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 mitogenactivated 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 mM. 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 nM 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.

Initiation of Oligodendrocyte Progenitor Cell Migration by a PDGF-A Activated Extracellular Regulated Kinase (ERK) Signaling Pathway

“…There are, however, numerous different MAPK isoforms, including ERK5, JNK2 and p38 MAPK . As stated above, the presence of SB203580, which inhibits the activity of p38 MAPK [30] did not inhibit PDGF-A induced OP migration in this study. This data indicates that p38 MAPK does not play a significant role in regulating PDGF-A induced migration of OP.…”

“…SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) is an inhibitor of both JNK and p38 MAPK [30,31]. Thirty minutes pretreatment of cells with SB203580 prior to the addition of PDGF-A had no significant effect on OP migration (Fig.…”

Activation of p38 MAP kinase and ERK are required for ultraviolet-B induced c-fos gene expression in human keratinocytes

“…The p38 inhibitor SB202190 does not a ect phosphorylation of p38 (data not shown) since the inhibitor only binds to the ATP pocket of p38 and blocks its intrinsic ATPase activity without a ecting its phosphorylation sites (Lee et al, 1994;Cuenda et al, 1995). In addition, the inhibition of p38 by SB202190 was reversible, therefore, the inhibitory e ect could not be detected by in vitro kinase assay of p38 (data not shown, Young et al, 1997;Wilson et al, 1997). Thus, we performed an in vivo kinase assay to determine p38 activity.…”

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

“…SB202190 specifically inhibits the activity of p38 MAPK by competing for the ATP-binding sites of the a and b isoforms. 32 This inhibitor has been used very extensively in the literature and has previously been shown to block p38 MAPK -mediated phosphorylation of ATF-2 in our laboratories. 33 Although both inhibitors dose-dependently increased TRAIL-induced death (Figure 6a), inhibition of NF-kB resulted in the highest degree of apoptosis in NHU cells, whereas in 253J cells, resistance was almost exclusively dependent on the p38 MAPK pathway (Figure 6a).…”