A small library of pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugates was screened for effects on morphology and growth characteristics of a human colon carcinoma cell line, and a compound was identified that causes cells to arrest in the G2/M stage of the cell cycle. Microarray analysis indicates that the histone H4c gene is significantly downregulated by this polyamide. RT-PCR and Western blotting experiments confirm this result, and siRNA to H4c mRNA yields the same cellular response. Strikingly, reduction of H4 protein by >50% does not lead to widespread changes in global gene expression. Sequence-specific alkylation within the coding region of the H4c gene in cell culture was confirmed by LM-PCR. The compound is active in a wide range of cancer cell lines, and treated cells do not form tumors in nude mice. The compound is also active in vivo, blocking tumor growth in mice, without obvious animal toxicity.

During the past 20 years, polyamides have evolved from the natural product distamycin to a new class of programmable heterocyclic oligomers that bind a broad repertoire of DNA sequences with high affinity and specificity. This chapter details recent advances in this field of research, focusing on molecular recognition of DNA, and biological applications such as modulating gene expression by small molecules. Work presented here represents efforts towards the modulation of specific cellular function by small molecules in an addressable fashion within the context of live cells.

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.

INTRODUCTION. Our laboratories have pioneered the use of sequencespecific pyrrole-imidazole (Py-Im) polyamides as regulators of mammalian gene expression [1]. Py-Im polyamides are the only available class of DNA ligands that can be designed to bind predetermined DNA sequences, with affinities and specificities comparable to those of natural eukaryotic DNA-binding transcription factors. DNA recognition depends on side-by-side amino acid pairing in the minor groove (Fig. 1). An Im/Py pair targets a G•C base pair, while Py/Im targets a C•G base pair. Py/Py, β-alanine (β)/β and β/Py pairs are degenerate and target both A•T and T•A base pairs. Motifs utilizing various combinations of rings have proved effective in targeting a wide range of DNA sequences [1].

Many diseases, such as cancer, are related to aberrant gene expression. Regulating transcription by chemical methods could be important in human medicine. Minor groove-binding polyamides offer one chemical approach to DNA recognition.

A series of hairpin pyrrole–imidazole polyamide–fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 μM polyamide–fluorescein conjugates for 10–14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a β-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the β-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole–imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an ≈10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

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.