Inferential NMR/X-ray-based structure determination of a dibenzo[a,d]cycloheptenone inhibitor-p38α MAP kinase complex in solution.
Inferential NMR/X-ray-based structure determination of a dibenzo[a,d]cycloheptenone inhibitor-p38α MAP kinase complex in solution.
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溶液中二苯并[a,d]环庚烯酮抑制剂-p38α MAP 激酶复合物的推理核磁共振/X 射线结构测定
DOI：
10.1002/anie.201105241
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发表时间：
2012
影响因子：
--
通讯作者：
M. Habeck
中科院分区：
文献类型：
--
作者：
V. S. Honndorf;N. Coudevylle;S. Laufer;S. Becker;C. Griesinger ;M. Habeck
p38丝裂原活化蛋白激酶（MAPK）的三磷酸腺苷（ATP）结合位点在其催化循环中发生了很大的构象变化。靶向活性位点的化合物，如吡啶基咪唑SB203580和4-苯基氨基二芳基酮[1,2]，已被证明与p38 MAPK结合具有高亲和力，但由于这些化合物具有高构象灵活性，特异性较低。因此，增加小分子抑制剂的刚性应该可以提高与p38 MAPK结合的特异性这导致了三环二苯并[a， d]环庚烯酮和二苯并[b， e]氧平酮抑制剂[3]的发展，它们含有稳定分子几何结构的凝聚环系统。p38α MAPK与三环抑制剂2-（2-氨基苯胺）- 10,11 -二氢二苯并[a， d]环庚烯-5- 1[3]配合物的晶体结构已由Koeberle等人以1.85分辨率测定（1，在支持信息中见图S1）。由于晶体结构，特别是激酶配合物的晶体结构，可能不能反映溶液中激酶的构象，因此我们使用核磁共振波谱法研究了1与p38α在室温下的结合模式。我们测量了与1配合物的氘化p38α的TROSY光谱（p38α - 1, IC50= 104 nm），并分配了62%的检测共振。游离p38α的TROSY-HSQC谱约有75%的预期信号，证实了之前的研究。[5,6]比较游离p38α和p38α - 1的光谱，发现抑制剂结合位点存在化学位移扰动。化合物1位于疏水性后袋中，与铰链区形成氢键在结合1时，由于谱线展宽，参与这些接触的大多数氨基酸的共振消失，这表明有几种构象处于中间交换；富含甘氨酸的环部分也以同样的方式受到影响。图1a显示了游离p38α和p38α - 1的TROSY-HN （CO）光谱的叠加图，它们依次被标记为13C和15N-Leu/15N-Met（另见辅助信息中的图S2）。加入1后，不再检测到Leu108-Met109和Leu86-Leu87之间的交叉峰。13C和15N-His/ 15n - leu标记的TROSY-HSQC光谱给出了类似的结果（图1B）。结合1后，来自连接N叶和C叶的铰链区域（His107, Leu108）的信号以及来自疏水口袋（Leu86, Leu87）的信号消失，而远离结合口袋的氨基酸（C叶中的Leu164和His142， N叶中的His80）则发生了化学位移。从信号的消失我们得出结论，p38α-1中存在中间交换时间尺度（μs-ms）上的运动，并且与p38α-SB203580配合物中的运动相似这一点特别值得注意，因为SB203580和1在结构上是不相关的。因此，对于柔性SB203580和刚性1，复合物在结合位点上比自由激酶更具动态性。为了表征激酶抑制剂复合物的溶液结构，我们测量了1H-15N残余偶极偶联（rdc），这是主链取向的敏感探针。58个rdc范围从À40到+ 50 Hz，可接受误差（< 5 Hz）被考虑用于进一步分析。我们比较了游离p38α和p38α - 1的rdc，而不参考结构（支持信息中的图S3），发现许多偶联非常相似。这表明p38α-1的溶液结构在很大程度上类似于自由形态（图S3）。
The adenosine triphosphate (ATP) binding site of the p38 mitogen-activated protein kinase (MAPK) undergoes a large conformational change during its catalytic cycle. Compounds that target the active site, such as the pyridinyl-imidazole SB203580 and 4-phenylaminodiarylketones,[1, 2] have been shown to bind to p38 MAPK with a high affinity but a low specificity as a result of the high conformational flexibility of these compounds. Increasing the rigidity of small-molecule inhibitors should, therefore, improve the specificity of binding to p38 MAPK.[3] This led to the development of tricyclic dibenzo [a, d] cycloheptenone and dibenzo [b, e] oxepinone inhibitors,[3] which contain condensed ring systems that stabilize the molecular geometry. The crystal structure of p38α MAPK in complex with the tricyclic inhibitor 2-(2-aminophenylamino)-10, 11-dihydrodibenzo [a, d] cyclohepten-5-one [3](1, Figure S1 in the Supporting Information) has been determined by Koeberle et al.[4] at 1.85 resolution. Because crystal structures, especially of kinase complexes, may not reflect the conformation of the kinase in solution,[5] we used NMR spectroscopy to study the binding mode of 1 to p38α in solution at ambient temperature. We measured TROSY spectra of deuterated p38α in complex with 1 (p38α–1, IC50= 104 nm) and assigned 62% of the detected resonances. The TROSY-HSQC spectrum of free p38α has about 75% of the expected signals, which confirms previous studies.[5, 6] Comparison of the spectra of free p38α and p38α–1 reveals chemical shift perturbations at the binding site of the inhibitor. Compound 1 is located in the hydrophobic back pocket and forms hydrogen bonds to the hinge region.[4] Upon binding of 1, the resonances of most of the amino acids that are involved in these contacts disappear as a result of line broadening, which indicates that several conformations are in intermediate exchange; the glycine-rich loop is partly affected in the same way. Figure1A shows an overlay of the TROSY-HN (CO) spectra of free p38α and p38α–1, which are sequentially labeled with 13C and 15N-Leu/15N-Met (see also Figure S2 in the Supporting Information). After the addition of 1, the cross-peaks between Leu108–Met109 and Leu86–Leu87 are no longer detected. TROSY-HSQC spectra with 13C and 15N-His/15N-Leu-labeling give similar results (Figure 1B). Upon binding of 1, the signals from the hinge region that connects the N lobe and the C lobe (His107, Leu108) as well as signals from the hydrophobic pocket (Leu86, Leu87) disappear, whereas amino acids that are distant from the binding pocket (Leu164 and His142 in the C lobe and His80 in the N lobe) have perturbed chemical shifts. From the disappearance of the signals we conclude that the motion on the intermediateexchange timescale (μs–ms) is present in p38α–1 and is similar to that in the p38α–SB203580 complex.[5] This is particularly remarkable because SB203580 and 1 are structurally unrelated. Thus, the complex is more dynamic in the binding site than the free kinase for both flexible SB203580 and rigid 1. To characterize the solution structure of the kinase–inhibitor complex, we measured 1H–15N residual dipolar couplings (RDCs), which are sensitive probes of the orientation of the backbone. 58 RDCs that ranged from À40 to+ 50 Hz with acceptable errors (< 5 Hz) were considered for further analysis. We compared the RDCs of free p38α and p38α–1 without reference to a structure (Figure S3 in the Supporting Information) and found that many of the couplings are very similar. This is an indication that the solution structure of p38α–1 resembles the free form in large parts (Figure S3 in the …
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