DNA-binding Polyamides Research Articles

Repression of the transcriptional activity of ERRα with sequence-specific DNA-binding polyamides.

The orphan nuclear receptors estrogen-related receptors (ERRs) bind to the estrogen-related receptor response element (ERRE) to regulate transcriptional programs in cellular metabolism and cancer cell growth. In this study, we evaluated the potential for a pyrrole-imidazole polyamide to block ERRα binding to ERREs to inhibit gene expression. We demonstrated that the ERRE-targeted polyamide 1 blocked the binding of ERRα to the consensus ERRE and reduced the transcriptional activity of ERRα in cell culture. We further showed that inhibiting ERRα transcriptional activity with polyamide 1 led to reduced mitochondrial oxygen consumption, a primary biological effect regulated by ERRα. Finally, our data demonstrated that polyamide 1 is an inhibitor for cancer cell growth.

4-Methyltrityl-Protected Pyrrole and Imidazole Building Blocks for Solid Phase Synthesis of DNA-Binding Polyamides.

DNA-binding polyamides are synthetic oligomers of pyrrole/imidazole units with high specificity and affinity for double-stranded DNA. To increase their synthetic diversity, we report a mild methodology based on 4-methyltrityl (Mtt) solid phase peptide synthesis (SPPS), whose building blocks are more accessible than the standard Fmoc and Boc SPPS ones. We demonstrate the robustness of the approach by preparing and studying a hairpin with all precursors. Importantly, our strategy is orthogonal and compatible with sensitive molecules and could be readily automated.

Toward encoding reactivity using double-stranded DNA. Sequence-dependent native chemical ligation of DNA binding polyamides

The coupling reaction between distamycin-related polyamides equipped with cysteine and thioester groups can be accelerated in the presence of double-stranded (ds) oligonucleotides with selected sequences. While the coupling reaction of reactive partners containing three pyrrole units is accelerated by dsDNAs containing ATTTTA or ATGTTA sites, the heterodimeric coupling between a tripyrrole and a polyamide equipped with two pyrroles and one imidazole, is accelerated by the latter DNA, but not by the other containing the A/T rich tract. These differences can be exploited for selecting preferred coupling pairs from a mixture of reactive monomers; therefore the reaction outcome depends on the instructions provided by the dsDNA sequence.

DNA binding polyamides and the importance of DNA recognition in their use as gene-specific and antiviral agents

DNA binding polyamides and the importance of DNA recognition in their use as gene-specific and antiviral agents

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents.

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of β-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

An ab-initio study of pyrrole and imidazole arylamides

Arylamide foldamers have been shown to have a number of biological and medicinal applications. For example, a class of pyrrole-imidazole polyamide foldamers is capable of binding specific DNA sequences and preventing development of various gene disorders, most importantly cancer. Molecular dynamics (MD) simulations can provide crucial details in understanding the atomic level events related to foldamer/DNA binding. An important first step in the accurate simulation of these foldamer/DNA systems is the reparametrization of force field parameters for torsion around the aryl-amide bonds. Here we highlight our Density Functional Theory (DFT) potential energy profiles and derived force field parameters for four aryl-amide bond types for the pyrrole and imidazole building blocks extensively used in foldamer design for the DNA-binding polyamides. These results contribute to developing of computational tools for an appropriate molecular modeling of pyrrole-imidazole polyamide/DNA binding, and provide an insight into the chemical factors that influence the flexibility of the pyrrole-imidazole polyamides, and their binding to DNA.

Construction of a Structurally Defined Double-Stranded DNA Catenane

Topologically interlocked structures like catenanes and rotaxanes are promising components for the construction of molecular machines and motors. Herein we describe the construction of double-stranded DNA catenanes for DNA nanotechnology. For this, C-shaped DNA minicircle fragments were equipped with sequence-specific DNA-binding polyamides and their respective binding site. Formation of catenanes is achieved by self-assembly of two of these fragments and subsequent addition of a ring-closing oligonucleotide.

Highly Efficient Synthesis of DNA-Binding Hairpin Polyamides via the Use of a New Triphosgene Coupling Strategy

A facile and highly efficient solid phase synthesis method is reported for the preparation of hairpin DNA-binding polyamides using the cost-effective triphosgene (BTC) activating agent. Difficult polyamide sequences were prepared from N-methylimidazole (Im) and N-methylpyrrole (Py) building blocks with high stepwise yields (>98%) using Boc chemistry. The versatility of the triphosgene coupling approach was also demonstrated for the first time on aryl hydrazine resins to afford biomedically relevant tail-truncated polyamides in excellent isolated yields.

Polyamide Struts for DNA Architectures

Owing to its well-established synthetic access, its well-known structural properties, and its size, DNA is a very interesting material for building nanoarchitectures in a bottom-up approach. This has already been proven in a number of studies. For example, cubes, octahedra, Borromean rings, as well as tubes (nanowires) have been built with DNA. However, DNA can not only be used as a structural material on a nanometer scale but also as a functional material, for example, to measure temperature force. However, as DNA is a linear polymer, it is difficult to build stable, branched three-dimensional architectures. Branched DNA duplexes offer a possible solution, but complex “tiles” have to be used for stable structures. Other approaches, for example, rely on the use of synthetic DNA derivatives to build trisoligonucleotidyl junctions. Polyamides derived from nonproteinogenic amino acids containing N-methlypyrrole, N-methylimidazole, or N-methylhydroxypyrrole groups can bind in a hairpin motif to the minor groove of double-stranded (ds) DNA. The base sequence to which they can bind selectively and with high affinity can be programmed by the sequence of the respective residues in the two antiparallel strands of the hairpin polyamide. A complete set of pairing rules is available for the straight-forward recognition of the majority of possible DNA sequences. DNA-binding polyamides of this type can be used for a variety of applications, like, for example, gene regulation or as sequence-selective nuclear stains. As most of the names for this class of compound are either lengthy or imprecise, we propose the term “Dervan-type polyamide” in honor of P. B. Dervan who made the major contributions in this field. Herein, we report on the use of Dervan-type polyamides as a possible second orthogonal structural element—besides Watson–Crick base pairing—for DNA-based architectures. Our rationale is that by connecting two hairpin polyamides with a long and flexible linker, we generate “DNA struts” that target their matching binding sites on different DNA duplex strands and can thus function as “sequence-selective glue”, which will make it easier to build up complex stable architectures. Only once before has a Dervan polyamide been shown to bind across twoDNAduplexes, but in this case, the two duplexes were preorganized next to each other in the nucleosome core particle. To test whether Dervan polyamides alone can be used to hold DNA objects together, we synthesized the heterobifunctional strut S-AB shown in Figure 1. It consists of the two

Improved nuclear localization of DNA-binding polyamides

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus ‘tail’ has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.

Exploring the limits of benzimidazole DNA-binding oligomers for the hypoxia inducible factor (HIF) site

The vascular endothelial growth factor (VEGF) and its receptors have been implicated as key-factors in tumor angiogenesis and are major targets in cancer therapy. New oligomers which mimic the architecture of DNA-binding polyamides have been designed to target the hypoxia inducible factor (HIF-1α) binding site on the promoter of VEGF gene. These oligomers incorporate an increasing number of six–five fused rings such as hydroxybenzimidazole–imidazole, benzimidazole–pyrrole, benzimidazole–chlorothiophene, and imidazopyridine–pyrrole, and bind the VEGF hypoxia response element (HRE) 5′-TACGT-3′ with high affinity and selectivity.

Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide

The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene.

Temperature-sensitive protein–DNA dimerizers

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.

Localization of DNA-binding polyamides in living cells.

Regulation of the processing of genes into nucleic acids and proteins is a substantial goal in medicine. Small molecules that could enter cells, localize to the nucleus, and bind chromosomal DNA sequence-specifically and with high affinity would be important tools for gene regulation. Pyrrole-imidazole polyamides are small molecules that bind the minor groove of DNA in a sequence-specific fashion according to a set of pairing rules, and with affinities rivaling natural transcription factors. Several in vitro experiments have shown that by directly competing with transcription factors for binding sites in gene promoter regions, polyamides can act to inhibit transcription of those genes. Polyamides bearing transcription activation domains can bind to promoter regions, recruit the transcriptional machinery to the gene, and activate transcription in vitro. Attempts to reproduce these results in vivo were largely unsuccessful, perhaps due to poor cellular trafficking properties of polyamides and polyamide-peptide conjugates. It was found that polyamides bearing the Bodipy fluorophore localize primarily to the cytoplasm of cells, or were excluded from cells altogether. In attempts to overcome this quality, peptides shown to improve cellular trafficking were appended to the polyamides. These peptides were generally not successful at inducing uptake, and were in many cases toxic to the cells. Small molecules were also appended to polyamides, likewise to improve uptake properties, but met with limited success. Surprisingly, the addition of a fluorescein or fluorescein-like fluorophore to polyamides permit them to localize to the nuclei of all cell lines tested, in a molecular content- and shape-dependent manner. This technology has been applied to several in vivo experiments, including the inhibition of androgen receptor binding to its cognate element in gene promoter regions.

Expanding the Repertoire of Heterocycle Ring Pairs for Programmable Minor Groove DNA Recognition

The discrimination of the four Watson-Crick base pairs by minor groove DNA-binding polyamides have been attributed to the specificity of three five-membered aromatic amino acid subunits, 1-methyl-1H-imidazole (Im), 1-methyl-1H-pyrrole (Py), and 3-hydroxy-1H-pyrrole (Hp) paired four different ways. The search for additional ring pairs that demonstrate DNA-sequence specificity has led us to a new class of 6-5 fused bicycle rings as minor groove recognition elements. The affinities and specificities of the hydroxybenzimidazole/pyrrole (Hz/Py) and hydroxybenzimidazole/benzimidazole (Hz/Bi) pairs for each of the respective Watson-Crick base pairs within the sequence context 5'-TGGXCA-3' (X = A, T, G, C) were measured by quantitative DNaseI footprinting titrations. The Hz/Py and Hz/Bi distinguish T.A from A.T. Hairpin polyamides containing multiple Hz/Py pairs were examined and were shown to mimic the Hp/Py pair with regard to affinity and specificity. Therefore, the Hz/Py pair may be considered a second-generation replacement for the Hp/Py pair.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Polyamides as artificial transcription factors: novel tools for molecular medicine?

Misregulation of gene expression often leads to severe defects in the development of an organism or to uncontrolled cell growth. Artificial transcription factors can control the expression of the genes concerned to restore normal cell regulation. Dervan and co-workers have synthesized several DNA-binding polyamides (see picture: TALE (yellow) is a class of homeobox protein) that show potential for the treatment of diseases such as cancer.

Paradoxical effects of DNA binding polyamides on HTLV-1 transcription.

Human T-cell leukemia virus type-1 (HTLV-1) depends on the virally encoded transcription factor Tax for efficient viral replication and gene expression. In a complex with CREB, Tax contacts the minor groove of the promoter DNA at guanine and cytosine rich sequences that flank three of the off-consensus cyclic-AMP response elements (CREs). In this study, we used six Tax-directed pyrrole-imidazole polyamides specifically designed to block Tax binding to DNA at each GC sequence of the three viral CREs. We found that four of these polyamides disrupt binding of the Tax/CREB complex in vitro, and that these same molecules also inhibit Tax-mediated transcription in vitro on chromatin-assembled templates. However, of these four Tax/CREB-specific polyamides, only one polyamide appears to be uniquely Tax specific. We show that polyamides can enter the nuclei of HTLV-1 infected T-cells, and two of the four polyamides down-regulated virion production in these cells. Together, these data illustrate the importance of studying polyamide inhibition of gene expression in vitro and in vivo, as the function of the polyamides in living cells is not fully understood. Finally, our data indicates that targeted disruption of the Tax/CREB complex, or other complexes which assemble on the HTLV-1 promoter, may provide a novel approach for inhibiting viral replication in vivo.

DNA-templated dimerization of hairpin polyamides.

Double-helical DNA accelerates the rate of ligation of two six-ring hairpin polyamides which bind adjacent sites in the minor groove via a 1,3-dipolar cycloaddition to form a tandem dimer. The rate of the templated reaction is dependent on DNA sequence as well as on the distance between the hairpin-binding sites. The tandem dimer product of the DNA-templated reaction has improved binding properties with respect to the smaller hairpin fragments. Since cell and nuclear uptake of DNA-binding polyamides will likely be dependent on size, this is a minimum first step toward the design of self-assembling small gene-regulating fragments to produce molecules of increasing complexity with more specific genomic targeting capabilities.

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.