Sequence-specific minor groove binding by bis-benzimidazoles: water molecules in ligand recognition

Abstract

The binding of two symmetric bis-benzimidazole compounds, 2,2-bis-[4'-(3"-dimethylamino-1"-propyloxy)phenyl]-5,5-bi-1H-benzimidazole and its piperidinpropylphenyl analog, to the minor groove of DNA, have been studied by DNA footprinting, surface plasmon resonance (SPR) methods and molecular dynamics simulations in explicit solvent. The footprinting and SPR methods find that the former compound has enhanced affinity and selectivity for AT sequences in DNA. The molecular modeling studies have suggested that, due to the presence of the oxygen atom in each side chain of the former compound, a water molecule is immobilized and effectively bridges between side chain and DNA base edges via hydrogen bonding interactions. This additional contribution to ligand-DNA interactions would be expected to result in enhanced DNA affinity, as is observed.

Figure 1

Structure of the drugs used in this study.

Figure 2

Sequence selective binding. The gels show DNase I footprinting with (A) 117mer and (B) 178mer PvuII–EcoRI restriction fragments cut from the plasmids pBS and pKS, respectively. In both cases, the DNA was labeled at the EcoRI site with [α-³²P]dATP in the presence of AMV reverse transcriptase. The products of nuclease digestion were resolved on an 8% polyacrylamide gel containing 7 M urea. Control tracks (Cont) contained no drug. The concentration (µM) of the drug is shown at the top of the appropriate gel lanes. Tracks labeled ‘G’ represent dimethylsulfate-piperidine markers specific for guanines. Numbers on the side of the gels refer to the standard numbering scheme for the nucleotide sequence of the DNA fragment.

Figure 3

Differential cleavage plots comparing the susceptibility of (A) the 117mer and (B) the 265mer pBS restriction fragments to DNase I cutting in the presence of the bis-benzimidazole compounds (2 µM each). Negative values correspond to a ligand-protected site and positive values represent enhanced cleavage. Vertical scales are in units of ln(fa) – ln(fc), where fa is the fractional cleavage at any bond in the presence of the drug and fc is the fractional cleavage of the same bond in the control, given closely similar extents of overall digestion. Each line drawn represents a 3-bond running average of individual data points, calculated by averaging the value of ln(fa) – ln(fc) at any bond with those of its two nearest neighbors. Only the region of the restriction fragment analyzed by densitometry is shown. Black boxes indicate the positions of inhibition of DNase I cutting in the presence of the drugs.

Figure 4

DNase I footprinting of compound 1 and Hoechst 33258 on the 174 bp PvuII–EcoRI DNA fragment from plasmid pKS. Numbers on the side of the gel refer to the standard numbering scheme for the nucleotide sequence of the DNA fragment, as indicated in Figure 5. Other details as for Figure 2.

Figure 5

Differential cleavage plots comparing the susceptibility of the 174mer pKS DNA fragment to DNase I cutting in the presence of compound 1 or Hoechst 33258 at (A) 1 or (B) 3 µM. Filled boxes indicate the positions of binding sites common to the two compounds. Open boxes refer to the position of binding sites specific to compound 1.

Figure 6

BIAcore SPR sensorgrams for the interaction of compounds 1 and 3 with the alternating AT and GC sequence DNA hairpins in HBS buffer at 25°C. Both compounds bind strongly to the AT sequence and reach saturation of the DNA in the concentration range of this experiment, 1–500 nM compound. In the same concentration range, no binding of compound 1 to the GC sequence is observed while significant binding of compound 3 can be detected. Fitting of results from these and additional experiments in the steady-state region provided data for determination of compound binding constants, as described in the Materials and Methods, and these are collected in Table 1.

Figure 7

BIAcore SPR sensorgrams for the complexes of compounds 1 and 3 with the AATT DNA minor groove in HBS buffer at 25°C. Both compounds bind strongly to the AATT sequence and reach saturation at concentrations below 500 nM. Global fitting of the curves to obtain association and dissociation kinetics constants was done with BIA Evaluation software and a single site interaction model (Materials and Methods). The best-fit lines through each experimental plot are also shown in the Figure and as can be seen, the global, single-site model provides excellent fits to all of the experimental curves. Similar fits with the other DNA samples provided the kinetics constants in Table 1.

Figure 8

Plots of the averaged MD structure of the complex of compound 1 with d(CGCGAATTCGCG)₂, showing the water molecule bridging between the ligand and base edges. Hydrogen bonds are shown as dashed lines.

Figure 9

Plots of the averaged MD structure of the complex of compound 3 with d(CGCGAATTCGCG)₂, showing the sole water molecule in the vicinity of the ligand–DNA interface.

Figure 10

Plots of the solvent-accessible surface of the d(CGCGAATTCGCG)₂ duplex together with the van der Waals surface of each ligand, viewed down one end of the minor groove for each averaged structure, (A) showing the complex with ligand 1, with the bound water molecule shown in green, and (B) showing the complex with ligand 3. A small void in the groove is apparent between this ligand and the DNA.

Figure 10

Plots of the solvent-accessible surface of the d(CGCGAATTCGCG)₂ duplex together with the van der Waals surface of each ligand, viewed down one end of the minor groove for each averaged structure, (A) showing the complex with ligand 1, with the bound water molecule shown in green, and (B) showing the complex with ligand 3. A small void in the groove is apparent between this ligand and the DNA.