Synthetic Pyrrole-Imidazole Polyamide Inhibits Expression of the Human Transforming Growth Factor-β1 Gene

Lai, Yuqin; Fukuda, Noboru; Ueno, Takayoshi; Matsuda, Hiroyuki; Saito, Satoshi; Matsumoto, Koichí; Ayame, Hirohito; Bando, Toshikazu; Sugiyama, Hiroshi; Mugishima, Hideo; Serie, Kazuo

doi:10.1124/jpet.105.089086

Cited by 57 publications

(47 citation statements)

References 31 publications

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“…The combination of Im/ Py recognizes GC in the DNA double-helix, Py/Py recognizes TA and AT and arrangements of these combinations made it possible to bind to a variety of sequence. Since they can bind DNA with higher affinity and specificity than the usual DNA binding proteins, PIPs designed to recognize the binding Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma (15)(16)(17)(18). Previously, we reported that PIP targeting for MMP-9 inhibit the migration and invasion of colon cancer cells in vitro and also inhibits their metastasis in vivo (19).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma

Sato¹,

Nagase²,

Obinata³

et al. 2013

International Journal of Oncology

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Abstract.We investigated the clinical significance of the expression levels of matrix metalloproteinase 9 (MMP-9) in renal cell carcinoma (RCC). In addition, we validated the efficacy of pyrrole imidazole polyamide (PIP) targeting MMP-9 on inhibiting proliferation and invasion of RCC. We evaluated the expression levels of MMP-9 in 249 RCC specimens by immunostaining and analyzed the association between MMP-9 expression levels and cancer-specific survival. Furthermore, in a human RCC cell line, Caki-2, we tested the effect of a couple of PIPs targeting MMP-9 one recognizing an NF-κB binding site (MMP-9-NF-κB PIP) and another for the AP-1 binding site (MMP-9-AP-1 PIP) in the MMP-9 promoter. The expression levels of MMP-9, proliferative activity and invasive capability were tested by quantitative PCR, WST8 assay and Matrigel invasion assay, respectively. By immunostaining of the clinical specimens, strong MMP-9 staining was proven to be a significant predictor of poor prognosis for cancer-specific survival (P<0.01). In Caki-2 cells, MMP-9-NF-κB PIP significantly reduced the expression levels of MMP-9 mRNA and inhibited cell invasion, but did not affect the cell proliferation activity. On the other hand, no effect was found in MMP-9-AP-1 PIP on MMP-9 mRNA expression, cell proliferation and invasion. We confirmed the inhibitory effects of MMP-9-NF-κB PIP on the expression of MMP-9 and subsequent invasion of Caki-2 cells. Since it was clearly shown that high MMP-9 expression levels were associated with poor prognosis of RCC, MMP-9 is a potential candidate target for RCC treatment. Transcription therapy using a minor groove binder, such as NF-κB PIP, may be a potential therapeutic agent for RCC, although further investigation is required.

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Section: Introductionmentioning

confidence: 99%

Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma

Sato¹,

Nagase²,

Obinata³

et al. 2013

International Journal of Oncology

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“…Binding site specificity is dependent on the side-byside pairing of pyrrole (Py) and imidazole (Im): the Py/Im pair targets the CG base pair, Py/Im recognizes the GC base pair, and Py/Py binds both AT and TA base pairs. [7][8][9] We demonstrated that PI polyamides targeting transforming growth factor-β1 (TGF-β1) potentially and transcriptionally improved progressive renal diseases, [10][11][12] arterial stenosis and hypertrophic scars.13) We also developed PI polyamides targeting lectin-like oxidative low-density lipoprotein (LDL) receptor-1 for atherosclerotic diseases.14) These PI polyamides were designed to bind to the transcription factor binding region in the promoter region of dsDNA and powerfully suppress the transcription of the target genes. It remains to be elucidated whether the pairing rules of PI polyamide for the DNA double helix are also available to bind to double-helical RNA.…”

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RNA Binding Properties of Novel Gene Silencing Pyrrole-Imidazole Polyamides

Iguchi

Fukuda

Takahashi

et al. 2013

Biological & Pharmaceutical Bulletin

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Pyrrole-imidazole (PI) polyamides are a novel group of gene-silencing compounds, which bind to a minor groove of double stranded (ds)DNA in a sequence-specific manner. To explore the RNA binding properties of PI polyamides targeting rat transforming growth factor-β1 (TGF-β1 Polyamide) and influenza A virus (PA polyamide), we designed dsRNAs with an identical sequence to the target DNA and analyzed RNA binding properties of the polyamide. Biacore assay showed fast binding of TGF-β1 Polyamide to the dsRNA, whereas mismatch polyamide did not bind to the dsRNA. Dissociation equilibrium constant (KD) value was 6.7×10 −7 of the target dsRNA. These results indicate that PI polyamide could bind to RNA with a 2 log lower binding affinity than its DNA-binding affinity. We designed a PI polyamide targeting the panhandle stem region of influenza A virus. KD value of the PI polyamide to dsRNA targeting influenza A virus was 4.6×10 −7. Gel-shift assay showed that TGF-β1 and PA polyamides bound to the appropriate dsDNA, whereas these PI polyamides did not show obvious gel-shift with the appropriate dsRNA. Structural modeling suggests that PI polyamide binds to the appropriate B-form dsDNA in the minor groove, whereas it does not fit in the minor groove to dsRNA. Thus PI polyamides have a lower binding affinity with target dsRNA than they do with dsDNA. The distinct binding properties of PI polyamides to dsRNA and dsDNA may be associated with differences of secondary structure and chemical binding properties between target RNA and DNA.Key words pyrrole-imidazole polyamide; double-stranded RNA; affinity; Biacore; gel-shift assay; influenza virus Engineered inactivation of gene function is important for elucidating the function of particular genes and may be used in gene therapy, treatment of viral infection, cancer and other diseases caused by aberrant gene expression. Gene function can be inactivated at the DNA level by nucleic acid medicines such as anti-gene 1) or antisense peptide nucleic acid, or at the RNA level by antisense oligodeoxynucleotides, 2) ribozymes, 3) small interfering RNA (siRNA), 4) and aptamers. 5)Since these nucleic acid agents are easily degraded by nucleases, suitable chemical modifications or drug-delivery systems, including vectors, are required for their therapeutic applications. Transcriptional regulation is essential for gene expression. Initiation of transcription requires binding of transcription factors to the cognate DNA response elements in the gene promoter. Thus, we developed pyrrole-imidazole (PI) polyamides as a novel gene-suppressing agent. PI polyamides were first identified from duocarmycin A and distamycin A and are small synthetic molecules that are composed of the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids. 6) Synthetic PI polyamides can bind to specific nucleotide sequences in the minor groove of double-stranded (ds) DNA with high affinity and specificity. PI polyamides are completely resistant to nucleases and could be delivered into tissues without any drug...

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“…We developed PI polyamides targeted to TGF-β1 as practical medicines that would transcriptionally inhibit the TGF-β1 gene (21,22). In the current study, to develop a PI polyamide targeted to the TGF-β1 promoter as a new therapeutic medicine for EPS, we examined the effects of our PI polyamide on expression by mesothelial cells of ECM mRNAs in vitro, and we evaluated EPS by histology and high-resolution regional elasticity mapping in rats in vivo.…”

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Pyrrole-Imidazole Polyamide Targeting Transforming Growth Factor β1 Ameliorates Encapsulating Peritoneal Sclerosis

et al. 2012

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♦ Objective: Encapsulating peritoneal sclerosis (EPS) is a devastating fibrotic complication in patients treated with peritoneal dialysis (PD). Transforming growth factor β1 (TGF-β1) is a pivotal factor in the induction of EPS. ♦ Methods: To develop pyrrole-imidazole (PI) polyamide, a novel gene silencer, targeted to the TGF-β1 promoter (Polyamide) for EPS, we examined the effects of Polyamide on messenger RNA (mRNA) expression of TGF-β1, vascular endothelial growth factor (VEGF), and extracellular matrix (ECM) in mesothelial cells in vitro, and on the thickness of injured peritoneum evaluated by histology and highresolution regional elasticity mapping in rats in vivo. ♦ Results: Polyamide significantly lowered mRNA expression of TGF-β1 and ECM in vitro. Polyamide labeled with fluorescein isothiocyanate was taken up into the injured peritoneum and was strongly localized in the nuclei of most cells. Polyamide 1 mg was injected intraperitoneally 1 or 3 times in rats receiving a daily intraperitoneal injection of chlorhexidine gluconate and ethanol (CHX) for 14 days. Polyamide significantly suppressed peritoneal thickening and the abundance of TGF-β1 and fibronectin mRNA, but did not affect expression of VEGF mRNA in the injured peritoneum. Elasticity distribution mapping showed that average elasticity was significantly lower in Polyamide-treated rats than in rats treated solely with CHX. ♦ Conclusions: Polyamide suppressed the stiffness, ECM formation, and thickening of the injured peritoneum that occurs during EPS pathogenesis. These data suggest that PI polyamide targeted to the TGF-β1 promoter will be a specific and feasible therapeutic strategy for patients with EPS.

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Synthetic Pyrrole-Imidazole Polyamide Inhibits Expression of the Human Transforming Growth Factor-β1 Gene

Cited by 57 publications

References 31 publications

Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma

Inhibition of MMP-9 using a pyrrole-imidazole polyamide reduces cell invasion in renal cell carcinoma

RNA Binding Properties of Novel Gene Silencing Pyrrole-Imidazole Polyamides

Pyrrole-Imidazole Polyamide Targeting Transforming Growth Factor β1 Ameliorates Encapsulating Peritoneal Sclerosis

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