Discovery and Characterization of a Substrate Selective p38 Inhibitor

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• 作者：Walter Davidson ; Lee Frego ; Gregory W. Peet ; Rachel R. Kroe ; Mark E. Labadia ; Susan M. Lukas ; Roger J. Snow ; Scott Jakes ; Christine A. Grygon ; Christopher Pargellis ; and Brian G. Werneburg
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：September 21, 2004
• 年：2004
• 卷：43
• 期：37
• 页码：11658 - 11671
• 全文大小：685K
• 年卷期：v.43,no.37(September 21, 2004)
• ISSN：1520-4995

文摘

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitoris substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 wasobserved to prevent the p38ages/gifchars/alpha.gif" BORDER=0>-dependent phosphorylation (K_i^app = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38ages/gifchars/alpha.gif" BORDER=0> andp38ages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">, but it did not prevent the phosphorylation of ATF-2 (K_i^app > 20 ages/entities/mgr.gif">M). In addition to kinetic studies,isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidatethe mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 bindsto p38ages/gifchars/alpha.gif" BORDER=0>, CMPD1 was not observed to compete with ATP for p38ages/gifchars/alpha.gif" BORDER=0>, nor was it able to interrupt thebinding of p38ages/gifchars/alpha.gif" BORDER=0> to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange massspectrometry (DXMS) was employed to study the p38ages/gifchars/alpha.gif" BORDER=0>·CMPD1 inhibitory complex, to provide newinsight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38ages/gifchars/alpha.gif" BORDER=0>·CMPD1 complex were compared to the data obtained for the p38ages/gifchars/alpha.gif" BORDER=0>·MK2a complex and a p38ages/gifchars/alpha.gif" BORDER=0>·activesite binding inhibitor complex. Alterations in the DXMS behavior of both p38ages/gifchars/alpha.gif" BORDER=0> and MK2a were observedupon complex formation, including but not limited to the interaction between the carboxy-terminal dockingdomain of MK2a and its binding groove on p38ages/gifchars/alpha.gif" BORDER=0>. Alterations in the D₂O exchange of p38ages/gifchars/alpha.gif" BORDER=0> produced byCMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38ages/gifchars/alpha.gif" BORDER=0>,resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues(E160 and D161), and a Mg²⁺ ion cofactor binding residue (D168). Although the exact mechanism ofsubstrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest thatCMPD1 binding in the active site region of p38ages/gifchars/alpha.gif" BORDER=0> induces perturbations that may result in the suboptimalpositioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38ages/gifchars/alpha.gif" BORDER=0>activity.