Synthetic Pyrrole-Imidazole Polyamide Inhibits Expression of the Human Transforming Growth Factor- 1 Gene

Pyrrole-imidazole (Py-Im) polyamides can bind to the predetermined base pairs in the minor groove of double-helical DNA with high affinity. These synthetic small molecules can interfere with transcription factor-DNA interaction and inhibit or activate the transcription of corresponding genes. In the present study, we designed and synthesized a Py-Im polyamide to target 545 to 539 base pairs of human transforming growth factor1 (hTGF1) promoter adjacent to the fat-specific element 2 (FSE2) to inhibit the expression of the gene. Gel mobility shift assay showed that the synthetic Py-Im polyamide binds to its corresponding double-strand oligonucleotides, whereas the mismatch polyamides did not bind. Fluorescein isothiocyanatelabeled Py-Im polyamide was detected in the nuclei of human vascular smooth muscle cells (VSMCs) after 2to 48-h incubation. Py-Im polyamide significantly decreased the promoter activity of hTGF1 determined by in vitro transcription experiments and luciferase assay. In cultured human VSMCs, Py-Im polyamide targeting hTGF1 promoter significantly inhibited expressions of hTGF1 mRNA and protein. These results indicate that the synthetic Py-Im polyamide designed to bind hTGF1 promoter inhibited hTGF1 gene and protein expression successfully. This novel agent will be used for the TGFrelated diseases as a gene therapy. Pyrrole (Py)-imidazole (Im) polyamides are small synthetic molecules composed of the aromatic rings of the N-methylpyrrole and N-methylimidazole amino acid (Trauger et al., 1996; White et al., 1997; Dervan, 2001). Synthetic polyamides can bind to specific nucleotide sequences in the minor groove of double-helical DNA with high affinity and specificity, suggesting that Py-Im polyamides could be useful tools for molecular biology and, potentially, medicine. Binding site specificity is dependent on the side-by-side pairing of Py and Im: the Py/Im pair targets the CG base pair, Im/Py recognizes the GC base pair, and Py/Py binds both AT and TA base pairs (Trauger et al., 1996; White et al., 1997; Dervan, 2001). Recent studies have shown that the AT degeneracy can be overcome by replacing one pyrrole ring of the Py/Py pair with 3-hydroxypyrrole (Hp); Hp/Py preferentially binds TA pairs (White et al., 1998). 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. Py-Im polyamides bind the minor groove and block binding of transcription factors inhibiting gene expression. Gottesfeld et al. (1997) reported inhibition of the transcription of 5S RNA gene by an eight-ring Py-Im polyamide designed to bind the recognition site of zinc-finger protein TFIIIA. To block activity of the human immunodeficiency virus type 1, two polyamides were designed to bind two transcription factor binding sites, and this inhibited virus replication by 99% (Dickinson et al., 1998). Thus, Py-Im polyamides designed to bind transcription factor binding sites can potentially suppress gene expression. Transforming growth factor1 (TGF1) represents a large family of cytokines that are involved in the regulation This work was supported in part by a grant-in-aid for the High-Tech Research Center from the Japanese Ministry of Education, Science, Sports, and Culture to Nihon University, and from the Ministry of Education, Science, Sports, and Culture of Japan (1550863). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.089086. ABBREVIATIONS: Py, pyrrole; Im, imidazole; TGF1, transforming growth factor1; VSMC, vascular smooth muscle cell; hTGF1, human transforming growth factor1; FSE2, fat-specific element 2; bp, base pair(s); DMF, N,N-dimethylformamide; FITC, fluorescein isothiocyanate; DMEM, Dulbecco’s modified Eagle’s medium; PMA, phorbol 12-myristate acetate. 0022-3565/05/3152-571–575$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 89086/3061418 JPET 315:571–575, 2005 Printed in U.S.A. 571 at A PE T Jornals on A uust 9, 2017 jpet.asjournals.org D ow nladed from of growth, differentiation, and morphogenesis in a wide range of cell types (Lyons and Moses, 1990; Sporn and Roberts, 1992). TGF1 has been reported to be involved in several cardiovascular diseases such as stroke, ischemic heart disease, and glomerulosclerosis, owing to its effects on the growth of vascular smooth muscle cells (VSMCs) and extracellular matrix formation (Grant et al., 1999; Joki et al., 2000; Kobayashi et al., 2001). TGF1 plays a pivotal role in chronic inflammatory changes of the interstitium and accumulation of extracellular matrix during renal fibrogenesis (Border and Noble, 1997; Blobe et al., 2000). We designed a Py-Im polyamide targeting the hTGF1 promoter adjacent to the fat-specific element 2 (FSE2) to inhibit expression of the human TGF1 (hTGF1) gene; we then examined the effect of this polyamide on hTGF1 gene expression. Materials and Methods General. Reagents and solvents were purchased from standard suppliers and used without further purification. NMR spectra were recorded with a JEOL JNM-A 500 nuclear magnetic resonance spectrometer (JEOL, Tokyo, Japan), and tetramethylsilane was used as the internal standard. Proton NMR spectra were recorded in parts per million (ppm) downfield relative to tetramethylsilane. The following abbreviations apply to spin multiplicity: s (singlet), d (doublet), t (triplet), q (quartet), qu (quintet), m (multiplet), and br (broad). Electrospray ionization mass spectrometry and electrospray ionization time-of-flight mass spectrometry were produced on a API 150 (PerkinElmerSciex Instruments, Boston, MA) and BioTOF II (Bruker Daltonics, Billerica, MA) mass spectrometer. Designing and Synthesis of Py-Im Polyamide Targeting the TGF1 Promoter. The structures of the match, FITC-labeled, and mismatch Py-Im polyamides used in this study are shown in Fig. 1. Py-Im polyamide was designed to bind bp 545 to 539 the hTGF1 promoter adjacent to the FSE2 binding site (Fig. 1A). One Im-Py substitution was induced to create the mismatch Py-Im polyamide. Machine-assisted automatic synthesis of Py-Im polyamides was performed with a Pioneer continuous-flow peptide synthesizer (Applied Biosystems, Foster City, CA) on a 0.1-mmol scale (200 mg of Fmoc-b-Ala-CLEAR Acid Resin, 0.50 mEq/g; Peptide Institute, Osaka, Japan). Automatic solid phase synthesis consisted of an N,Ndimethylformamide (DMF) wash, removal of the Fmoc group with 20% piperidine/DMF, a methanol wash, coupling with monomer in the presence of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine (4 Eq each) for 60 min, a methanol wash, protection with acetic anhydride/ pyridine if necessary, and a final DMF wash. We generally obtained moderate yields (10–39%) of Py-Im polyamides. After removal of Fmoc group of Fmoc-b-alanine-CLEAR Acid Resin, the resin was washed successively with methanol. The coupling step was performed with Fmoc-amino acid followed by a wash with methanol. These steps were repeated several times until all sequences were introduced. After the coupling steps were completed, the N-terminal amino group was protected or coupled with FITC and washed with DMF, and the reaction vessel was drained. The synthetic polyamides were isolated after the acidic (5 ml of 91% trifluoroacetic acid, 3% triisopropylsilane, 3% dimethylsulfide, 3% water/0.1 mM resin) or basic (5 ml N,N-dimethyl-1,3-propanediamine/0.1 mM resin, 55°C overnight) cleavage step. Polyamides were purified by high-performance liquid chromatography using a Chemcobond 5-ODS-H column (Chemco Scientific, Osaka, Japan), 0.1% AcOH/CH3CN, 0 to 50% linear gradient, 0 to 40 min, 254 nm. Ac-ImPyPy-b-ImPyPy-g-PyPyPy-bPyPy-bCOOH (Fig. 1B): 29 mg (17%); H NMR [dimethyl sulfoxide (DMSO)-d6] d: 1.50 (4H, m), 1.78 (2H, t, J 7.0 Hz), 2.01 (3H, s), 2.21 (4H, t, J 7.0 Hz), 2.27 (4H, m), 2.59 (4H, t, J 7.0Hz), 3.77 (3H, s), 3.80 (3H, s), 3.80 (6H, s), 3.81 (3H, s), 3.82 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 3.84 (3H, s), 3.93 (3H, s), 3.95 (3H, s), 6.75 (1H, s), 6.82 (1H, d, J 2.0 Hz), 6.85 (3H, m), 6.90 (1H, d, J 2.0 Hz), 7.01 (1H, d, J 2.0 Hz), 7.11 (1H, d, J 2.0 Hz), 7.12 (1H, d, J 2.0 Hz), 7.16 (2H, d, J 2.0 Hz), 7.17 (3H, d, J 2.0 Hz), 7.19 (1H, d, J 2.0 Hz), 7.21 (1H, d, J 2.0 Hz), 7.25 (2H, d, J 2.0 Hz), 7.41 (1H, s), 7.45 (1H, s), 8.03 (3H, m), 8.22 (1H, m), 9.83 (1H, s), 9.86 (2H, s), 9.86 (1H, s), 9.87 (1H, s), 9.88 (2H, s), 9.91 (1H, s), 9.93 (1H, s), 10.21 (1H, s), 10.28 (1H, s); electrospray ionization-mass spectrometry mass-to-charge ratio (m/e) calcd for C79H90N28O17 (M H) 1703.7, found 1703.7. FITC-b-ImPyPy-b-ImPyPy-g-PyPyPy-b-PyPy-bDp (Fig. 1D): 7.0 mg (3.0%); H NMR [dimethyl sulfoxide (DMSO)-d6] d: 1.50 (4H, m), 1.78 (2H, t, J 7.5 Hz), 2.08 (6H, s), 2.17 (4H, t, J 7.5 Hz), 2.29 (4H, m), 2.59 (2H, t, J 7.5 Hz), 2.70 (2H, t, J 9.5 Hz), 3.04 (4H, dd, J 7.5 Hz, 9.5 Hz), 3.43(6H, m), 3.78 (3H, s), 3.79 (3H, s), 3.80 (6H, s), 3.81 (3H, s), 3.82 (3H, s), 3.83 (3H, s), 3.84 (6H, s), 3.95 (6H, s), 6.55 (2H, dd, J 2.5 Hz, 7.5 Hz), 6.59 (2H, d, J 7.5 Hz), 6.66 (2H, d, J 2.5 Hz), 6.79 (1H, d, J 2.5 Hz), 6.85 (4H, m), 6.90 (1H, s), 7.12 (2H, s), 7.16 (8H, m), 7.21 (2H, d, J 2.5 Hz), 7.25 (3H, s), 7.45 (1H, s), 7.46 (1H, s), 7.83 (1H, t, J 5.0 Hz), 7.95 (1H, s), 9.94 (2H, s), 10.09 (1H, s), 10.26 (1H, s), 10.35 (1H, s); electrospray ionization-mass spectrometry mass-to-charge ratio (m/e) calcd for C106H116N32O21S (M H) 2205.87, found 2205.8. Cell Culture and Distribution of Py-Im Polyamide. Human VSMCs (Cambrex Bio Science Rockland, Inc., Rockland, ME) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) and 50 mg/ml streptomycin (Invitrogen). To examine the distribution of Fig. 1. Structure and target sequence of the Py-Im polyamide targeting hTGF1 promoter. Py-Im polyamide was designed to bind bp 545 to 539 of the hTGF1 promoter adjacent to the FSE2 (A). Structures of the hTGF1-specific (B), mismatch (C), and FITC-conjugated (D) Py-Im polyamide are shown. 572 Lai et al. at A PE T Jornals on A uust 9, 2017 jpet.asjournals.org D ow nladed from Py-Im polyamide in cells, VSMCs were plated on two-well chamber slides (LabTek; Nalge Nunc International, Tokyo, Japan) at a density of 3000/cm. Cells were incubated with 1 nM FITC-conjugated Py-Im polyamide in DMEM for 2 h and fixed for 20 min (glutaraldehyde). Nuclear staining was achieved with Hoechst 33324 and visualized with an Olympus microscope (Olympus, Tokyo, Japan) using appropriate filters. hTGF1 Promoter Activity. A 2.2-kb fragment of the hTGF1 promoter was inserted into a pGL3 basic (Promega, Madison, WI) vector (pGL3-TGF1). One microgram of pGL3-TGF1 was transfected into human VSMCs in serum-free medium with the LipofectAMINE reagent for 6 h. Twenty-four hours after transfection, cells were incubated with 1 M Py-Im targeting human TGF1 or mismatch polyamide in the presence or absence of 1 M phorbol 12-myristate acetate (PMA) in DMEM containing 0.5% calf serum for 24 h. Luciferase activity was measured in cell extracts with the Dual-Luciferase reporter gene assay system (Promega). Gel Mobility Shift Assay. A double-stranded DNA fragment corresponding to bp 548 to 537 of human TGF1 promoter was labeled with [ P]ATP by T4 polynucleotide kinase (Promega) according to the standard method (Sambrook et al., 1989). The labeled double-stranded DNA was then incubated with 10 nM mismatch polyamide or 1, 2, 4, and 10 nM Py-Im polyamides at 37°C for 15 min. DNA-Py-Im complexes were separated by electrophoresis on 20% polyacrylamide gels and visualized by autoradiography. Reverse Transcription-Polymerase Chain Reaction and Western Blot Analysis. Total RNA was extracted from cultured cells as described previously (Mocharla et al., 1990) and was reversetranscribed with oligo dT (Takara Biochemicals, Osaka, Japan) and avian myeloblastoma virus reverse transcriptase (Takara Biochemicals) at 37°C for 40 min. Then, 1.5 l of reverse-transcribed material was amplified with TaqDNA polymerase (Takara Biochemicals), as described previously using each specific primers (Ando et al., 2004). Western blot analysis was performed as described previously using each specific antiserum (Ando et al., 2004). Statistical Analysis. Values are reported as mean standard error of the mean (S.E.M.). Statistical analysis was done with Student’s t test for unpaired data or with two-way analysis of variance or Duncan’s multiple range test. p 0.05 was considered statistically significant.