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Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns.

Meier JL, Montgomery DC, Dervan PB - Nucleic Acids Res. (2011)

Bottom Line: Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude.Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation.Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

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Inhibition of nuclear receptor-mediated gene expression by β-aryl polyamides. (A) General schematic of nuclear receptor-mediated gene expression and polyamide inhibition of the protein–DNA interface. (B) Effect of β-turn polyamides 2 and 4 on DEX-induced GILZ gene expression as measured by qRT-PCR analysis. I = DEX-induced. NI = non-induced. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. (C) Effect of β-substituted polyamides 2 and 4 on dihydrotestosterone (DHT)-induced PSA gene expression as measured by qRT-PCR analysis. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. I = induced. NI = not induced.
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gkr970-F3: Inhibition of nuclear receptor-mediated gene expression by β-aryl polyamides. (A) General schematic of nuclear receptor-mediated gene expression and polyamide inhibition of the protein–DNA interface. (B) Effect of β-turn polyamides 2 and 4 on DEX-induced GILZ gene expression as measured by qRT-PCR analysis. I = DEX-induced. NI = non-induced. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. (C) Effect of β-substituted polyamides 2 and 4 on dihydrotestosterone (DHT)-induced PSA gene expression as measured by qRT-PCR analysis. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. I = induced. NI = not induced.

Mentions: A549 lung epithelial cells have been widely applied as a model for inflammatory gene expression mediated by the transcription factor glucocorticoid receptor (GR) (24–26). GR is a member of larger family of nuclear receptors that utilize activation by small molecule ligands in order to effect release from cytoplasmic inhibitory complexes, after which they traffic to the nucleus, multerimize with their cognate protein partners, and activate (or repress) transcription (Figure 3A). Hairpin polyamides have been previously shown to inhibit nuclear receptor–DNA interactions in cell culture, making them promising agents for mechanistic studies of nuclear receptor–DNA binding and therapeutic modulation of nuclear receptor activity in diseases such as prostate cancer. Polyamides 1–12 target the 5′-WGWWCW-3′ sequence found in the consensus glucoroticoid response element (GRE). Therefore, as an initial test of whether the increased activity of β-aryl polyamides might be extended to gene regulatory studies, we analyzed the effects of β-aryl polyamide 4 and parent polyamide 2 on endogenous GR-mediated transcription in A549 cells. Following 48-h treatment with 2, 4 or vehicle DMSO, A549 cells were induced with DEX before isolation of total RNA and analysis by qRT-PCR. As seen in Figure 3B, β-aryl polyamide 4 blunts GR-driven transcription of the canonical GR-regulated gene GILZ (24,27) in a dose-dependent fashion. Remarkably, inhibition of gene expression by 4 occurs at polyamide concentrations 100× lower than parent compound 2. Similar results are seen for expression of FKBP5, another prototypical GR target (Supplementary Figure S1) (28). Notably, these effects are not expected to be due to cytotoxicity, as these experiments utilize a fivefold higher cell plating density than cytotoxicity analyses and are normalized to a housekeeping gene (GUSB) that remains stable to polyamide treatment (Supplementary Figure S2). Time course experiments show substantial inhibition of DEX-induced transcription as early as 12 h after polyamide treatment, far prior to the onset of cytotoxicity (Figure 4). Since the sequence targeted by 4, 5′-WGWWCW-3′, is also found in the androgen response element (ARE), we next tested whether 4 showed similarly enhanced inhibition of androgen receptor (AR) regulated gene expression (7). LNCaP prostate cancer cells were exposed to polyamides 2 or 4 for 48 h prior to induction with the AR-activating ligand 4,5α-dihydrotestosterone (DHT). Quantitative PCR analysis of reverse-transcribed mRNA shows a drastic decrease in transcription of the known AR target gene prostate specific antigen (PSA, also known as KLK3) following exposure to 4 (Figure 3C) (29,30). Once again, this inhibition is greater than that observed when parent compound 2 is applied at 100× greater concentrations, reducing PSA mRNA to below basal (non-induced) levels. These results highlight the activity of β-aryl polyamides as potent antagonists of nuclear receptor-mediated gene expression in living cells.Figure 3.


Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns.

Meier JL, Montgomery DC, Dervan PB - Nucleic Acids Res. (2011)

Inhibition of nuclear receptor-mediated gene expression by β-aryl polyamides. (A) General schematic of nuclear receptor-mediated gene expression and polyamide inhibition of the protein–DNA interface. (B) Effect of β-turn polyamides 2 and 4 on DEX-induced GILZ gene expression as measured by qRT-PCR analysis. I = DEX-induced. NI = non-induced. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. (C) Effect of β-substituted polyamides 2 and 4 on dihydrotestosterone (DHT)-induced PSA gene expression as measured by qRT-PCR analysis. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. I = induced. NI = not induced.
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Related In: Results  -  Collection

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Show All Figures
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gkr970-F3: Inhibition of nuclear receptor-mediated gene expression by β-aryl polyamides. (A) General schematic of nuclear receptor-mediated gene expression and polyamide inhibition of the protein–DNA interface. (B) Effect of β-turn polyamides 2 and 4 on DEX-induced GILZ gene expression as measured by qRT-PCR analysis. I = DEX-induced. NI = non-induced. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. (C) Effect of β-substituted polyamides 2 and 4 on dihydrotestosterone (DHT)-induced PSA gene expression as measured by qRT-PCR analysis. Polyamide 2 concentrations: 100, 1000, 10 000 nM, polyamide 4 concentrations: 1, 10, 100 nM. I = induced. NI = not induced.
Mentions: A549 lung epithelial cells have been widely applied as a model for inflammatory gene expression mediated by the transcription factor glucocorticoid receptor (GR) (24–26). GR is a member of larger family of nuclear receptors that utilize activation by small molecule ligands in order to effect release from cytoplasmic inhibitory complexes, after which they traffic to the nucleus, multerimize with their cognate protein partners, and activate (or repress) transcription (Figure 3A). Hairpin polyamides have been previously shown to inhibit nuclear receptor–DNA interactions in cell culture, making them promising agents for mechanistic studies of nuclear receptor–DNA binding and therapeutic modulation of nuclear receptor activity in diseases such as prostate cancer. Polyamides 1–12 target the 5′-WGWWCW-3′ sequence found in the consensus glucoroticoid response element (GRE). Therefore, as an initial test of whether the increased activity of β-aryl polyamides might be extended to gene regulatory studies, we analyzed the effects of β-aryl polyamide 4 and parent polyamide 2 on endogenous GR-mediated transcription in A549 cells. Following 48-h treatment with 2, 4 or vehicle DMSO, A549 cells were induced with DEX before isolation of total RNA and analysis by qRT-PCR. As seen in Figure 3B, β-aryl polyamide 4 blunts GR-driven transcription of the canonical GR-regulated gene GILZ (24,27) in a dose-dependent fashion. Remarkably, inhibition of gene expression by 4 occurs at polyamide concentrations 100× lower than parent compound 2. Similar results are seen for expression of FKBP5, another prototypical GR target (Supplementary Figure S1) (28). Notably, these effects are not expected to be due to cytotoxicity, as these experiments utilize a fivefold higher cell plating density than cytotoxicity analyses and are normalized to a housekeeping gene (GUSB) that remains stable to polyamide treatment (Supplementary Figure S2). Time course experiments show substantial inhibition of DEX-induced transcription as early as 12 h after polyamide treatment, far prior to the onset of cytotoxicity (Figure 4). Since the sequence targeted by 4, 5′-WGWWCW-3′, is also found in the androgen response element (ARE), we next tested whether 4 showed similarly enhanced inhibition of androgen receptor (AR) regulated gene expression (7). LNCaP prostate cancer cells were exposed to polyamides 2 or 4 for 48 h prior to induction with the AR-activating ligand 4,5α-dihydrotestosterone (DHT). Quantitative PCR analysis of reverse-transcribed mRNA shows a drastic decrease in transcription of the known AR target gene prostate specific antigen (PSA, also known as KLK3) following exposure to 4 (Figure 3C) (29,30). Once again, this inhibition is greater than that observed when parent compound 2 is applied at 100× greater concentrations, reducing PSA mRNA to below basal (non-induced) levels. These results highlight the activity of β-aryl polyamides as potent antagonists of nuclear receptor-mediated gene expression in living cells.Figure 3.

Bottom Line: Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude.Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation.Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.

Show MeSH