294002TGX-221Wortmannin

EC50LY2940022.852e-006

TGX-2211.949e-006

Wortmannin4.011e-009

Log Drug Conc [M]

% in

hibi

tion

PI3K-gamma inhibition

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3-10

0102030405060708090

100110

LY294002

Wortmannin

TGX-221

EC50LY2940021.165e-005

Wortmannin1.264e-009

TGX-2212.732e-006

Log Conc [M]%

inhi

bitio

n

PI3K-alpha inhibition

-12 -11 -10 -9 -8 -7 -6 -5 -4 -30

102030405060708090

100

LY294002

Wortmannin

TGX-221

EC50LY2940022.308e-006

Wortmannin5.173e-010

TGX-2211.799e-006

Log Conc [M]

% in

hibi

tion

A

B

C

D

E

Fig.4. (A) Me2SO effect. ATP, 25 lM; [c-32P]ATP, 3 lCi; PIP2:PtdIns, 1 lg/well (1:1 ratio); Me2SO concentration, variable; temperature, 30 �C; enzyme concentration, 50 ng/well/reaction; time, 2 h; buffer, Mops. (B) Reference compound screening. ATP, 25 lM; [c-32P]ATP, 3 lCi; PIP2:PI, 1 lg/well; temperature, 30 �C; enzyme concentration,50 ng/well/reaction; time for the reaction, 2 h; buffer, Mops. (C) Reference compound inhibition in other PI3K isoforms. IC50 determination of PI3Kd with LY294002, TGX-221,and wortmannin. (D) IC50 determination of PI3Kc with LY294002, wortmannin, and TGX-221. (E) IC50 determination of PI3Ka with LY294002, wortmannin, and TGX-221. TheIC50 was generated in a sigmoidal dose–response curve (variable slope) by nonlinear regression in GraphPad Prism software.

Table 1IC50 values of PI3K isoforms using the phosphocellulose radioactive method.

Enzyme Wortmannin (nM) TGX-221 (nM) LY294002 (nM)

PI3Ka 0.5 1,800 2,312PI3Kb 9 4.7 900PI3Kc 1.2 2,700 11,657PI3Kd 4 1,949 2,800

136 Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138

to three different pH variants: pH 6.0, pH 7.0, and pH 8.0 (Fig. 2a).There were significant differences in PI3K activity with differentpH and different buffers. The best activity was seen at pH 7.0 inboth the Mops and the Tris buffer, with Mops buffer showing bet-ter activity in 2 h incubation time.

To further investigate the activity of PI3K, time kinetics wasperformed up to 3 h using Mops, pH 7.0, buffer and a fixed enzymeconcentration of 100 ng/well (Fig. 2B). The enzyme activity

Assay performance with day to day variation

1 2 3 4 5 6 7 8 9 10 11 12 13 141

10

100

% CV of Background

% CV of signal

Days

% C

Vs

Z- factor performance with day to day variation

1 2 3 4 5 6 7 8 9 10 11 12 13 140.450.500.550.600.650.700.750.800.850.900.95 Z-factor

Days

Z-Fa

ctor

A

B

Fig.5. Assay performance. (A) The x axis denotes individual experiments; y axisdenotes the %CV generated in separate experiments, %CV for signal and %CV forbackground in a particular experiment, calculated from standard deviations andmean of the counts (signal or background). (B) The x axis denotes individualexperiments; y axis denotes the Z factor generated in separate experiments. The Zfactor was calculated as determined according to Zhang et al. [8].

Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138 137

exhibited good linearity up to the time tested. Two hours was cho-sen for future experiments. This exemplified conversion of sub-strate into product that was captured on phosphocellulose paper.

To optimize the enzyme concentration required for the assay,substrate was used in excess (10 lg/well; 1:1 ratio of PIP2 and Ptd-Ser in the complete reaction mix). Different amounts of enzymewere taken for the assay, ranging from 1 to 250 ng/well (Fig. 2C).The assay was performed in Mops buffer and terminated after2 h. Based on the dose response of the enzyme titration depicting>20-fold S:B with 50 ng enzyme (Fig. 2C) and <20% substrate utili-zation at 2 h reaction time (data not shown), 50 ng was chosen forfuture characterization and profiling experiments.

It has been reported that PtdSer contributes to the associationof PI3K to its substrate PtdIns [9,10]. We examined the optimal ra-tio of PIP2 and PtdSer in the substrate mix by varying the relativepercentage composition of each (Fig. 3A). The bell-shaped curveobtained clearly demonstrated that PtdSer plays an important rolein PIP2 phosphorylation by PI3K enzyme. The best signal was ob-tained with a 1:1 ratio of PIP2 and PtdSer.

To ascertain the percentage of radioactive phosphate incorpora-tion in PIP3 formed from PIP2, an experiment was conducted usingvarious concentrations of cold ATP and a 1:1 ratio of PtdIns andPtdSer, starting from 0.01 to 5 lg of lipid in each well. The exper-iment was done in triplicate in 96-well phosphocellulose plateswith 2 h incubation. The reaction mixture was then processedand allowed to bind to the phosphocellulose filter plate. The cpmvalue obtained from only 5 lCi [c-32P]ATP (devoid of cold ATP)was normalized as 100% and the cpm obtained from the reactionwells containing different amounts of cold ATP + 5 lCi [c-32P]ATPwere used to deduce the percentage of incorporation into PIP3 cap-tured on the phosphocellulose plate. Cold ATP concentrations wereplotted on the x axis and the relative percentages of radiolabeledphosphate incorporation were plotted on the y axis (Fig. 3B). Theincorporation was linear and it exemplified the incorporation ofc-32P into the lipid substrate in an ATP dose–response manner,thereby validating this assay format. Decrease in cold ATP concen-tration showed a linear increase in the counts with respect to in-crease in lipid concentration. The percentage of conversion toradiolabeled PIP3 was clearly trapped on the phosphocellulose fil-ter plate and was directly proportional to the count (cpm). This isunambiguous evidence proving that PtdIns upon protonation bindsto the phosphocellulose filter plate. The positive charge on the ki-nase substrate peptides facilitates its binding to the phosphocellu-lose paper. Likewise, PtdIns also acquired a positive charge,correlating to the counts generated in the experiment.

An apparent Km for ATP was determined from the Michaelis–Menten kinetic analysis using 1 lg of 1:1 lipid substrate(PtdIns:PtdSer). The Km was determined to be 23.4 lM (±3.5) gen-erated by calculating in GraphPad Prism software using theMichaelis–Menten template (Fig. 3C).

To gain further insight into the kinetic data of the dual-sub-strate system, the lipid Km was generated with 25 lM cold ATPusing different concentrations of 1:1 PtdIns:PtdSer ranging from0.01 to 5 lg. The lipid Km was determined to be 0.98 lg (Fig. 3D).Me2SO tolerance tested at Km suggested that the assay can tolerateup to 3% Me2SO (Fig. 4A).

PI3K reference inhibitor compounds, TGX-221, LY294002, andwortmannin, were tested at various concentrations in the currentassay format. Fifty nanograms of enzyme and 1 lg of 1:1PtdIns:PtdSer lipid substrate were subjected to 2 h incubation withreference compounds. The IC50 values obtained for TGX-221,LY294002, and wortmannin in the PI3Kb assay were 4 ± 3 nM,1 ± 2.5 lM, and 7 ± 5 nM, respectively (Fig. 4B). These values werein excellent agreement with the existing literature on the referencecompounds wherein IC50 values for LY294002 were in the range

1 ± 4 lM and for wortmannin in the range 5 ± 10 nM in differentassay formats [11–13,16].

Isoform selectivity: pan-PI3K inhibition (a, c, and d)

The assay was extended to other isoforms of PI3K. Referencecompounds LY294002, wortmannin, and TGX-221 were studiedon a, d, and c isoforms in dose-dependent concentrations andIC50 were generated. Wortmannin showed a universal dose re-sponse (IC50) with all isoforms, whereas TGX-221, a very specificinhibitor of PI3Kb, showed a clear difference in inhibitory profiles(IC50) across PI3Kb vis-a-vis other isoforms. LY294002 showed rel-atively higher IC50 across all isoforms with the highest in PI3Kc(Fig. 4C–E and Table 1). This clearly demonstrates the extensionof this methodology for the development of inhibitors of all PI3Kisoforms (pan-PI3K) and can be further modified for other lipidkinases.

Robustness of the assay and reliability of the phosphocellulose plate forthe library screening

Finally the assay was examined for performance reliability androbustness by determining the %CV and Z factor. The %CVs calcu-lated from the averages and standard deviations of the positivecontrol (signal/activity) and negative control (noise/background)for each run spanning several experiments appear in Fig. 5A. TheZ factor determined from every run across several individualexperiments emphasizes the data quality and assay robustness.

138 Development of phosphocellulose paper-based screening of inhibitors/M. Yanamandra et al. / Anal. Biochem. 449 (2014) 132–138

The assay system in this study demonstrated acceptably high Zfactor (greater than 0.6) for all the plates (Fig. 5B).

The main advantage of this assay methodology arises from thefact that potential artifacts resulting in false positives can beavoided. Colored agents, chemical quenching agents, or fluorescentchemical agents that might show effects in scintillations do notshow any effect in this format.

The assay was used to screen small molecules (data not shown)wherein LY294002 was used as an internal reference in every plate.LY294002 IC50 showed an excellent reproducibility across the platesand the S:B ratio was impressively high, in the range of 20–30.

The SAR for a compound library was successfully generated(unpublished data), emphasizing the regular usage of this methodin compound library screening during hit identification and hit tolead generation for drug discovery purposes. A detailed character-ization of compound mechanisms of action was deemed to beundertaken to unravel compound properties while carrying themfurther in the discovery process. This might require tweaking ofthe assay to suit the purpose.

Our study reports the development and validation of a protocolin 96-well format for the identification of inhibitors of PI3Kb that iseasily amenable to any environment including HTS applications.This method confirms the universal concept of the ‘‘gold standardmethod for radioactive detection of kinase activity.’’ In comparisonto other existing assay formats, this method is inexpensive androbust. Additionally it demonstrates an acceptably high S:B ratioand Z values.

Enzyme kinetic data in this phosphocellulose paper demon-strate the assay adaptability to any other lipid kinases in the familythat are attractive targets for drug discovery. The simple adaptabil-ity of this application enables the identification of potential inhib-itors of the PI3K family as well as other classes of molecules. Theassay was sensitive enough to detect a low nanomolar range ofIC50. SAR-related compound profiling and isoform selectivity canbe investigated easily in this format.

References

[1] P.T. Hawkins, K.E. Anderson, K. Davidson, L.R. Stephens, Signalling throughClass I PI3Ks in mammalian cells, Biochem. Soc. Trans. 34 (2006) 647–662.

[2] S.F. Jackson, S.M. Schoenwaelder, Type I phosphoinositide 3-kinases: potentialantithrombotic targets?, Cell Mol. Life Sci. 63 (2006) 1085–1090.

[3] S.P. Jackson, S.M. Schoenwaelder, I. Goncalves, W.S. Nesbitt, C.L. Yap, C.E.Wright, V. Kenche, K.E. Anderson, S.M. Dopheide, Y. Yuan, S.A. Sturgeon, H.Prabaharan, P.E. Thompson, G.D. Smith, P.R. Shepherd, N. Daniele, S. Kulkarni,B. Abbott, D. Saylik, C. Jones, L. Lu, S. Giuliano, S.C. Hughan, J.A. Angus, A.D.Robertson, H.H. Salem, PI 3-kinase p110[beta]: a new target for antithrombotictherapy, Nat. Med. 11 (2005) 507–514.

[4] Z.A. Knight, M.E. Feldman, A. Balla, T. Balla, K.M. Shokat, A membrane captureassay for lipid kinase activity, Nat. Protoc. 2 (2007) 2459–2466.

[5] K. Fuchikami, H. Togame, A. Sagara, T. Satoh, F. Gantner, K.B. Bacon, P.Reinemer, A versatile high-throughput screen for inhibitors of lipid kinaseactivity: development of an immobilized phospholipid plate assay forphosphoinositide 3-kinase gamma, J. Biomol. Screen. 7 (2002) 441–450.

[6] C. Stankewicz, F.H. Rininsland, A robust screen for inhibitors and enhancers ofphosphoinositide-3 kinase (PI3K) activities by ratiometric fluorescencesuperquenching, J. Biomol. Screen. 11 (2006) 413–422.

[7] J. Vidugiriene, H. Zegzouti, S.A. Goueli, Evaluating the utility of abioluminescent ADP-detecting assay for lipid kinases, Assay Drug Dev.Technol. 7 (2009) 585–597.

[8] J.H. Zhang, T.D. Chung, K.R. Oldenburg, A simple statistical parameter for use inevaluation and validation of high throughput screening assays, J. Biomol.Screen. 4 (1999) 67–73.

[9] G.M. Carman, R.A. Deems, E.A. Dennis, Lipid signaling enzymes and surfacedilution kinetics, J. Biol. Chem. 270 (1995) 18711–18714.

[10] R.A. Deems, Interfacial enzyme kinetics at the phospholipid/water interface:practical considerations, Anal. Biochem. 287 (2000) 1–16.

[11] T.A. Klink, K.M. Kleman-Leyer, A. Kopp, T.A. Westermeyer, R.G. Lowery,Evaluating PI3 kinase isoforms using Transcreener ADP assays, J. Biomol.Screen. 13 (2008) 476–485.

[12] T. Lingaraj, J. Donovan, Z. Li, P. Li, A. Doucette, S. Harrison, J.A. Ecsedy, L.Dang, W. Zhang, A high-throughput liposome substrate assay withautomated lipid extraction process for PI 3-kinase, J. Biomol. Screen. 13(2008) 906–911.

[13] B. Boldyreff, T.L. Rasmussen, H.H. Jensen, A. Cloutier, L. Beaudet, P. Roby, O.G.Issinger, Expression and purification of PI3 kinase alpha and development ofan ATP depletion and an alphascreen PI3 kinase activity assay, J. Biomol.Screen. 13 (2008) 1035–1040.

[14] S. McLaughlin, J. Wang, A. Gambhir, D. Murray, PIP(2) and proteins:interactions, organization, and information flow, Annu. Rev. Biophys. Biomol.Struct. 31 (2002) 151–175.

[15] J.A. Engelman, Targeting PI3K signalling in cancer: opportunities, challengesand limitations, Nat. Rev. Cancer 9 (2009) 550–562.

[16] A. Gray, H. Olsson, I.H. Batty, L. Priganica, C. Peter, Downes, Nonradioactivemethods for the assay of phosphoinositide 3-kinases and phosphoinositidephosphatases and selective detection of signaling lipids in cell and tissueextracts, Anal. Biochem. 313 (2003) 234–245.

[17] S.P. Jackson, C.L. Yap, K.E. Anderson, Phosphoinositide 3-kinases and theregulation of platelet function, Biochem. Soc. Trans. 32 (2004) 387–392.