title: Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.
abstract: 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.
authors.affiliation: Division of Nephrology Hypertension and Endocrinology, Department of Medicine, School of Medicine, Nihon University, Tokyo, Japan.
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Wan
authors.forename: Noboru
authors.initials: N
authors.lastname: Fukuda
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ueno
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsuda
authors.forename: Kosuke
authors.initials: K
authors.lastname: Saito
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nagase
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsumoto
mesh_terms: D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000037:antagonists & inhibitors*; D004247:DNA / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D024202:Electrophoretic Mobility Shift Assay; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects*; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D050527:Mesangial Cells; D058990:Molecular Targeted Therapy; D018218:Neoplasms, Fibrous Tissue / Q000503:physiopathology / Q000628:therapy; D009757:Nylons / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010704:Phorbols / Q000032:analysis / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011758:Pyrroles / Q000737:chemistry / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: C526317:CCN2 protein, human; D006028:Glycosphingolipids; D007093:Imidazoles; D009757:Nylons; D009841:Oligonucleotides; C097562:PGL3a glycosphingolipid; D010704:Phorbols; D011758:Pyrroles; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D004247:DNA; D016650:Fluorescein-5-isothiocyanate