Trials for development of novel inhibitors for intracellular signaling.

PLC- γ在其SH2和SH3基序附近具有一个磷脂酶C抑制剂（PCI）区域，该区域在体外强烈抑制纯化PLC同工异构体的PIP_2水解活性。与PCI区氨基酸序列相同的合成肽（PCI肽）可抑制各PLC亚型诱导的PIP_2水解。已经证明，各种合成肽的酰化增强了它们对质膜的渗透性，使肽在完整细胞内成为潜在的特异性效应物或抑制剂。基于这些信息，我们合成了各种酰基化PCI肽，并使用正常成纤维细胞和癌细胞检测了它们对细胞内PLC活性的影响。在本研究中，我们得到了以下结果：1)我们合成了50种酰基化PCI肽，其中35种酰基化肽用于本研究。2)乙酰化并未改变PCI肽的特定二级结构，这一结构在所有PCI肽中都是保守的，可能是PLC抑制所必需的，并强烈增强了它们的PLC抑制效力。这些肽的酰化显著增强了它们对质膜的渗透性，用酰化的肽对细胞进行预孵育可抑制细胞内PLC的激活。不同类型的酰化对多肽的抑制作用不同。4) n -肉豆芽酰基化PCI肽是最有效的PCI肽之一，可抑制3Y1细胞（ED_<50>=1muM）中配体依赖性IP_3的形成和某些癌细胞的生长。5)本研究表明，结合多种N端和c端修饰可以合成更有效的PCI肽，但很难合成与PCI肽结构相关的化合物。

PLC-gamma possesses a phospholipase C inhibitor (PCI) region adjacent to its SH2 and SH3 motifs that strongly suppresses the PIP_2 hydrolyzing activity of purified PLC isoforms in vitro. Synthetic peptides identical to the amino acid sequence of the PCI region (PCI peptides) inhibit PIP_2 hydrolysis induced by each PLC isoforms. It has been demonstrated that the acylation of various synthetic peptides enhances their permeability to the plasma membrane, rendering the peptides potentially specific effectors or inhibitors inside intact cells. Based on this information, we synthesized various kinds of acylated PCI peptides, and examined their effects on intracellular PLC activity using normal fibroblasts and cancer cells. In this study, we obtained following results. 1) We synthe sized 50 kinds of acylated PCI peptides, among which 35 acylated peptides were used in this study. 2) Acylation of the PCI peptides did not alter their specific secondary structure which is conserved among all PCI peptides and probably required for PLC inhibition, and strongly enhanced their PLC inhibitory potency. Acylation of the peptides significantly enhanced their permeability to the plasma membrane, and preincubation of cells with acylated peptides resulted in inhibition of intracellular PLC activation. The inhibitory effects of the peptides varied among types of acylation.4) N-myristoylated PCI peptides, one of the most potent PCI peptides, suppressed the ligand-dependent IP_3 formation in 3Y1 cells (ED_<50>=1muM) and growth of some carcinoma cells. 5) This study indicated a possibility to produce more potent PCI peptides by combination of various N- and C-terminal modifications and a difficulty to produce synthetic compounds structurally related to PCI peptides.