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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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Quantitative fluorescence analysis of β-turn substitution on Py–Im polyamide nuclear uptake. (A) Nuclear localization of β-aryl polyamide 13, as verified by colocalization with Hoechst 33342. (B) Influence of incubation time on fluorescence for A549 cells treated with 100 nM polyamide 13 or 14. X-axis: relative median fluorescence (FL1: FITC channel); Y-axis: hours of polyamide treatment. (C) Influence of dosage concentration on nuclear accumulation of polyamides. Polyamide 13 concentration: 100, 1000, 10 000 nM. Polyamide 14 concentration: 100, 1000 nM.
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gkr970-F5: Quantitative fluorescence analysis of β-turn substitution on Py–Im polyamide nuclear uptake. (A) Nuclear localization of β-aryl polyamide 13, as verified by colocalization with Hoechst 33342. (B) Influence of incubation time on fluorescence for A549 cells treated with 100 nM polyamide 13 or 14. X-axis: relative median fluorescence (FL1: FITC channel); Y-axis: hours of polyamide treatment. (C) Influence of dosage concentration on nuclear accumulation of polyamides. Polyamide 13 concentration: 100, 1000, 10 000 nM. Polyamide 14 concentration: 100, 1000 nM.

Mentions: Hypothetically, the increased biological activity of β-aryl turn polyamides could be attributed to either (i) an improved ability to impede protein–DNA interactions, (ii) increased uptake or (iiii) reduced efflux. The former seemed unlikely given that our initial structure–activity analysis showed no correlation between steric bulk of the β-aryl turn, which would be expected to affect the interaction of groove-binding proteins, and cytotoxicity (Figure 2). Therefore, to examine cellular uptake in a systematic fashion we synthesized fluorescent analogues of polyamides 2 and 4 and analyzed their accumulation by confocal microscopy and flow cytometry (11,31). Polyamide-FITC conjugates 13 and 14 show similar trends in terms of biological activity compared to parent compounds, although the observed gap in cytotoxicities is decreased from ~100× to ~10× (Supplementary Figure S3). Following addition to growth media, polyamides 13 and 14 penetrate the membrane and localize to the nucleus of A549 cells, as verified by colocalization with the well-known DNA stain Hoechst (Figure 5A). However, flow cytometry analysis reveals quantitative differences in the kinetics and degree of uptake. Cells treated with 100 nM β-aryl polyamide 14 demonstrate a rapid increase in fluorescence intensity between 0–12 h, compared to much slower accumulation of β-amino polyamide 13 dosed under identical conditions (Figure 5B). Analyzing the overall percentage of fluorescently labeled cells as compared to a DMSO-treated control shows that treatment with 100 nM 14 results in fluorescent labeling of ~88% of A549 cells after 6 h, while cells treated with 100 nM 13 show labeling of only ~5% of cells over the same time period (Supplementary Figure S4). In order to gain a more quantitative view of the fluorescence increase, we calculated the nuclear concentrations of fluorescent polyamides 13 and 14 through comparison to fluorescent beads functionalized with known amounts of the FITC fluorophore (14,31). Using this methodology, at 48 h we observe a >4× greater accumulation of 14 than 13 in A549 nuclei when dosed at identical concentrations (100 nM). However, these concentrations can be shifted by increasing polyamide concentration, as a 10× greater dosage concentrations results in a ~3× increase in polyamide concentration values over 48 h (Figure 5C). Notably, this is not due merely to decreased cell growth, as analysis of cell count and viability prior to flow cytometry revealed no differences between treated samples. Finally, to differentiate uptake and efflux, we compared the effect of verapamil on uptake of β-aryl and β-amino polyamides. Verapamil is an inhibitor of the p-glycoprotein transporter that has previously been implicated in cellular efflux of polyamides (32). If β-aryl polyamide 4 is attaining higher concentrations through reduced efflux, verapamil treatment should have little or no effect on intracellular polyamide concentration, whereas if β-aryl polyamide 4 is attaining higher concentrations through enhanced uptake, verapamil will have an additive effect on nuclear accumulation. Our results are consistent with the latter mechanism, as we observed similarly higher fluorescent labeling by both 2 and 4 in A549 cells co-treated with a non-toxic (10 µM) dose of verapamil (Supplementary Figure S5). Overall these findings have two implications: (i) β-aryl turns can significantly increase the rate of polyamide uptake at submicromolar concentrations and (ii) polyamide cytotoxicity and cellular uptake are well correlated, as the 10× increase in cytotoxicity of 14 relative to 13 is mirrored by its accumulation in cells at 10× lower concentrations.Figure 5.


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

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

Quantitative fluorescence analysis of β-turn substitution on Py–Im polyamide nuclear uptake. (A) Nuclear localization of β-aryl polyamide 13, as verified by colocalization with Hoechst 33342. (B) Influence of incubation time on fluorescence for A549 cells treated with 100 nM polyamide 13 or 14. X-axis: relative median fluorescence (FL1: FITC channel); Y-axis: hours of polyamide treatment. (C) Influence of dosage concentration on nuclear accumulation of polyamides. Polyamide 13 concentration: 100, 1000, 10 000 nM. Polyamide 14 concentration: 100, 1000 nM.
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Related In: Results  -  Collection

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Show All Figures
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gkr970-F5: Quantitative fluorescence analysis of β-turn substitution on Py–Im polyamide nuclear uptake. (A) Nuclear localization of β-aryl polyamide 13, as verified by colocalization with Hoechst 33342. (B) Influence of incubation time on fluorescence for A549 cells treated with 100 nM polyamide 13 or 14. X-axis: relative median fluorescence (FL1: FITC channel); Y-axis: hours of polyamide treatment. (C) Influence of dosage concentration on nuclear accumulation of polyamides. Polyamide 13 concentration: 100, 1000, 10 000 nM. Polyamide 14 concentration: 100, 1000 nM.
Mentions: Hypothetically, the increased biological activity of β-aryl turn polyamides could be attributed to either (i) an improved ability to impede protein–DNA interactions, (ii) increased uptake or (iiii) reduced efflux. The former seemed unlikely given that our initial structure–activity analysis showed no correlation between steric bulk of the β-aryl turn, which would be expected to affect the interaction of groove-binding proteins, and cytotoxicity (Figure 2). Therefore, to examine cellular uptake in a systematic fashion we synthesized fluorescent analogues of polyamides 2 and 4 and analyzed their accumulation by confocal microscopy and flow cytometry (11,31). Polyamide-FITC conjugates 13 and 14 show similar trends in terms of biological activity compared to parent compounds, although the observed gap in cytotoxicities is decreased from ~100× to ~10× (Supplementary Figure S3). Following addition to growth media, polyamides 13 and 14 penetrate the membrane and localize to the nucleus of A549 cells, as verified by colocalization with the well-known DNA stain Hoechst (Figure 5A). However, flow cytometry analysis reveals quantitative differences in the kinetics and degree of uptake. Cells treated with 100 nM β-aryl polyamide 14 demonstrate a rapid increase in fluorescence intensity between 0–12 h, compared to much slower accumulation of β-amino polyamide 13 dosed under identical conditions (Figure 5B). Analyzing the overall percentage of fluorescently labeled cells as compared to a DMSO-treated control shows that treatment with 100 nM 14 results in fluorescent labeling of ~88% of A549 cells after 6 h, while cells treated with 100 nM 13 show labeling of only ~5% of cells over the same time period (Supplementary Figure S4). In order to gain a more quantitative view of the fluorescence increase, we calculated the nuclear concentrations of fluorescent polyamides 13 and 14 through comparison to fluorescent beads functionalized with known amounts of the FITC fluorophore (14,31). Using this methodology, at 48 h we observe a >4× greater accumulation of 14 than 13 in A549 nuclei when dosed at identical concentrations (100 nM). However, these concentrations can be shifted by increasing polyamide concentration, as a 10× greater dosage concentrations results in a ~3× increase in polyamide concentration values over 48 h (Figure 5C). Notably, this is not due merely to decreased cell growth, as analysis of cell count and viability prior to flow cytometry revealed no differences between treated samples. Finally, to differentiate uptake and efflux, we compared the effect of verapamil on uptake of β-aryl and β-amino polyamides. Verapamil is an inhibitor of the p-glycoprotein transporter that has previously been implicated in cellular efflux of polyamides (32). If β-aryl polyamide 4 is attaining higher concentrations through reduced efflux, verapamil treatment should have little or no effect on intracellular polyamide concentration, whereas if β-aryl polyamide 4 is attaining higher concentrations through enhanced uptake, verapamil will have an additive effect on nuclear accumulation. Our results are consistent with the latter mechanism, as we observed similarly higher fluorescent labeling by both 2 and 4 in A549 cells co-treated with a non-toxic (10 µM) dose of verapamil (Supplementary Figure S5). Overall these findings have two implications: (i) β-aryl turns can significantly increase the rate of polyamide uptake at submicromolar concentrations and (ii) polyamide cytotoxicity and cellular uptake are well correlated, as the 10× increase in cytotoxicity of 14 relative to 13 is mirrored by its accumulation in cells at 10× lower concentrations.Figure 5.

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