Intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses

The invention relates to an intact cell assay for direct quantitation of protein tyrosine phosphatase (PTP) activity using the baculovirus expression system. A PTP expressed in transformed host insect cells is processed and localized in their predicted subcellular compartments. Assays are performed on the PTP expressing host cells challenged with a substrate such as, p-nitrophenyl phosphate. This substrate is hydrolysed to p-nitrophenol by expressed phosphatase activity. Emergence of p-nitrophenol is determined spectrophotometrically and is a measure of PTP activity. Further, the assay and transformed host cells of this invention are particularly useful in a screening method for selecting potential inhibitors to PTPs. Basically, transformed host cells are pre-incubated with or without inhibitors and the assay conducted as described. This invention relates to an intact cell assay with a direct readout of PTP activity that is useful in a cell-based screening method for selecting inhibitors of PTPs.

BACKGROUND OF THE INVENTION
 Protein tyrosine phosphatases (PTPs) (EC 3.1.3.48) dephosphorylate tyrosyl
 residues, acting in concert with protein tyrosine kinases (PTKs) to
 control tyrosine phosphorylation of proteins in the cell. This reversible
 phosphorylation at tyrosyl residues has been found in many signal
 transduction pathways, such as those involved in the action of growth
 factors, control of cellular proliferation, differentiation and metabolism
 (1, 2). Although PTPs are very different in size and structural
 organization, they contain a unique and highly conserved active site
 sequence (1,2,6). Representative examples of PTPs are CD45 and PTP1B.
 CD45, is a transmembrane receptor-type PTP that activates protein tyrosine
 kinase p561ck and p59fyn, and is involved in T cell (3,4) and B cell(5)
 signaling. PTP1B is a nonreceptor PTP containing a single catalytic domain
 and has been implicated in insulin-resistant states and type 2 diabetes
 (6,7-9).
 Specific and selective PTP inhibitors have a potential value as biological
 tools to reveal the function of individual phosphatases in cellular signal
 transduction pathways and as disease-modulating drugs. To date, sodium
 orthovanadate (vanadate), pervanadate and phenylarsine oxide are known PTP
 inhibitors. None of these exhibit tyrosine phosphatase selectivity. Small
 peptides containing the hydrolytically stable difluorophosphonomethyl
 phenylalanine were found to be inhibitors of PTP1B (10). Available
 cell-based systems that can be used for the identification of a broad
 range of selective PTP inhibitors employ downstream readouts for the
 quantitation of PTP activity.
 A limitation in this type of assay is that test inhibitors can interact at
 sites in the signal transduction pathway other than at the enzyme of
 interest, thus giving rise to an erroneous outcome, such as a false
 positive result for a test inhibitor. To date there are no known assays
 without intermediate steps, having an immediate end-point and that allow
 for a direct quantitation of PTP activity.
 Previously, an in vitro intact cell assay for screening nonsteroidal
 anti-inflammatory drug inhibition of human cyclooxygenase was developed.
 The assay utilizes the baculovirus expression system in sf9 insect cells
 (11).
 Consequently, one object of the present invention was to develop an assay
 for PTP activity that overcomes the defects in the prior art.
 Another object was to provide an assay for PTP activity that is reliable
 and reproducible.
 More particularly, an object of the present invention was to select
 cell-permeable PTP substrates such that the hydrolysis of these substrates
 provides a direct readout of the phosphatase activity.
 These and other objects will become apparent to those of ordinary skill
 from the teachings provided herein.
 The application refers to a number of publications, the content of which is
 hereby incorporated by reference in their entirety.
 SUMMARY OF THE INVENTION
 A method for assaying protein tyrosine phosphatase (PTP) activity in a
 eukaryotic host cell is disclosed comprising the steps of:
 (a) transforming a host cell with a vector containing a nucleotide sequence
 encoding a PTP to provide a transformed host cell line;
 (b) incubating said transformed host cell with a substrate to said PTP
 activity; and
 (c) comparing the amount of PTP activity in the transformed host cell to a
 standard.

DETAILED DESCRIPTION
 Protein tyrosine phosphatases are present in high amounts within cells. As
 a consequence, it is difficult to evaluate the activity of specific
 PTPases above the background of total cellular PTPases. PTPases have been
 overexpressed in mammalian cells (sometimes to the detriment of cellular
 function) using various overexpression systems. To date, the only way to
 discriminate between a specific PTPase of interest and all other cellular
 PTPases is to monitor a downstream readout parameter. For example, in the
 case of CD45, the amount of IL-2 secreted by Jurkat cells is an indication
 of the activity of this PTPase. This downstream readout may lead to the
 introduction of variables that may result in an erroneous outcome.
 As used herein the term "transform", refers to a general term for the
 introduction of a nucleotide sequence into host cells including infecting
 host cells with a recombinant virus. A representative example is a
 baculovirus vector engineered with a nucleotide sequence of interest used
 to infect an insect cell for the expression of the peptide encoded by the
 nucleotide sequence.
 The present invention concerns the development of a cell-based method for
 measuring the activity of a specific PTP. Particularly, the method relates
 to a direct measurement of PTP activity in a transformed eukaryotic host
 cell expressing exogenous PTP activity.
 Accordingly the present invention provides a method to directly assay PTP
 activity, thus eliminating the effect of downstream variables.
 Thus, the first embodiment of the present invention is directed to an
 intact cell assay for a PTP. The PTP of interest is expressed in
 transformed eukaryotic host cells, the transformed cells are incubated
 with a substrate and a direct measure of phosphatase activity is obtained.
 In a particular preferred embodiment there is provided a eukaryotic host
 cell transformed with an appropriate expression vector having a nucleotide
 sequence encoding a PTP. The nucleotide sequence encoding PTP optionally
 includes any derivatives, variants or fragments capable of expressing the
 encoded PTP. All these are within the scope of this invention. In a
 non-limiting example representative PTPs selected for the purpose of this
 invention include CD45 and PTP1B having GenBank Accession numbers Y00062
 and G190741, respectively (Hooft van Huijsduijnen, 1998, Gene 225:1-8).
 Other examples of PTPs useful for the purpose of this invention are listed
 in Hooft van Huijsduijnen (supra).
 The eukaryotic host cells used for the transformation include mammalian,
 fungal (including yeast) and insect cells. In a particular embodiment,
 insect cells are the preferred host cells. Non-limiting examples of insect
 cells include sf21, sf9, High Five (BT1-TN-5B1-4), BT1-Ea88, Tn-368,
 mb0507, Tn mg-1, Tn Ap2, and whole insect larvae.
 In a specific embodiment of this invention, the host cell is the insect
 cell sf9, which is transformed with appropriate insect cell recombinant
 virus. These recombinant viruses that are able to transform and infect an
 insect cell line are well known to a person skilled in the art and can be
 selected and used accordingly. These are all within the scope of this
 invention.
 In a most preferred embodiment of this invention, the cell-based assay
 comprises sf9 insect cells transformed with a specific PTP recombinant
 baculovirus for the expression of the specific PTP by the sf9 cells.
 In a further aspect, the substrates used for the purpose of this invention
 comprise compounds that are cell permeable and have a hydrolysable
 phosphate. Non-limiting examples include fluorescein monophosphate,
 fluorescin diphosphate, ELF 97 (obtained from Molecular Probes Inc.) and
 p-nitrophenyl phosphate (pNPP).
 In an additional aspect of this invention, PTP activity can be evaluated
 either by a decrease in substrate concentration or an emergence of
 reaction product(s). Different means for detecting the result of the
 phosphatase activity include spectrophotometric, fluorescence, ELISA
 (enzyme linked immunoassay), RIA (radioimmunoassay) and radioactivity (by
 tagging the substrate).
 Of the different substrates tried by the Applicant, the substrate, pNPP
 produced consistently reproducible and reliable results, with an endpoint
 that can be easily measured.
 Therefore in a specific embodiment of this invention, the preferred
 substrate is pNPP. This substrate is cell permeable and is hydrolysed by
 PTP resulting in the product p-nitrophenol. As described above
 p-nitrophenol can be measured by means including spectrophotometric.
 In a preferred embodiment the emergence of the product of pNPP,
 p-nitrophenol is measured spectrophotometrically.
 According to another embodiment, the present invention provides a screening
 method to identify test compounds as inhibitors to PTP. Particularly, this
 invention provides a screening method for identifying PTP inhibitors that
 are cell permeable.
 In this method, the assay of the present invention is performed in the
 presence or absence of a test compound and the amounts of hydrolysed
 substrate compared. A variance in the results may be an indication of the
 efficacy of a test compound as a candidate inhibitor to PTP.
 Therefore, this invention provides a screening method for identifying a
 test compound as an inhibitor to PTP activity in a eukaryotic host cell,
 which comprises the steps of:
 a) incubating a eukaryotic host cell transformed with a vector containing a
 nucleotide sequence encoding a PTP in the presence of a PTP substrate and
 the test compound under conditions which effect PTP activity;
 (b) incubating the eukaryotic host cell transformed with a vector
 containing a nucleotide sequence encoding a PTP in the absence of the test
 compound under conditions which effect PTP activity; and
 (c) comparing the results of step (a) and step (b).
 This invention further provides host cells transformed with nucleotide
 sequences encoding PTPs. These transformed hosts may be used in the
 methods and assays of this invention.
 The methods and assays of the present invention, for measuring PTP activity
 can be comprised in a kit, such a kit includes: eukaryotic cells
 transformed with a vector containing a nucleotide sequence encoding PTP,
 substrate for PTP activity and appropriate buffers.
 In an aspect of the present invention, the screening method can be scaled
 up and adapted for a highthroughput system. Such a system is useful for
 identifying an inhibitor to PTP for use as a research tool and for
 therapeutic applications in mammals in need of such therapy.
 EXAMPLES
 Cell Culture
 sf9 cells (Invitrogen, San Diego, Calif., U.S.A.) were cultured in spinner
 flasks at 28.degree. C. in Graces supplemented medium (Gibco-BRL,
 Mississauga, Ontario, Canada) with 10% heat-inactivated fetal bovine serum
 (Gibco-BRL) following the protocol of Summers and Smith (12).
 Example 1
 Construction of Recombinant Baculovirus Transfer Vectors
 The cDNA for PTP1B came from Dr. R. L. Erikson, Harvard University, USA.
 The CD45 cDNA was obtained from Dr. Frank Jirik, University of British
 Columbia, Canada. The recombinant baculoviruses were prepared using the
 Bac-to-Bac Baculovirus Expression System (Gibco-BRL, Mississauga, Ontario,
 Canada). Briefly, the genes of interest were cloned into the pFASTBAC
 donor plasmid, which had been engineered to include a FLAG sequence at the
 5' end of the cDNA. The FLAG sequence allows for easy identification and
 purification of the expressed protein of interest using the anti-FLAG M2
 antibody (Intersciences Inc., Markham, Ontario). The resultant plasmids
 were transformed into competent DH10BAC E. coli cells. Following
 transposition and antibiotic selection, the recombinant bacmid DNA was
 isolated from selected E. coli colonies and then used to transfect sf9
 insect cells. The virus found in the supernatant media was amplified three
 times up to a total viral stock volume of 500 mL.
 Example 2
 Production of Recombinant Proteins
 Baculovirus infection of 500-mL spinner cultures of sf9 cells was done
 essentially as described by Summers and Smith (12). Sf9 cells at a density
 of 1-3.times.10.sup.6 cells/mL were pelleted by centrifugation at 300 g
 for 5 min, the supernatant was removed, and the cells were resuspended at
 a density of 1.times.10.sup.7 cells/mL in the appropriate recombinant
 viral stock (MOI of 10). Following gentle shaking for 1.5 hr at room
 temperature, fresh medium was added to adjust the cell density to
 .about.1.times.10.sup.6 cells/mL and the cells were cultured in suspension
 at 28.degree. C. for the indicated times, post-infection.
 Example 3
 Cellular Fractionation and Whole Cell Extracts from Infected sf9 Cells
 At various times post-infection, aliquots of the infected cells were
 removed, and protein expression analyzed by SDS-PAGE and western blot
 analysis. Cellular fractionation was performed as follows. Infected sf9
 cells were centrifuged at 300 g for 5 min and the pelleted cells washed
 with cold PBS. The cell pellets were then resuspended in 1 mL of cold 0.1
 M Tris, pH 7.4, 5 mM EDTA and sonicated 3.times.10 sec on ice. Samples
 were centrifuged for 10 min at 500 g at 4.degree. C. and the resulting
 supernatants retained. The pellets were resuspended in 1 mL of cold
 Tris/EDTA buffer, sonicated, and recentrifuged as described above. The two
 supernatant fractions were pooled and centrifuged at 4.degree. C. for 60
 min at 100,000 g. The microsomal pellets were resuspended by a 2-sec pulse
 sonication in 0.1 M Tris, pH 7.4, 5 mM EDTA.
 Whole cell extracts were prepared from 1 mL aliquots of infected sf9 cells
 at the specified times post-infection. Pelleted cells (300 g, 5 min) were
 washed once in PBS (4.degree. C.), resuspended in 50 .mu.L of water and
 cell membrane disrupted by freezing and thawing the pelleted cells.
 Protein concentrations were determined using Coomasie Protein Assay
 Reagent Kit (Pierce, Rockford, Ill., USA) with bovine serum albumin as the
 standard.
 Example 4
 SDS-PAGE/Western Blot Analysis
 The expression and production of specific protein, time course of
 expression and subcellular localization of the individual phosphatases
 from the recombinant baculovirus infected sf9 cells was determined by
 western blot analysis. Briefly, whole cell lysate and 100,000 g fractions
 (pellet or supernatant) were mixed with SDS sample buffer (60 mM Tris.Cl
 [6.8], 10% SDS, 6% glycerol, 5% .beta.-mercaptoethanol, 0.05% bromophenol
 blue), heated to 95.degree. C. for 4 min, and electrophoresed on 10%
 Tris-glycine acrylamide denaturing gels (Novex, San Diego, Calif.,
 U.S.A.). Proteins were transferred electrophoretically to nitrocellulose
 membranes for 16 hr at 100 mA constant current using a Novex transfer
 apparatus. The membranes were reacted with the following antibodies.
 FLAG M2 Antibody
 The nitrocellulose membranes were blocked for 30 min with 5% powdered milk
 in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20, followed by 2
 washes in the Tris/NaCl buffer containing (v/v) 1% Tween 20. The blots
 were then incubated with 1/2000 final dilution of FLAG M2 Antibody
 (Intersciences Inc., Markham, Ontario) in Tris/NaCl buffer. Immunoreactive
 proteins were visualized by using a 1/2000 dilution of anti-mouse Ig,
 horseradish peroxidase (Amersham, Oakville, Ontario) as the second
 antibody and development by ECL as described by the manufacturer (Amersham
 Life Science, Oakville, Ontario).
 CD45 Antibody
 The nitrocellulose membranes were blocked for 60 min with 5% powdered milk
 in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween20. The blots were
 immediately incubated with 1/500 dilution of anti-CD45 antibody
 (Transduction Laboratories, Lexington, Ky., U.S.A.) in blocking buffer for
 1 h. Following 6 washes in Tris/NaCl buffer, the blots were incubated for
 1 h with a 1/2000 dilution of anti-mouse Ig, horseradish peroxidase. The
 immunoreactive protein was visualized by ECL.
 The results shown in FIG. 1A, indicate that FLAG-tagged PTP1B-FL (.about.52
 kDa) is expressed by about 24 hpi, exponentially increases between 26 and
 48 hpi, and then begins to be cleaved or degrade by 72 hpi. PTP1B-FL is
 present in the membrane fraction of the sf9 cells at about 29 hpi and by
 about 48 hpi it appears intact in the cytosol. However, by about 72 hpi, a
 50 kDa cleavage product predominates in the cytosol while the higher
 molecular weight form remains present in the membrane fraction.
 The expression pattern of FLAG-tagged CD45-cat is very similar to that of
 PTP1B and is shown in FIG. 1B. The protein is observed beginning at about
 24 hpi with an apparent molecular weight of 98 kDa and continues to be
 exponentially expressed to about 48 hpi. Extracts from the mock-infected
 (HCOX-1) sf9 cells at 25 and 30 hpi, as expected show no FLAG M2 antibody
 cross reacting with any proteins. Since FLAG affinity purification on the
 M2 antibody column yields CD45-cat protein in the supernatant fraction of
 lysed cells (data not shown), it is postulated that the recombinant
 FLAG-tagged CD45-cat is localized in the cytosol of sf9 cells.
 The expression of the full length CD45 shown in FIG. 1C, is significantly
 lower than that of the other two phosphatases, presumably reflecting the
 membrane localization of this protein. CD45-FL is just visible at about 29
 hpi in whole cell extract. At later time points of infection, about 49 and
 80 hpi, the .about.150 kDa protein species predominates, but presumably
 many proteolytic degradation products cross react with the anti-CD45
 transmembrane antibody, especially when protein gels are overloaded as is
 the case of the later timepoints. When infected cells are fractionated at
 about 29 hpi there is no visible CD45-FL, however at about 80 hpi the full
 length protein is clearly visualized intact and non-degraded, and as
 expected appears to be localized mainly in the microsomal fraction.
 Results of the western analysis demonstrate that the PTPs are expressed at
 various but detectable levels at about 29 hpi. Further evaluation of the
 infected sf9 cells by trypan blue at this time point, indicate that the
 cells are still highly viable and demonstrating that the development of a
 cell-based assay for PTPs activity is feasible.
 Example 5
 Intact Cell Assay Development
 Sf9 cells were infected with PTP1B-FL, CD45-FL, CD45-cat or HCOX-1 (11)
 (mock-infected) recombinant baculovirus. Infected cells were collected 29
 hpi by centrifugation in Beckman GS-6R at 460 rpm (48 g) for 5 min, washed
 once in assay buffer (Hanks' solution buffered with 15 mM Hepes, pH 7.4
 (Sigma, St. Louis, Mo., U.S.A.)) and recentrifuged at 300 rpm (21 g) for
 10 min. The cells were gently resuspended in assay buffer and examined
 using a hemacytometer for cell density and viability by trypan blue
 exclusion. Assays were performed using a Tomtec Quadra 96 pipeting robot,
 programmed to mix gently after each addition as follows. Aliquots of
 2.times.10.sup.5 PTP expressing cells in 200 .mu.L of Hanks' solution were
 dispensed into each well of 96-well polypropylene plates and pre-incubated
 either with inhibitor or DMSO vehicle (3 .mu.L) for 15 min at 37.degree.
 C. The pre-incubated cells were challenged with different concentrations
 of tissue culture grade pNPP (Sigma-Aldrich Canada Ltd., Oakville,
 Ontario) for 15 min then pelleted by centrifugation for 3 min at 410 g and
 4.degree. C. Aliquots of 100 .mu.L of supernatant were transferred to
 fresh clear polystyrene 96-well plates and the amount of hydrolysis
 product determined spectrophotometrically at OD.sub.405. Phosphatase
 activity attributed to the recombinant PTP is the difference in the amount
 of pNPP hydrolysis between the PTP expressing and mock-infected cells.
 Initial characterization of the PTP insect cell-based assays was carried
 out at 29 hpi using PTP1B-FL and CD45-cat recombinant baculoviruses.
 Although many substrates were analyzed (data not shown), pNPP was chosen
 for its consistent and reproducibile results. The rate of hydrolysis of
 pNPP, as measured spectrophotometrically at OD.sub.405, correlated with
 substrate concentration, time of substrate challenge, cell number, viral
 titer and final tyrosine phosphatase protein expression. Using
 2.times.10.sup.5 PTP-expressing cells/well and varying the concentration
 of pNPP between 0 and 50 mM, the hydrolysis of the substrate by PTP1B-FL
 and CD45-cat during a 15 minute incubation followed very similar profiles.
 FIG. 2 illustrates typical pNPP hydrolysis patterns of these two
 phosphatases. In the absence of cells, increasing concentrations of pNPP
 led to a slight increase in the baseline absorbance at OD.sub.405.
 Mock-infected (HCOX-1) cells contain background levels of PTPs and
 therefore exhibit increased hydrolysis of substrate with increasing
 substrate concentration. Sf9 cells infected with either PTP1B-FL or
 CD45-cat recombinant virus showed marked hydrolysis of pNPP at low
 substrate concentration with maximal levels of substrate utilization
 occurring at 10 mM pNPP for PTP1B-FL and CD45-cat. The window between PTP
 hydrolysis and mock-infected cells represents the amount of substrate
 utilization due to the recombinant tyrosine phosphatase.
 FIG. 3, shows the time course of different concentrations of pNPP
 hydrolysis by sf9 cell expressing CD45-cat or PTP1B-FL. The hydrolysis
 appears to be linear with respect to time, irrespective of substrate
 concentration or phosphatase present in the sf9 cells.
 FIG. 4 demonstrates that in this assay the rate of substrate hydrolysis is
 linear with respect to cell number.
 A summary of the total output of PTP activity is shown in FIG. 5. Intact
 uninfected sf9 cells, challenged with 10 mM pNPP have a basal level of
 phosphatase activity as shown by the increase in the absorbance at 405
 when compared to 10 mM pNPP/Hanks alone. Intact sf9 cells infected with
 PTP1B-FL, CD45-cat and CD45-FL have significantly increased rate of pNPP
 hydrolysis of 8.3, 7.1 and 2.4 fold respectively when compared to
 mock-infected cell (background). These levels of phosphatase activity
 correlate with the relative amount of protein visualized at 29 hpi in
 western blot analysis (FIG. 1), with the level of expression being the
 highest in PTP1B-FL followed by CD45-cat and CD45-FL. In the case of
 PTP1B-FL and CD45-cat, the amount of pNPP hydrolyzed by enzymes inside the
 intact sf9 cells represents .about.30% of the total hydrolysis that can
 occur if the cells are disrupted and the enzymes released to be in direct
 contact with the substrate (data not shown).
 The following conditions for subsequent experimentation were used. Infected
 sf9 cells expressing either PTP1B-FL, CD45-cat or CD45-FL are harvested at
 29 hpi by gentle centrifugation and resuspended in Hepes-buffered Hanks'
 solution, pH 7.4 at 37.degree. C. An aliquot of 2.times.10.sup.5
 PTP-infected cells are challenged with a final concentration of 10 mM pNPP
 for 15 min, centrifuged at 4.degree. C. and the amount of substrate
 hydrolysis determined spectrophotometerically at OD.sub.405.
 Example 6
 Inhibitor Titration and Characterization
 The inhibition of PTP activity was determined by comparing the amount of
 pNPP hydrolyzed in the presence of an inhibitor and in the DMSO treated
 PTP expressing cells. Briefly, potential inhibitors are preincubated with
 infected sf9 cells for 15 min prior to a 15 min challenge with 10 mM pNPP.
 The inhibitors studied include ouabain (Sigma-Aldrich Canada, Oakville,
 Ontario), an inhibitor of Na--K ATPase known also to exhibit p-nitrophenyl
 phosphatase activity; okadaic acid (Gibco-BRL, Gaithersburg, Md., U.S.A.),
 a known serine/threonine phosphatase inhibitor; 30% H.sub.2 O.sub.2
 (Aldrich), phenylarsine oxide (Sigma, St. Louis, Mo., U.S.A.), vanadate,
 and pervanadate. Vanadate and pervanadate were prepared as described (13).
 This represents four known protein tyrosine phosphatase inhibitors. The
 diflurophosphonomethyl phenylalanine containing peptide inhibitor, a
 potent and selective PTP1B-FL inhibitor obtained from the Merck sample
 collection.
 FIG. 6 tabulates the results of the potency and selectivity of the tested
 inhibitors. Neither ouabain at 1 mM or okadaic acid at 6.7 .mu.M had any
 effect on the CD45-cat or background phosphatases in infected sf9 cells.
 As predicted, hydrogen peroxide and phenylarsine oxide showed limited
 potency and selectivity with IC.sub.50 values in the range of 7-16 .mu.M.
 Vanadate is not very potent on background tyrosine phosphatases in
 mock-infected sf9 cells with an IC.sub.50 of 1.6 .mu.M, but appears to be
 very effective at inhibiting the expressed PTPs with slightly greater
 selectivity (4.5.times.) towards CD45 than PTP1B-FL. Of the tested
 compounds, pervanadate appears to be the most potent inhibitor of CD45 and
 PTP1B-FL with IC.sub.50 values between 5-20 nM. Pervanadate is a general
 term for a variety of complexes formed between vanadate and hydrogen
 peroxide. Pervanadate inhibits PTPs by the irreversible inactivation of
 the active site cysteine, a mechanism distinct from the competitive
 inhibitory nature of vanadate (13). The diflurophosphonomethyl
 phenylalanine peptide is a proprietary compound that has been found to
 inhibit PTP1B-FL with an IC.sub.50 of 0.3 .mu.M, a value that is similar
 to that of vanadate. This compound, however, is 33-fold more selective for
 PTP1B-FL than CD45-FL, CD45-cat or background phosphatases found in
 mock-infected sf9 cells.
 Discussion
 The role of protein tyrosine phosphatases (PTPs) in the modulation of
 signal transduction by tyrosine-kinase containing receptors and oncogene
 products has been gaining more investigational attention. PTP1B, a
 cytosolic non-receptor PTP, is one of the first PTPs identified and is
 implicated in the regulation of the insulin signaling pathway (7-9). CD45
 is a transmembrane receptor-type PTP expressed on all hematopoietic cells
 except erythrocytes (14), is required for normal T and B cell signaling
 (3,4,5). Thus, inhibition of PTP1B or CD45 could potentially have
 therapeutic benefits in the areas of NIDDM or immunosuppression,
 respectively. The successful development of intact sf9 cell-based assays
 for COX-1 and COX-2 (11) that could identify potent and isozyme selective
 NSAIDs prompted us to investigate the use of the baculovirus system again.
 In order to define potent, selective and cell-permeable inhibitors for
 intracellular phosphatases, a cell-based assay utilizing sf9 cells
 infected with specific PTP recombinant baculoviruses was developed.
 An alternative method for discriminating between the activity of a PTP of
 interest and endogeneous phosphatase activity is monitoring downstream
 readout parameter. For example, in the case of CD45, the amount of IL-2
 secreted by Jurkat cells can be used as a measure of the activity of this
 PTP. As documented previously (11) and the results presented in this study
 show, the baculovirus expression system can be developed into cell-based
 assays for use in drug screening. We describe here a "mix and read" intact
 cell assay for the activity of specific protein tyrosine phosphatase,
 which eliminates potential interference of downstream sites in the signal
 transduction pathway when evaluating a test compound. The PTP of interest
 is overexpressed in sf9 cells for a short period of time which is not
 detrimental to cellular function, challenged with an inhibitor then a
 substrate and direct read out of inhibition is obtained. Thus, the
 advantage of such a cell-based assay is that there are no intermediate
 steps between PTP enzyme inhibition and the endpoint readout. Using intact
 sf9 cells expressing PTP1B or CD45, we have characterized known PTP
 inhibitors, as well as, demonstrated that the assay can identify potent
 and highly selective compounds inhibitory for a PTP of interest.
 The sf9 insect cell line used in the baculovirus expression system is
 derived from the ovarian tissue of the fall armyworm Spodoptera (12).
 Infection of this cell line with recombinant baculoviruses for PTP1B and
 CD45 results in the expression of these proteins.
 The genome of the baculovirus Autographa californica encodes a 19 kDa
 protein tyrosine phosphatase (15). Sf9 cells appear to offer the cellular
 environment and factors necessary for both PTP1B and CD45 phosphatase
 activity. The cellular localization of the expressed PTPs was as expected,
 since the catalytic domain of CD45 (CD45-cat) does not contain a
 transmembrane domain as the full length CD45, it was expressed at high
 concentrations in cytosolic fractions. CD45-FL is found to be minimally
 expressed (as compared to the general expression level of many proteins)
 in the membrane fraction of sf9 cells, this is probably due to the fact
 that it is a transmembrane protein and therefore proceeds through the
 Golgi prior to insertion into the plasma membrane. The level of expression
 of the full length form of CD45 at 29 hpi is the same as that found in an
 equivalent number of Jurkat cells, which express very high amounts of CD45
 per cell surface area (data not shown). The full length PTP1B is anchored
 to the endoplasmic reticulum at its C-terminal (16). PTP1B-FL is expressed
 at very high levels and found to be associated with the membrane fraction
 at early time points post-infection. At about 48 hpi, a time when the
 viability of the infected cells begins to decline, PTP1B-FL is found in
 the cytosol and at greater than 72 hpi, a 50 kDa N-terminal fragment
 appears both in the membrane and cytosolic fractions. This is presumably
 due to proteolytic breakdown of the protein. The demonstration that the
 expressed PTPs are found in the expected cellular location provides an
 advantage for drug screening. Proper compartmentalization may be necessary
 for PTP specificity of cellular substrate and selectivity of some
 potential inhibitors based on intracellular drug distribution.
 It was important to identify an appropriate time point for this assay, ie;
 harvesting the recombinant cells at a timepoint when a sufficient amount
 of PTP is produced and the cells remain healthy and viable (prior to the
 deleterious effects associated with later stages of viral infection). The
 infected sf9 cells are harvested at 29 hpi, a timepoint when each of the
 PTPs is fully post-translationally modified, properly targeted and active.
 The cells expressing the appropriate PTP were assayed in 96-well plates and
 mechanization was facilitated using the Tomtec Quadra 96 pipetting device.
 The effect of inhibitors on PTP activity is a measure of the change in the
 hydrolysis of para-nitrophenyl phosphate (pNPP) when compared to a
 control. Different substrates were tested, pNPP yielded the most
 consistent results.
 The uptake of phosphate is important and vital in the metabolic
 requirements of growth and replication in eukaryotic cells. The substrate,
 pNPP, is presumed to be taken up into the sf9 cells by a phosphate
 transporter. The assay demonstrates that the rate of hydrolysis of pNPP
 correlates well with the viral titer used to infect the sf9 cells, as well
 as, cell number and substrate concentration. Further, the window of
 phosphatase activity between the PTP of interest and the background
 activity in mock-infected cells correlates with amount of protein
 expressed. Of the three PTPs tested PTP1B-FL and CD45-cat were most highly
 expressed with a 6 and 5-fold window of pNPP hydrolysis activity,
 respectively. CD45-FL was barely detected by Western and concomitantly
 showed a 1.5-fold window of activity.
 The profiles of the inhibitors shown in FIG. 6, demonstrated sigmoidal
 shaped IC.sub.50 curves in this assay.
 Na--K-ATPase is known to exhibit pNPP hydrolyzing activity. To demonstrate
 that the amount of p-nitrophenol detected in the present assay was due to
 phosphatase activity, ouabain, an inhibitor of Na--K-ATPase, was tested
 and showed no inhibition up to 1 mM for both the background infected cells
 and CD45 expressing cells. Serine/threonine phosphatases do not hydrolyse
 pNPP since okadaic acid (17-19) had no effect. H.sub.2 O.sub.2 has been
 shown to inactivate recombinant PTP1B in vitro by oxidizing its catalytic
 site cysteine (20). Direct exposure of cells to H.sub.2 O.sub.2 activates
 signal transduction pathways by increasing protein tyrosine
 phosphorylation (21-23). The testing of H.sub.2 O.sub.2 in our cell-based
 assay indeed showed that non-selective inhibition of PTP1B or CD45 in the
 IC.sub.50 range of 10-15 .mu.M is obtained. Phenylarsine oxide is a
 documented membrane permeable inhibitor of protein phosphotyrosine
 phosphatases (24), the results confirm that it is a non-potent,
 non-selective inhibitor of PTP1B and CD45 expressed within sf9 cells.
 Vanadate is a general PTP inhibitor (25) found to be competitively and
 reversibly bound at the active site (13) with insulin-mimetic properties
 (26), and in human clinical trials its been shown to be potentially useful
 in treating NIDDM (27). Vanadate was chosen to validate the assay
 described herein. It was found to be more potent than H.sub.2 O.sub.2 and
 phenylarsine oxide at inhibiting the expressed phosphatases and appears to
 be slightly more selective for CD45 than PTP1B. Pervanadate, a complex of
 vanadate and H.sub.2 O.sub.2, is also an insulin-mimetic and is documented
 as being more potent at increasing the levels of cellular tyrosine
 phosphorylation (28-32). It was found to be an irreversible inhibitor of
 PTP1B (13). Our data correlates and substantiates these observations.
 Pervanadate was 7-15 times more potent than vanadate in inhibiting CD45
 and PTP1B expressed in the sf9 cell assay, though this inhibition is non
 selective. PTP inhibitors have value as biological research tools in the
 understanding of signal transduction pathways and as therapeutic agents
 with the potential of treating diseases and conditions needing the
 inhibition of specific protein tyrosine phosphatases.
 In a successful application of this invention, the screening effort has
 uncovered a test compound, diflurophosphonomethyl phenylalanine. This
 compound containing peptide is cell permeable and &gt;33-fold selective,
 potent PTP1B inhibitor. This compound is a nonhydrolyzable phosphotyrosine
 mimic, which functions as a competitive inhibitor at the catalytic site.
 The baculovirus expression system can supply recombinant protein for
 enzymatic characterization, X-ray crystallographic studies, in vitro
 purified enzyme and microsomal assays and as reported previously (11) and
 here, reliable cell-based assays. The PTP1B and CD45 assays presented
 herein are mere representative examples of PTPs. It is the intent of the
 Applicant, that this assay be used for any PTP. Therefore, this invention
 encompasses any PTP for the purpose of providing an opportunity to uncover
 potentially useful inhibitors to a PTP that may be useful as a research
 tool and in the treatment of many yet to be identified therapeutic
 targets.
 REFERENCES
 1. Charbonneau H and Tonks N K, 1002 protein phosphatases? Annu Rev Cell
 Biol 8: 463-493, 1992.
 2. Neel B G and Tonks N K, Protein tyrosine phosphatases in signal
 transduction, Current Opinions in Cell Biology 9: 193-204, 1997.
 3. Trowbridge I S, Ostergaard H L and Johnson P, CD45: a leukocyte-specific
 member of the protein tyrosine phosphatase family, Biochim Biophys Acta
 1095: 46-56, 1991.
 4. Cahir McFarland E D, Hurley T R, Pingel J T, Sefton B M, Shaw A and
 Thomas M L, Correlation between src family member regulation by
 protein-tyrosine-phosphatase CD45 and transmembrane signaling through the
 T-cell receptor, Proc Natl Acad Sci 90: 1402-1406, 1993.
 5. Lin J, Brown V K and Justement L B, Regulation of Basal Tyrosine
 Phosphorylation of the B Cell Antigen Receptor Complex by the Protein
 Tyrosine Phosphatase, CD45, J Immunol 149: 3182-3190, 1992.
 6. Zhang Z Y, Protein-Tyrosine Phosphatases: Biological Function,
 Structural Characteristics and Mechanism of Catalysis, Critical Reviews in
 Biochemistry and Molecular Biology 33: 1-52, 1998.
 7. Cicirelli M F, Tonk N F, Diltz C D, Weiel J E, Fischer E H and Krebs E
 G, Microinjection of protein-tyrosine-phosphatase inhibits insulin action
 in Xenopus oocytes, Proc Natl Acad Sci 87: 5514-5518, 1990.
 8. Ahmad F, Li P M, Meyerovitch J and Goldstein B J, Osmotic loading of
 neutralizing antibodies demonstrates a role of
 protein-tyrosine-phosphatase-1B in negative regulation of the insulin
 action pathway, J Biol Chem 270: 20503-20508, 1995.
 9. Seely B L, Staubs P A, Reichart D R, Berham P, Milarski K L, Salteil A
 R, Kusari J and Olefsky J M, Protein tyrosine phosphatase-1B interacts
 with the activated insulin receptor, Diabetes 45: 1379-1385, 1996.
 10. Burke T R, Kole H K and Roller P P, Potent Inhibition of Insulin
 Receptor Dephosphorylation by a Hexamer Peptide Containing the
 Phosphotyrosyl Mimetic F2Pmp, Biochem Biophys Res Commun 204: 129-134,
 1994.
 11. Cromlish W and Kennedy B, Selective Inhibition of Cyclooxygenase-1 and
 -2 Using Intact Insect Cell Assays, Biochem Pharm 52: 1777-1785, 1996.
 12. Summers M D and Smith G E, A manual for Methods for Baculovirus Vectors
 and Insect Culture Procedures(Bulletin No. 1555). Texas A & M University,
 Texas Agricultural Experiment Station, College Station, Tex., 1987.
 13. Huyer G, Lui S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailus G,
 Gresser M J and Ramachandran C, Mechanism of Inhibition of
 Protein-tyrosine Phosphatases by Vanadate and Pervanadate. J Biol Chem
 272: 843-851, 1997.
 14. Yanagi S, Sugawara H, Kurosaki M, Sabe H, Yamamura H and Kurosaki T,
 CD45 Modulates Phosphorylation of Both Autophosphorylation and Negative
 Regulatory Tyrosines of Lyn in B Cells, J Biol Chem 271: 30487-30492,
 1996.
 15. Sheng Z and Charbonneau H, The Baculovirus Autographa californica
 encodes a Protein Tyrosine Phosphatase, J Biol Chem 268: 4728-4733, 1993.
 16. Frangioni J V, Beahm P H, Shifrin V, Jost C A and Neel B G, The
 nontransmembrane tyrosine phosphatase PTP-1B localizes to the Endoplasmic
 Reticulum via its 35 amino acid C-terminal Sequence, Cell 68: 545-560,
 1992.
 17. McVicar D W, Mason A T, Bere E W and Ortaldo J R, Activation of
 peripheral large granular lymphocytes with the seine/threonine phosphatase
 inhibitor, okadaic acid, Eur J Immunol 24: 165-170, 1994.
 18. Fujiki H and Suganmura M, Tumor necrosis factor-alpha, a new tumor
 promoter, engendered by biochemical studies of okadaic acid, J Biochem
 (Tokyo) 115: 1-5, 1994.
 19. Yu J S and Yang S D, Okadaic acid, a serine/threonine phosphatase
 inhibitor, induces tyrosine dephosphorylation/inactivation of protein
 kinase FA/GSK-3 alpha in A431 cells, J Biol Chem 269: 14341-14344, 1994.
 20. Lee S-R, Kwon K-S, Kim S-R and Rhee S G, Reversible Inactivation of
 Protein-tyrosine Phosphatase 1B in A431 Cells stimulated with Epidermal
 Growth Factor, J Biol Chem 273: 15366-15372, 1998.
 21. Sundaresan M, Yu Z-X, Ferrans, V J, Irani K and Finkel T, Requirement
 of generation of H2O2 for platelet-derived growth factor signal
 transduction, Science 270: 296-299, 1996.
 22. Bae Y S, Kang S W, Seo M S Baines I C, Tekle E, Chock P B and Rhee S G,
 Epidermal growth factor (EGF)-induced generation of hydrogen peroxide.
 Role in EGF receptor-mediated tyrosine phosphorylation, J Biol Chem 272:
 217-221, 1997.
 23. Heffetz D, Bushkin I, Dror R and Zick Y, The insulinomimetic agents
 H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact
 cells, J Biol Chem 265: 2896-2902, 1990.
 24. Li J, Elberg G and Shechter Y, Phenylarsine oxide and vanadate:
 apparent paradox of inhibition of protein phosphotyrosine phosphatases in
 rat adipocytes, Biochim Biophys Acta 1312: 223-230, 1996.
 25. Swarup G, Cohen S and Garbers D L, Inhibition of membrane
 phosphotyrosyl-protein phosphatase activity by vanadate, Biochem Biophys
 Res Comm 107: 1104-1109,1982.
 26. Heyliger C E, Tahiliani A G and McNeil J H, Effect of vanadate on
 elevated blood glucose and depressed cardiac performance of diabetic rats,
 Science 227: 1474-1477, 1985.
 27. Goldfine A B, Simonson D C, Folli F, Path M E and Kahn C R, Metabolic
 effects of sodium metavanadate in humans with insulin-dependent and
 noninsulin-dependent diabetes melitus in vivo and in vitro studies, J Clin
 Endocrinol & Metab 80: 3311-3320, 1995.
 28. Kadota S, Fantus I G, Deragon G, Guyda H J, Hersh B and Posner B I,
 Peroxides of vanadium: a novel and potent insulin-mimetic agent which
 activates the insulin receptor kinase, Biochem Biophys Res Comm 147:
 259-266, 1987.
 29. Fantus I G, Kadota S, Deragon G, Foster B and Posner B I,
 Pervanadate[peroxide(s) of vanadate] mimics insulin action in rat
 adipocytes via activation of the insulin receptor tyrosine kinase,
 Biochemistry 28: 8864-8871, 1989.
 30. Posner B I, Faure R, Burgess J W, Bevan A P, Lachance D, Zhang-Sun G,
 Fantus I G, Ng J B, Hall D A, Lum B S and Shaver A, Peroxovanadium
 compounds. A new class of potent phosphotyrosine phosphatase inhibitors
 which are insulin mimetics, J Biol Chem 269: 4596-4604, 1994.
 31. Bevan A P, Drake P G, Yale J-F, Shaver A and Posner B I, Peroxovanadium
 compounds: biological actions & mechanism of insulin-mimesis, Mol Cell
 Biochem 153: 49-58, 1995.
 32. Yale J-F, Vigeant C, Nardolillo C, Chu Q, Yu J-Z, Shaver A and Posner B
 I, In vivo effects of peroxovanadium compounds in BB rats, Mol Cell
 Biochem 153: 181-190, 1995.