Inhibition of human papilloma virus E2 DNA binding protein by covalently linked polyamides

Abstract

Polyamides are a class of heterocyclic small molecules with the potential of controlling gene expression by binding to the minor groove of DNA in a sequence-specific manner. To evaluate the feasibility of this class of compounds as antiviral therapeutics, molecules were designed to essential sequence elements occurring numerous times in the HPV genome. This sequence element is bound by a virus-encoded transcription and replication factor E2, which binds to a 12 bp recognition site as a homodimeric protein. Here, we take advantage of polyamide:DNA and E2:DNA co-crystal structural information and advances in polyamide synthetic chemistry to design tandem hairpin polyamides that are capable of displacing the major groove-binding E2 homodimer from its DNA binding site. The binding of tandem hairpin polyamides and the E2 DNA binding protein to the DNA site is mutually exclusive even though the two ligands occupy opposite faces of the DNA double helix. We show with circular permutation studies that the tandem hairpin polyamide prevents the intrinsic bending of the E2 DNA site important for binding of the protein. Taken together, these results illustrate the feasibility of inhibiting the binding of homodimeric, major groove-binding transcription factors by altering the local DNA geometry using minor groove-binding tandem hairpin polyamides.

Figure 1

Schematic representation of single and tandem hairpin polyamides designed to target the HPV E2-DNA binding site. The N-methylpyrrole and N-methylimidazole rings are represented by open circles and filled circles, respectively. The flexible β-alanine monomer is represented by a diamond symbol. The γ-turn monomer, R-2,4-diaminobutyric acid, is indicated by a curved line. The dimethylaminopropylamide (Dp) at the C-terminus is represented by a plus sign enclosed within a half-circle. The position of the various flexible linkers (mono-, di-, tetra- and hexaethylene) used in this study is indicated by an open rectangle. The relationship between the polyamide nomenclature and type of linker is indicated in Table 1.

Figure 2

Quantitative DNase I footprinting titrations with the tandem hairpin PA1 and its component single hairpin polyamides. The composite schematic relationship between these molecules is shown to the left of the autoradiogram. The footprinting analysis was performed on a DNA restriction fragment encoding six identical copies of the HPV-18 E2 binding site 4 (BS4) (18). Lane 1 contained no polyamide. The DNA was incubated with increasing concentrations of tandem hairpin PA1: 0.01 (lane 2), 0.1 (lane 3), 1 (lane 4), 10 (lane 5) and 100 nM (lane 6). Concentrations of polyamides PA3 and PA2 surveyed were 0.1 (lanes 7 and 12), 1 (lanes 8 and 13), 10 (lanes 9 and 14), 100 (lanes 10 and 15) and 1000 nM (lanes 11 and 16). Note that the concentration ranges tested for the tandem hairpin and single hairpins differ by an order of magnitude. Vertical bars on the right of the figures represent the six tandem BS4 sites.

Figure 3

Order of addition experiments with E2dbd and polyamides. (A) An EMSA was performed in which the radiolabeled HPV E2 BS4 probe was preincubated with polyamide (or the vehicle DMSO) under equilibrium conditions prior to the addition of E2dbd as described in Materials and Methods. DNA was incubated in the absence of either polyamide (lane 1) or E2dbd (lanes 2–6) or preincubated with DMSO (lane 2) or 0.13 (lane 3), 0. 64 (lane 4), 3.2 (lane 5) or 16 nM PA1 (lane 6) prior to the addition of a fixed concentration of the DNA binding domain of E2 (10 nM). (B) An EMSA was performed in parallel in which the radiolabeled HPV E2 BS4 probe was preincubated with the DNA binding domain of E2 under equilibrium conditions prior to the addition of 10 nM polyamide PA1 (or the vehicle DMSO) as described in Materials and Methods. DNA was preincubated with E2dbd (lanes 1–5) prior to the addition of a variable concentration of DMSO (lane 1) or 0.13 (lane 2), 0. 64 (lane 3), 3.2 (lane 4) or 16 nM PA1 (lane 5).

Figure 4

Competition experiments with E2dbd and polyamides. (A) EMSA was performed with a radiolabeled HPV E2 BS4 probe in reactions lacking E2dbd (lane 1) or containing a constant concentration of E2dbd (10 nM; lanes 2–25). Reactions were supplemented with the vehicle DMSO (lane 2) or polyamide PA1 (lanes 3–10), PA3 (lanes 11–18) or PA2 (lanes 19–26) diluted in DMSO. Polyamide concentrations tested were 0.13 (lanes 3, 11 and 19), 0.64 (lanes 4, 12 and 20), 3.2 (lanes 5, 13 and 21), 16 (lanes 6, 14 and 22), 80 (lanes 7, 15 and 23) and 400 nM (lanes 8, 16 and 24) and 2 (lanes 9, 17 and 25) and 10 µM (lanes 10, 18 and 26). (B) Quantitative DNase I footprinting assays were performed with the radiolabeled restriction fragment described in the legend to Figure 2. Reactions were performed in the absence of E2dbd (lanes 1–4) or in the presence of a constant concentration of E2dbd (lanes 5–9). Polyamide PA1 was titrated at the following concentrations: 0 (lane 5), 0.1 (lanes 1 and 6), 1 (lanes 2 and 7), 10 (lanes 3 and 8) and 100 nM (lanes 4 and 9). A magnification of lanes 4 and 5 in the main autoradiogram is shown as an inset to the left. Horizontal arrows indicate unique hypersensitivity bands characteristic of polyamide PA1 binding (lane 4) or E2dbd binding (lane 5) to a BS4 DNA site.

Figure 5

The effect of linker length on tandem hairpin functionality was assayed by EMSA. The assay was performed as described in the legend to Figure 3A. Control reactions were performed in the absence or the presence of E2dbd (lanes 1 and 2, respectively). Competition experiments with tandem polyamides PA5 (lanes 3–10), PA6 (lanes 11–18) and PA1 (lanes 19–26) are shown. Netropsin, a natural product containing two pyrroles, is used as a reference standard in this experiment (lanes 27–34). Polyamide concentrations surveyed were 0.13 (lanes 3, 11, 19 and 27), 0.64 (lanes 4, 12, 20 and 28), 3.2 (lanes 5, 13, 21 and 29), 16 (lanes 6, 14, 22 and 30), 80 (lanes 7, 15, 23 and 31) and 400 nM (lanes 8, 16, 24 and 32) and 2 (lanes 9, 17, 25 and 33) and 10 µM (lanes 10, 18, 26 and 34).

Figure 6

A circular permutation assay was utilized to determine the effect of tandem hairpin PA1 on the intrinsic bend of a probe encoding the E2-DNA binding site. In this variation on an EMSA, a DNA fragment with a centralized bend will result in a lower mobility fragment whereas a DNA with a terminally positioned bend will result in a higher mobility fragment (57). (A) A schematic showing the relative positions of the tandemly duplicated restriction sites found on the radiolabeled probe encoding the BS4 E2-DNA binding site. (B) A circular permutation assay showing the radiolabeled probe restricted with the following enzymes: MluI (lane 1), NheI (lane 2), SpeI (lane 3), PvuII (lane 4), XhoI (lane 5), EcoRV (lane 6), NruI (lane 7), RsaI (lane 8) and BamHI (lane 9). (C) The circular permutation assay described in (B) is supplemented with 10 nM tandem hairpin polyamide PA1.

Figure 7

Model for the inhibition of DNA binding by the major groove-binding HPV-18 E2 homodimer by a reversal of an intrinsic bend in the DNA by a minor groove-binding tandem hairpin polyamide. (A) A hypothetical model is shown for the HPV-18 E2 accentuation of an intrinsic bend in the DNA binding site is based on the enhanced affinity of HPV-16 E2 protein for pre-bent DNA sequences containing adenosine tracts (31) and the increased DNA bending observed in X-ray crystallography studies on BPV-1 E2:DNA co-crystals (33,47). (B) A model for the reversal of the intrinsic bend of the adenosine tract-containing the E2-DNA binding site by the minor groove-binding tandem hairpin polyamide is based on the inhibition of E2 binding experiments shown in Figure 3 and the circular permutation experiment shown in Figure 6.