Assay method

An assay is provided for the identification and comparison of compounds having activity as modulators of the expression and release of TNFa, IL-1 or IL-6 in humans and animals in which modulation of the catalytic activity of PRAK (p38-regulated/activated protein kinase) is employed for identification and comparison of test compounds.

EXAMPLE Inhibitors of PRAK, a Downstream Kinase of p38a, have Anti-inflammatory Properties. Prak was recently identified as a substrate of p38a (New et al. ibid). Prak is a kinase homologous to MapkapK2, MapkapK3, Mnk1 and Mnk2, and is able to efficiently phosphorylate the small heat shock protein HSP27 in vitro (New et al. ibid). Further in vivo substrates have not been identified yet, and the biological function of Prak is unknown to date. Below we provide evidence that Prak is involved in the expression of TNF&agr;, IL-1 or IL-6 in human peripheral blood mononuclear cells or dermal fibroblasts, based on the fact that small molecular weight inhibitors of the catalytic activity of Prak inhibit the production of the inflammatory mediators TNFa, IL-1, or IL-6 in vitro. Thus, Prak is apparently involved in the inflammatory response in vivo, and inhibition of this kinase provides a new anti-inflammatory principle. Materials and Methods 1. In vitro Kinase Assay Recombinant human Prak was expressed in E. coli as His6-tagged protein substantially as described for Mnk1 by Tschopp et al. (Tschopp et al. (2000) Phoshorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies. Molecular Cell Biology Res. Comm. 3, 205-211). Phospho-p38a and His6-Hsp27 was prepared substantially as described (New et al. and Tschopp et al. ibid). All kinase reactions were performed with the following reaction buffer. Kinase buffer (5×): 125 mM Hepes (Stock at 1M; Gibco &num;15630-056), 125 mM &bgr;-glycerophosphate (Sigma &num;G-6251):125 mM MgCl 2 (Merck &num;5833); 0.5 mM Sodium orthovanadate (Sigma &num;5-6508), 10 mM DTT (Boehringer Mannheim &num;708992), pH 7.4. The (5×) kinase buffer is freshly prepared from 25× stock solution. DTT is stored frozen at −20° C. in small aliquots and added immediately before use. Prior to the assay, recombinant Prak is phosphorylated by incubation with phospho-p38. The kinase mix is prepared by mixing phospho-p38 (final concentration: 30 &mgr;g/ml) and Prak (final concentration: 150 &mgr;g/ml) in 1× kinase buffer (containing 5 &mgr;M ATP. The reaction mixture is incubated for 30 min at RT and chilled on ice until further use. For the assay, the activated kinases are prediluted in 1× kinase buffer/0.00025% TWEEN20 to 15 &mgr;g/ml. The compounds to be tested are prediluted in 10% DMSO to 10× the final assay concentration. The substrate mix is prepared by mixing His-HSP27 (71.5 &mgr;g/ml) with ATP (1.43 &mgr;M) and 3 33 P-ATP (7.15 &mgr;Ci/ml) in 1× kinase buffer/0.00025% TWEEN20. To this mix, 5 &mgr;l of compound are added, and the reaction is started by addition of 10 &mgr;l of kinase mix. The assay is stopped after one hour of incubation at room temperature by the addition of 5 &mgr;l EDTA 0.5M. The label incorporated into protein is separated from labelled ATP by filtration through an Immobilon-P menbrane. After washing and drying the membrane, incorporated label is quantified by liquid scintillation counting. Percent inhibition is calculated relative to control reactions which do not contain inhibitor. 2. TNF&agr; and IL-1 Release from Human PBMCs Cell Separation and Culture All cell culture and ELISA work was done on 96 well plates (Costar). Mononuclear cells were prepared from the blood of healthy volunteers using ficoll-hypaque density separation according to the method of Hansel et al (Hansel TT et al. (1991) An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Imm. Methods 145, 105-110) and used at a concentration of 100,000 cells/well in RPMI 1640, 5% FCS (Gibco). Serial dilution of the test compound were made in DMSO and diluted with culture medium. Final DMSO concentration in the culture medium was 0.1%. Compound were preincubated with the cells for 30 minutes at 37° C., 5% CO 2 . Then, the cells were stimulated with LPS (5 &mgr;g/ml, Sigma) and &ggr;-interferon (100 U/ml, Boehringer Mannheim) and cultured for 3 hours (for TNF&agr;-determination) or 4.5 hours (for Il-1 &bgr; and Il-6 determination). The plates were centrifuged, the supernatants were removed and stored at −80° C. until ELISA determination. TNF&agr;-ELISA For ELISA determination, supernatants were diluted 1:1 and added into a plate coated with the anti humanTNF monoclonal antibody (357-101-4, clone 92030603 from European Collection of Animal Cell Cultures, antibody produced and purified within Novartis Pharma, 1 &mgr;g/ml in PBS) After overnight incubation, the plates were washed (4×) and biotinylated anti humanTNF monoclonal antibody 2-179-E11 (clone 92030602 (European Collection of Animal Cell Cultures), produced and purified in Novartis Pharma) was added to a final concentration of 1 &mgr;g/ml (100 &mgr;l/well). After 4 hours of incubation, plates were washed 4× and streptavidin-alkaline phosphatase (Jackson Immunoresearch, &num;016-050-084, 1:7500 dilution) was added. The p-nitrophenyl phosphate substrate was prepared immediately before the assay from tablets (1 mg/ml in buffer pH 9.8, 100 &mgr;l/well). Color formation was monitored in a Bio-Tec ELx 808 plate reader at 405 nm in the kinetic mode for 30 minutes. A set of humanTNF standards (31.1 pg-2000 pg) is included on each plate and used to calculate TNF concentrations from the slopes for each individual well. IC 50 -values were calculated using the Origin software package by weighted fitting of the means of percent inhibition for each inhibitor concentration to the logistic function. Hu IL-1 2 ELISA ELISA microtiter plates were coated with a murine anti-human IL-1 2 MAb (Human IL-1 2 Cytoset (Biosource International Inc., &num;CHC1214), 100 &mgr;l at 1 &mgr;g/ml) in PBS 0.02% NaN 3 and incubated overnight at &plus;4° C., The following day, microtiter plates were washed 4 times with PBS/0.05% Tween/0.02% NaN 3 and blocked with 300 &mgr;l of PBS/2% bovine serum albumin (BSA)/0.02% NaN 3 for 3 h. Plates were washed again (4 times) and 100 &mgr;l of supernatant (final dilutions of 1:15 and 1:30) or of the recombinant human IL-1 2 standard (titration curve ranging from 640 to 10 pg/ml in 2 fold dilution steps) was added in duplicate together with a biotin-labelled murine anti-human IL-1 2 MAb (50¼ l at 0.4 &mgr;g/ml). The plates were incubated for 2 h at room temperature with shaking (700 rpm). After incubation the plates were washed 4 times, and streptavidin alkaline phosphatase conjugate (Jackson Immunoresearch, &num;016-050-084) was added at a final dilution of 1/3000 (100 ¼ l/well; 30 min at room temperature). After washing (4 times) the substrate (p-nitrophenylphosphate in diethanolamine buffer; 100 &mgr;l ) was added for 30 min. Reaction was blocked by the addition of 50 &mgr;l/well of NaOH. Plates were read in a microtiter reader (Bio-Rad) using filters of 405 and 490 nm. 3. Cytotoxicicty THP-1 cells are obtained from ATCC and stored in liquid nitrogen. Medium for growth of THP-1 cells: RPMI 1640(GIBCO)&plus;10% fetal bovine serum (GIBCO)&plus;L-glutamine (2 mM)&plus;penicillin/streptomycin (100 U/ml) &plus;&bgr;-mercaptoethanol (0.05 mM). Cell culture medium for THP-1 incubation: RPMI 1640(GIBCO)&plus;5% fetal bovine serum (GIBCO)&plus;L-glutamine (2 mM)&plus;penicillin/streptomycin (100 U/ml)&plus;&bgr;-mercaptoethanol (0.05 mM). The solution for stimulation is prepared by mixing equal volumes of IFN&ggr; (Boehringer Mannheim, 2000 U/ml) and LPS (Sigma, L8274, 100 &mgr;g/ml). The dye solution is prepared from 500 mg MTT (Sigma, M2128) in 100 ml phosphate buffered saline (PBS) and kept under sterile conditions at 4° C. Sodium dodecyl sulfate (SDS) solution (10%) is made from 50 g SDS (Bioprobe Systems) and 5 ml 1N HCl. The solution is made up to 500 ml with distilled water and kept at room temperature (precipitates in the fridge). All incubations are done at 37° C. in 5% CO 2 . Test compounds are dissolved in dimethylsulfoxide (DMSO) at 10 mM and further diluted in DMSO to 3 mM and 1 mM. The DMSO stock solutions are further diluted in RPMI 1640 to 300 &mgr;M, 100 &mgr;M and 30 &mgr;M. The final DMSO concentration is 0.1 or 0.3%. The 96-well plates for the incubation are prepared as follows: In duplicate wells, 60 &mgr;l cell culture medium, 20 &mgr;l compound and 100 &mgr;l THP-1 cell culture (500,000 cells/ml) are pre-incubated for 30 min. To stimulate the cells, 20 &mgr;l of the LPS/&bgr;-IFN solution is added and the cells are incubated for 24 hours. At the end of the incubation period, the plates are centrifuged and 100 &mgr;l medium is removed. Dye solution (10 &mgr;l) is added to each well and the plates are incubated for 4 hours or overnight. SDS-solution (100 &mgr;l) is added to each well and the plates are again incubated for at least 10 hours. The absorbance at 540 nm is read with a microplate reader (Biotec 808 Elx). Blue-colored wells indicate living cells, whereas yellow wells indicate a high percentage of dead cells. Calculation of cytotoxicity: The absorbance corresponding to 100% cytotoxicity equals the absorbance of wells without cells (no MTT converted into the blue dye). The absorbance corresponding to no cytotoxicity is taken from wells without substance, but with control solvent. Percent cytotoxicity is calculated for each substance concentration. 4. IL-6 Production in Human Dermal Fibroblasts Neutralization Assay Human dermal foreskin fibroblasts were obtained from Clonetics (CC-2509) and grown in FBM (Clonetics, CC-3131 ) including bFGF (1 ng/ml, CC-4065), insulin (5 &mgr;g/ml, CC-4021), and 2% FCS (CC-4101). For induction of IL-6, cells were seeded at a density of 10 4 cells per well in a 48 well tissue cluster. The following day, cells were starved for 6-7 h in FBM containing 2% FCS. Compounds were added at final concentrations of 10 and 1 &mgr;M 30 min prior to stimulation with recombinant human IL-1&bgr; (100 pg/ml). Cell supernatant was taken 16-17 h after stimulation and the amount of released IL-6 determined in a sandwich ELISA. IL-6 ELISA ELISA microtiter plates were coated with a murine anti-human IL-6 MAb (314-14 (Novartis Pharma; batch EN23,961, 5.5 mg/ml); 100 &mgr;l at 3 &mgr;g/ml) in PBS 0.02% NaN 3 and incubated overnight at &plus;4° C., The following day, microtiter plates were washed 4 times with PBS/0.05% Tween/0.02% NaN 3 and blocked with 300 &mgr;l of PBS/3% bovine serum albumin (BSA)/0.02% NaN 3 for 3 h. Plates were washed again (4 times) and 100 &mgr;l of supernatant (final dilutions of 1:20) or of the recombinant human IL-6 standard ((Novartis Pharma &num;91902), titration curve ranging from 1 to 0.0156 ng/ml in 2 fold dilution steps) was added in duplicate. After an overnight incubation at RT the plates were washed (4 times) and a different murine anti-human IL-6 MAb (110-14, Novartis Pharma; 6.3 mg/ml); 100 ¼ l at 1 &mgr;g/ml; 3 h at room temperature) was added. After additional 4 washes, a biotin-labelled goat anti-mouse IgG2b antiserum (Southern Biotechnology; &num;1090-08) was added at the final dilution of 1/10000 (100 &mgr;l/well; 3 h at room temperature). After incubation plates were washed 4 times and streptavidin coupled to alkaline phosphatase (Jackson Immunoresearch, &num;016-050-084) was added at a final dilution of 1/3000 (100 ¼ l/well; 30 min at room temperature). After washing (4 times) the substrate (p-nitrophenylphosphate in diethanolamine buffer; 100 &mgr;l) was added for 30 min. Reaction was blocked by the addition of 50 &mgr;l/well of 1.5 M NaOH. Plates were read in a microtiter reader (Bio-Rad) using filters of 405 and 490 nm. IL-6 levels in culture supernatants were calculated in reference to the standard curve using the cubic curve fit. Percentage inhibitions and IC 50 values were calculated Results As shown in the Table, small molecular weight compounds which inhibit the kinase activity of Prak in vitro, inhibit with a similar IC50 the release of TNFa and IL-1b from LPS-treated human peripheral mononuclear cells. In addition, the production of IL-6 is inhibited in human dermal fibroblasts by these compounds. 1 In vitro kinase IL-1&bgr; Inhibition assay TNF&agr; release Cyto- of IL-6 &commat; Prak release IC50 toxicity 1 &mgr;M Compound IC50 &lsqb;nM&rsqb; IC50 &lsqb;nM&rsqb; &lsqb;nM&rsqb; IC50 &lsqb;nM&rsqb; &lsqb;%&rsqb; Compound 1100 2200 1100 >30000 100 A Compound A has the following structural formula 1 Compound A is included per se within the scope of the present invention.