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Quantitating the concentration of Py-Im polyamide-fluorescein conjugates in live cells.

ABSTRACT: Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1 and 0.5microM, which is significantly lower than the 2microM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.

SUBMITTER: Hsu CF

PROVIDER: S-EPMC2587135 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Quantitating the concentration of Py-Im polyamide-fluorescein conjugates in live cells.

Hsu Carey F CF Dervan Peter B PB

Bioorganic & medicinal chemistry letters 20080520 22

Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1 and 0.5microM, which is significantly lower than the 2microM dosage c ...[more]

PMID: 18524590

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Oligomerization route to Py-Im polyamide macrocycles.

Project description:Cyclic eight-ring pyrrole-imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solution-phase methods. A rapid solution-phase oligomerization approach to eight-ring symmetrical cyclic polyamides yields 12- and 16-membered macrocycles as well. A preference for DNA binding by the 8- and 16-membered oligomers was observed over the 12-ring macrocycle, which we attributed to a conformational constraint not present in the smaller and larger systems.

| S-EPMC2736337 | biostudies-literature

Synthesis of cyclic Py-Im polyamide libraries.

Project description:Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.

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An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors.

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Activity of a Py-Im polyamide targeted to the estrogen response element.

Project description:Pyrrole-imidazole (Py-Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py-Im polyamides with differing DNA sequence specificity for activity against 17β-estadiol (E2)-induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5'-WGGWCW-3' (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity.

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Structural basis for cyclic Py-Im polyamide allosteric inhibition of nuclear receptor binding.

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A C-14 labeled Py-Im polyamide localizes to a subcutaneous prostate cancer tumor.

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Conformational modulation of DNA by polyamide binding: structural effects of f-Im-Py-Im based derivatives on 5'-ACGCGT-3'.

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| S-EPMC3814215 | biostudies-literature

Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture.

Project description:A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 microM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an approximately 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

| S-EPMC218713 | biostudies-literature

Sequence specific suppression of androgen receptor-DNA binding in vivo by a Py-Im polyamide.

Project description:The crucial role of androgen receptor (AR) in prostate cancer development is well documented, and its inhibition is a mainstay of prostate cancer treatment. Here, we analyze the perturbations to the AR cistrome caused by a minor groove binding molecule that is designed to target a sequence found in a subset of androgen response elements (ARE). We find treatment with this pyrrole-imidazole (Py-Im) polyamide exhibits sequence selectivity in its repression of AR binding in vivo. Differentially changed loci are enriched for sequences resembling ARE half-sites that match the Py-Im polyamide binding preferences determined in vitro. Comparatively, permutations of the ARE half-site bearing single or double mismatches to the Py-Im polyamide binding sequence are not enriched. This study confirms that the in vivo perturbation pattern caused by a sequence specific polyamide correlates with its in vitro binding preference genome-wide in an unbiased manner.

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Gene Expression Changes in a Tumor Xenograft by a Py-Im Polyamide

Project description:Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5’-WGGWWW-3’ with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. A FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which were interrogated by qRT-PCR and correlated with cell culture expression levels.

2012-10-02 | GSE40218 | GEO

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