Discovery and characterization of a substrate selective p38α inhibitor
Discovery and characterization of a substrate selective p38α inhibitor
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DOI：
10.1021/bi0495073
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发表时间：
2004-09-21
期刊：
影响因子：
2.9
通讯作者：
Werneburg, BG
中科院分区：
文献类型：
--
作者：
Davidson, W;Frego, L;Werneburg, BG
本文描述了通过高通量筛选鉴定的p38促分裂原活化蛋白激酶（p38）CMPD 1的新抑制剂。与先前描述的p38抑制剂不同，该抑制剂具有底物选择性，并且与ATP无竞争性。在稳态动力学实验中，观察到CMPD 1阻止含有p38 α和p38 β对接结构域的促分裂原活化蛋白激酶活化蛋白激酶2（MK 2a）的剪接变体的p38 α依赖性磷酸化（Ki（app）= 330 nM），但它不阻止ATF-2的磷酸化（Ki（app）> 20 μ M）。除了动力学研究，等温滴定量热法和表面等离子体共振实验进行，以阐明抑制机制。虽然等温滴定量热分析表明CMPD 1结合p38 α，但未观察到CMPD 1与ATP竞争p38 α，也不能通过表面等离子体共振观察到CMPD 1中断p38 α与MK 2a的结合。因此，采用氘交换质谱法（DXMS）来研究p38 α·CMPD 1抑制复合物，以提供对底物选择性抑制机制的新见解。将从p38 α CMPD 1复合物获得的DXMS数据与从p38 α MK 2a复合物和p38 α活性位点结合抑制剂复合物获得的数据进行比较。在复合物形成后观察到p38 α和MK 2a的DXMS行为的改变，包括但不限于MK 2a的羧基末端对接结构域与其在p38 α上的结合沟之间的相互作用。由CMPD 1产生的p38 α的D2 O交换的改变表明，底物选择性抑制剂在p38 α的活性位点附近结合，导致对含有核苷酸结合口袋残基、对接沟残基（E160和D161）和Mg 2+离子辅因子结合残基（D168）的区域的扰动。虽然这种新型抑制剂的底物选择性抑制的确切机制尚未公开，但结果表明，在p38 α的活性位点区域中的CMPD 1结合诱导扰动，这可能导致底物和辅因子在过渡态中的次优定位，从而导致p38 α活性的选择性抑制。
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 inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-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 p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K-i(app) > 20 muM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D2O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, 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 of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.