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
Department of Comparative Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939, USA.
J Biol Chem. 1997 May 2;272(18):12116-21. 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, 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.
已确定一系列吡啶基咪唑化合物的作用位点，这些化合物在体外是p38丝裂原活化蛋白激酶的选择性抑制剂，在体内可阻断促炎细胞因子的产生。通过埃德曼测序表明，125I-SB206718可在Thr175处与非磷酸化的大肠杆菌表达的p38激酶发生交联，该位点靠近ATP结合位点。对大肠杆菌表达的p38激酶进行滴定热分析研究表明，SB203580以1:1的化学计量比结合，并且p38激酶与ATP类似物FSBA（5'-[对-(氟磺酰基)苯甲酰基]腺苷）预孵育可阻断结合，FSBA可共价修饰ATP结合位点。SB203580抑制非磷酸化酶的内在ATP酶活性，其Km为9.6 mM。使用肽底物对活性、磷酸化的酵母表达的p38激酶进行动力学研究表明，SB203580与ATP竞争，Ki为21 nM，并且激酶抑制与结合及生物学活性相关。诱变表明，125I-SB206718的结合依赖于ATP口袋中的催化残基K53和D168。这些发现表明，吡啶基咪唑在体内通过与ATP竞争来抑制p38激酶活性发挥作用，并且它们的选择性可能由ATP结合口袋内或附近非保守区域的差异决定。
