53

OLIGONUCLEOTIDESVolume 19, Number 1, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/oli.2008.0169

Benzimidazoles: A Minor Groove-Binding Ligand-Induced Stabilization of Triple Helix

Akash K. Jain, Sharad K. Gupta, Urmila Tawar, Sneh K. Dogra, and Vibha Tandon

Nonintercalating minor groove-binding ligands netropsin, Hoechst 33258, and DAPI are reported to destabilize the triplex. Ligands with different substitutions on the phenyl ring of bis- and terbenzimidazoles were evalu-ated for their effect on the stability of C+.GC triplex and Hoogsteen duplex. We found that newly synthesized benzimidazoles stabilize the triplex as shown by fl uorescence and melting studies. Modeling studies showed that these molecules bind in the Watson–Crick minor groove of triplex, which can exert a profound impact on the properties of the host triplex. Circular dichroism–binding studies indicate 5.77 base triplets/ligand as an apparent binding site for bis- and 8.66 for terbenzimidazoles. The stabilization of triplex can be attributed to the protonation of nitrogens and amines of benzimidazoles at pH 5.2 that compensate the negative charge of phos-phate backbone to reduce the repulsion between the strands resulting in the stabilization.

Introduction

Triplex DNA is of immense interest as a target for small molecule therapeutic agents (Knauert and Glazer, 2001;

Vasquez et al., 2002). Two types of triplexes are known, which vary in composition and orientation of third strand (TFO). Pyrimidine-rich third strand bind parallel to the purine strand of the duplex and identifi ed by the forma-tion of T.AT and C+.GC triplex, whereas purine rich third strand bind in antiparallel orientation generating A.AT and G.GC triplets (Keppler and Fox, 1997). The C+.GC triplet can be formed at low pH (<6.0), required for the protonation of third strand cytosine. The C+.GC triplets are particularly unstable as a result of repulsion between adjacent positive charges (Kiessling et al., 1992). In an attempt to stabilize C+.GC triplex two kinds of approaches were taken, fi rst, modifi cation of nucleic acid bases (Xiang et al., 1994; Bates et al., 1996) and second to use ligands that bind to triplex (Mergny et al., 1992).

DNA minor groove-binding ligands (MGBLs) are also shown to bind to triplex (Park and Breslauer, 1992). Few MGBLs can be used to induce the formation of triplex, for example, binding of berenil or DAPI (Pilch et al., 1994). Some MGBLs like distamycin and Netropsin destabilize the triplex (Park and Breslauer, 1992). Hoechst 33258 were also found to destabilize the triplexes containing T.A.T triplets (Durand et al., 1994; Kim et al., 1996). But it stabilizes the triplex when conjugated at the 5′ end of the TFO (Robles et al., 1997).

It appears from most of the data that minor groove bind-ers can modulate the affi nity and selectivity of the interac-tion of the major groove-bound third strand (Fortsch et al., 1996). Hoogsteen duplexes (HDs) are part of pyrimidine-motif triplexes, so their studies are helpful to understand the properties of triplexes (van de Sande et al., 1988).

This paper investigates the interaction of triplex, HD and Watson–Crick (WC)-duplex with novel bis- and terbenz-imidazoles along with the parent compound Hoechst 33258. Three new terbenzimidazoles are 5-(4-methylpiperazin-1-yl)-2-[2′-{2″-(4-hydroxy-3-methoxyphenyl)-5″-benz-imidazolyl} -5′-benzimidazolyl] benzimidazole (TBZ), 5-(4-methylpiperazin-1-yl)-2-[2′-{2″-(3,4-dimethoxyphenyl)-5″-benzimidazolyl}-5′-benzimidazolyl]-benzimidazole (DMTBZ) and 5-(4-methylpiperazin-1-yl)-2-[2′-{2″-(4-hydroxy-3-ethoxyphenyl) -5″-benzimidazolyl}-5′-benzimidazolyl]-be-nzimidazole (ETTBZ) and three new bisbenzimidazoles are 5-(4-methylpiperazin-1-yl)-2-[2′-(3-ethoxy,4-hydoxyphenyl)-5′-benzimidazolyl]-benzimidazole(ETBBZ), 5-(4-met- hylpiperazin-1-yl) -2-[2′-(4-hydroxy,3-methoxyphenyl)-5′-benzimidazolyl]- benzimidazole (MMBBZ), and 5-(4-methylpiperazin-1-yl)-2-[2′-(4-cyanophenyl)-5′-benzimidazolyl]-benzimidazole (BBZCN) synthesis of these ligands has been communicated to Bioorganic and Medicinal Chemistry, (Gupta et al., 2008). We have observed that all benzimidazoles including parent compound Hoechst 33258 stabilize triplex, while these ligands do not have any

Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.

07-OLI_19-1_0169.indd 53 2/26/2009 4:24:39 AM

JAIN ET AL.54

AGA AG-3′ (oligo-2), 5′-CTT CTT TCT TTT TTC TTC TTT TCT CT-3′ (oligo-3). Watson–Crick–duplex was prepared by mixing equimolar solutions of oligo-2 and oligo-3, HD from oligo-1 and oligo-2 and triplex from all three oligos in sodium cacodylate buffer (20 mM sodium cacodylate, 100 mM NaCl, at pH 5.2, pH 6.5, and pH 7.2) heated to 75°C for 3 minutes, cooled slowly to room temperature (r.t.) (Nagaich et al., 1994; Liu et al., 1993).

Electrophoretic mobility shift assay

Oligo-1, Oligo-2, Oligo-3, and 24, 26, 28-mer markers were 5′ end labeled with [γ−32P] ATP using T4 polynucleotide kinase (MBI Fermentas) and 20% denaturing gel (containing 8 M urea) was run to check the purity of oligonucleotides in 89 mM trisborate (pH 8) at 8 V/cm for 2 hours and exposed to Kodak fi lm at −80°C (Fig. 1A).

The labeled oligonucleotide was hybridized with equimo-lar amount of complementary strands to make the labeled duplex and triplex (oligo-1* and oligo-2 for HD oligo-2 and oligo-3* for WC duplex, oligo-1*, oligo-2, and oligo-3 for tri-plex,* indicate labeled strand) in 3 μL of 3X buffer (135 mM Tris-acetate, pH 5.2 containing 30 mM MgCl2) to a fi nal 10 μL reaction volume. Solutions were heated to 75°C for 3 minutes. and then cooled slowly to r.t. Electrophoresis was carried out onto a 10% native polyacrylamide gel in 50 mM tris-acetate, pH 5.2 having 10 mM MgCl2 at 8 V/cm for 10 hours at 10°C and developed as above (Fig. 1B).

apparent effect on HD stability, in our experimental condi-tions. We hereby conclude by our experiments that benzimi-dazoles nitrogens get protonated at acidic pH 5.2; hence the positive charge on benzimidazoles reduces overall repul-sion between the duplex and the TFO, and thus stabilize the TFO. To prove electrostatic interactions that play major role in triplex stabilization, we have performed thermal melt-ing experiments at different pH. (pH-5.2, 6.5, and 7.2). Our results clearly suggest that the stability of triplex is decreas-ing gradually as deprotonation of benzimidazoles nitrogens takes place when increasing pH from 5.2 to pH-7.2.

Materials and Methods

Characterization of ligands by IR, NMR, and mass spectroscopy

ETTBZ IR: 3436, 3196, 1632, 1561, 1413, 1282, 1132, 824; 1H NMR (DMSO-d6): δ 13.4–13.55 (b, 3H), 9.5 (s, 1H), 8.45 (s, 1H), 8.39 (s, 1H), 8.03–8.08 (m, 4H), 7.9 (d, 1H), 7.68 (d, 1H), 7.38 (d, 1H), 6.91–7.05 (m, 3H), 3.92 (q, 2H), 3.23 (t, 4H), 2.60 (t, 4H), 2.58 (s, 3H), 1.25 (t, 3H); m/z (MALDI): 586.2 (M+1), calculated 585.

DMTBZ IR: 3438, 3195, 1633, 1562, 1410, 1285, 1130, 825; 1H NMR (DMSO-d6): δ 13.4–13.55 (b, 3H, 9.5 (s, 1H), 8.45 (s, 1H), 8.39 (s, 1H), 8.03–8.08 (m, 4H), 7.9 (d, 1H), 7.68 (d, 1H), 7.38 (d, 1H), 6.91–7.05 (m, 3H), 3.92 (s, 3H), 4.09 (s, 3H), 2.60 (t, 4H), 2.58 (s, 3H); m/z (MALDI): 585.1 (M+), calculated 585.

ETBBZ IR: 3405, 2922, 1636, 1497, 1446, 1371, 1281, 1131, 821 cm-1; 1H NMR (DMSO-d6): δ 13.0 (bs, 2H), 8.3 (s, 1H), 8.17 (d, 2H), 8.03 (m, 1H), 7.81 (d, 1H), 7.64 (d, 1H), 7.13 (d, 2H), 6.77 (d, 1H), 3.98 (q, 2H), 3.5 (t, 4H), 2.33 (s, 3H), 1.25 (t, 3H); m/z (FAB): 469 (M+1), calculated 468.

MMBBZ IR: 3401, 2922, 1635, 1497, 1446, 1371, 1281, 1131, 819 cm-1; 1H NMR (DMSO-d6): δ 2.33 (s, 3H), 3.5 (t,4H), 3.91 (s,3H), 6.77 (d,1H), 7.13 (d,2H), 7.64 (d,1H), 7.81 (d,1H), 8.03 (m,1H),8.17(d,2H), 8.3 (s,1H), 13.2(bs,2H); m/z (FAB mass): 455 (M+1), calculated 454.

BBZCN IR: 3410, 2228, 2922, 1635, 1495,1375, 1280, 1133, 820 cm-1; 1H NMR (DMSO-d6): δ 13.1(bs, 2H), 8.3 (s, 1H), 8.17 (d, 2H), 8.03 (m, 1H), 7.81 (d, 1H), 7.64 (d, 1H), 7.13 (d, 2H), 6.77 (d, 1H), 3.5 (t, 4H), 2.33 (s, 3H); m/z (FAB): 434 (M+), calcu-lated 434.

Molar extinction coefficients of ligands

ε345 = 16,650 for TBZ, ε345 = 25,500 for DMTBZ, ε350 = 29,400 for ETTBZ, ε340 = 31,600 for MMBBZ, ε342 = 25,200 for ETBBZ, and ε342 = 22,400 for BBZCN.

Molar extinction coefficients (ε260) for oligos

5′-TCT CTT TTC TTC TTT TTT CTT TCT TC-3′ (Oligo-1)—196,500.

5′-AGA GAA AAG AAG AAA AAA GAA AGA AG-3′ (Oligo-2)—337,100.

5′-CTT CTT TCT TTT TTC TTC TTT TCT CT-3′ (Oligo-3)—196,500.

Oligonucleotides

Oligonucleotides were purchased from Microsynth, Switzerland: 5′-TCT CTT TTC TTC TTT TTT CTT TCT TC-3′ (oligo-1), 5′-AGA GAA AAG AAG AAA AAA GAA

1 2 3 4 5 6A

B 1 2 3 4

FIG. 1. (A) Electrophoretic mobility shift assay (EMSA) of oligonucleotides. Lane 1 26-mer marker, lane 2 oligo-1 (TCT CTT TTC TTC TTT TTT CTT TCT TC), lane 3 oligo-2 (AGA GAA AAG AAG AAA AAA GAA AGA AG), lane 4 oligo-3 (CTT CTT TCT TTT TTC TTC TTT TCT CT), lane 5 28-mer marker, lane 6 24-mer marker. (B) Electrophoretic mobility shift assay (EMSA) of different DNA structures. 10% PAGE was run in buffer 50 mM Tris-acetate having 10 mM MgCl2, pH 5.2 at 8 V/cm for 10 hours at 10°C. Lane 1 26-mer duplex, lane2 WC-duplex, lane 3 HD, lane 4 triplex made from oligo-1, oligo-2, and oligo-3.

07-OLI_19-1_0169.indd 54 2/26/2009 4:24:40 AM

INTERACTION OF BENZIMIDAZOLES WITH DIFFERENT DNA STRUCTURES 55

Auto Dock run

Individual docking runs for all ligands were performed with WC-duplex, HD, and triplex using Auto Dock 3.0.5. 70, 70, and 126 number of points were used to build a grid box from the center of oligonucleotide in X, Y, and Z dimensions. The Grid spacing of 0.586 Å was used. Lamarckian genetic algorithm was applied as a docking search parameter. This program keeps the macromolecule rigid that allows tor-sional fl exibility for the ligand. Standard values for all dock-ing parameters were used in Auto Dock calculations. The results were analyzed by Auto Dock Tools.

Results

Gel electrophoretic mobility shift assays

The DNA structures showed the following order of mobility; WC-duplex > HD > triplex (Fig. 1B). At pH 5.2, HD showed retarded mobility than the WC-duplex because the positive charges of the C+ residues in the former reduce the net negative charge on the helix (Raghunathan et al., 1994; Escude et al., 1996). This behavior also refl ects the differ-ent helical structure of the HD compared to the WC-duplex (Escude et al., 1996). The lowest mobility was observed for triplex because of higher molecular weight.

Absorption and emission studies

The binding properties of the ligands with WC-duplex, HD, and triplex were studied using absorption and emis-sion titrations at r = 0.05, 0.10, and 0.15 (Table 1). Among the bisbenzimidazoles, Hoechst 33258 showed bathochromic shift of 7 nm while MMBBZ, ETBBZ, and BBZCN showed a bathochromic shift of 10 nm, 8 nm, and 10 nm, respectively with all the three DNA structures. Terbenzimidazoles, TBZ, and DMTBZ showed a bathochromic shift of 10 nm while ETTBZ showed a shift of 20 nm with all the three DNA structures.

Further, interaction of ligands with three DNA structures was studied by emission spectroscopy (Table 1). Hoechst 33258 showed a blue shift of 15 nm with all the three DNA structures at r = 0.05, 0.10, and 0.15. MMBBZ, ETBBZ, and BBZCN showed a blue shift of 22 nm, 10 nm, and 11 nm, respectively with three DNA structures. TBZ gave a blue shift of 17 nm with WC-duplex and triplex and blue shift of 18 nm and 19 nm with HD at r = 0.1 and 0.15. ETTBZ gave a blue shift of 11 nm with WC-duplex and 16 nm–17 nm with HD and triplex. DMTBZ gave a blue shift of 15 nm with WC-duplex and 18 nm–19 nm with HD and triplex.

Circular dichroism spectroscopy

The benzimidazoles showed induced CD signals in the range of 340–380 nm with WC-duplex and triplex (Figs. 2 and 3). Intensities of CD signals increased on increasing the amount of drug and became constant at a particular ligand/DNA ratio (r).

All bisbenzimidazoles showed an induced ellipticity at 356 nm with WC-duplex and this value gradually increased with increase in “r” and became constant at r = 4.5 (Fig. 2C). Similar with triplex, where ellipticity values became constant at r = 4.5. Hence the value of “n” [ Stoichiometry = Number

Absorption and emission spectroscopy

Approximately 10−6 M solutions of all the ligands were prepared in 20 mM sodium cacodylate buffer, 100 mM NaCl, pH5.2, and the absorbance spectra were recorded using a UV-Vis spectrophotometer (Cary Varian 400). Every ligand was equilibrated for 25 minutes with each DNA structure (WC duplex, Hoogsteen duplex, and triplex) at three ligand/DNA ratios 0.05, 0.10, 0.15; spectral measure-ments were recorded using a 1 cm path length quartz cell at 25°C.

Fluorescence spectra were recorded on a Perkin Elmer LS50B spectrofl uorimeter using 1 cm path length quartz cells at 25°C. DNA concentration was 1 μM for each strand and ligand/DNA ratio (r) were kept at 0.05, 0.10, and 0.15 for each DNA structure. The instrument parameters were as follows Ex. slit = 10 mm, Em. slit = 10 mm, scan speed = 50 nm/min.

Circular dichroism spectroscopy

Circular dichroism (CD) spectra were recorded on a Jasco J -810 CD spectropolarimeter in a 2 mm path length quartz cell at 25°C. All three DNA structures were made by anneal-ing the complimentary oligonucleotide strands (5 μM each strand) in 20 mM sodium cacodylate buffer containing 100 mM NaCl at pH 5.2. The ligand was added to DNA at in-creasing ligand/DNA ratio, and incubated for 25 minutes.

Thermal-melting studies

Tm measurements were performed at 260 nm on a Cary Varian-400 UV-Visible spectrophotometer using 1 cm path length quartz cells with 1 μM of each DNA strand in 20 mM sodium cacodylate containing 100 mM NaCl, at pH 5.2, pH 6.5, and pH 7.2, while the temperature was raised from 15°C to 90°C at the rate of 0.25°C/minute. The melting tempera-ture was determined by the derivative method. The ligand/DNA ratio was kept at 5 for all three DNA structures.

Preparation of oligonucleotides for molecular docking

Three-dimensional structures of protonated triplex, HD, and WC-duplex DNA for the previously mentioned sequence were built using Biopolymer module of Insight II (Accelrys version 2005; Accelrys Inc., San diego, CA, USA). AMBER99 partial atomic charges were added and optimized until RMS gradient 0.5 Kcal/(å mol) using HyperChem 7.5 (Hypercube, Gainesville, FL, USA). We used a dialog based utility HIN2PDBQS (Sobhani et al., 2006) for merging nonpolar hydrogens and assigning AMBER86 partial atomic charges to make the system acceptable to Auto Dock v3.0.5 (Morris et al., 1998). Solvation parameters were added by Auto Dock Tools (Michel and Sanner, 1999).

Preparation of ligands for molecular docking

Three-dimensional structures of all seven ligands were built by the Builder module of Insight II. AMBER99 partial atomic charges were assigned and optimized until conver-gence of 0.001Kcal/(Å mol) on HyperChem. Auto Dock Tools assigned Gasteiger charges and ligand’s translation, rota-tion, and internal torsions before docking.

07-OLI_19-1_0169.indd 55 2/26/2009 4:24:40 AM

JAIN ET AL.56

cases two or more than two maxima were obtained between these range.

Thermal-melting studies

Stabilization of triplex was studied by the effect of ligands on the melting temperature of the three DNA structures at r = 5 as the DNA sequences used in this study are 26-mer. For

of base duplex/Ratio “r” (ligand/DNA ratio)] for bisbenz-imidazoles with both WC duplexes and the triplex was 5.77, (26/4.5) ,that is, 5.77 base duplets of WC-duplex and 5.77 base triplets of triplex were bound per molecule (Fig. 2C) but for terbenzimidazoles, the stoichiometry was found to be 8.66 (26/3) with both WC-duplex and triplex (Fig. 2E).

In the case of HD-duplex, all benzimidazoles show in-duced CD signals in the range of 300 nm–400 nm. In all

E

Elli

pticity (

mdeg)

Drug/DNA ratio (r)

WC-TBZ

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5

C

Elli

pticity (

mdeg)

Drug/DNA ratio (r)

WC-ETBBZ

n = 5.77

356 nm

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5

0.51

1.52

2.53

3.54

0

0.5

1

1.5

2

2.5

B

Wavelength (nm)

CD

(m

deg)

WC-ETBBZ

r = 4.5 to 6.0

‒3‒2‒10

200 220 240 260 280 300 320 340 360 380 400 420 440

1

2

3

4

5

D

Wavelength (nm)

CD

(m

deg)

WC-TBZ

r = 3.0 to 5.0

‒3.5

‒2.5

‒1.5

‒0.5200 225 250 275 300 325 350 375 400 425 450

0.5

1.5

2.5

3.5

4.5

A

Wavelength (nm)

CD

(m

deg)

Watson-Crick duplex

Hoogsteen duplex

Triple Helix

‒8

‒6

‒4

‒2

0

200 220 240 260 280 300 320 340 360 380 400 420 440

2

4

6

8

FIG. 2. Circular dichroism (CD) spectra of (A) WC-duplex, HD, and triplex. (B) WC-duplex with ETBBZ at different ligand–DNA ratio r. (C) Ellipticity at 356 nm vs. r plot for the titration of WC-duplex with ETBBZ. (D) WC-duplex with TBZ at differ-ent r. (E) Ellipticity at 356 nm vs. r plot for the titration of WC-duplex with TBZ.

Table 1. Excitation/Emission Wavelengths (λex/em) of New Ligands in Free State/on Binding with Different DNA Structures (WC-duplex, HD, and triplex)

Compound

WC-duplex HD Triplex

r = 0.05 r = 0.1 r = 0.15 r = 0.05 r = 0.1 r = 0.15 r = 0.05 r = 0.1 r = 0.15

H- 33258 352/469 352/470 352/470 352/470 352/470 352/470 352/470 352/470 352/470TBZ 355/463 355/463 355/463 355/462 355/461 355/461 355/463 355/463 355/463ETTBZ 370/469 370/465 370/465 370/464 370/464 370/465 370/463 370/463 370/463DMTBZ 355/465 355/465 355/466 355/462 355/464 355/464 355/461 355/462 355/463ETBBZ 350/472 350/472 350/472 350/472 350/471 350/471 350/471 350/471 350/471MMBBZ 350/471 350/471 350/472 350/472 350/472 350/472 350/472 350/472 350/472BBZCN 352/470 352/470 352/470 352/470 352/470 352/470 352/470 352/470 352/470

r = drug/DNA ratio.

λex/em for free ligand (Hoechst 33258, 345/485; TBZ, 345/480; ETTBZ, 350/480; DMTBZ, 345/480; ETBBZ, 342/481; MMBBZ, 340/494;

BBZCN, 342/481).

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INTERACTION OF BENZIMIDAZOLES WITH DIFFERENT DNA STRUCTURES 57

transition) and 9.5°C (duplex to coil transition) has been found. BBZCN showed only ΔTm of 1 and 4.5°C for triplex-to-duplex and duplex-to-coil transition, respectively (Table 2). Similarly, the ΔTm of 8.5°C–10°C (triplex-to-duplex transition) and 13°C–14°C (duplex-to-coil transition) was observed with TBZ, ETTBZ, and DMTBZ with the triplex at pH 5.2 (Table ).

At pH 6.5 in triplex, the ΔTm for triplex to duplex tran-sition (Table 2) was reduced with all the ligands (bis- and terbenzimidazoles) while duplex-to-coil transition showed similar change in the ΔTm when compared with pH at 5.2.

Tm study, higher ligand/DNA ratio r = 5 was used to satu-rate the binding sites of WC-duplex, HD, and triplex because a stoichiometry of n = 5.77 was observed for bisbenzimidazoles with the three DNA structures and n = 8.66 was observed for terbenzimidazoles with the help of CD experiments. In Table 2, at pH-5.2, the bisbenzimidazoles ETBBZ, MMBBZ showed slightly higher ΔTm as compare to the parent compound Hoechst 33258 on binding to triplex. For example ΔTm of 6°C (triplex to duplex transition) and 10°C (duplex to coil transi-tion) while for Hoechst 33258 ΔTm of 5°C (triplex to duplex

A4

CD

(m

de

g)

HD-Hoechst-33528

Wavelength (nm)

r = 4.5 to 6.0

2

0200 225 250 275 300 325 350 375 400 425 450

‒2

‒4

B3.5

CD

(m

de

g)

HD-ETBBZ

Wavelength (nm)

r = 4.5 to 5.0

1.5

0.5200 220 240 260 280 300 320 340 360 380 400 420 440

‒2.5

‒4.5

D 6

4

CD

(m

deg)

Tr-Hoechst-33528

Wavelength (nm)

r = 4.5 to 6.0

2

0

200

220

240

260

280

300

320

340

380

360

400

420

440

‒6‒4‒2

‒8‒10

E

Elli

pticity (

mdeg)

n = 5.771.5

22.5

33.5

4

10.5

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5Drug/DNA (r)

Tr-Hoechst-33528

356 nm

F 65

CD

(m

deg)

Tr-TBZ

Wavelength (nm)

r = 3.0 to 6.043

200 220 240 260 280 300 320 340 380360 400 420 440‒2‒3

‒1

12

0

‒4‒5‒6‒7

G

Elli

pticity (

mdeg)

n = 8.66

1.5

2

2.5

3

1

0.5

0

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6

Drug/DNA (r)

Tr-TBZ

356 nm

C3.5

CD

(m

de

g)

HD-TBZ

Wavelength (nm)

r = 3.0 to 5.0

1.5

0.5200 220 240 260 280 300 320 340 360 380 400 420 440

‒2.5

‒4.5

FIG. 3. Circular dichroism (CD) spectra of (A) HD with Hoechst 33258 at different ligand/DNA ratio r. (B) HD with ETBBZ at different ligand/DNA ratio r. (C) HD with TBZ at different r. (D) Triplex with Hoechst 33258 at different drug/DNA ratio r. (E) Ellipticity at 356 nm vs. r plot for the titration of triplex with Hoechst 33258. (F) Triplex with TBZ at different r. (G) Ellipticity at 356 nm vs. r plot for the titration of triplex with TBZ.

07-OLI_19-1_0169.indd 57 2/26/2009 4:24:41 AM

JAIN ET AL.58

relation to the oligonucleotide. This procedure was repeated until we found the conformations in good harmony show-ing the position of ligand in minor groove of sequence in question. To meet aspects of calculation time and data size on one hand, and convergence criteria and statistical rele-vance on the other hand, 10 independent docking runs were performed in each case. Out of 10 conformations obtained for each ligand with either of three DNA structures, the docked ligand–DNA complex with minimum docked en-ergy and free energy were chosen for evaluation of results (Table 4). The docked energy and free energy of terbenzimi-dazoles is lower than that of bisbenzimidazoles suggesting that the triplex– terbenzimidazole complex is much more stable than triplex–bisbenzimidazoles. Similarly, the docked energy of triplex–ligand complexes is lower than that of HD–ligand complexes but higher than that of WC–ligand complexes. Therefore, it is inferred that the ligands stabilize the DNA structures in the following order: WC-duplex > Triplex > HD.

Discussion

Oligonucleotide sequences were chosen to fulfi ll the fol-lowing conditions: (1) 26-Mer oligonucleotide sequences

With triplex, at pH 7.2, all bis- and terbenzimidazoles viz TBZ, ETTBZ, DMTBZ, ETBBZ, MMBBZ showed ΔTm

3°C, 3°C, 3.5°C, 1°C, 1°C (triplex-to-duplex transition) while for duplex-to-coil transition, all bis- and terbenzimidazoles showed ΔTm in between 10°C–14°C (Table 2).

For WC-duplexes (Table 3), all benzimidazoles except BBZCN have shown signifi cantly higher ΔTm (9°C–14°C) while we did not observed any apparent change in the ΔTm

of duplexes at different pH (Table 3).At all three pH range, each bis- and terbenzimidazole

showed very little change (0.5°C–1.5°C) in ΔTm with HD duplex (data not shown).

Docking simulation results

In docking simulations, seven ligands (bis- and terben-zimidazoles) were docked onto duplex, HD, and triplex made from oligo-1, 2, and 3 according to the above docking protocol. Quantitative and qualitative considerations were taken to select the most probable conformations of the com-plexes given by Auto Dock. First, the conformations with the lowest free docked energy were chosen as the start-ing point, later these conformations were analyzed qual-itatively based on the location/orientation of the ligand in

Table 2. UV Melting Temperatures of Triplex with Hoechst 33228 and Newly Synthesized Benzimidazoles at Drug/DNA Ratio r = 5 in Sodium Cacodylate Buffer (20 mM Sodium Cacodylate Containing 100 mM NaCl)

Compound

pH 5.2 pH 6.5 pH 7.2

Tm°C (Aa, Ba)

ΔTm°C (A, B)

Tm°C (Aa, Ba)

ΔTm°C (A, B)

Tm°C (Aa, Ba)

ΔTm°C (A, B)

Triplex 41 ± 0.57, 53 ± 0.43 36 ± 0.39, 54 ± 0.47 34 ± 0.28, 54 ± 0.37Hoechst 33258 46 ± 0.37, 62.5 ± 0.44 5, 9.5 38 ± 0.52, 63.5 ± 0.59 2, 9 35 ± 0.43, 64.5 ± 0.35 1,10.5TBZ 49.5 ± 0.29, 66.5 ± 0.50 9, 13.5 41 ± 0.35, 67 ± 0.45 5, 13 37 ± 0.45, 67 ± 0.60 3, 13ETTBZ 49 ± 0.41, 66 ± 0.35 8.5, 13 41 ± 0.22, 66.5 ± 0.51 5, 12.5 37 ± 0.36, 67 ± 0.59 3, 13DMTBZ 50 ± 0.24, 66.5 ± 0.37 10, 14 41.5 ± 0.47, 67 ± 0.34 5.5, 13.5 37.5 ± 0.41, 68 ± 0.29 3.5, 14ETBBZ 46 ± 0.51, 63 ± 0.45 6, 10 38.5 ± 0.55, 64.5 ± 0.29 2.5, 10.5 35 ± 0.49, 65 ± 0.65 1, 11MMBBZ 46 ± 0.33, 63 ± 0.60 6, 10 38 ± 0.36, 64 ± 0.33 2, 10 35 ± 0.39, 65 ± 40 1, 10.5BBZCN 42 ± 0.53, 57.5 ± 0.36 1, 4.5 36.5 ± 0.48, 58 ± 0.26 0.5, 4 34±38, 58±30 0.0, 4

A, triplex-to-duplex transition; B, duplex to coil transition.aData are means ± SD of three independent measurements.

Table 3. UV Melting Temperatures of WC-duplex with HOECHST 33528 and Newly Synthesized Benzimidazoles at Drug/DNA Ratio r = 5 in Sodium

Cacodylate Buffer (20 mM Sodium Cacodylate Containing 100 mM NaCl)

Compound

pH-5.2 pH-6.5 pH-7.2

Tm°Ca ΔTm°C Tm°Ca ΔTm°C Tm°Ca ΔTm°C

WC-duplex 53 ± 0.45 54.5 ± 0.40 55 ± 0.33Hoechst 33258 62 ± 0.32 9 63 ± 0.29 8.5 64 ± 0.39 9TBZ 65 ± 0.61 12 67 ± 0.36 12.5 67.5 ± 0.51 12.5ETTBZ 65 ± 0.44 12 66.5 ± 0.62 12 68 ± 0.43 13DMTBZ 66.5 ± 0.39 13.5 68 ± 0.58 13.5 69 ± 0.47 14ETBBZ 63 ± 0.69 10 64.5 ± 0.30 10 65 ± 0.31 10MMBBZ 63 ± 0.50 10 64.7 ± 0.25 10.2 65.2 ± 0.54 10.2BBZCN 57 ± 0.35 4 58 ± 0.47 3.5 59 ± 0.32 4

aData are means ± SD of three independent measurements.

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INTERACTION OF BENZIMIDAZOLES WITH DIFFERENT DNA STRUCTURES 59

to ~80% of the helical turn (n = 8.66) and Bisbenzimidazoles bind with half helical turn of these DNA structures (n = 5.77), thereby stabilizing the triplex greater than bisbenz-imidazoles. Similarly, terbenzimidazoles provide more sta-bilization to WC-duplex (13°C–14°C) than bisbenzimidazoles (9.5°C–10°C for WC-duplex) (Table 2).

The electron-donating groups at the phenyl ring increases the electron density at nitrogens of imidazole ring, thus in-creasing ligand binding with DNA (Tawar et al., 2003). All the ligands in this study have electron-donating groups on the phenyl ring except for BBZCN that has electron with-drawing cyano group on its phenyl ring (Table 5). It showed ΔTm = 4°C with WC-duplex, 2°C (triplex-to-duplex transi-tion) and 4.5°C (duplex-to-coil transition) with triplex and 1°C with HD at pH 5.2, indicating the effect of phenyl ring substituents on the stability of DNA structures by these ligands.

The bisbenzimidazole derivative Hoechst 33258 was found to be amphiphilic with four basic and three acidic sites. It is shown that imidazole nitrogen (N-7) of benzimid-azole easily gets protonated at pH 5.24, generating a charged species (Krishnamurthy et al., 1986; Ladinig et al., 2005). Similarly, the nitrogens and amine groups of the benzimida-zoles would get protonated under our experimental condi-tions (pH 5.2). In the case of triplex, the positive charge of the protonated benzimidazole molecules bound to WC-groove, compensate the repulsion between the negatively charged polyphosphate backbones of the target duplex and the TFO. This hypothesis supported by fact that (1) At different pH, ΔTm was decreases as pH increases from 5.2 to 7.2, (2) None of the benzimidazoles showed signifi cant binding with HD-duplex, (3) This observation is also supported by CD

were chosen to form stable HD and pyrimidine motif triplex so that the effect of ligands on the stability of these struc-tures, whether positive or negative, can be monitored easily. (2) Oligonucleotides have A6T6 stretch, the bisbenzimidazole binding sites in the middle of sequence to accommodate the bis- and terbenzimidazoles. (3) One strand contains only py-rimidine and other only purine bases to constitute HD. (4) Sequences used to form HD had extensive mismatching for WC pairing on polarity reversal.

At pH 5.2, HD showed retarded mobility than the WC-duplex because the positive charges of the C+ residues in the former reduce the net negative charge on the helix (Raghunathan et al., 1994; Escude et al., 1996). This behavior also refl ects the different helical structure of the HD com-pared to the WC-duplex (Escude et al., 1996). Ligands also get protonated at pH 5.2 and terbenzimidazole had higher molecular weight and more positive charge. Hence ligand–DNA complexes traveled more slowly. Triple helix made from oligo-1, oligo-2, and oligo-3 showed lower mobility because of higher molecular weight and its complexes with ligands had shown further retardation in mobility.

Minor groove binders were reported to destabilize the TAT triplex by modulating the affi nity and selectivity of the major groove-bound third strand (Fortsch et al., 1996). Under our experimental conditions, none of the ligands showed de-stabilization of triplex. Triplex without drugs did not show sharp biphasic transition, but with all the ligands triplex showed clear biphasic transition because ligand increases the ΔTm of duplex-to-coil transition more than that of triplex-to-duplex transition. The ΔTm for triplex to duplex transition was higher with terbenzimidazoles than bisbenzimidazoles. This can be attributed to the fact that terbenzimidazoles bind

Table 4. Results of Docking of the Ligands with the WC-Duplex, HD, and Triplex

TBZ ETTBZ DMTBZ Hoechst-33258 BBZCN ETBBZ

Triplex (protonated)

Docked Energy −17.12 −17.78 −16.77 −14.11 −14.57 −13.8 Ref RMS 50.77 62.57 26.55 65.43 46.79 56.29 Free energy −15.05 −15.28 −14.85 −12.71 −12.85 −12.25 KI 9.25 × 10−12 6.25 × 10−12 1.30 × 10−11 4.85 × 10−10 3.80 × 10−10 1.03 × 10−9

Intermolecular energy −16.61 −17.15 −16.72 −13.64 −14.1 −13.5 Internal energy −0.52 −0.63 −0.05 −0.47 −0.47 −0.29HD (protonated) Docked energy −11.64 −12.25 −11.1 −10.48 −9.89 −8.85 Ref RMS 28.5 60.92 69.38 60.8 23.9 37.17 Free energy −9.67 −9.75 −8.72 −9.33 −8.23 −7.24 KI 8.09 × 10−8 7.05 × 10−8 4.01 × 10−7 1.45 × 10−7 9.23 × 10−7 4.86 × 10−6

Intermolecular energy −11.23 −11.62 −10.59 −10.26 −9.48 −8.49 Internal energy −0.41 −0.62 −0.5 −0.22 −0.41 −0.36WC-duplex Docked energy −20.31 −18.94 −19.59 −14.6 −17.18 −16.01 Ref RMS 52.81 49.12 47.08 56.04 20.34 70.1 Free energy −18.96 −18.16 −18.83 −13.34 −15.45 −14.76 KI 1.26 × 10−14 4.89 × 10−14 1.56 × 10−14 1.68 × 10−10 4.68 × 10−12 1.50 × 10−11

Intermolecular energy −20.52 −20.03 −20.7 −14.27 −16.7 −16.01 Internal energy 0.21 1.09 1.11 −0.34 −0.47 0.01

All energies are given in kcal/mol.

07-OLI_19-1_0169.indd 59 2/26/2009 4:24:42 AM

JAIN ET AL.60

and docking results, which showed that binding of benzimi-dazoles to HD-duplex is much less stable than compared to triplex.

Apart from hydrogen bonding other factors such as elec-trostatic, van der Waals interactions, and nonbonded con-tacts with the walls of minor groove have strong roles to play in binding process between benzimidazole derivatives and DNA (Kakkar et al., 2005). Hence, it appears that the sta-bilization provided by the benzimidazoles to HD and triplex was the result of electrostatic and van der Waals interactions. These observations are further confi rmed on the basis of pH experiments, which clearly suggest that the electrostatic interactions were reduced due to reduction in the charge at increasing pH from 5.2 to 7.2.

The benzimidazoles showed induced CD signal with WC-duplex and triplex at 356 nm indicating that these ligands bind to the above DNA structures in similar manner (Figs. 2 and 3). These ligands bind to HD-duplex nonspecifi cally

A C

B D

E G

F H

I K

J L

FIG. 4. Docking simulation results of the ligands with differ-ent DNA structures. The images are of the minimum docked energy of the complex. (A) WC-duplex-Hoechst 33258. (B) WC-duplex-BBZCN. (C) WC-duplex-DMTBZ. (D) WC-duplex-ETTBZ. (E) Triplex-Hoechst 33258. (F) triplex-BBZCN. (G) Triplex-DMTBZ. (H) Triplex-ETTBZ. (I) HD-Hoechst 333258. (J) HD-BBZCN. (K) HD-DMTBZ. (L) HD-ETTBZ.

Table 5. Structure of Ligands

S. No. Compound n R1 R2

1 Hoechst 33258 2 H OH2 TBZ 3 OCH3 OH3 ETTBZ 3 OCH2CH3 OH4 DMTBZ 3 OCH3 OCH3

5 ETBBZ 2 OCH2CH3 OH6 MMBBZ 2 OCH3 OH7 BBZCN 2 H CN

N

N

NH

N

n

R1

R2

H3C

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INTERACTION OF BENZIMIDAZOLES WITH DIFFERENT DNA STRUCTURES 61

DNA as a rigid structure. That is why the docking energy, free energy, and KI obtained from Auto Dock or any other docking software for benzimidazoles docked to DNA struc-tures may not be suffi ciently accurate.

But the docking results clearly suggest that all six benzimidazoles stabilize the DNA structures in the fol-lowing order: WC-duplex > Triplex > HD and terben-zimidazole stabilized triplex, WC-duplex more than bisbenzimidazoles.

The similar binding mode of ligands with WC-duplex and triplex can be explained as the minor groove of both WC-duplex and triplex are easily accessible for the ligands. Helical groove of the HD has different helical structure than WC-duplex (Raghunathan et al., 1994), thus benzimi-dazoles were expected to have different kind of binding with HD, which is further supported by the molecular mod-eling that bis- and terbenzimidazoles bind nonspecifi cally to HD (Fig. 5E and F). Because HD is the component of the pyrimidine motif triplex (Liu et al., 1993), the ability of a ligand to bind with triplex could be related to its ability to bind with HD (Escude et al., 1996). However, our results do not correlate with this theory. In the HD, the minor groove is narrower than triplex and WC-duplex, because of which ligands do not have a preferred stable conformation in the narrow groove of HD. These results give new insights about interaction between ligands and triple helices and defi ne a starting point for the design of new triplex-specifi c molecules.

Acknowledgments

Authors are thankful to DST, Government of India, New Delhi for providing the funds. S.K.G. is thankful to ICMR for Senior Research Fellowship.

as these ligand showed induced CD signal at more than one place in between 340 nm and 380 nm (Fig. 2). As we could not infer much from CD data, molecular dynamic simulations studies were used to understand the binding of ligands with DNA. The docking results further supported the CD data that all the ligands bind in the minor groove of WC-duplex, WC-groove of triplex whereas all benzimid-azole bind nonspecifi cally with HD-duplex (Fig. 4). The free energy, docking energy, and KI obtained for docked complex of BBZCN-DNA structures are slightly higher in comparison to parent molecule Hoechst 333258 (Table 4), which is not supported by the ΔTm. It is well accepted that all molecular docking methods have severe limitations and because of which they provide wrong energy calculations. There may be two major reasons of the energies calculated for the compound BBZCN do not fi t the corresponding observed ΔTm are (1) inaccuracies in the energy models used to score potential ligand–receptor complexes, and (2) the inability of current methods to account for conformational changes that occur during the binding process not only for the ligand, but also for the receptor (Teodoro et al., 2001). In addition to above, some more inaccuracy is added in the results during the docking process, by making compromise between calculation time and data size on one hand, and convergence criteria and statistical relevance on the other hand. The Auto Dock method is a rigid docking method and in which DNA (receptor) is being considered in a single dominant conformation. But DNA backbone plays a criti-cal role just like protein backbone and a number of confor-mations are possible for the backbone of DNA leading to number of conformations of DNA, and these non-negligible changes in conformation during binding process results on faulty scoring, calculation of docking energy (Brem and Dill, 1999), and free energy because we are considering

A

C

E F

D

B

FIG. 5. Docking simulation results of the ligands with different DNA structures. (A) WC-duplex-ETBBZ. (B) WC-duplex-TBZ. (C) Triplex-ETBBZ. (D) Triplex-TBZ. (E) HD-ETBBZ. (F) HD-TBZ.

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Dr. B.R. Ambedkar Center for Biomedical ResearchUniversity of Delhi

Delhi 110007 India

E-mail: vtandon@acbr.du.ac.in

Received for publication November 7, 2008; accepted after revision December 5, 2008.

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