Pyridinyl Imidazole Inhibitors of p38 Mitogen-activated Protein Kinase Bind in the ATP Site

• Published in: The Journal of biological chemistry Vol. 272; no. 18; pp. 12116 - 12121
• Main Authors: Young, P R, McLaughlin, M M, Kumar, S, Kassis, S, Doyle, M L, McNulty, D, Gallagher, T F, Fisher, S, McDonnell, P C, Carr, S A, Huddleston, M J, Seibel, G, Porter, T G, Livi, G P, Adams, J L, Lee, J C
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02.05.1997
• Subjects: Adenosine - analogs & derivatives; Adenosine - pharmacology; Adenosine Triphosphate - metabolism; Affinity Labels; Amino Acid Sequence; Binding Sites; Binding, Competitive; Calcium-Calmodulin-Dependent Protein Kinases - antagonists & inhibitors; Calcium-Calmodulin-Dependent Protein Kinases - chemistry; Calcium-Calmodulin-Dependent Protein Kinases - isolation & purification; Calorimetry; Cloning, Molecular; Cross-Linking Reagents; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Escherichia coli; HeLa Cells; Humans; Imidazoles - metabolism; Imidazoles - pharmacology; Kinetics; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; p38 Mitogen-Activated Protein Kinases; Pyridines - metabolism; Pyridines - pharmacology; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.18.12116

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125 I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli -expressed p38 kinase at Thr 175 , which is proximal to the ATP binding site. Titration calorimetric studies with E. coli -expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5′-[ p -(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a K m of 9.6 m m . Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a K i of 21 n m and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125 I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.