DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 muM 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 muM concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G(2)/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers

Accessibility of nuclear chromatin by DNA binding polyamides

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes

Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates

Background: Pyrrole–imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA–protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. Results: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the γ turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide–CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. Conclusions: The ability of polyamide–CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms

Inhibition of DNA Binding by NF-κB with Pyrrole-Imidazole Polyamides

Synthetic ligands that bind to predetermined DNA sequences will offer a chemical approach to gene regulation if inhibition of a broad range of transcription factors can be achieved. NF-κB is a transcription factor that regulates a multitude of genes, including those involved in immune, inflammatory, and anti-apoptotic responses. NF-κB binds as heterodimer predominantly in the major groove. We report the design of polyamides that bind in the minor groove and target overlapping portions of an NF-κB binding site (5‘-GGGACTTTCC-3‘). We find that compounds that target the 5‘-GGGACT-3‘ portion of the site can inhibit DNA binding by NF-κB while those that target the 5‘-ACTTTCC-3‘ portion do not. Addition of NF-κB to the list of protein−DNA complexes that can be disrupted by minor groove binding ligands potentially increases the utility of polyamides as regulators of gene expression

Inhibition of DNA Binding by NF-κB with Pyrrole-Imidazole Polyamides

Synthetic ligands that bind to predetermined DNA sequences will offer a chemical approach to gene regulation if inhibition of a broad range of transcription factors can be achieved. NF-κB is a transcription factor that regulates a multitude of genes, including those involved in immune, inflammatory, and anti-apoptotic responses. NF-κB binds as heterodimer predominantly in the major groove. We report the design of polyamides that bind in the minor groove and target overlapping portions of an NF-κB binding site (5‘-GGGACTTTCC-3‘). We find that compounds that target the 5‘-GGGACT-3‘ portion of the site can inhibit DNA binding by NF-κB while those that target the 5‘-ACTTTCC-3‘ portion do not. Addition of NF-κB to the list of protein−DNA complexes that can be disrupted by minor groove binding ligands potentially increases the utility of polyamides as regulators of gene expression

Inhibition of major groove DNA binding bZIP proteins by positive patch polyamides

Cell permeable synthetic ligands that bind to predetermined DNA sequences offer a chemical approach to gene regulation, provided inhibition of a broad range of DNA transcription factors can be achieved. DNA minor groove binding polyamides containing aminoalkyl substituents at the N-1 of a single pyrrole residue display inhibitory effects for a bZIP protein which binds exclusively in the DNA major groove. For major groove protein inhibition, specific protein-DNA contacts along the phosphate backbone were targeted with the positively charged dimethylamino substituent on the backbone of a minor groove binding polyamide hairpin. Remarkably, these polyamides bind DNA with enhanced affinity and uncompromised specificity when compared to polyamides with the aminoalkyl moiety at the C-terminus. By adding bZIP transcription factors to the class of protein-DNA complexes that can be disrupted by minor groove binding ligands, these results may increase the functional utility of polyamides as regulators of gene expression

Inhibition of major groove DNA binding bZIP proteins by positive patch polyamides

Cell permeable synthetic ligands that bind to predetermined DNA sequences offer a chemical approach to gene regulation, provided inhibition of a broad range of DNA transcription factors can be achieved. DNA minor groove binding polyamides containing aminoalkyl substituents at the N-1 of a single pyrrole residue display inhibitory effects for a bZIP protein which binds exclusively in the DNA major groove. For major groove protein inhibition, specific protein-DNA contacts along the phosphate backbone were targeted with the positively charged dimethylamino substituent on the backbone of a minor groove binding polyamide hairpin. Remarkably, these polyamides bind DNA with enhanced affinity and uncompromised specificity when compared to polyamides with the aminoalkyl moiety at the C-terminus. By adding bZIP transcription factors to the class of protein-DNA complexes that can be disrupted by minor groove binding ligands, these results may increase the functional utility of polyamides as regulators of gene expression