Mishra, K., Bhardwaj, R. and Chaudhury, N. K. Netropsin, a Minor Groove Binding Ligand: A Potential Radioprotective Agent.
Minimizing radiation-induced damages in DNA is an important aspect in the development of chemical radioprotectors. The aim of this study was to evaluate the possible radioprotective ability of the DNA minor groove binding ligand netropsin in an aqueous solution of plasmid DNA (pBR322) and to compare its efficacy with that of Hoechst 33258, a known radioprotector. The radiochemical parameters D0, GSSB and DMF were calculated in pBR322 DNA. Based on a comparison of the DMFs of netropsin and Hoechst 33258, netropsin appeared to be the better radioprotector. The ligand binding site accessibility of the restriction enzyme EcoRI at the ligand-pBR322 complex was assessed using a restriction-digestion assay in irradiated solutions. A distinct ligand-bound site protection in netropsin-DNA was observed in irradiated solutions. However, no site protection was observed in the presence of Hoechst 33258. The possible role of ligand-induced structural stabilization in irradiated aqueous solutions was also investigated using netropsin-calf thymus DNA melting temperature measurements. The greater radioprotective ability of netropsin in solutions of DNA was suggested to be due to its higher binding affinity and its ability to provide higher structural stabilization. EcoRI digestion revealed that hydroxyl radical (OH•) generated by ionizing radiation is not able to radiolyse the netropsin-DNA complex. These results will help in developing better radioprotectors.
INTRODUCTION
DNA is the prime target of ionizing radiation in cells, and thus it is important to protect it from the deleterious effects of ionizing radiation (1). Several antitumorigenic, antiviral and antibacterial agents interact with DNA, and drug designers are focusing small molecules that bind to specific sequences of DNA (2,3). Molecules that bind with specific AT- and GC-rich sequences as well as specific conformation of DNA are important in the development of nucleic acid-targeted drugs (4–6). Bis-benzimidazole derivative Hoechst 33258-DNA complexes have been used in several studies of the nature of the interaction, binding forces and thermodynamics using both theoretical and experimental tools. In 1984, Smith and Anderson (7) reported a radioprotective effect of a more cell-permeable variant, Hoechst 33342, at a very low concentration (8.75 µM) in HT29 human adenocarcinoma cells; the observed dose-modifying factor (DMF) was ∼1.7. In subsequent studies, Young and Hill (8) reported radioprotection in KHT fibrosarcoma, B16F10 melanoma and SCCVII squamous cell carcinoma cells. Similarly, Lyubimova et al. (9) found a DMF of about 3 in mouse endothelial cells. Simultaneously, Denison et al. (10) and Martin et al. (11) elucidated the radiochemical basis of this novel observation. The important finding was the high radioprotective efficacy compared to classical thiol-based radioprotectors, for example WR-1065 (12). Based on these radiochemical findings, methylproamine, a more potent (about 40 times higher) derivative of Hoechst 33342, was developed and synthesized. The higher efficacy of methylproamine was attributed to electron donation from the substituted N-CH3)3 group on the phenyl ring to the site of DNA damage (13,14).
This class of DNA-binding molecules possesses certain specific physicochemical and thermodynamic properties related to the recognition of binding sites in DNA (15). Thus the molecular mechanism of Hoechst-mediated radioprotection is expected to be multidimensional. Because we were interested in developing chemical radioprotectors, we focused on the possible role of physicochemical interactions (6,16). In an earlier study, we investigated the role of Hoechst 33258-induced stabilization of DNA and the implications for radioprotection (17). This information is an addition to the known radiochemical properties (scavenging of free radicals and quenching of DNA radicals) of Hoechst 33342 (10). The binding characteristics were elucidated with synthesized Hoechst analogues (6,16) and appeared to correlate with radioprotection of DNA in solutions (18). However, the radioprotective efficacy, i.e. the DMF, was found to be minimal in cellular (in vitro) studies. Thus there is a need for improvement of the design of these DNA minor groove ligands for developing better radioprotective agents.
The physicochemical properties of ligand-DNA interactions are expected to contribute to the structural stabilization in irradiated ligand-DNA complexes. Previous studies from our laboratory implicated a role of physicochemical properties (binding affinity) using Hoechst 33258 in irradiated Hoechst 33258-DNA solutions. In the present studies, we examined similar minor groove binder, netropsin, that binds with DNA in the AT-rich minor groove region.
MATERIALS AND METHODS
Chemicals and Biochemicals
Netropsin and Hoechst 33258 were obtained from Sigma Chemical Co. (St. Louis, MO). The structures of these ligands are shown in Fig. 1. Plasmid DNA (pBR322, of 4361 bp, CsCl purified) and EcoRI (kit) were obtained from Bangalore Genei Pvt. Ltd. (Bangalore, India). Plasmid DNA was supplied in 10 mM phosphate buffer. Calf thymus DNA was obtained from Sigma. Tris-HCl was of Excela“R” grade and was obtained from Qualigen Glaxo Fine Chemicals (Mumbai, India). EDTA, NaH2PO4•2H2O and Na2HPO4 were from E. Merck (Germany). Agarose was of low EEO grade for electrophoresis from Bangalore Genei Pvt. Ltd. All other chemicals were of analytical grade and were used without further purification.
Gamma-Radiation Sources
Gamma irradiation was carried out using two different sources: a 60Co teletherapy unit (model Eldorado 75, Canadian Atomic Energy, Canada) at dose rate of 0.80–0.75 Gy/min and a γ-ray chamber model GC-5000, Ver. 1.2 (DAE, BRIT Mumbai, India) at a dose rate of 54.5–3.3Gy/min. For calculation of the D0, a source with a lower dose rate (Eldorado model 75, AECL, Canada) was used.
Preparation of Netropsin-pBR322 Solution
The stock of pBR322 (50 µg/200 µl) in phosphate buffer obtained from Bangalore Genei was diluted further, and an appropriate aliquot was taken to obtain a working concentration of 40 µM per bp. The ligand solution of netropsin was prepared at two different concentrations (20 and 40 µM), and the DNA and ligand solutions were mixed at equal volumes so that the final concentration of DNA was 20 µM per bp and that of netropsin was 10 and 20 µM.
Preparation of Netropsin-Calf Thymus DNA Solution
A stock solution of calf thymus DNA of 200 µM per bp was prepared in phosphate buffer (pH 7.2, 10 mM). The ligand solution of netropsin was prepared at a concentration of 20 µM. The netropsin-DNA solutions were prepared by mixing equal volumes by slowly adding the required ligand solutions dropwise with continuous stirring. The final concentration of DNA was 100 µM in each solution.
Gamma Irradiation of Ligand-pBR 322
All samples were irradiated in 1.5-ml Eppendorf tubes kept in ice in aerated 10 mM phosphate buffer (pH 7.2). The concentration of DNA in the agarose gel electrophoresis experiment was 20 µM per bp in each lane, and the final volume was 15 µl. The required amounts of Hoechst 33258 and netropsin were added to the plasmid prior to irradiation. These samples were loaded in the respective wells with 5 µl loading buffer.
Gamma Irradiation of Netropsin-DNA (calf thymus) Solutions and Thermal Denaturation Temperature Measurements
Freshly prepared netropsin-DNA (calf thymus) solutions were irradiated with 120 Gy using a Gamma 5000 at a dose rate of 54.5–53.3 Gy/min. The double-helix to single-coil phase transition temperature (Tm) of DNA and netropsin-DNA complexes was measured from the midpoint of the phase transition region of a sigmoidal curve showing an increase in absorbance at 260 nm with increasing temperature. Absorbance as a function of temperature was recorded using a UV-Vis double-beam Spectrophotometer (model GBC 916 UV-Vis, Australia). The temperature was controlled to an accuracy of ±0.2°C with a software-controlled thermo-electrical device.
Agarose Gel Electrophoresis Assay
Separation of the different forms of pBR322 (20 µM per bp) as a function of radiation dose was performed on 1.2% agarose gels in Tris-acetate-EDTA (pH 8) mixed with 5 µl of bromophenol blue-glycerol solution using a horizontal submarine gel electrophoresis system (18,19). Electrophoresis was performed at 50 V, 22 mA, for 3–4 h. Gels were then stained in TAE buffer containing 0.75 µg/ml ethidium bromide for 1.5 h followed by distaining for 0.5 h. The gel was scanned and quantified by densitometry (model GEL DOC XR and analyzed by using Quantity one, Bio-Rad Instruments).
Calculation of D0 and GSSB
The data obtained from densitometry were plotted as the logarithm of the fraction of intact supercoiled DNA as a function of radiation dose using a least-mean-squares straight line of the form y = ae−bx, where y is the fraction of intact supercoiled plasmid and a and b are the intercept and slope, respectively (20,21). The D0 was calculated from the reciprocal of b and is the dose required to induce one SSB per plasmid. Therefore, at the D0, the concentration of SSBs is equal to the concentration of DNA expressed as plasmid (molecular mass = 650 g mol−1 bp−1 × no. of base pairs). From the definition, G value for the formation of SSBs is
where the density of solution ρ was assumed to be unity. The G value is in µmol J−1 (21).
pBR 322 Digestion by EcoRI
pBR322 (200 ng) as substrate for EcoRI and the amounts of ligands indicated in Fig. 5 were incubated with the restriction endonuclease EcoRI (10 U, obtained from Bangalore-Genei, India) at 37°C in 20 µl volume containing 10 mM phosphate buffer (pH 7.2), 50 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM MgCl2 and 5 mM β-mercaptothiol. Reactions were stopped after 1 h by cooling the samples to 0°C followed by the addition of gel loading buffer (5 µl) containing 0.25% (w/v) bromophenol blue, 0.25% (v/v) xylenecynol, and 30% (v/v) glycerol in water (pH 8). The extent of plasmid DNA digestion was determined by agarose gel electrophoresis. The details of electrophoresis and analysis were as described above section.
RESULTS
Yield of Single-Strand Breaks (GSSB)
pBR322 was chosen to assess the yield of single-strand break (GSSB). Supercoiled and nicked circular plasmid DNA were clearly observed in agarose gel electrophoresis images stained with ethidium bromide (Fig. 2A). The supercoiled form disappeared with the simultaneous increase in the nicked circular form with increasing radiation dose. The figure shows the dose-dependent radiation-induced changes in the relative amounts of the two forms in the absence of any ligands. From the reciprocal of the slope of the semilogarithmic plot of the fraction of the intact supercoiled form as a function of radiation dose, the D0 of pBR322 DNA was calculated as 8.2 Gy (Fig. 2B). The GSSB in the case of pBR322 was 6.11 × 10−8 Gy−1 Da−1. These values are in the range reported in earlier studies (22–28) and are shown in Table 1.
Figure 2
Panel A: Radiation-induced single-strand breaks in plasmid DNA (20 µM per bp). The fraction of the supercoiled form (the front band) gradually decreased with increasing dose. The radiation doses in lanes 1 to 8 were 0, 2.5, 5, 7.5, 10, 12.5, 15 and 20 Gy, respectively. The dose rate was 0.8 Gy/min. Panel B: Radiation-induced single-strand breaks in plasmid DNA. Disappearance of the fraction of supercoiled form as a function of dose: The fraction of supercoiled DNA was fitted with the equation y = ae−bx.
Table 1
Yields of Single-Strand Breaks (GSSB)
Radioprotection by Netropsin
To investigate the possible radioprotective effects of netropsin on pBR322, different concentrations of netropsin (10 and 20 µM) were used, and the solutions containing pBR 322 (20 µM per bp) were irradiated with 20 and 30 Gy. At 20 Gy, most of the supercoiled DNA (Fig. 3A, lane 2) had disappeared, whereas with increasing concentrations of netropsin the relative percentage of this form increased proportionately. This is clearly evident from the analysis of the band intensities in Fig. 3B. Interestingly, at 30 Gy the presence of netropsin protected the supercoiled form in a concentration-dependent manner (Fig. 3B).
Figure 3
Panel A: Radioprotection by netropsin against radiation-induced scission of plasmid DNA. Lane 1, unirradiated control; lane 2, pBR322 + 20 Gy; lane 3, pBR322 + 10 µM netropsin; lane 4, pBR322 + netropsin (10 µM) + 20 Gy; lane 5, pBR322 + netropsin (10 µM) + 30 Gy; lane 6, pBR322 + netropsin (20 µM); lane 7, pBR322 + netropsin (20 µM) + 20 Gy and lane 8, pBR322 + netropsin (20 µM) + 30 Gy. Panel B: Graphical representation of the numerical values for the fraction of the intact supercoiled form of the corresponding agarose gel image in panel A.
Comparison of Radioprotective Effects of Netropsin and Hoechst 33258
Denison et al. (10) demonstrated the radioprotective efficacy of Hoechst 33258 using the same DNA model in similar experimental conditions. Therefore, we attempted to compare the radioprotective efficacy of netropsin with Hoechst 33258. The results in Fig. 3 clearly indicated the radioprotective ability of netropsin in aqueous solutions of pBR322. Concentrations similar to those in earlier reports were used and were irradiated with different doses up to 20 Gy (10). As shown in Fig. 4A and B, the supercoiled form decreased more at comparatively lower radiation doses in the presence of Hoechst 33258. The plot (intact supercoiled form as a function of radiation dose) for both the DNA minor groove binding ligands clearly showed the better efficacy of netropsin (Fig. 4C). The radiochemical yield (GSSB) and D0 were calculated from the slopes of the fitted curves corresponding to netropsin and Hoechst 33258. The D0 for pBR322 DNA without ligand (control) was 8.2 Gy, whereas in the presence of Hoechst 33258 and netropsin, the D0 increased to 28.0 and 42.7 Gy, respectively. The calculated D0 for pBR322 DNA for Hoechst 33258 was similar to the value reported by Denison et al. (10). The ratio D0 (netropsin)/D0 (control) is the dose-modifying factor (DMF) and is 5.2 ± 0.2 at 10 µM of netropsin; the corresponding ratio for Hoechst 33258 is 3.4 ± 0.3. The GSSB values for netropsin and Hoechst 33258 were equivalent to 1.17 × 10−8 SSB Gy−1 Da−1 and 1.785 × 10−8 SSB Gy−1 Da−1, respectively. These findings suggested better radioprotection by netropsin than by Hoechst 33258.
Figure 4
Protection of pBR322 DNA from radiation-induced strand breaks by 10 µM netropsin (panel A) and Hoechst 3325810 µM (panel B). Representative agarose gel electrophoresis image showing radiation dose response (0–20 Gy) of netropsin and Hoechst 33258. Lanes from left: lane 1, unirradiated control (pBR322); lane 2, pBR 322 + ligand + 2.5 Gy; lane 3, pBR322 + ligand + 5 Gy; lane 4, pBR 322 + ligand + 10 Gy; lane 5, pBR322 + ligand + 15 Gy and lane 6, pBR322 + ligand + 20 Gy. Panel C: Comparison of radioprotection in the presence of netropsin and Hoechst 33258. Induction of DNA single-strand breaks was quantified in terms of loss of intact supercoiled plasmid DNA: (♦) irradiated control; (▪) 10 µM Hoechst 33258; (▴) 10 µM netropsin. The data points for irradiated control pBR 322 are same as in Fig. 2B.
Inhibition of EcoRI Accessibility at 5′-GAATTC-3′ Site before and after Irradiation Confirms Site-Specific Radioprotection by Netropsin
The purpose of this study was to investigate the possibility of site-specific protection of DNA by netropsin and Hoechst 33258. Since netropsin and Hoechst 33258 needed 4–6 bp of DNA for binding, and the detailed nature of complexation of these ligands with 5′GAATTC3′ sequence was known, we probed this canonical DNA sequence with EcoRI, which has a similar DNA recognition sequence for its cleavage activity (29,30). pBR322 has a single site for the action of this enzyme, and after cleavage it converts supercoiled forms into linear forms (Fig. 5A). Netropsin-pBR322 DNA complex inhibited the cleavage (supercoiled and nicked circular forms were not converted to linear) of DNA in a concentration-dependent manner at its site of action (5′G↓AATTC 3′) as shown in lanes 1 and 2 in Fig. 5B. Interestingly, in irradiated solutions of netropsin-pBR322 complex, this enzyme could not convert the supercoiled and nicked circular forms to linear; moreover, the amount of the supercoiled form remaining after protection by netropsin was unaltered (lanes 3, 4). This suggested the protection of a specific site by netropsin and also indicated that netropsin was still bound at this site even after irradiation. The presence of Hoechst 33258, on the other hand, did not hinder the accessibility of EcoRI in either irradiated or unirradiated complexes, as is evident from the observation of the linear form in lanes 5–8. The reasons for the observed differences in response to catalytic actions of EcoRI is not clear, and various physicochemical factors like binding affinity, structural stabilization upon binding with ligand, and water of hydration could be contributing factors.
Figure 5
Panel A: Linearization of pBR322 DNA by EcoRI restriction endonuclease. Lane 1, Control; and lane 2, pBR322 + EcoRI. Panel B: Site-specific radioprotection by netropsin: Inhibition of EcoRI accession at the binding site 5′G↓AATTC3′. Lane 1, pBR322 + netropsin (10 µM) + EcoRI; lane 2, pBR322 + netropsin (20 µM) + EcoRI; lane 3, pBR322 + netropsin (10 µM) + 20 Gy + EcoRI; lane 4, pBR 322 + netropsin (20 µM) + 20 Gy + EcoRI; lane 5, pBR322 + Hoechst 33258 (10 µM) + EcoRI; lane 6, pBR322 + Hoechst 33258 (20 µM) + EcoRI; lane 7, pBR322 + Hoechst 33258 (10 µM) + 20 Gy + EcoRI and lane 8, Hoechst 33258 (20 µM) + 20 Gy + EcoRI. The dose rate was 54.5–53.3 Gy/min.
Measurement of Melting Temperature (Tm) or Helix-Coil Transition
The binding of minor groove ligands increases the stability of DNA, which can be measured from DNA melting profiles (17,31,32). To investigate the possible role of stability induced by netropsin in irradiated DNA solutions, the melting temperature of the ligand-calf thymus DNA complex was determined from the melting profiles at same concentration (10 µM) of ligand (netropsin and Hoechst 33258). The measured Tm's are shown in Table 2. The melting temperature of unirradiated calf thymus DNA was 72.8°C and increased to 80.5°C in the presence of netropsin. The melting temperature decreased (ΔTm) by −1.7°C in irradiated (120 Gy) solutions. In the case of free DNA (without netropsin), this difference was −9.7°C. This was expected because the ligand-induced stability in DNA is a cooperative phenomenon. This increased ΔTm clearly indicated that netropsin protected DNA from thermal denaturation in irradiated solutions. The corresponding ΔTm with Hoechst 33258 has been observed as −4.5°C, as reported in our earlier study (17). Comparison of the ΔTm values with Hoechst 33258 and netropsin suggested that the radiation-induced decrease in melting temperature was greater with Hoechst 33258.
Table 2
Thermal Denaturation Temperature, Tm, of Ligand-DNA (calf thymus) Complex
DISCUSSION
Figure 4C, which shows the intact supercoiled form of pBR322 as a function of radiation dose in the presence of netropsin and Hoechst 33258, indicates that netropsin demonstrated better radioprotective efficacy. The GSSB values of netropsin and Hoechst 33258 were calculated as 1.17 ×10−8 SSB Gy−1 and 1.785 × 10−8 SSB Gy−1 Da−1, respectively. This value supported the observations related to better radioprotection by netropsin.
Role of Binding Characteristics of Netropsin and Hoechst 33258 with DNA and Increased Radioprotection by Netropsin
Netropsin and Hoechst 33258 are well-characterized DNA minor groove binding ligands that serve as model compounds for studying drug-DNA interactions (34). Both netropsin and Hoechst 33258 bind similarly at the AT-rich minor groove region in DNA. Crystallographic studies on netropsin-DNA complexes revealed that the amide NH groups of netropsin make bifurcated hydrogen bonds with N3 and O2 of A and T bases of the opposite strands of DNA. These hydrogen bonds promote sequence specificity and result in high binding affinity (35). Netropsin binds tightly in the AT-rich sequence of DNA with a Kd of 4 × 109 M−1 and increases the stability of DNA. Similar hydrogen bonding interaction occurs in the case of Hoechst 33258; the two benzimidazole nitrogen atoms N-2 and N-3 of Hoechst 33258 form bifurcated hydrogen bonds with N3 and O2 of the A and T bases of the opposite strand (36). The Kd for Hoechst 33258 is one order of magnitude less. Differences in the sequence selectivity between Hoechst and netropsin have been reported (37). Netropsin requires about 5.5 bp for binding to DNA (34) while Hoechst 33258 requires 4–5 bp. The binding of netropsin is thermodynamically favorable and entropically driven. The energetics (ΔG) of the binding interactions of both these ligands is also reported to be similar (37). These differences indicate that binding of netropsin and Hoechst 33258 is thermodynamically favorable but that the association constant for netropsin is higher than that for Hoechst 33258 and is more entropically driven. Therefore, these differences in the physicochemical properties related to binding appear to be associated with the observed greater radioprotective abilities of netropsin.
Role of Ligand-Induced Structural Stabilization and Radioprotection
Binding affinity in the minor groove is also known to contribute stability toward thermal denaturation in irradiated DNA. This is reflected in DNA melting temperature (Tm) measurements in the presence of different ligand-DNA complexes in irradiated solutions (6,17). Tm has been used to correlate the decrease in stability based on the extent of strand breaks induced by ionizing radiation. A decrease in Tm has been correlated with the extent of DNA strand breaks (33). In our earlier study, we also observed that Hoechst 33258 increased the stability of calf thymus DNA in a ligand concentration-dependent manner (17) and contributed to an increase in stability in γ-irradiated Hoechst 33258-DNA solutions. In this study, we therefore used calf thymus DNA to investigate the effect of radiation on the netropsin-DNA complex. A comparison of Tm measurements of netropsin and Hoechst 33258 with calf thymus DNA is shown in Table 2. Both netropsin and Hoechst 33258 appeared to provide a similar extent of stability in calf thymus DNA, but in irradiated solutions the lowering of Tm (ΔTm) was less in netropsin-calf thymus DNA: −1.7°C compared to −4.5°C in Hoechst-DNA. This further supports our previous findings regarding the role of structural stability in the observed radioprotection by netropsin in irradiated DNA solution (17). Structural stabilization may simply protect DNA by blocking radical attacks at the ligand binding sites.
Other physicochemical properties such as DNA hydration are also closely related to the damages induced by radiation (39,40). A specific comparison in relation to netropsin- or Hoechst 33258-mediated radiation protection and hydration had not been attempted previously. Though both ligands bind similarly with DNA, the extent of the displacement of water molecules from the site of bindings was higher by Hoechst 33258 (2,38). Further alterations resulting from neutralization of charges associated with phosphate ion in the backbone of DNA are expected to be greater for the dicationic ligand netropsin than for the monocationic ligand Hoechst 33258, and this in turn will influence the water of hydration. Therefore, multiple factors appear to contribute to minor groove ligand-mediated radioprotection in DNA in solutions. The relative differences between netropsin and Hoechst 33258 observed in the present study in terms of radioprotection are thought be a combined effect of radiochemical and physicochemical processes and require further investigation.
Conclusions
Our results demonstrated the radioprotective ability of netropsin in solution. The mechanisms appears to be complex; protection of the DNA binding site from radiolytic attacks of reactive species, higher binding affinity and structural stabilization appear to contribute in addition to the radiochemical properties of this ligand. Other physicochemical properties such as hydration and charge neutralization need to be investigated in the future. However, based on this study, netropsin is projected as a new molecule in the category of DNA minor groove binder as a possible candidate for a chemical radioprotector. The present findings will provide useful information for designing better minor groove binding agents for radioprotection.
Acknowledgments
The authors are thankful to Dr. R. P. Tripathi, Director, Institute of Nuclear Medicine and Allied Sciences, for his interest in this area of research. Support from the Institute of Genomics and Integrative Biology, Delhi and ACBR, Delhi University for gel documentation and analysis is gratefully acknowledged. The authors also acknowledge the suggestions provided by Dr. B. S. Dwarakanath, Head of Division of Radiation Biosciences, during the course of this work. One of the authors (K. Mishra) is thankful to the university grant commission (UGC), New Delhi for a junior research fellowship. This work was funded by project no. INM 301 of DRDO, Govt. of India.
