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note
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Virtual screening: proteasome X-ray structure (PDB ID 1IRU) was used for virtual screening due to the sequence identity within the chymotryptic subpocket of the bovine proteasome located on the L and M chain and the human proteasome X and HC5 chains. Protein-ligand docking was used for virtual screening with the binding pocket defined around the catalytically active threonine (THR1) containing the subpockets S1, S2, and S3. Virtual high-throughput screening was done using the docking engine of 4SCan/ProPose (Seifert, M. H.; Wolf, K.; Vitt, D. DDT Biosilico 2003, 1, 143.; Seifert, M. H.; Schmitt, F.; Herz, T.; Kramer, B. J. Mol. Model. 2004, 10, 342) on 4SC's database of five million chemical compounds. The 5000 best docking hits were submitted to filtering: a distance cutoff of 10 Å to THR1 was applied, molecular properties (molecular weight, topological polar surface area, number of rotational bonds) were filtered, unwanted functional groups were omitted, and finally a visual inspection of the docked structures yielded a ranking list. A representative set of 236 compounds was selected for purchasing and biochemical testing.
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12
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34548842876
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note
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50 values data were fitted to a four parameter logistic function using SigmaPlot.
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13
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34548858039
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note
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4/well and allowed to attach overnight. The cells were subsequently incubated for 5 h with described compounds at 100, 30, 10, 3, 1, 0.3, 0.1, and 0 μM and then stimulated with 10 ng/ml TNF-α for 22 h. The supernatant of the cell was analyzed for SEAP activity using a chemiluminescent SEAP reporter gene assay (Roche, Mannheim, Germany), and a cell viability assay was prepared using a CellTiter-BluTM Cell Viability Assay (Promega, Mannheim, Germany). For each concentration of the compound four replicates were measured.
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15
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34548844303
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All compounds were characterized by MS and NMR (300 MHz) and exhibited satisfactory properties.
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Ohnishi, H.; Kosuzume, H.; Mizota M.; Suzuki, Y.; Mochida, E. European patent EP 1984/0116421, 1984.
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