Eldon E. Baird

Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands

The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach for the regulation of gene expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity,. Although oligonucleotides and their analogues have been shown to interfere with gene expression,, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G·C from C·G and both of these from A·T/T·A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish A·T from T·A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A·T from T·A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole–imidazole–pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson–Crick base pairs in the minor groove of DNA.

On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides.

BACKGROUND Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (Im) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G.C and A.T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G.C base pairs. RESULTS Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G.C and A.T base pairs in the central position. We observed that a G.C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A.T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im. CONCLUSIONS An Im/Im pairing is energetically disfavored for the recognition of both A.T and G.C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.

Stereochemical Control of the DNA Binding Affinity, Sequence Specificity, and Orientation Preference of Chiral Hairpin Polyamides in the Minor Groove

Three-ring polyamides containing pyrrole (Py) and imidazole (Im) amino acids covalently coupled by γ-aminobutyric acid ( γ) form six-ring hairpins that recognize five-base-pair sequences in the minor groove of DNA. Selective chiral substitution of the “ γ-turn” enhances the properties of polyamide hairpins with regard to DNA affinity and sequence specificity. Polyamides of core sequence composition ImPyPyγPy which differ by selective stereochemical substitution of the prochiral R-position in theγ-turn were prepared. The DNA binding properties of two enantiomeric polyamides were analyzed by footprinting and affinity cleavage on a DNA fragment containing two match sites (5 ′-TGTTA-3′ and 5′-ACATT-3′) and one 5′-TGTCA-3′ mismatch site. Quantitative footprint titrations demonstrate that replacement of γ-aminobutyric acid by ( R)2,4-diaminobutyric acid enhances DNA binding affinity for the 5 ′-TGTTA-3′ match site 13-fold ( Ka ) 3.8× 109 M-1). The enhanced affinity is achieved without a compromise in sequence selectivity, which in fact increases and is found to be 100-fold higher relative to binding at a single base pair mismatch sequence, 5′-TGTCA-3′. An (S)-2,4-diaminobutyric acid linked hairpin binds with 170-fold reduced affinity relative to theR-enantiomer and only 5-fold sequence specificity versus a 5 ′-ACATT-3′ reversed orientation site. These effects are modulated by acetylation of the chiral amine substituents. This study identifies structural elements which should facilitate the design of new hairpin polyamides with improved DNA binding affinity, sequence specificity, and orientational selectivity.

Inhibition of Ets-1 DNA Binding and Ternary Complex Formation between Ets-1, NF-kappa B, and DNA by a Designed DNA-binding Ligand

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-κB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1·NF-κB·DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-κB also inhibits NF-κB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Inhibition of major-groove-binding proteins by pyrrole-imidazole polyamides with an Arg-Pro-Arg positive patch.

BACKGROUND Gene-specific targeting of any protein-DNA complex by small molecules is a challenging goal at the interface of chemistry and biology. Polyamides containing N-methylimidazole and N-methylpyrrole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to many naturally occurring DNA-binding proteins. It has been shown that an eight-ring hairpin polyamide targeted to a specific minor-groove contact within a transcription factor binding site can inhibit protein-DNA binding and gene transcription. Polyamides and certain major-groove-binding proteins have been found to co-occupy the DNA helix, however. To expand the number of genes that can be targeted by pyrrole/imidazole polyamides, we set out to develop a class of polyamides that can selectively inhibit major-groove-binding proteins. RESULTS An eight-ring hairpin polyamide conjugated to a carboxy-terminal Arg-Pro-Arg tripeptide was designed to deliver a positive residue to the DNA backbone and interfere with protein-phosphate contacts. Gel mobility shift analysis demonstrated that a polyamide hairpin-Arg-Pro-Arg binding in the minor groove selectively inhibits binding of the transcription factor GCN4 (222-281) in the adjacent major groove. Substitution within the Arg-Pro-Arg revealed that each residue was required for optimal GCN4 inhibition. CONCLUSIONS A pyrrole-imidazole polyamide that binds to a predetermined site in the DNA minor groove and delivers a positive patch to the DNA backbone can selectively inhibit a DNA-binding protein that recognizes the adjacent major groove. A subtle alteration of the DNA microenvironment targeted to a precise location within a specific DNA sequence could achieve both gene-specific and protein-specific targeting.

Tandem Hairpin Motif for Recognition in the Minor Groove of DNA by Pyrrole–Imidazole Polyamides

Linking hairpin recognition units “tail-to-turn” in the minor groove of DNA results in the extension of hairpin polyamide binding site size to 11 base pairs. A twelve-ring tandem hairpin (see diagram) binds its match DNA site at picomolar concentrations with unprecedented sequence specificity.

Promoter Scanning for Transcription Inhibition with DNA-Binding Polyamides

ABSTRACT When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.

DNA Binding Ligands with Excellent Antibiotic Potency Against Drug-Resistant Gram-Positive Bacteria

An efficient synthesis of DNA binding molecules consisting of four heterocyclic carboxamide units and various substituents at both termini is described. The minor-groove binding ligands showed excellent activity against a broad range of Gram-positive bacteria; no cross-resistance to known antibacterial drugs was observed.

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole.

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.

Cooperative hairpin dimers for recognition of DNA by pyrrole-imidazole polyamides

A doubling of the length of binding site for the same size of ligand is achieved by the title compound by formation of a cooperative hairpin dimer on binding to DNA (depicted schematically below). The binding affinity and selectivity are unaffected by this new binding pattern. Circles represent heterocyclic rings, and diamonds and curved lines represent β-alanine and (R)-2,4-diaminobutyric acid residues, respectively.