title: Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.
abstract: 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, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, 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 Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-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.
authors.affiliation: Department of Comparative Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939, USA. Peter_R_Young@sbphrd.com
authors.forename: P R
authors.initials: PR
authors.lastname: Young
authors.forename: M M
authors.initials: MM
authors.lastname: McLaughlin
authors.forename: S
authors.initials: S
authors.lastname: Kumar
authors.forename: S
authors.initials: S
authors.lastname: Kassis
authors.forename: M L
authors.initials: ML
authors.lastname: Doyle
authors.forename: D
authors.initials: D
authors.lastname: McNulty
authors.forename: T F
authors.initials: TF
authors.lastname: Gallagher
authors.forename: S
authors.initials: S
authors.lastname: Fisher
authors.forename: P C
authors.initials: PC
authors.lastname: McDonnell
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: M J
authors.initials: MJ
authors.lastname: Huddleston
authors.forename: G
authors.initials: G
authors.lastname: Seibel
authors.forename: T G
authors.initials: TG
authors.lastname: Porter
authors.forename: G P
authors.initials: GP
authors.lastname: Livi
authors.forename: J L
authors.initials: JL
authors.lastname: Adams
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D002151:Calorimetry; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases; D016297:Mutagenesis, Site-Directed; D011725:Pyridines / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; D011994:Recombinant Proteins; C090943:SB 206718; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000241:Adenosine; C093642:SB 203580; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole