Allosteric modulation of DNA by small molecules

Abstract

Many human diseases are caused by dysregulated gene expression. The oversupply of transcription factors may be required for the growth and metastatic behavior of human cancers. Cell permeable small molecules that can be programmed to disrupt transcription factor-DNA interfaces could silence aberrant gene expression pathways. Pyrrole-imidazole polyamides are DNA minor-groove binding molecules that are programmable for a large repertoire of DNA motifs. A high resolution X-ray crystal structure of an 8-ring cyclic Py/Im polyamide bound to the central 6 bp of the sequence d(5'-CCAGGCCTGG-3')2 reveals a 4 A widening of the minor groove and compression of the major groove along with a >18 degrees bend in the helix axis toward the major groove. This allosteric perturbation of the DNA helix provides a molecular basis for disruption of transcription factor-DNA interfaces by small molecules, a minimum step in chemical control of gene networks.

Fig. 1.

Chemical structure of the cyclic polyamide and DNA sequence. Cyclic polyamide 1 targeting the sequence 5′-WGGCCW-3′ shown with ball-and-stick model superimposed onto the DNA oligonucleotide used for crystallization. Black circles represent imidazoles, open circles represent pyrroles, and ammonium substituted half circles at each end represent the (R)-α-amine-γ-turn.

Fig. 2.

Comparison of native DNA to polyamide/DNA complex. (A) Native DNA crystal structure at 0.98 Å resolution. (B) Comparison to DNA/polyamide co-crystal structure at 1.18 Å resolution. (Both structure solved by direct methods.) (C) Analysis of native DNA (yellow) compared to polyamide complexed DNA (blue). Chart on the top left shows variation in the minor groove width for native DNA (yellow) and polyamide-complexed DNA (blue) over the central core sequence 5′-GGCC-3′. Chart on the bottom left shows variation in the major groove width for native DNA (yellow) and polyamide complexed DNA (blue) over the central core sequence 5′-GGCC-3′. Overlay of the curves calculated geometric helix model from each structure showing a DNA bend of >18° in the polyamide/DNA complex compared to native DNA.

Fig. 3.

Conformation of the α-amino substituted GABA turn. Two possible Conformations A and B are shown with conformation A directing the β-methylene up and away from the minor groove floor while orienting the α-ammonium toward the minor groove wall. Conformation B presents the β-methylene down toward the minor-groove floor while orienting the α-ammonium up and out of the minor-groove, relieving possible steric interaction with the sugar-phosphate backbone (minor-groove wall). View looking down the DNA minor-groove, showing the (R)-α-amine-γ-turn conformation observed in the X-ray crystal structure, which matches that of conformation B. Electron density map is contoured at the 1.0 σ level.

Fig. 4.

Direct and water-mediated noncovalent molecular recognition interactions. (A) Geometry of the α-amino turn interacting with the AT base pair through water-mediated hydrogen bonding interactions. Structural basis for the turn preference for AT versus GC is demonstrated by the β-methylene conformational preference, which points down toward the DNA minor-groove floor within van der Waals contact distance of the adenine base. (B) Isolated view of one-half of the macrocyclic-polyamide showing hydrogen bond distances made to the DNA minor groove floor by the imidazoles and amides of compound 1. (C) Im-Py pair showing the mechanism for GC specificity. (D) Interaction of the O4′ oxygen of a deoxyribose sugar with the terminal imidazole aromatic ring through a lone-pair-π interaction. The sugar conformation is C2′-endo at the N-terminal imidazole of the polyamide with the sugar oxygen lone-pair pointing directly to the centroid of the imidazole ring. The distance between the sugar oxygen and the ring centroid is 2.90 Å, which is less than the sum of the van der Waals radii to any atom in the imidazole ring. Electrostatic potential maps calculated at the HF/3–21g level of theory show the slightly electropositive nature of the imidazole ring under these conditions (Fig. S6). (E) View of the O4′ deoxyribose oxygen atom looking through the imidazole ring showing the ring centroid superimposed on the oxygen atom. All distances are reported in angstroms (Å), and all electron density maps are contoured at the 1.0 σ level (Im, imidazole; Py, pyrrole).