DNA-binding studies of a series of novel water-soluble derivatives of 1 , 4-dihydropyridine

E. Leonova, E. Rostoka, L. Baumane © 2018 E. Leonova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 547.82 + 577.1


Introduction
Synthetic derivatives of 1,4-dihydropyridine (1,4-DHP) possess important biochemical and pharmacological properties.They show modulating effect on cardiovascular and neuronal processes as well as anticancer, geroprotective and radioprotective effects.Some 1,4-DHP manifest antimutagenic activity and anticlastogenic effects and stimulate DNA repair [1][2][3][4].Lacidipine, ramipril and valsartan protect DNA against oxidative damage in the heart infarction zone [5].1,4 DHP can act also as free radical scavengers [6,7] and increase bioavailability of nitric oxide [7].Another group of 1,4-DHP derivatives generates DNA breaks, via radical or other mechanisms [8].1,4-DHP with positively charged groups are used as vectors for DNA delivery inside the cells [9].Most biological effects of this class of the compounds are usually ascribed to blocking calcium channels, this can lead to multiple biological effects following different intracellular pathways, resulting in weakening of oxidative stress [10].
However, in the present investigation we have focused our attention on a group of 1,4-DHP derivatives considered to be "unusual".These are the water soluble molecules with no or very weak blocking activity towards calcium channels.Some of them manifest different biological activities.Our recent results revealed DNA binding capacity of another water-soluble 1,4-DHP , the antimutagene AV-153 [11] and some other 1,4-DHPs [12].Aim of the present work was to reveal structure-functional relations of DNA-binding capacity of water-soluble1,4-DHPs, to study the mode of the compound and DNA interactions of the most active compounds and to compare DNA-binding capacity with other activities of the compounds.

Chemicals
Water-soluble derivatives of 1,4-DHP were synthesized in the Laboratory of Membrane Active Compounds of the Latvian Institute of Organic Synthesis.The compounds structures are given in Table 1.Tris base, sucrose, ethidium bromide, acridine orange, Triton-X-100, ethidium bromide (EtBr), Na 2 EDTA, LiCl, NaCl, CaCl 2 and other inorganic salts were purchased from Sigma-Aldrich.2-mercaptoethanol was obtained from Ferak Berlin, sodium dodecyl sulphate was supplied by Acros Organics, isoamylic alcohol was obtained from Stanlab, and 6×Orange loading solution, RNase A and Proteinase K were purchased from Fermentas.Peroxynitrite was synthesized as described [13].

Isolation of DNA
The pTZ57R plasmid was isolated from Escherichia coli DH5alpha strain transformed with this plasmid, sonicated and purified essentially as described [11].

UV/VIS spectroscopic measurements
UV-VIS spectra were recorded with a Perkin Elmer Lambda 25 UV/VIS spectrophotometer in the absence of DNA and in the presence of increasing amounts of DNA in 5 mM NaCl and 5 mM Tris HCl at pH 7.4 or other buffer.A 30 μM solution of the tested compound was diluted out of a 1 mM stock solution in the buffer in a quartz cell (2 ml).A reference cell was filled with 1 ml of the buffer.The mixture was stirred thoroughly and titrated by 1.2 mM DNA solution, 10 μM each time to both sample and reference cells.DNA molar concentration was calculated on the basis of absorbance of the solution at 260 nm and molar extinction coefficient for DNA.Spectra were recorded in the 400-200 nm interval at room temperature.
Binding constants were calculated by applying the formula according to [14], where A 0 is absorption of the free substance, A is absorption in [the] presence of DNA, and c DNA is DNA concentration.
Fluorescence spectra of a 25 μM solution of the 1,4-DHP in 5 mM Tris HCl; 5 mM NaCl at pH 7.4 or other buffer were recorded over a range of 240-700 nm at an excitation wavelength of 350 nm.DNA was sequentially added up to 225 µM, 10 µM at each step until saturation.EtBr displacement assay were carried out as described [11], with minor modifications: fluorescence intensity of the DNA-EtBr complex was recorded at 600 nm using an indirect excitation wavelength of EtBr at 260 nm.

The melting temperature
The melting temperature (Tm) of DNA and

Cyclic voltammetry
Voltammetric experiments were performed using an EcoChemie Autolab PGSTAT 302Т potentiostat/galvanostat (Utrecht, The Netherlands) with the electrochemical software package Nova 2.0.A three-electrode system was used: a 2 mm-sized Pt disk working electrode, an Ag/AgCl reference electrode (3 M KCl) and a Pt wire counter electrode.Electrodes were purchased from Metrohm Co (Herisau, Switzerland).1,4-DHP solution was added to 0.1 M Tris-HCl (pH = 7.4) solution up to a final concentration 5 mM, and voltammograms were recorded.After that 10 µM of DNA was added to solution and measurements were repeated.The step was repeated at least twice.A scan rate of 100 mV/s was used throughout the experiments.All electrodes were washed with double distilled water prior to each measurement.Oxygen-free nitrogen was bubbled through the solution for 5 min before each experiment.All experiments were carried out at 25 °C.
The binding constant was determined according to the following equation: log (1/DNA) = log (K) + log [I free /(I free -I bond )], where K -the apparent binding constant; I freethe peak current of free compound; and I bondthe peak current of compound in the presence of DNA (Feng et al. 1997).
The number of the binding sites was determined according to the equation: The number of electrons (n) was calculated using equation: Ep -Ep/2 = 47.7 mV/αn where Ep -peak potential of compound, mV; Ep/2 -half wave potential of compound, mV; α -the assuming value = 0,539; n -number of electrons [10].

Fenton reaction -DNA protection assay
pTZ57R DNA was treated with 0.003% hydrogen peroxide and 0.01mM iron(II) sulphate in PBS for 30 min at 37°C.Induction of singlestrand breaks was monitored by electrophoresis in agarose gels in neutral conditions following conventional protocols.Briefly, 0.2 μg of DNA was incubated with hydrogen peroxide and iron(II) sulphate in the presence or absence of 1,4-DHP at room temperature.Following incubation, the samples were mixed with 6×Orange loading solution and loaded onto 0.8% agarose gel containing 40 mM Tris, 20 mM sodium acetate and 2 mM EDTA and electrophoresed in a horizontal slab gel apparatus in Tris/acetate/EDTA gel buffer at 30V. Results were presented as percentage of supercoiled and open circular DNA ( [15]).Data were normalized according Kolmogorov-Smirnov, statistical analysis was performed by one direction ANOVA and Tuckey post-test.

Absorption studies
Absorption titration was carried out to monitor the interaction of the compounds with sonicated plasmid DNA.In a series of water-soluble monocyclic derivatives of 1,4-dihydropyridine with carboxylate groups in position 4 the compounds manifested different affinity to DNA determined mainly by substituents in positions 3 and 5.The compounds with cyano group or acetyl groups in position 3 and 5 (J-3-183 and AV-154 correspondingly) did not interact with DNA.Replacement of 3,5-acetyl groups (AV-154) with methoxycarbonyl groups (J-7-53-B) did not improve the DNA binding, however ethoxycarbonyl groups (AV-153 Na) made the compound able to interact with DNA: a pronounced hyperchromic and bathochromic effects were observed as described previously ([11]; Table 1).Further modification of positions 3 and 5 decreased the DNA binding capacity of the compounds, it decreased almost five-fold when aromatic rings were added to ethoxycarbonyl groups (J-8-120; Table 1), and a drastic 30-fold difference was observed between compounds with ethoxycarbonyl (AV-153 Na) and propoxycarbonyl groups (J-4-96) in positions 3 and 5. Interestingly, the ethoxycarbonyl groups in positions 3 and 5 appear to determine the DNA-binding capacity in the series of our compounds with no cyclic side groups.At the same time, the same ethoxycarbonyl groups in positions 3 and 5 determine antimicrobial activity in another series of 1,4-DHP [18], thus antimicrobial activity might be due to the compound capability of binding DNA.
Capacity of interactions with DNA was strongly dependent also on substituents in position 4. Addition of alanine in position-4 as amide of 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydroisonicotinic acid (alapyrone) abolished an ability to bind DNA, however taurine in the same position (tauropyrone) maintained the ability to interact with DNA (Table 1).
Tricyclic fused 1,4-DHP derivatives -decahydroacridine-1,8-diones (PP-150-Na and PP-544-NH 4 ; B-5-Na) produced hyperchromic effect without any shifts (Fig. 1).PP-544-NH 4 was the most effective DNA binder, replacement of carboxylic group in position 4 with an aromatic ring and addition of aliphatic chains ending with a carboxylic group to nitrogen in position 1 drastically decreased the effectiveness of DNA binding (Fig. 2, Table 1).Binding constant of the PP-544-NH 4 with DNA depended on the ionic strength of the solution: it increased when ionic strength raised from 10 mM to 50 mM and abruptly decreased at 150 and 300 mM NaCl (not shown).
Formerly it was shown that AV-153-Na binds to DNA via intercalation ( [11]).For further studies we have chosen two compounds -PP-544-NH 4 , as it turned out to be the strongest binder among the studied and J-4-96, differing from AV-153 by length of the groups in positions 3 and 5, the latter modification drastically decreased its affinity to DNA (Table 1).

Fluorescence assay
Interactions with DNA of the PP-544-NH 4 , the strong DNA binder revealed by UV/VIS spectroscopy, was also confirmed by fluorescence measurements.When irradiated with excitation light at 255 nm, the compound emitted fluorescent light at 462 nm, intensity of the fluorescence increased when DNA was added to the solution (Fig. 2A).These results confirm again the fact of the direct interaction between the compound and DNA, indicating decrease of fluorescence quenching effect of solvent molecules after penetration of the molecule in hydrophobic environment [19].Interestingly, two other tricyclic compounds B-5-Na and PP-150-Na produced a different effect.Excitation maximum was at 400 nm, emission peak -at 515 nm.After DNA addition, the maximum emission peak underwent a red shift to 520 nm, but without the fluorescence intensity enhancement (Fig. 2B).Compound J-4-96 with longer propoxycarbonyl groups in positions 3 and 5 compared to AV-153 and much lower affinity to DNA manifested similar changes in fluorescence spectra after addition of DNA.Besides an increase of the fluorescence intensity a red shift was observed (Fig. 3A).However unlike AV-153 the compound did not extrude EtBr out of DNA, as intensity of fluorescence of EtBr and DNA complex did not decrease significantly in presence of the compound (Fig. 3C).Apparently, the 1,4-DHP can bind DNA by different modes -intercalation, as AV-153 and minor groove binding, as weaker binders (J-4-96).Indeed, good binders could probably interact with DNA by both intercalation and DNA minor groove binding, like the berberine [20], modifications of the molecules decreasing the affinity to DNA could mainly abolish ability to intercalate.

Cyclic voltammetry
Interaction of the PP-544-NH 4 with DNA was confirmed electrochemically.Figure 4B shows cyclic voltammograms of 5 mM PP-544-NH 4 alone and presence of increasing concentrations of DNA in 0.1 M Tris-HCl buffer, pH = 7.4.The peak current decreases upon the addition of increasing concentrations of DNA, owing to the binding of the 1,4-DHP.The compound exhibited a single well defined anodic peak, which corresponds to the oxidation of dihydropyridine ring [22].In reverse scan no one peak was observed indicating that oxidation of the compound is an irreversible process.The binding constant of the compound calculated on the basis of electrochemical experiments was equal to 1.11 × 10 4 , the compound should interact with 2 base pairs.

Other biological activities of DNA-binding 1,4-DHP
It was interesting to compare DNA-binding capacities of the 1,4-DHP with other activities of the compounds.

Decomposion of peroxynitrite in the presence of 1,4-DHPs
It was revealed that the decomposition of peroxynitrite was slightly accelerated in [the] presence of J-4-186, J-7-53-B and J-8-120 (Table 1).The strong DNA binder tricyclic fused 1,4-DHP derivative PP-544-NH 4 produced a paradoxical kinetic curve -the optical density of peroxynitrite and DHP mixture increased with time.Apparently, the compound interacts chemically with peroxynitrite, the reaction product has absorbance peak in the area of peroxynitrite absorbance maximum (Fig. 5).However, we could not reveal any correlation between DNA binding and peroxynitrite scavenging capacities.

Radical scavenging -EPR measurements
The ability of the 1,4-DHP to scavenge other free radicals, namely OH radical produced in the Fenton reaction was tested by EPR method.
We have tested both strong (PP-150-Na) and weak DNA binders (AV-154-Na, J-7-53, J-8-120) at 1000 μM concentration.The signals of the second component of EPR spectra were measured on the 3rd min (I 3 ) and 5th min (I 5 ) and the difference between them I 3 -I 5 was calculated (Fig. 6A).Scavengers of OH radicals should increase the difference between I 3 and I 5. Representative kinetics of the decrease of EPR signal intensity is shown in Fig. 6B, results are summarized in Table 1.As seen in Figure 6B AV-154 does not interfere with the rate of the reaction, an impact of PP-150-Na is modest.Similar results were obtained for most other compounds.Thus, a correlation between radical scavenging and DNA-binding capacities was not observed.Moreover, some compounds, including one of the strongest DNA binders PP-544-NH 4 increased intensity of the hydroxyl radical signal, indicating their pro-oxidant effect.It also turned out that J-4-96 and J-

DNA protection in vitro against damage by radical produced in Fenton reaction
Ability of the tested compounds to protect plasmid DNA against induction of singlestrand breaks produced by radicals generated in Fenton reaction was tested by means of DNA electrophoresis in neutral conditions.The reaction produced a significant DNA damage (p < 0.0001 n = 35-91).Some of the tested compounds produced a protecting effect.Some weak binders (J-7-53 and J-4-96) even enhanced the level of DNA breakage to some extent, however statistical significance was not reached (not shown).Thus in these series of experiments we could not detect a pronounced correlation between DNA-binding capacity and DNA protection.Taken together, our data indicate that several 1,4-DHP derivatives can bind efficiently to DNA, affinity to DNA strongly depends on the structure of the derivative.1,4-dihydropyridine with carboxylate groups in position-4 and with ethoxycarbonyl groups in positions 3 and 5 (AV-153) and fused tricyclic molecules appear to be the best DNA binders.Both intercalative and minor grove binding mechanisms are possible.No evident correlations between DNA binding and other activities of the compounds could be revealed.

(
I -I DNA )/I DNA = K [DNA]/2s where I -the peak potential of compound in the absence of DNA; A, I DNA -the peak potential of compound in the presence of DNA; A, K -the binding constant of compound-DNA complex; [DNA] -concentration of DNA, mol/L; s -number of binding sites (Aslanoglu 2006; Carter et al. 1989).

Fig. 1 .
Fig. 1.UV/VIS spectra of PP-544-NH4 in absence and presence of DNA added by 12.5 µM at each step.

Fig. 2 .
Fig. 2. A -Fluorescence spectra of PP-544-NH 4 with excitation at λ=250 in presence of different concentrations of DNA; B -Fluorescence spectra of PP-150-Na with excitation at 400 nm, 12.5 µM DNA was added at each step until saturation (125 µM).Concentration of the compound was 25 µM.

Fig. 5 .
Fig. 5. Changes of the spectrum of PP-544-NH 4 in presence of peroxynitrite with time (0 -25 min).Peroxynitrite was added also to the control cuvette.

Fig. 6 .
Fig. 6.A -EPR spectra of DMPO-OH radicals generated in Fenton reaction in presence of DMPO. 1 -EPR spectra of DMPO-OH radicals 3 min after mixing the components for Fenton reaction.2 -EPR spectra of DMPO-OH radicals 5 min after mixing the components for Fenton reaction.I 3 and I 5 -intensities of EPR signals used for quantification of DMPO-OH radicals at corresponding time.3 -difference between 3 min and 5 min spectra indicating decrease of the signal intensity and lack of generation of other radicals.B -time course of decrease of intensity of DMPO-OH radical spectra. 1 -control mixture; 2 -in presence of AV-154; 3 -in presence of PP-150-Na.