Molecular basis and generation of new compounds for probing phosphorylation-mediated signaling pathways

有证据表明，蛋白激酶参与了多种细胞过程，如去极化偶联的平滑肌收缩，促分泌剂刺激的分泌细胞释放生物活性物质，以及有丝分裂原激活的细胞生长。蛋白激酶抑制剂在研究蛋白激酶的生理学意义上似乎是有用的，而且部分蛋白激酶可能用作药物。这一假设已经被我们的H系列蛋白激酶抑制剂H-89、H-7、KN-62和HA-1077所证实。本研究的目的是阐明它们的作用，并为蛋白激酶抑制剂的分子设计提供理论依据。所取得的结果总结如下1.测定了蛋白激酶A与H-7，8，89的络合物的晶体结构(Bossmeyer et.艾尔JBC，1996)。这表明，蛋白质的腺嘌呤口袋很好地容纳了H系列化合物中的异奎诺激动素磺酰胺。似乎很有可能t…学位添加到异奎诺激动磺酰胺上的化学结构对化合物结合的贡献更多地决定了它们对蛋白激酶的选择性。HA-1077对包括肌球蛋白轻链激酶在内的多种蛋白激酶具有非特异性抑制作用，用于治疗蛛网膜下腔出血后脑血管痉挛。通过在HA-1077上添加一些化学基团，例如它的甲基化，这些新化合物被发现显著地改变为某些蛋白激酶的特异性抑制剂。结合上述发现，有可能通过改变连接在异喹啉磺酰胺核心上的化学基团来修饰H系列化合物。根据构效关系，我们发现异喹啉磺酰胺对KN-62细胞的钙/钙调蛋白依赖性蛋白激酶抑制也是必不可少的，虽然该化合物与钙调蛋白竞争，但不与ATP竞争。我们认为，H系列化合物是新的蛋白激酶抑制剂的种子化合物，当获得更多关于蛋白激酶结构的信息时，它们将结出更多的果实。较少

Evidence accum ulated that protein kinases are involved in a variety of cellular processes such as depolarization -coupled smooth muscle contraction, secretagogues-stimulated release of biologically active substances from secretory cells, and mitogen-activated cell growth. Protein kinase inhibitors seem to be usful in studying physiological significance of protein kinases and part of tham probably works as medicines. This hypothesis has been, if any, proved with our H-series protein kinase inhibitors, H-89, H-7, KN-62, and HA-1077. Our goal of this research is to elucidate their actions and to construct some theory for molecular designing of protein kinase inhibitors. The results obtained are summarized as described below.1. The crystal structure of protein kinase A complex with H-7,8,89 was deterimined (Bossmeyer et. al. JBC,1996). This revealed that the adenine pocket of the protein accommodates well the isoquinokinesulfonamide of H-series compounds. It seems likely that the degree t … More o which the chemical structure added to the isoquinokinesulfonamide contributes to binding of the compounds determines their selectivity for protein kinases.2. HA-1077 shows a non-specific inhibition for a variety of protein kinases including myosin light chain kinase and is used as medicine for the treatment of cerebral vasospasm after subarachnoidal hemorrhage. By addition of some chemical groups to HA-1077, for example its methylation, such new compounds were found to change remarkably into specific inhibitors of some protein kinase. Combined with the findings described above, it is possible to amend H-series compounds by altering their chemical groups attached to the isoquinolinesulfonamide core.3. Based on structure-activity relationship, it was found that an isoquinolinesulfonamide was also indispensable for KN-62 to show calcium/calmodulin-dependent protein kinase inhibition, although the compound competed with calmodulin, but not with ATP.We consider that H-series compounds are seed compounds for new protein kinase inhibitors, and that they will bear more fruits when more information conceruing the structures of protein kinases are available. Less