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

ABSTRACT: Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1: has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1: in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1: distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1: show no increase in HIF1? and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1?-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608-17. ©2015 AACR.

SUBMITTER: Szablowski JO

PROVIDER: S-EPMC4873353 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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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.

| S-EPMC3538502 | biostudies-literature

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

Project description:Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. In this paper we describe a high-resolution X-ray crystal structure of a β-amino turn-linked eight-ring cyclic Py-Im polyamide bound to the central six base pairs of the sequence d(5'-CCAGTACTGG-3')(2), revealing significant modulation of DNA shape. We compare the DNA structural perturbations induced by DNA-binding transcripton factors, androgen receptor and glucocorticoid receptor, in the major groove to those induced by cyclic polyamide binding in the minor groove. The cyclic polyamide is an allosteric modulator that perturbs the DNA structure in such a way that nuclear receptor protein binding is no longer compatible. This allosteric perturbation of the DNA helix provides a molecular basis for disruption of transcription factor-DNA interfaces by small molecules, a minimum step in chemical control of gene networks.

| S-EPMC2954530 | 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.

| S-EPMC6486638 | biostudies-literature

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

Project description:The DNA sequence 5'-ACGCGT-3' is in the core site of the Mlu 1 cell-cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido-imidazole-pyrrole-imidazole-amine(+) (f-Im-Py-Im-Am(+) ) can target the minor groove of 5'-ACGCGT-3' as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f-Im-Py-Im-Am(+) derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis-ligation ladders assay was used to evaluate the conformational effects of f-Im-Py-Im-Am(+) and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide-DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B-form like overall structure. After binding with f-Im-Py-Im-Am(+) derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed.

| S-EPMC3814215 | biostudies-literature

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

Project description: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.

| S-EPMC2587135 | biostudies-literature

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.

| S-EPMC3651785 | biostudies-literature

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

Project description:In an effort to quantitate Py-Im polyamide concentrations in vivo, we synthesized the C-14 radioactively labeled compounds 1-3, and investigated their tumor localization in a subcutaneous xenograft model of prostate cancer (LNCaP). Tumor concentrations were compared with representative host tissues, and exhibited a certain degree of preferential localization to the xenograft. Compound accumulation upon repeated administration was measured. Py-Im polyamide 1 was found to accumulate in LNCaP tumors at concentrations similar to the IC50 value for this compound in cell culture experiments.

| S-EPMC4134956 | 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 in prostate cancer development is well documented, and its inhibition is a mainstay of prostate cancer treatment. Here we analyze the perturbations to the androgen receptor cistrome caused by a minor groove binding molecule that is designed to target a sequence found in a subset of androgen response elements. We find treatment with this pyrrole-imidazole polyamide exhibits sequence selectively in its repression of androgen receptor 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 ARE half-site bearing single or double mismatches to the Py-Im polyamide binding sequence are not enriched. This study represents an indirect determination of Py-Im polyamide binding preference in vivo using an unbiased approach.

2019-02-22 | GSE125552 | GEO

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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