Synthesis and PKC binding of conformationally restricted indolactams by aza-Claisen rearrangement

蛋白激酶C （PKC）是参与细胞信号转导和肿瘤促进的关键酶家族。它由一个蛋白质磷酸化的催化结构域和一个结合肿瘤启动子如磷酯和远杀细胞素的调节结构域组成。PKC中的肿瘤启动子结合位点是位于n端调控区域的富含半胱氨酸结构域（CRD）。随着11个PKC同工酶的发现，对同工酶功能的特异性分析日益受到重视。同工酶选择性激活剂或抑制剂是分析PKC作用的有力工具，成为治疗与PKC激活相关疾病（如高血糖血管并发症）的新药物线索。然而，由于纯PKC同工酶不易从天然来源中获得，因此此类化合物的开发受到阻碍。我们采用固相策略合成了约50个氨基酸组成的PKCgamma、delta和eta的CRD，建立了天然PKCs的模型。这些肽在锌处理后有效折叠，产生PKC调节结构域替代物，该替代物结合[^3H] phorbol 12,13-二丁酸酯（PDBu），具有与天然PKC本身相当的高亲和力，表明这些肽可作为天然PKC γ， delta和eta的有效模型。远杀菌素及其核心结构(-)-吲哚内酰胺- v是最有希望作为PKC同工酶选择性激活或抑制的先导化合物，因为它们的化学性质非常稳定且易于合成。利用PKC替代肽，基于aza-Claisen重排设计构象限制性吲哚内酰胺衍生物的策略，我们鉴定了两个新的具有高PKCgamma CRD选择性的吲哚内酰胺衍生物。在(-)-吲哚内酰胺- v的第5位和第13位之间形成桥接是将分子固定在活性构象和开发具有高同工酶选择性的新型PKC激活剂的最有效方法之一。

Protein kinase C (PKC) is a key enzyme family involved in cellular signal transduction and tumor promotion. It consists of a catalytic domain for protein phosphorylation and a regulatory domain which binds tumor promoters like phorbol esters and teleocidins. The tumor promoter-binding site in PKC is a cysteine-rich domain (CRD) at the N-terminal regulatory region. With the discovery of eleven PKC isozymes, increasing importance is placed on isozyme specific analysis of function. Isozyme-selective activators or inhibitors represent powerful tools to analyze the role of PKC and become new medicinal leads for diseases related to PKC activation like the vascular complications by hyperglycemia.However, the development of such compounds has been hampered since pure PKC isozymes are not easily obtainable from natural sources. We have synthesized the CRD's of PKCgamma, delta, and eta consisting of ca.50 amino acids by solid phase strategy to establish the models of native PKCs. These peptides were efficiently folded upon zinc treatment to produce PKC regulatory domain surrogates that bind [^3H] phorbol 12,13-dibutyrate (PDBu) with high affinity comparable to native PKC itself, suggesting that these peptides serve as effective models for native PKCgamma, delta, and eta.Teleocidins and its core structure (-) -indolactam-V are most promising as lead compounds for isozyme selective activation or inhibition of PKC since they are chemically very stable and easily synthesized. Using the PKC surrogate peptides, we have identified two new indolactam derivatives with high PKCgamma CRD selectivity based on our strategy for the design of conformationally restricted indolactam derivatives by aza-Claisen rearrangement. Bridge formation between position 5 and 13 of (-) -indolactam-V was proved to be one of the most effective methods to fix the molecule to the active conformation and to develop new PKC activators with high isozyme selectivity.