Prevention of MKK6-dependent activation by binding to p38α MAP kinase
Prevention of MKK6-dependent activation by binding to p38α MAP kinase
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DOI：
10.1021/bi051714v
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发表时间：
2005-12-20
期刊：
影响因子：
2.9
通讯作者：
Ward, WHJ
中科院分区：
文献类型：
--
作者：
Sullivan, JE;Holdgate, GA;Ward, WHJ
抑制p38 α MAP激酶是治疗炎性疾病的潜在方法。p38 α激活环上的MKK 6依赖性磷酸化增加了其催化活性和对ATP的亲和力。抑制剂BIRB 796在ATP的嘌呤部分所使用的位点处结合，并延伸到ATP不使用的“选择性口袋”中。它取代了激活环起始处的Asp 168-Phe 169-Gly 170基序，促进了“DFG-out”构象。一些其他抑制剂仅结合在嘌呤位点，p38 α保持“DFG-in”构象。我们现在证明，选择性口袋化合物除了抑制活化的p38 α的催化作用外，还能防止MKK 6依赖的p38 α活化。仅使用嘌呤位点的抑制剂不能阻止MKK 6依赖性活化。我们提出了动力学分析的七种抑制剂，其晶体结构与p38 α的复合物已被确定。这项工作包括四种新的晶体结构和一种新的测定方法来测量非活化p38 α的Kd。选择性口袋化合物与p38 α的结合比嘌呤位点化合物慢30倍以上，这显然是由于DFG-外构象的低丰度。在抑制炎性细胞因子TNF α的细胞产生的浓度下，选择性口袋化合物降低磷酸化p38 α和β的水平。DFG-out构象的稳定似乎干扰MKK 6对作为底物的p38 α的识别。ATP在阻止活化方面的竞争不如在抑制催化方面的竞争有效。通过与酶的不同构象结合，防止活化的化合物提供了调节p38 α的替代方法。
Inhibition of p38 alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38 alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38 alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38 alpha in addition to inhibiting catalysis by activated p38 alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38 alpha have been determined. This work includes four new crystal structures and a novel assay to measure K-d for nonactivated p38 alpha. Selectivity pocket compounds associate with p38 alpha over 30-fold more slowly than purine site Compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNF alpha, selectivity pocket compounds decrease levels of phosphorylated p38 alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38 alpha as a Substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, Compounds that prevent activation offer an alternative approach to modulation of p38 alpha.