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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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Biological activity and DNA-binding of β-substituted hairpin polyamides. Cytotoxicity analyses were conducted 96 h following polyamide treatment in the A549 lung carcinoma cell line. IC50 values representing the mean of three biological replicates. ΔTm denotes the shift in melting temperature following polyamide treatment for the 5′-WGWWCW-3′ duplex DNA sequence shown.
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gkr970-F2: Biological activity and DNA-binding of β-substituted hairpin polyamides. Cytotoxicity analyses were conducted 96 h following polyamide treatment in the A549 lung carcinoma cell line. IC50 values representing the mean of three biological replicates. ΔTm denotes the shift in melting temperature following polyamide treatment for the 5′-WGWWCW-3′ duplex DNA sequence shown.

Mentions: Following an unanticipated observation that a hairpin polyamide conjugated to an aryl group at the β-amino position showed greater activity in cell culture, we synthesized a small panel of β-aryl substituted polyamides (4–12) targeting the sequence 5′-WGWWCW-3′ and benchmarked their DNA-binding affinities and biological activity against unsubstituted parent (1), β-amino (2) and β-acetylated (3) GABA turns in the A549 human lung carcinoma cell line (Figure 2). We used cytotoxicity at 96 h as a proxy for uptake following the observation that polyamide uptake, gene regulatory activity and cytotoxicity are often highly correlated (13). Thermal denaturation analysis of a DNA duplex of the sequence 5′-TTGCTGTTCTGCAA-3′ (polyamide match site in bold) shows all polyamides containing a β-amino GABA group (2–14) similarly increase the melting temperature by ~13–15°C, suggesting no substantial energetic penalty for appendage of the bulky β-aryl groups (Figure 2). Cytotoxicity analyses demonstrate that β-amino GABA incorporating polyamide 2 (IC50 = 3.2 µM) is considerably more cytotoxic than its unsubstituted counterpart 1 (IC50 > 30 µM). While this trend mirrors the relative duplex stabilization of these molecules (ΔTm1 = 8.8°C; ΔTm2 = 13.3°C), simple N-acetylation of the β-amino turn (3) results in another order of magnitude increase in growth inhibition while not greatly affecting binding affinity (ΔTm3 = 13.2°C). Replacement of the acetyl unit of 3 with a benzoyl functionality (4) results in approximately another order of magnitude increase in cytotoxicity (IC50 = 35 nM), again without concomitant change in the duplex stabilizing ability of this minor groove binder. Within the aryl series (4–12) several trends are seen, including increased cytotoxicity of p-substituted benzoic acids (compare 9 and 10) and a preference for electron-withdrawing groups at the p-position (compare 6 and 7). Remarkably, significantly increasing the steric bulk of the β-aryl turn, as in polyamides 8 and 11, does not greatly affect either DNA-binding or cytotoxicity, arguing against interaction of the β-aryl turn with a small pocket of a specific protein partner as a mechanism of cytotoxicity. Since previous studies have noted that polyamide activity can be strongly influenced by cell type (12), we tested the generality of the increased cytotoxicity of 4 and 9 in LNCaP prostate cancer, MCF-7 breast cancer and HCT-116 colon cancer cell lines. All three cell lines showed a similar increase in potency for β-aryl compared to β-amino polyamides as was observed in A549 cells (Table 1).Figure 2.


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

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

Biological activity and DNA-binding of β-substituted hairpin polyamides. Cytotoxicity analyses were conducted 96 h following polyamide treatment in the A549 lung carcinoma cell line. IC50 values representing the mean of three biological replicates. ΔTm denotes the shift in melting temperature following polyamide treatment for the 5′-WGWWCW-3′ duplex DNA sequence shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300022&req=5

gkr970-F2: Biological activity and DNA-binding of β-substituted hairpin polyamides. Cytotoxicity analyses were conducted 96 h following polyamide treatment in the A549 lung carcinoma cell line. IC50 values representing the mean of three biological replicates. ΔTm denotes the shift in melting temperature following polyamide treatment for the 5′-WGWWCW-3′ duplex DNA sequence shown.
Mentions: Following an unanticipated observation that a hairpin polyamide conjugated to an aryl group at the β-amino position showed greater activity in cell culture, we synthesized a small panel of β-aryl substituted polyamides (4–12) targeting the sequence 5′-WGWWCW-3′ and benchmarked their DNA-binding affinities and biological activity against unsubstituted parent (1), β-amino (2) and β-acetylated (3) GABA turns in the A549 human lung carcinoma cell line (Figure 2). We used cytotoxicity at 96 h as a proxy for uptake following the observation that polyamide uptake, gene regulatory activity and cytotoxicity are often highly correlated (13). Thermal denaturation analysis of a DNA duplex of the sequence 5′-TTGCTGTTCTGCAA-3′ (polyamide match site in bold) shows all polyamides containing a β-amino GABA group (2–14) similarly increase the melting temperature by ~13–15°C, suggesting no substantial energetic penalty for appendage of the bulky β-aryl groups (Figure 2). Cytotoxicity analyses demonstrate that β-amino GABA incorporating polyamide 2 (IC50 = 3.2 µM) is considerably more cytotoxic than its unsubstituted counterpart 1 (IC50 > 30 µM). While this trend mirrors the relative duplex stabilization of these molecules (ΔTm1 = 8.8°C; ΔTm2 = 13.3°C), simple N-acetylation of the β-amino turn (3) results in another order of magnitude increase in growth inhibition while not greatly affecting binding affinity (ΔTm3 = 13.2°C). Replacement of the acetyl unit of 3 with a benzoyl functionality (4) results in approximately another order of magnitude increase in cytotoxicity (IC50 = 35 nM), again without concomitant change in the duplex stabilizing ability of this minor groove binder. Within the aryl series (4–12) several trends are seen, including increased cytotoxicity of p-substituted benzoic acids (compare 9 and 10) and a preference for electron-withdrawing groups at the p-position (compare 6 and 7). Remarkably, significantly increasing the steric bulk of the β-aryl turn, as in polyamides 8 and 11, does not greatly affect either DNA-binding or cytotoxicity, arguing against interaction of the β-aryl turn with a small pocket of a specific protein partner as a mechanism of cytotoxicity. Since previous studies have noted that polyamide activity can be strongly influenced by cell type (12), we tested the generality of the increased cytotoxicity of 4 and 9 in LNCaP prostate cancer, MCF-7 breast cancer and HCT-116 colon cancer cell lines. All three cell lines showed a similar increase in potency for β-aryl compared to β-amino polyamides as was observed in A549 cells (Table 1).Figure 2.

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
Related in: MedlinePlus