Pyridinyl Imidazole Inhibitors of p38 Mitogen-activated Protein Kinase Bind in the ATP Site
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 nonphosphory...
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Published in | The Journal of biological chemistry Vol. 272; no. 18; pp. 12116 - 12121 |
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Main Authors | , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
02.05.1997
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Subjects | |
Online Access | Get full text |
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Summary: | 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 (5â²-[ 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.272.18.12116 |