Aminocyanopyridine inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

Bioorganic and Medicinal Chemistry Letters

Volumn 15, Issue 6, 2005, Pages 1587-1590

  Anderson, David R ^a   Hegde, Shridhar ^a   Reinhard, Emily ^a,c   Gomez, Leslie ^a,d   Vernier, William F ^a   Lee, Len ^a   Liu, Shuang ^b   Sambandam, Aruna ^b   Snider, Patricia A ^a,e   Masih, Liaqat ^b  

^a PFIZER GLOBAL RESEARCH AND DEVELOPMENT   (United States)

^b ALBANY MOLECULAR RESEARCH INC   (United States)

^c H LUNDBECK A S   (Denmark)

^d JOHNSON AND JOHNSON PHARMACEUTICAL RESEARCH AND DEVELOPMENT   (United States)

^e ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES   (United States)

Author keywords

Kinase inhibitor; MK 2; TNF

Indexed keywords

AMINOCYANOPYRIDINE INHIBITOR; ANTIINFLAMMATORY AGENT; BENZOPYRAN DERIVATIVE; ISOENZYME; MITOGEN ACTIVATED PROTEIN KINASE ACTIVATED PROTEIN KINASE 2; PROTEIN KINASE INHIBITOR; TUMOR NECROSIS FACTOR ALPHA; UNCLASSIFIED DRUG;

ARTICLE; CELL STRAIN U937; CONTROLLED STUDY; CORRELATION ANALYSIS; ENZYME INHIBITION; HUMAN; HUMAN CELL; IC 50; PROTEIN EXPRESSION; STRUCTURE ACTIVITY RELATION; STRUCTURE ANALYSIS;

EID: 20044384349     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2005.01.067     Document Type: Article

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9. 50 value determination: Recombinant MAPKAPK2 was phosphorylated at a concentration of 42-78 mM by incubation with 0.23 mM of active p38a in 50 mM HEPES, 0.1 mM EDTA, 10 mM magnesium acetate, and 0.25 mM ATP, pH 7.5 for 1 h at 30°C. The phosphorylation of HSP-peptide (KKKALSRQLSVAA) by MAPKAPK2 was measured using an anion exchange resin capture assay method. The reaction was carried out in 50 mM β-glycerolphosphate, 0.04% BSA, 10 mM magnesium acetate, 2% DMSO, and 0.8 mM dithiotheritol, pH 7.5 in the presence of the HSP-peptide with 0.2 μCi [g33P]ATP and 0.03 mM ATP. The reaction was initiated by the addition of 15 nM MAPKAPK2 and was allowed to incubate at 30°C for 30 min. The reaction was terminated and [g33P]ATP was removed from solution by the addition of 150 mL of AG 1X8 ion exchange resin in 900 mM sodium formate pH 3.0. A 50 mL aliquot of head volume was removed from the quenched reaction mixture and added to a 96-well plate, 150 mL of Microscint-40 (Packard) was added and the amount of phosphorylated-peptide was determined. The assay is performed at a final concentration of 2% DMSO. The error for this assay is taken to be 0.2 log units
10. 2. Differentiation of U937 to monocytic/macrophage-like cells is induced by the addition of phorbol12-myristate 13-acetate (Sigma) at final concentration of 20 ng/mL to a culture of U937 cells at ∼0.5 million cells/mL and incubated for 24 h. The cells are centrifuged, washed with PBS and resuspended in fresh media without PMA and incubated for 24 h. Cells adherent to the culture flask are harvested by scraping, centrifugation, and resuspended in fresh media to 2 million cells/mL, and 0.2 mL is aliquoted to each of 96 wells in flat-bottom plate. Cells are then incubated for an additional 24 h to allow for recovery. The media is removed from the cells, and 0.1 mL of fresh media is added per well. 0.05 mL of serially diluted compound or control vehicle (Media with DMSO) is added to the cells. The final DMSO concentration does not exceed 1%. After 1 h incubation, 0.05 mL of 400 ng/mL LPS (E. coli serotype 0111:B4, Sigma) in media is added for final concentration of 100 ng/mL. Cells are incubated at 37°C for 4 h. After 4 h incubation, supernatants are harvest and assayed by ELISA for the presence of TNFα. The error for this assay is taken to be 0.5 log units
11. Adult male 225-250 g Lewis rats (Harlan Sprague-Dawley) were used. Rats were fasted 18 h prior to oral dosing, and allowed free access to water throughout the experiment. Each treatment group consisted of five animals. Compounds were prepared as a suspension in a vehicle consisting of 0.5% methylcellulose, 0.025% Tween-20 in PBS. Compounds or vehicle were orally administered in a volume of 1 mL using an 18-gauge gavage needle. LPS (E. coli serotype 0111:B4, Lot #39H4103, Cat. # L-2630, Sigma) was administered 1-4 h later by injection into the penile vein at a dose of 1 mg/kg in 0.5 mL sterile saline. Blood was collected in serum separator tubes via cardiac puncture 1.5 h after LPS injection, a time point corresponding to maximal TNFα production. After clotting, serum was withdrawn and stored at -20°C until assay by ELISA