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WO1998030718A1 - Systeme d'expression stable d'une phosphodiesterase de camp a affinite elevee et son utilisation - Google Patents

Systeme d'expression stable d'une phosphodiesterase de camp a affinite elevee et son utilisation Download PDF

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WO1998030718A1
WO1998030718A1 PCT/CA1998/000024 CA9800024W WO9830718A1 WO 1998030718 A1 WO1998030718 A1 WO 1998030718A1 CA 9800024 W CA9800024 W CA 9800024W WO 9830718 A1 WO9830718 A1 WO 9830718A1
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enzyme
pde
rolipram
camp
iva
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Douglas J. Pon
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Merck Frosst Canada and Co
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Definitions

  • This invention related to a system for stably expressing a low-Km cAMP phosphodiesterase (PDE) and the use of the system for assessing inhibitors of PDE.
  • PDE low-Km cAMP phosphodiesterase
  • this invention related to a system for the stable expression of a low-Km PDE rV and the use of the stable system in the evaluation of inhibitors of PDE TV.
  • This invention also relates to an improved in vitro assay for the evaluation of inhibitors of PDE rV directed against a "high-affinity" state enzyme with respect to rolipram.
  • Phosphodiesterases are a family of enzymes that metabolize 3', 5' cyclic nucleotides to inactive metabolites, thereby terminating their second messenger role in mediating the cellular responses to various hormones and neurotransmitters.
  • PDEs Phosphodiesterases
  • seven families of PDE enzymes have been identified and each isoenzyme has been mapped to a distinct gene locus. These isoenzymes exhibit different substrate specificities for cAMP and/or cGMP, catalytic activities, tissue and cellular distributions, and sensitivity to different endogenous activators and inhibitors.
  • selective inhibitors of some of these isoenzymes have been synthesized, and have been validated as useful tools for examining the biological function(s) of these enzymes in various tissues (See reference (1)).
  • the low-Km cAMP specific, type IV PDE family of enzymes has generated considerable interest as potential targets for the development of novel antiasthmatic and antiinflammatory drugs (2).
  • this family of PDEs exist at least four isoenzymes, each of which is encoded by a distinct gene (3). Additionally, the mRNA of each gene product is thought to undergo alternative splicing, thereby giving rise to isoforms for each isoenzyme, respectively (4,5).
  • the four known PDE IV gene products (a,b,c,d) exhibit different tissue distributions (6), but in general are expressed in a wide number of cells that play a role in allergic and inflammatory responses (7).
  • PDE r enzyme(s) have been shown to block antigen-induced airway eosinophilia in allergic guinea pigs (8), inhibit superoxide production by human neutrophils (9), suppress LPS induction of TNFa release from human monocytes (10), inhibit arachidonate release from human neutrophils (11) and to block ozone-induced airways hyperreactiviy in a variety of species (12).
  • This invention related to a system for stably expressing a low-Km cAMP phosphodiesterase (PDE) and the use of the system for assessing inhibitors of PDE.
  • PDE low-Km cAMP phosphodiesterase
  • this invention related to a system for the stable expression of a low-Km cAMP PDE TV and the use of the stable system in the evaluation of inhibitors of PDE IV.
  • This invention also relates to an improved in vitro assay for the evaluation of inhibitors directed against a "high-affinity" state of the enzyme with respect to rolipram.
  • a CHO-K1 cell line stably expressing a recombinant full- length human PDE IVa (rhPDElVa) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis which revealed the presence of a 117 kDa immunoreactive band using rabbit anti-PDE IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 x g supernatant (soluble enzyme preparation) obtained from the same cell line.
  • FIG. 1 Kinetic characteristics of PDE Ja expressed in CHO-K1 cells
  • Figure 2b Kinetic characterization of HSPDE4A4B expressed in CHO-
  • FIG 3b Immunocytochemistry of transfected CHO-K1
  • Figure 4b Immunocytochemistry of transfected CHO-K1
  • Figure 4b Immunocytochemistry of transfected CHO-K1
  • the invention encompasses a method of assessing the capacity of an inhibitor of phosphodiesterase IV to inhibit phosphodiesterase IV, comprising the steps of:
  • test compound (2) test compound; and (3) a suitable enhancer of ionic strength;
  • the soluble full length low-Km cAMP phosphodiesterase IVa enzyme shall be defined as phosphodiesterase enzyme that has a K m of 5 ⁇ M or less and is at least
  • the amount of test compound used in the method may range from about 1 ⁇ Mol/L to 0.3 nMol/L.
  • the enhancer of ionic strength includes, but is not limited to MgCl2, NaCl, Choline Chloride,
  • the amount of enhancer of ionic strength used in the method may range from 0 mM to about 600 mM, preferably about 300 mM.
  • the incubation period may range from about 5 to 20 min, preferably about 10 min.
  • the temperature of incubation may range from 25 to 37°C , preferably about 30°C.
  • the means of measuring phosphodiesterase activity shall include, but is not limited to binding of 5' AMP to Scintillation Proximity
  • Assay Beads such as manufactured by AMERSHAM,UK.
  • the invention encompasses a system for stably expressing a soluble low-Km cAMP phosphodiesterase IV enzyme (PDE r ) comprising: CHO-K1 cells transfected with an expression vector for expressing human PDE IV DNA, the expression vector comprising pEE7.
  • PDE r soluble low-Km cAMP phosphodiesterase IV enzyme
  • the system for stably expressing high affinity PDE IV comprises CHO-K1 cells as identified by accession number ATCC CRL 9618, an expression plasmid comprising vector pEE7 and human PDE TV cDNA.
  • the invention encompasses a method of assessing the capacity of an inhibitor of phosphodiesterase IV to inhibit phosphodiesterase IV, comprising the steps of: (a) preparing a reaction mixture comprising: (1) CHO-K1 cells stably expressing full length low-Km cAMP phosphodiesterase IVa enzyme;
  • test compound (b) incubating said reaction mixture;
  • the amount of CHO-K1 cells used in the method may range from 0.1 million/ml to 0.4 minllion/ml, preferably about0.2 million/ml.
  • the amount of prostaglandin 12 used in the method may range from 5 ⁇ Mol/L to 20 ⁇ Mol/L, preferably about 10 ⁇ Mol/L.
  • the amount of test compound used in the method may range from 0.0003 ⁇ M to 10 ⁇ M.
  • the incubation period may range from 5 min to 20 min, preferably about 10 min.
  • the temperature of incubation may range from 20 to 30 °C, preferably about 25 °C.
  • the means of measuring phosphodiesterase activity shall include, but is not limited to determination of cellular cAMP by radioimmune assay.
  • CHO-K1 cells were transfected with an expression vector containing a 2700 bp cDNA insert encoding for a full-length PDE /a enzyme as described in Materials and Methods.
  • the expression plasmid is based on the vector pEE7. The following abbreviations are used to describe the features of this plasmid.
  • HCMV MIE human cytomegalovirus major immediate early gene promoter/enhancer, hyg: hygromycin resistance gene, TK: thymidine kinase, SV40 origin: SV40 origin of replication.
  • the vector also includes an E.coli replicon and beta-lactamase gene from the plasmid pBR322.
  • Figure 2a Kinetic characteristics of PDE IVa expressed in CHO-K1 cells
  • PDE IVa activity was assayed in the 100,000 x g supernatant obtained from sonicated CHO-K1 cells stably expressing the enzyme.
  • the enzyme activity was assayed in duplicate at substrate concentrations ranging from 0.2 ⁇ M to 30 ⁇ M using the Amersham SPA beads as described in Materials and Methods. Shown are the saturation kinetics for the enzyme.
  • the inset graph is a double-reciprocal plot of the kinetic data. Average of 3 separate experiments +/- SD .
  • Figure 2b Kinetic characterization of HSPDE4A4B expressed in CHO- Kl cells.
  • Panel A Shown are the saturation kinetics for the enzyme.PDE IVA activity was assayed in the 100,000 x g supernatant obtained from sonicated CHO-K1 cells stably expressing the enzyme. The enzyme activity was assayed in duplicate at substrate concentrations ranging from 0.2 mM to 10 mM using the Amersham
  • Filled circles represent enzyme activity measured as described in Materials and Methods. Filled squares are data obtained in the presence of 300 mM KCl.
  • Panel A Western blot analysis for PDE IVa enzyme expression was performed on the soluble portion of CHO-K1 cells transfected with a vector missing the PDE IVa cDNA insert (Lane 1) and CHO-K1 cells expressing high PDE IV activity (Lane 2). The blot was probed with a rabbit anti-PDE ⁇ Va antibody raised against the C-terminus of the enzyme.
  • Panel B Northern blot analysis for PDE IVa mRNA transcripts. Poly-A RNA was extracted from control CHO-K1 cells grown in hygromycin B selection media (Lane 3) and clones stably expressing PDE IV activity (Lane 4).
  • the blot was probed with a 32P- labelled 420 bp oligonucleotide obtained by PCR amplification of a stretch of PDE IVa cDNA from nucleotide 2392 to 2859.
  • the blot was washed with 2XSSC containing 0.1% SDS.
  • CHO-K1 cells transfected with either the pEE7 expression either containing (panel A) or not containing (panel B) the cDNA insert encoding for HSPDE4A4B were grown, under both hygromycin B and G418 selection .
  • the cells were fixed with an ice cold solution of 100% methanol for 10 minutes on ice.
  • Rabbit antisera raised against the NH2-terminus of HSPDE4A4B was diluted 1/500 and incubated at 4o C overnight.
  • the cells were washed with 3 changes of PBS.
  • a donkey anti-rabbit secondary antibody conjugated to C y 3 (indocarbocyanine) was incubated with the cells.
  • the labeled cells were rinsed and visualized with an Olympus fluorescence microscope. Similar data were obtained using an antisera raised against an epitope contained within the catalytic domain of the enzyme.
  • Binding of [3H](R)-rolipram to purified FLAG PDE INA enzyme was examined in the presence of the indicated concentrations of KCl and/or 3 mg/ml HSA. Shown is the specific saturable binding to the purified FLAG enzyme with increasing concentrations of ligand from 0.2 to 100 nM (panel A). Each point represents the mean of 3 separate experiments performed in duplicate ⁇ SEM. Scatchard analyses of specific [ 3 H](R)-rolipram binding in the presence of 0 mM KCl (panel B) and 150 mM KCl + 3 mg/ml HSA (panel C). Each point represents the average of 3 separate experiments.
  • Compounds evaluated include the following:
  • a human PDE IVa cDNA was obtained from a human frontal cortex cDNA library (CLONETECH) by hybridization with a partial cDNA isolated from the human monocytic cell line U937 (23).
  • a rabbit polyclonal antiserum was raised to a fusion protein comprised of glutathione-S transferase linked to the carboxyl terminal 162 amino acids of human PDE IVa.
  • a fusion protein comprised of glutathione-S transferase linked to the carboxyl terminal 162 amino acids of human PDE IVa.
  • FBS heat inactivated fetal bovine serum
  • penicillin/streptomycin 25 mM Hepes, pH 7.4
  • the cells were placed in an incubator for 24 hr at 37°C and 5% CO2- The cells were then washed with warmed sterile phosphate buffered saline (PBS) and incubated with 2 ⁇ g/ml DNA, and 9 ⁇ g/ml lipofectamine reagent in Opti- MEM for 7 hr at 37° C and 5% CO2- The incubation solution was diluted
  • CHO-Kl cells were plated at a density of 10 ⁇ cells/175 cm 2 containing complete media with 500 ⁇ g/ml hygromycin. The flasks were maintained in an incubator at 37°C with 5.0% CO2 for 72 hr. The media was changed and the cells were allowed to grow overnight. The cells were washed and dissociated from the plate with PBS containing 0.5 mM EDTA. Cellular cAMP content was measured by centrifuging the cell suspension at 150 g x 10 min and resuspending the cells in a Hanks buffered salt solution at a density of 0.2 x 10 ⁇ cells/ml.
  • the cells were preincubated at room temperature for 15 min and then incubated with 10 ⁇ M prostaglandin 12 (PGI2) and the indicated compound for an additional 10 min.
  • Basal cAMP levels were determined by incubating the cells in 0.1% DMSO. The incubations were terminated by the addition of HCl (0.1 N final) and the cells measured for cAMP.
  • Determinations of whole-cell cAMP content were performed by incubating 100 ⁇ l reconstituted rabbit anti-succinyl cAMP serum with 100 ⁇ l of the whole-cell reaction or known cAMP standard and 30 pmol of 1251-cAMP TME in a SCINTISTRIPTM we n (300 ⁇ l final volume) at room temperature for 18 h. Total cpm (Bo) was determined in the absence of sample or cAMP standard. The reaction mixture was then aspirated out of the well, and individual wells were counted in a BECKMAN LS 6000SC with the window open from 10-999 for 1 min.
  • %B/Bo [(standard or sample cpm - non-specific cpm) / (Bo cpm - non-specific cpm)] x 100.
  • Non-specific cpm were determined by incubating only the 1251-cAMP TME with assay buffer (50 mM acetate; pH 5.8) in the ScintiStripTM well. All determinations were performed in triplicate.
  • CHO-Kl cells were grown and resuspended as described above except that the Hank's buffer was replaced by a solution containing 0.25 M sucrose; 10 mM Tris, pH 7.5; and 0.5 mM EDTA. The cells were preincubated and incubated with 1 ⁇ M PGI2 and the indicated compounds at room temperature as described above. The incubations were terminated by the addition of 0.05% Triton X-100 (final) and incubating the cells for 5 min on ice.
  • Protein kinase activity was measured in a medium containing 0.83 mM 32p ATP (sodium salt); 100 ⁇ g histone F2B; 30 mM MgCl2; 1 ⁇ M IBMX; 10 mM DTT; and 8 mM PO4 buffer, pH 6.6. This mixture (final volume 220 ⁇ l) was incubated in a waterbath for 30 min at 35°C and terminated by adding 5 ml of ice cold 5% TCA. The precipatated protein was applied to a Whatman GF/B filter under vacuum and the filter was subsequently washed with 3 x 5 ml ice cold TCA solution. The amount of -32P incorporation was measured by placing the filters in Aquasol and counting in a
  • Basal cAMP independent protein kinase activity was determined by the inclusion of 1 ⁇ M Protein Kinase A Peptide Inhibitor (19) in the reaction mixture. Maximal cAMP protein kinase activity was determined in the presence of 10 ⁇ M cAMP. All determinations were performed in triplicate and activity ratios were calculated as the [(kinase activity in the presence of PDE inhibitor minus cAMP independent kinase activity)/(maximal cAMP kinase activity minus cAMP independent kinase activity)] x 100.
  • CHO-Kl cells were lysed by sonication for 10 sees at a power setting of 50% (BRAUNSONIC Model 2000) in an ice cold solution containing 50 mM Tris, pH 7.5; 1 mM EDTA; and 200 ⁇ M - mercaptoethanol.
  • the soluble and particulate fractions of the cell were obtained by centrifuging the sonicate for 90 min at 100,000 x g at 4°C.
  • PDE activity was measured in a solution containing 50 mM Tris, pH 7.5; 10 mM MgCl2; 1 mM EDTA; and 100 nM (or indicated) 3H-cAMP (100 ml final volume) in the presence of varying concentrations of inhibitor.
  • the reaction mixture containing enzyme was incubated for 10 min at 30°C in 96-well View Plates (PACKARD), and terminated by the addition of 50 ⁇ l Phosphodiesterase Scintillation Proximity Assay (SPA) Beads (AMERSHAM) containing 18 mM ZnSO
  • SPA Phosphodiesterase Scintillation Proximity Assay
  • CHO-Kl cells stably expressing epitope FLAG PDE IV constructs were lysed by sonication for 10 sees at a power setting of 50% (Braunsonic
  • the affinity matrix was washed with column buffer until all unbound protein was eluted from the column as determined with a UV-2 flow cell. Bound material was eluted with column buffer containing 0.5 mg/ml. FLAG peptide at a flow rate of 2 ml/min. The eluted material was monitored for (R)-rolipram- sensitive phosphodiesterase activity and M2 immunoreactivity.
  • Membrane bound or soluble protein was subjected to SDS- PAGE with 4-12% precast Tris-glycine gels using a NOVEX slab gel apparatus. Prior to SDS-PAGE, samples were incubated in a buffer containing 2% SDS with 10 mM -mercaptoethanol at 95°C for 2 min. Samples (2-10 ⁇ g) were electrophoresed in the gels using a Laemmli buffer system at 100 V for 2 hr. Immunoprobing of PDE IVa enzyme was performed by transferring proteins to nitrocellulose membrane at 30 V for 12 hr in a SDS-free Towbin buffer using a NOVEX transblot apparatus.
  • the transfer-membranes were blocked with 10% skim milk powder in Tris buffered saline and 0.1% Tween 20 (TBS+T) for 24 hr.
  • TBS+T Tris buffered saline and 0.1% Tween 20
  • the membranes were washed twice in TBS+T and incubated with anti- PDE IVa (1:200 dil) in TBS+T and 1% BSA for 1 hr at room temperature.
  • the membranes were again washed in TBS+T and then incubated with an anti-rabbit IG horseradish linked whole Ab from donkey for 1 hr at room temperature.
  • Excess secondary antibody was washed from the membrane with TBS+T and the immunoconjugates were visualized with the AMERSHAM ECL Western blotting detection reagents.
  • CHO-Kl cells transfected with either the pEE7 expression containing or not containing the cDNA insert encoding for HSPDE4A4B were grown, under both hygromycin B and G418 selection .
  • the cells were fixed with an ice cold solution of 100% methanol for 10 minutes on ice.
  • the methanol was removed and the cells were rinsed with 3 changes of PBS.
  • the cells were incubated for 10 min in Biogenx Universal Blocking reagent.
  • the cells were again washed with 2 changes of PBS.
  • the antisera was diluted 1/1000 and incubated at 4°C overnight.
  • the cells were washed with 3 changes of PBS.
  • a donkey anti-rabbit secondary antibody conjugated to C y 3 (indocarbocyanine) was incubated with the cells at room temperature for 1 hr.
  • the labeled cells were rinsed and visualized with an Olympus fluorescence microscope.
  • CHO-Kl cells stably expressing the prostaglandin IP receptor (20) were transfected with a vector containing a cDNA insert encoding for a full-length human PDE IVa enzyme (Fig. 1) and grown in the presence of hygromycin B. Forty colonies were selected and examined for the stable expression of PDE r a enzyme. Two of the 40 clones selected displayed at least a 30-fold increase in cAMP hydrolytic activity compared to those cells which were transfected with a vector not containing the PDE IVa cDNA insert. As shown in Fig. 2a, the Km of the enzyme for cAMP as a substrate was determined to be 3.5 ⁇ M.
  • Western blot analysis of the soluble fraction using an anti PDE r a antibody demonstrates the full-length expression of the PDE TVa enzyme with an apparent molecular mass of 125 KDa (Fig 3a).
  • Northern blot analysis of poly A RNA prepared from the stable transfectant revealed the presence of a 2700 bp transcript. No immunoreactive protein or mRNA transcripts encoding for the PDE IVa enzyme was detected in the VH2 control CHO-Kl cells by Western or Northern blot analysis, respectively.
  • CDP 840 and (S)- rolipram were the least potent of the compounds tested, and a maximal "plateau" response was not obtained. An estimated 30-fold stereoselectivity between the (R) and (S) isomers of rolipram was noted. No significant changes in the VH2 CHO control cellular content of cAMP was observed with the inhibitors in either the presence or the absence of 10 ⁇ M PGI2-
  • the rank order potencies of the compounds to activate PKA in the cell was very similar to the rank order for their ability to elevate cAMP in the cells. It was determined that RS 14203 was more potent than (R)-rolipram > CDP 840 > (S)-rolipram in mediating an activation of PKA phosphotransferase activity.
  • the apparent EC50s of the four compounds to activate PKA were calculated to be 10 nM, 168 nM, 319 nM, and 1 ⁇ M, respectively. From these data, the lack of correlation between the rank potencies of the compounds when comparing the whole-cell data with the measured enzyme activity cannot be accounted for by cAMP compartmentalization. Effect of Ionic Strength on PDE IV Inhibitor Potency
  • PDE rVa enzyme versus the ability of this same group of compounds to inhibit the recombinant enzyme in a broken-cell preparation is shown in figures 10a and b.
  • FIG. 9 There is a lack of concordance (Fig. 9) between the ability of a compound to elevate cAMP in the CHO-Kl cells and its biochemical potency as determined at a MgCl2 concentration of 10 mM
  • the FLAG PDE IVA met 330 enzyme also appears as a single band but with a molecular mass of 85 KDa. These same protein bands are recognized by both antibodies directed against the catalytic domain of the PDE IVA enzyme and antibodies directed against the FLAG peptide as shown in figs, lib and lie, respectively.
  • the FLAG PDE IVA enzyme exhibits first-order kinetics with respect to substrate (fig. 12a) with a K ⁇ of 2.9 ⁇ 0.4 mM cAMP and a V max of 9.7 + . 0.2 mMol cAMP hydrolyzed/mg prot/min when assayed in the presence of 150 mM KCl.
  • the truncated FLAG PDE IVA- ⁇ ,, enzyme exhibits the same first-order kinetics (fig. 12b) with a K,.. of 2.6 ⁇ 0.2 mM cAMP and a V max of 6.7 + 0.8 mMols cAMP hydrolyzed/mg prot/min in the presence of 150 mM KCl.
  • IC 50 25 nM
  • the shift in sensitivity of the enzyme to (R)-rolipram occured in concentrations of HSA as low as 0.1 mg/ml and was essentially maximal when the concentration of HSA in the enzyme assay buffer was increased to 1 mg/ml (not shown).
  • the presence of 150 mM KCl in combination with 3 mg/ml HSA provided an additional 3-fold increase in enzyme sensitivity to (R)-rolipram (25 nM to 9 nM).
  • the purified FLAG PDE r A enzyme exhibited an 8-fold increase in the saturable binding of [ 3 H](R)-rolipram in the combined presence of 3 mg/ml HSA and 150 mM KCl.
  • Scatchard analysis of the data again reveals the binding of ligand to a single class of high- affinity sites (fig. lOf) with a Kd of 0.4 + 0.1 nM and B max of 240 fmol 1.6 pmol purified enzyme.
  • No specific binding of [ 3 H](R)-rolipram to the HSA was noted either in the presence or absence of 150 mM KCl.
  • presoaking the filtermats for 15 minutes in a solution containing 1 % PEI, 150 mM KCl and 3 mg/ml HSA had no effect upon the B max of the enzyme preparation.
  • cAMP second messengers
  • Agents are able to modulate cAMP levels in target tissues by either altering the rate of its synthesis through an activation of adenylyl cyclase or by affecting the rate of its metabolism via phosphodiesterase enzymes (23). Since cAMP is ubiquitously distributed throughout all mammalian tissues, indiscriminate pharmacological manipulation in the cellular content of this molecule would clearly lead to undesirable effects in a patient. A rekindling of interest has arisen in this field with the identification of a family of phosphodiesterase enzymes which are selective for cAMP and which are primarily expressed in cells thought to play a role in the inflammatory process(es).
  • the PDE IV enzyme family is considered to be the biochemical target for a class of compounds typified by rolipram, and therefore, a clear biochemical and pharmacological understanding of how these agents act in vitro and in vivo would lead potentially to the development of the next generation of antiasthmatic and antiinflammatory drugs (2).
  • CDP 840 is as potent as RS 14203 and more potent than (R)-rolipram when assayed against the recombinant full-length PDE r a enzyme.
  • CDP 840 is consistently less potent than RS 14203 and (R)- rolipram at elevating whole-cell cAMP levels in eosinophils from guinea pigs, human neutrophils, and in HL-60 and U937 cells (data not presented).
  • a CHO-Kl cell line stably expressing high levels of the PDE rVa enzyme was established.
  • the PKA activation assay was found to be more sensitive than assaying changes in whole-cell cAMP levels.
  • the inhibitors were able to mediate an activation of the kinase in the presence of 1 ⁇ M PGI2, whereas at least 10 ⁇ M PGI2 was required to observe any increase in whole-cell cAMP with the inhibitors.
  • the EC50S obtained for the compounds from the two assays were closely similar.
  • CDP 840 was not appreciably affected with increasing amounts of salt in the assay.
  • the rank order of potencies of the inhibitors to inhibit the phosphodiesterase enzyme under assay conditions resembling the ionic strength found in the intact cell closely reflects their ability to elevate cAMP in the whole-cell assay.
  • the apparent stereoselectivity of (R) and (S)-rolipram observed in the whole-cell is recovered with the soluble enzyme under high salt conditions. It would appear, therefore, that the lack of correlation between the biochemical potencies of the inhibitors on the enzyme and their ability to elevate cAMP in the whole-cell results from the inability the recreate the intracellular environment in which the PDE IVa enzyme exists normally.
  • cAMP second messengers
  • Agents can modulate cAMP levels in target tissues either by altering the rate of cAMP synthesis through an activation of adenylyl cyclase or by affecting the rate of cAMP metabolism via phosphodiesterase enzymes (47). Since cAMP is ubiquitous to all mammalian tissues, indiscriminate pharmacological manipulation in the cellular content of this second messenger would clearly lead to undesirable effects in a patient. A rekindling ofinterest in the cAMP field has arisen with the identification of a family of phosphodiesterase enzymes which are predominately expressed in cells thought to play a role in asthma and inflammatory processes.
  • the PDE IV enzyme family is considered to be the biochemical target for a class of compounds typified by rolipram.
  • the biochemical mechanism by which rolipram is thought to act in the treatment of endogenous depression is via selective inhibition of cAMP dependent phosphodiesterase activity (49). It was shown that micromolar concentrations of rolipram inhibited the cAMP PDE activity found in membraneous and cytosolic fractions prepared from rat brain.
  • rat forebrain membranes contained a high density of saturable high-affinity ( ⁇ )[3H] rolipram binding sites. These sites bound ( ⁇ )[3H] rolipram with a Kd of 1.2 nM and B max of 19.3 pmol/g rat forebrain. It was thought that this binding site was related to PDE enzymes confined to the CNS, but the >1000-fold difference between the measured Kd and IC 50 for rolipram in the binding and enzymatic assays, respectively, raised doubt as to the involvement of a single common enzyme.
  • UCR1 region appears to harbor distinct hydrophillic and hydrophobic domains whereas UCR2 is predominately hydrophillic.
  • yeast two hybrid systems Houslay et al.(50) have reported that UCR1 and UCR2 are able to interact suggesting that the N-terminus may be involved in establishing distinct tertiary structures. These tertiary structures may then impact upon the catalytic conformation of the enzyme with respect to activity and inhibitor sensitivity. Switching of the PDE IV enzyme from low-affinity to high-affinity with respect to rolipram has also been shown by treating the enzyme with vanadate/glutathione (39).
  • the ratPDE3/TvO phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein-kinase. J. Biol. Chem.: 269, 18271-18274, 1994.

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Abstract

On a déterminé une lignée cellulaire CHO-K1 exprimant de façon stable une enzyme humaine PDE IVa de recombinaison à longueur totale (rhPDEIVa) par sélection d'hygromycine B. On a évalué l'inhibition de l'activité de PDE IVa exprimée par des inhibiteurs sélectifs de PDE IV. L'ordre de classification des puissances des inhibiteurs en élevant cAMP dans la méthode de détermination de cellules entières s'est avéré totalement différent de celui obtenu avec l'enzyme soluble. L'ordre de classification des puissances s'est également maintenu quand on a mesuré des rapports d'activité de PKA à la place de niveaux de cAMP. Quand on a examiné l'inhibition de l'activité de PDE IVa soluble en présence de 100 mM de MgCl2, l'ordre de classification des puissances des inhibiteurs s'est avéré être identique à celui qu'on avait obtenu pour les méthodes de détermination de cellules entières (à la fois cAMP et PLA). La conformation observée d'affinité élevée de l'enzyme ne dépendait pas d'un cation ou d'un anion spécifique. Le déplacement de sensibilité de l'enzyme rhPDE IVa pour (R)-rolipram en présence d'une force ionique accrue était accompagné par une capacité améliorée de la préparation d'enzyme soluble à se fixer de façon spécifique et avec une affinité élevée à [3H](R)-rolipram.
PCT/CA1998/000024 1997-01-09 1998-01-07 Systeme d'expression stable d'une phosphodiesterase de camp a affinite elevee et son utilisation Ceased WO1998030718A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1041157A3 (fr) * 1999-03-29 2000-10-11 Warner-Lambert Company Méthodes pour le criblage des lignes cellulaires non-recombinantes capables d'exprimer une isoenzyme PDE4 unique et pour le criblage des inhibiteurs de PDE4

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