Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression

The glucocorticoid receptor (GR) affects the transcription of genes involved in diverse processes, including energy metabolism and the immune response, through DNA-binding dependent and independent mechanisms. The DNA-binding dependent mechanism occurs by direct binding of GR to glucocorticoid response elements (GREs) at regulatory regions of target genes. The DNA-binding independent mechanism involves binding of GR to transcription factors and coactivators that, in turn, contact DNA. A small molecule that competes with GR for binding to GREs could be expected to affect the DNA-dependent pathway selectively by interfering with the protein-DNA interface. We show that a DNA-binding polyamide that targets the consensus GRE sequence binds the glucocorticoid-induced zipper (GILZ) GRE, inhibits expression of GILZ and several other known GR target genes, and reduces GR occupancy at the GILZ promoter. Genome-wide expression analysis of the effects of this polyamide on a set of glucocorticoid-induced and -repressed genes could help to elucidate the mechanism of GR regulation for these genes

Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules

Commercials, careers and culture: travelling salesmen in Britain 1890s-1930s

Within the lower middle-class, British commercial travellers established a strong fraternal culture before 1914. This article examines their interwar experiences in terms of income, careers, and associational culture. It demonstrates how internal labour markets operated, identifies the ways in which commercial travellers interpreted their role, and explores their social and political attitudes

Characterization and Solubilization of Pyrrole–Imidazole Polyamide Aggregates

To optimize the biological activity of pyrrole–imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied polyamides were found to form measurable particles 50–500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-β-cyclodextrin (HpβCD). In mice, the use of HpβCD allowed for improved injection conditions and subsequent investigations of the availability of polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im polyamides and facilitate their study in animal models

Microwave Assisted Synthesis of Py-Im Polyamides

Microwave synthesis was utilized to rapidly build Py-Im polyamides in high yields and purity using Boc-protection chemistry on Kaiser oxime resin. A representative polyamide targeting the 5′-WGWWCW-3′ (W = A or T) subset of the consensus Androgen and Glucocorticoid Response Elements was synthesized in 56% yield after 20 linear steps and HPLC purification. It was confirmed by Mosher amide derivatization of the polyamide that a chiral α-amino acid does not racemize after several additional coupling steps

Structural Basis for Cyclic Py-Im Polyamide Allosteric Inhibition of Nuclear Receptor Binding

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

Guiding the Design of Synthetic DNA-Binding Molecules with Massively Parallel Sequencing

Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10^(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5′-WCGCGW-3′ from 5′-WGCGCW-3′. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications

DNA Repair Interference with Sequence Specific DNA Binding Pyrrole Imidazole Polyamides; a Novel Radiation Sensitizer

Pyrrole (Py)—Imidazole (Im) polyamides are a class of non-genotoxic DNA-binding small molecules with modular sequence recognition and high affinity. A polyamide targeted to the Androgen Receptor (AR) Response Element (ARE) half-site, suppresses AR positive prostate tumor xenografts. It is hypothesized that polyamides may displace DNA repair elements recruited after genotoxic insult and thereby sensitize cells to ionizing radiation. DNA double strand repair was assessed in a cell-free system using PC3 cell lysate, and a linearized pGEM3Z plasmid containing an insert with a BamH1 restriction site flanked by polyamide binding site motifs, and analyzed by qPCR. Rejoining of the linearized plasmid was reduced by 85 % and inhibited rejoining by DNA ligase compared to untreated controls. Neutral comet assay revealed no genomic fragmentation after 48 h of 10 µM polyamide treatment. Genomic fragmentation was statistically similar 1 h post radiation with 10 Gy, +/− polyamide. However, at 24 h post radiation, cells pretreated with 10 µM polyamide had persistently elevated tail lengths (181 vs. 129 p < 0.001) and tail moment (103 vs 74 p < 0.001). After radiation, greater than 95 % of cells +/− 10 µM polyamide pretreatment demonstrated increased γ-H2AX phosphorylation. 24 h post RT, 32% of polyamide treated cells continued to have elevated γ-H2AX phosphorylation, whereas only 19% of untreated cells continued to have elevated γ-H2AX phosphorylation. Polyamide treatment with 10 mM resulted in a sensitization enhancement ratio of 2.48 at 4 Gy by clonogenic assay. In conclusion Py-Im polyamides are non-genotoxic and sequence specific; potentially offering a strategy for sequence-directed interference with DNA repair and other DNA-depended processes