Division of Nephrology Hypertension and Endocrinology, Department of Medicine, School of Medicine, Nihon University, Tokyo, Japan.
Biol Pharm Bull. 2011;34(10):1572-7. doi: 10.1248/bpb.34.1572.
Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-β1 (TGF-β1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-β1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-β1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.
吡咯并咪唑（PI）聚酰胺可以与双螺旋 DNA 小沟中的特定序列结合，并抑制基因的转录。我们设计并合成了一种针对人结缔组织生长因子（hCTGF）启动子区域邻近 Smads 结合位点的 PI 聚酰胺。在产生 PI 聚酰胺的偶联活化剂中，1-[双（二甲氨基）亚甲基]-5-氯-1H-苯并三唑-1-氧化物 3-氧化物六氟磷酸盐（HCTU）在 PI 聚酰胺的总产率中最有效。凝胶迁移分析表明，专门针对 hCTGF 设计的 PI 聚酰胺（PI 聚酰胺至 hCTGF）结合了适当的双链寡核苷酸。一种异硫氰酸荧光素（FITC）缀合的 CTGF PI 聚酰胺穿透细胞膜并在培养的人肾小球系膜细胞（HMC）的核内积累，并在那里保留 48 小时。PI 聚酰胺至 hCTGF 显著降低了培养的 HMC 中佛波醇 12-肉豆蔻酸酯（PMA）或转化生长因子-β1（TGF-β1）刺激的 hCTGF 启动子的荧光素酶活性。PI 聚酰胺至 hCTGF 以剂量依赖性方式显著降低了 PMA 或 TGF-β1 刺激的 hCTGF mRNA 的表达。PI 聚酰胺至 hCTGF 显著降低了 HMC 中 PMA 或 TGF-β1 刺激的 hCTGF 蛋白水平。这些结果表明，开发的针对 hCTGF 的合成 PI 聚酰胺可能是一种用于纤维化疾病的新型基因沉默剂。
