Programmable oligomers targeting 5'-GGGG-3' in the minor groove of DNA and NF-kappaB binding inhibition

Abstract

A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened to target 5'-WGGGGW-3', a core sequence in the DNA-binding site of NF-kappaB, a prolific transcription factor important in biology and disease. Five Bi and Ip containing oligomers bound to the 5'-WGGGGW-3' site with high affinity. One of the oligomers (Im-Im-Im-Im-gamma-Py-Bi-Py-Bi-beta-Dp) was able to inhibit DNA binding by the transcription factor NF-kappaB.

Figure 1

Crystal structure of the p50/p65 NF-κB heterodimer bound to the DNA duplex 5′-TGGGGACTTTCC-3′ (ref. 13). The p50 and p65 monomers are represented as gold and dark blue ribbons, respectively. a) Top view looking down the DNA double helix. b) Side view showing the 5′-GGGG-3′ oligomer binding site. GC rich regions are shown in red and AT rich regions are shown in light blue. c) Sequence of DNA bound to NF-κB.

Figure 2

Structures of the (a) pyrrole-benzimidazole internal dimer (-Py-Bi-), (b) imidazole-imidazopyridine internal dimer (-Im-Ip-), and (c) imidazole-imidazopyridine cap (Im-Ip-) in comparison with their respective five membered ring systems. Hydrogen-bonding surfaces to the DNA minor-groove floor are bolded.

Figure 3

Postulated hydrogen-bonding models for the 1:1 polyamide-DNA complexes with their matched sequence and the ball-and-stick representation for compounds 1 and 6 over the 6-base-pair matched binding site (variable region W = A or T). a) Im-Im-Im-Im-γ-Py-Py-Py-Py-β-Dp (1); b) Im-Im-Im-Im-γ-Py-Bi-Py-Bi-β-Dp (2), Im-Im-Im-Ip-γ-Py-Bi-Py-Bi-β-Dp (3), Im-Im-Ip-Im-γ-Py-Bi-Py-Bi-β-Dp (4), Im-Ip-Im-Im-γ-Py-Bi-Py-Bi-β-Dp (5), Im-Ip-Im-Ip-γ-Py-Bi-Py-Bi-β-Dp (6).

Figure 4

Illustration of NF-kB:DNA binding inhibition by oligomer 2.

Figure 5

Quantitative DNase I footprinting experiments in the hairpin motif for polyamides 1, 2, and 3 respectively, on the 278 bp, 5′-end-labelled PCR product of plasmid pEF16: lane 1, intact DNA; lane 2, G and A sequencing reaction; lane 3, DNase I standard; lanes 5–15, 1 pM, 3 pM, 10 pM, 30 pM, 100 pM, 300 pM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM polyamide concentration, respectively. Each footprinting gel is accompanied by the following: (top) Ball-and-stick models of the parent polyamide; and (bottom) Binding isotherms for the four designed sites. θ_norm values were obtained according to published methods. A binding model for the hairpin motif is shown centered at the top as a ball-and-stick model with the polyamide bound to its target DNA sequence. Imidazoles and pyrroles are shown as filled and non-filled circles, respectively; Dimer units are represented as rectangles containing either Bi enclosed in a circle representing benzimidazole or Ip in a black box representing imidazopyridine; Beta alanine is shown as a diamond; the gamma-aminobutyric acid turn residue is shown as a semicircle connecting the two subunits.

Figure 6

Quantitative DNase I footprinting experiments in the hairpin motif for polyamides 4, 5, and 6 respectively, on the 278 bp, 5′-end-labelled PCR product of plasmid pEF16: lane 1, intact DNA; lane 2, G and A sequencing reaction; lane 3, DNase I standard; lanes 5–15, 1 pM, 3 pM, 10 pM, 30 pM, 100 pM, 300 pM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM polyamide concentration, respectively. Each footprinting gel is accompanied by the following: (top) Ball-and-stick models of the parent polyamide; and (bottom) Binding isotherms for the four designed sites. θ_norm values were obtained according to published methods. A binding model for the hairpin motif is shown centered at the top as a ball-and-stick model with the polyamide bound to its target DNA sequence. Imidazoles and pyrroles are shown as filled and non-filled circles, respectively; Dimer units are represented as rectangles containing either Bi enclosed in a circle representing benzimidazole or Ip in a black box representing imidazopyridine; Beta alanine is shown as a diamond; the gamma-aminobutyric acid turn residue is shown as a semicircle connecting the two subunits.

Figure 7

a) Matched DNA sequence with the p50 protein of NF-κB overlapping the oligomer binding site. Asterisks indicate the location of radio-labeled nucleotides in the probe sequence. b) Mismatch DNA sequence. c) Gel shift screen for compounds 1–6 at concentrations of 10 and 100 nM. d) Plot of relative NF-κB inhibition for compounds 1–6.

Figure 8

(Top) Ball-and-stick model for 2 and EC50 value from gel shift experiment. (Middle) Representative NF-κB titration gel (n=3) for 2. (Bottom) Binding isotherm for 2.

Figure 9

Antibody supershift on Match DNA. The NF-κB band is shifted in the presence of anti-p50 or anti-p65 antibody.

Scheme 1

Synthesis of imidazopyridine-imidazole dimers Im-Ip-OH (9) and Boc-Im-Ip-OH (12). i) 8, PhNO₂, 140°C; ii) Oleum, red-fuming HNO₃; iii) 8, PhNO₂, 140°C; iv) H₂, Pd/C, DMF; (Boc)₂O, DIEA, DMAP, DMF; 4N KOH, MeOH.

Scheme 2

Representative solid-phase synthesis of polyamide 6 along with a table of the amino acid building blocks used for the synthesis. Reaction conditions: (i) 80% TFA/DCM; (ii) Boc-Py-Bi-OH (13), HBTU, DIEA, DMF; (iii) Ac₂O, DIEA, DMF; (iv) repeat i – iii; (v) 80% TFA/DCM; (vi) Boc-γ-OH (14), HBTU, DIEA, DMF; (vii) Ac₂O, DIEA, DMF to provide BR1; (viii) 80% TFA/DCM; (ix) Boc-Im-Ip-OH (12), HBTU, DIEA, DMF; (x) 80% TFA/DCM; (xi) Im-Ip-OH, HBTU, DIEA, DMF; (xii) dimethylaminopropylamine (Dp), 80 °C 2 h; (xiii) preparative HPLC to give 6.