Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular

Jul 5, 2013 - *Phone: +91-33-2429-8861. ... Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different or...
0 downloads 10 Views 5MB Size
Download PDF
Article pubs.acs.org/jmc

Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular Cytotoxicity with Involvement of C‑7-OH and −NH Functional Groups Suman K. Samanta,† Devawati Dutta,† Sarita Roy,‡ Kaushik Bhattacharya,† Sayantani Sarkar,† Anjan K. Dasgupta,‡ Bikas C. Pal,§ Chhabinath Mandal,§ and Chitra Mandal*,† †

Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial ResearchIndian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India ‡ Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India § National Institute of Pharmaceutical Education and Research, Kolkata, 4 Raja S. C. Mullick Road, Kolkata 700032, India S Supporting Information *

ABSTRACT: Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure−activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxymahanine-treated cells, indicating significant contribution of the C-7-OH group. OMethylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.

■

INTRODUCTION Phytochemicals hold a high potential for the treatment of a variety of cancers.1 Many phytochemicals like resveratrol, (−) epigallocatechin gallate (EGCG), 6-gingerol, taxol, and myricetin have been demonstrated to inhibit cancer cell proliferation and to induce cell death through apoptosis.1 Various parts of an edible plant, Murraya koenigii, have been used in conventional and folk medicine for the treatment of rheumatism, traumatic injury, influenza, and other diseases.1 Leaf extract of this plant is a rich source of carbazole alkaloids.2 Mahanine, a carbazole alkaloid, was purified from the leaves of this plant and exhibits antimutagenicity, cytotoxicity, and antimicrobial activity.2−4 It induces apoptosis in acute lymphoid and chronic myeloid leukemia, histiocytic lymphoma, promyelocytic leukemia, prostate, and pancreatic cancer.2,4−6 Thus, mahanine is a highly promising candidate for chemotherapy. Many anticancer drugs including belotecan from camptothecin are known to interact with DNA directly by preventing the proper relaxation of DNA by topoisomerases.7,8 The modes of interaction of these small molecules to DNA are generally through intercalation in which the planar molecules stack between DNA base pairs.8 This decrease in the DNA helical twist causes lengthening of DNA and considerable change in DNA conformation. Although the creation of the intercalation cavity is energetically unfavorable, the binding stability of such © XXXX American Chemical Society

complex is achieved by higher contribution from stacking interactions of the flat unsaturated rings of the intercalator. Ellipticine, 9-aminoacridine, and ethidium bromide are known as typical intercalators.9 Alternatively, some molecules are groove binders which place in the minor groove of DNA. Intermolecular interactions between a small molecule and the two strands of a DNA duplex stabilize the binding, usually showing higher association constants in comparison with intercalators. Mitomycin and distamycin are known minor groove binding agents.10 Both types of binding are known to impart anticancer and antibacterial properties.7 A variety of spectroscopic studies like UV−visible, infrared (IR), and circular dichroism (CD) have been used to characterize the interaction of the intercalators and groove binders to DNA, which can provide some light on the structural aspects of these complexes.11 Here, we have chemically modified the functional groups of mahanine and examined their involvement in the induction of cell death in 19 different cancer cell lines from 7 different types of cancers in comparison to naturally occurring derivatives. We have also analyzed how such modification influences its mode of interaction with DNA by several biophysical analyses. The Received: February 25, 2013

A

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Chemical modifications of mahanine and dehydroxy-mahanine isolated from leaves of Murraya koenigii..

in a concentration dependent manner as compared to dehydroxy-mahanine in all the cases (Figure 2A). The time dependent activity of mahanine and dehydroxy-mahanine in a representative cell line (T98G) also showed a similar trend (Supporting Information Figure S3). Approximately 7.0−18.0 μM of mahanine was required to inhibit 50% growth inhibition (IC50) of all these 19 cancer cells. On the other hand, the IC50 values of dehydroxy-mahanine were in the range between 25− 50 μM under identical condition. The sole structural difference between these two compounds is the presence of a hydroxyl group at C-7 position in mahanine which is absent in dehydroxy-mahanine. These results suggested that C-7-OH group has a significant contribution toward the elicitation of the cytotoxic effect of mahanine. Next we compared the concentration (0−30 μM) dependent effects of mahanine and dehydroxy-mahanine with three other chemically modified compounds (O-Me-mahanine, O-Acmahanine, and N-Me-dehydroxy-mahanine) on the inhibition of cell proliferation in a representative set of five cell lines derived from five different cancers namely human glioma (T98G), pancreatic (Panc 1), lung (A549), colorectal carcinoma (HCT116), and chronic myelogenous leukemic cells (K562) after 24 h treatment (Figure 2B). IC50 values of mahanine were much lower than those of O-Me-mahanine in all cells tested, indicating that the methylation of the C-7-OH group reduced its antiproliferative activity. Interestingly, the IC50 values of mahanine and O-Acmahanine were very close to each other, apparently indicating that the O-acetylation did not reduce the antiproliferative activity of mahanine. This might be due to the deacetylation of this derivative by esterase present indigenously in cells. It was further confirmed by incubating acetylated mahanine only with medium at physiological condition. The result demonstrated that acetylated form of the mahanine could not convert to its native form (mahanine) in medium only (data not shown).

interaction of mahanine with DNA was evaluated by UV−vis, Fourier transform infrared spectroscopy (FTIR), CD spectroscopy, and isothermal titration calorimetry (ITC) study. Additionally, we have performed molecular docking and dynamics simulation to evaluate the nature and strength of binding of mahanine and its derivatives to DNA. In conclusion, our observations demonstrate potential identification of functional groups of a novel promising herbal molecule to treat an array of cancers. Such studies would be helpful in developing new agent with higher efficacy.

■

RESULTS Synthesis of Different Analogues of Mahanine. Both the purified molecules, mahanine and dehydroxy-mahanine, were chemically modified at C-7 and N-9 positions in their carbazole moieties (Figure 1). A methyl, acetyl, and biotinyl group was added to the C-7 hydroxyl group of mahanine to get O-Me-mahanine, O-Ac-mahanine, and biotinylated mahanine, respectively. In addition, the imino group at N-9 of dehydroxymahanine was also modified by methylation to get N-Medehydroxy-mahanine. The structures of these derivatives were identified by MS and NMR (1H and 13C) (Supporting Information Figure S2). It was observed that the synthetic derivatives were soluble only up to a certain concentration (200 μM) in experimental buffer solution at 25 °C compared to both mahanine and dehydroxy-mahanine. The biological activities of these derivatives were evaluated using different cancer cells. However, biotinylated mahanine was not soluble in experimental buffer, possibly due to the presence of a bulky functional group. Modified Mahanine Shows Differential Antiproliferative Activity on Cancer Cells. Growth inhibitory potential of mahanine and its natural derivative (dehydroxy-mahanine) were evaluated on the proliferation of 19 different cell lines derived from 7 human malignant tissues after 48 h treatments. Mahanine showed a significantly better antiproliferative activity B

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. continued the mean ± SD of three independent experiments. *, P < 0.05, significant difference between two test groups.

It might be noted that the IC50 values of N-Me-dehydroxymahanine were significantly higher than those of dehydroxymahanine, suggesting the presence of the imino hydrogen at N9 position was essential for higher antiproliferative activity of dehydroxy-mahanine. In contrast, mahanine showed very little cytotoxic effect on Vero and HEK-293T cells, used as normal control (Figure 2C). This was demonstrated by >85% viability in case of Vero and >65% viability in case of HEK-293T even at 60 μM concentration of mahanine after 24 h of treatment. Mahanine Exhibits Changes of the Shape and Density of Cancer Cells Compared to Other Derivatives. Morphological changes of a representative cancer cell (glioma, U373MG) was evaluated after treatment of mahanine, O-Memahanine, and O-Ac-mahanine by phase contrast microscopy after 3 and 12 h of treatment at 30 μM concentration (Figure 2D). Mahanine-treated cells exhibited a complete collapse of their shape and density at 12 h, while some damage could be noticed after 3 h. In contrast, the cells appear to be unaltered in presence of O-Me-mahanine even after 12 h of exposure, indicating weaker cytotoxic effect than mahanine, suggesting the importance of C-7-OH. When we compared the effect of O-Ac-mahanine on the cells with that of mahanine at 3 h of exposure, the cells appear to be less affected in O-Ac-mahanine. But, after 12 h of exposure of O-Ac-mahanine, the cells were considerably damaged although not to the same extent as mahanine treated cells. If the exposure was extended up to 24 h, O-Ac-mahanine showed almost similar effect as mahanine (data not shown). This might be due to the deacetylation of O-Ac-mahanine with time through hydrolysis by esterase present indigenously in cells at physiological condition and therefore regeneration of active C7-OH. This was more clear at earlier time points; the percentage of viable cells upon treatment of O-Ac-mahanine was much higher than mahanine treatment, indicating that Oacetylation of C-7-OH capable of blocking the effect of mahanine at least up to 3 h (Figure 2D). Mahanine Shows Enhanced DNA Damage Compared to Other Derivatives. Cellular DNA damage by comet assay is a common biomarker for apoptosis.12 Mahanine (20 μM) treated T98G resulted in extensive DNA damage, as reflected from the tail length of the comet after 24 h (Figure 3A). In contrast, other derivatives under identical condition resulted in reduced amount of DNA damage or fragmentation. The extent of mahanine-induced DNA damage was ∼70% compared to other derivatives as determined by measuring the tail length of the comet under microscope. The data of tail lengths (μm) were represented as mean ± SD from at least 20 cells or comets in each treatment group. Enhanced Apoptosis in Mahanine-Treated T98G and Panc-1 Cells Compared to Dehydroxy-mahanine. Cell death in T98G and Panc-1 cells induced by mahanine and dehydroxy-mahanine (30 μM) were monitored by 7-AAD after 24 h of treatment (Figure 3B). Mahanine-treated significant cell death of T98G and Panc-1 was indicated by 75.6% and 91.2% 7-AAD positivity respectively. In contrast, dehydroxy-mahanine triggered only 10.13% and 17.06% cell death than that of

Figure 2. Effect of mahanine and its derivatives on inhibition of cancer cell proliferation. (A) Inhibition of proliferations of 19 cell lines from 7 different cancers in presence of mahanine and dehydroxy-mahanine as determined by MTT assay after 48 h of treatment. The concentration needed for 50% reduction of viability (IC50) is shown. (B) IC50 values of mahanine, dehydroxy-mahanine, and three chemically modified compounds on selected cancer cells after 24 h of treatment. Inhibition of proliferations of human chronic myelogenous (K562) leukemic, glioma (T98G), pancreatic (Panc 1), lung (A549), and colorectal (HCT116) cancer cells were assayed by MTT in presence and absence of five compounds. (C) Comparison study of cytotoxic activity of representative cancer cell lines with the normal cell lines in presence of 0, 15, 30, 45, and 60 μM mahanine at 24 h determined by MTT assay. (D) Morphological changes in glioblastoma cells (U373MG) before and after exposure of mahanine, methylated mahanine (Memahanine), and acetylated mahanine (Ac-mahanine) (30 μM) after 3 and 12 h of treatment by phase contrast microscopy. Each value is C

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. continued treatment as described in Material and Methods. The relative ratio of 585/530 nm fluorescence (ΔΨm), i.e., J-aggregates in mitochondria vs monomers in the cytosol represents ΔΨm. The ratio of emission of red (585 nm) and green (530 nm) fluorescence from mitochondria and cytosol, i.e., polarization of ΔΨm, was analyzed immediately by flow cytometry. (E) Mitochondria-mediated death cascade activation in T98G and Panc 1 cells induced by mahanine (25 μM) and O-Memahanine (25 μM), dehydroxy-mahanine (50 μM), and N-Medehydroxy-mahanine (50 μM) after 24 h treatment. The status of Bclxl, Bid, caspase 7, caspase 8, caspase 9, and PARP were determined in immunoblot analysis as described in Materials and Methods. In each Western blot analysis, β-actin was the loading control. Each value is the mean ± SD of three independent experiments. *, P < 0.05, significant difference between two test groups.

control in respective cells, thus substantiating the importance of C-7-OH group of mahanine in cytotoxicity. Induction of apoptosis of T98G and Panc-1 cells were further determined using dUTP-FITC staining after 24 h (Figure 3C). The significant extent of dUTP-FITC positive cells confirmed that growth inhibition was due to apoptosis. Exposure of mahanine (30 μM) led to 60.72% (T98G) and 45.9% (Panc-1) cells toward the TUNEL positivity. On the other hand, dehydroxy-mahanine, under identical conditions, showed marginal increase in dUTP-FITC positive cells, being only 9.58% and 1.13% positivity for respective cancers. These observations authenticated the potential contribution of −OH group at C-7 of mahanine, which is absent in dehydroxymahanine. Enhanced Mitochondrial Transmembrane Depolarization (ΔΨm) in Mahanine-Treated T98G and Panc-1 Cells Compared to Dehydroxy-mahanine. Our earlier studies demonstrated that increased ROS production in mahanine-treated leukemic and pancreatic cancer cells raised the possibility of mitochondrial dysfunction.4,6 Hence, the mitochondrial membrane potential was examined using different derivatives of mahanine on T98G and Panc-1 cells at 24 h by JC1 (Figure 3D). In mahanine (30 μM) treated cells, the ratio of 585/530 nm fluorescence (ΔΨm), i.e., J-aggregates in mitochondria vs monomers in the cytosol exhibited 82.4% (FL1) than that of the control (10.26%), suggesting enhanced mitochondrial transmembrane depolarization. In contrast, mitochondrial depolarization was only 16.72% in dehydroxymahanine-treated cells at identical condition. Mahanine-Induced Alteration in Several Pro- and Anti-apoptotic Mitochondrial Proteins in Cancer Cells Compared to Its Other Derivatives. Enhanced mitochondrial depolarization in mahanine-treated cancer cells prompted us to examine the alteration of the expression of several mitochondrial proteins like antiapoptotic protein Bcl-xL and pro-apoptotic protein Bid. Those are two essential elements involved in cell survival and death.13 Decrease in the total Bid suggests that it is cleaved and thus the activated form of Bid is generated and that can initiate mitochondrial death cascade.14 Western blot analysis revealed significant down regulation of Bcl-xL and total Bid in mahanine (25 μM) treated (24 h) cells (Figure 3E). Methylation at the effective functional group (OH) at C-7 of mahanine could not decrease Bcl-xL or total Bid. Therefore, decrease in the survival rate of mahaninetreated cells was associated with the decrease in the Bcl-xL and total Bid, suggesting a role of this C-7-OH group in mitochondrial damage by affecting these Bcl-2 family proteins.

Figure 3. Effect of mahanine and its derivatives in cancer cells. (A) DNA damage visualized with the comet assay in T98G cells incubated for 24 h with mahanine and its derivatives at 20 μM dose compare to untreated cells. Tail lengths in μm were represented as mean ± SD from at least 20 cells or comets in each treatment groups. (B) Induction of apoptosis in glioma (T98G) and pancreatic (Panc 1) cancer cells in absence and presence of mahanine and dehydroxymahanine (30 μM) after 24 h of treatment as determined by 7-ADD positivity. (C) Effects of mahanine and dehydroxy-mahanine (30 μM) on T98G and Panc 1 cells as determined by flow cytometry using dUTP-FITC after 24 h of treatment. (D) Mitochondrial involvement in mahanine and dehydroxy-mahanine (20 and 30 μM) treated T98G and Panc 1 cells was characterized using a dye JC 1 after 24 h of D

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Similar kind of down regulation of Bcl-xL and Bid proteins were also observed in dehydroxy-mahanine-treated T98G and Panc-1 cells only at higher concentrations (50 μM), suggesting additional contribution of −NH group in apoptosis. However, N-Me-dehydroxy-mahanine even at 50 μM concentration could not exhibit such down-regulation, further confirming the role of imino group at N-9 present in dehydroxy-mahanine. Methylation of Effective Functional Groups of Mahanine Reduces Activation of Mitochondria-Mediated Caspase-Dependent Death Cascade. The apoptosis induced by mahanine and its derivatives were further compared by measuring the levels of pro-caspases using immunoblot analysis (Figure 3E). At 24 h, mahanine (25 μM) was effective to degrade pro-caspases 7, 8, and 9 in both T98G and Panc-1 cells whereas no significant decrease of these protein levels was observed in Me-mahanine-treated cells demonstrating the role of −OH group at C-7. On the other hand, dehydroxy-mahanine showed comparable level of pro-caspase activation like mahanine only at higher concentration (50 μM). In contrast, N-Me-dehydroxy-mahanine-treated cells failed to activate the caspases at the same concentration, indicating additional contribution of −NH functional group at N-9. Enhanced Cleavage of PARP in Mahanine-Treated T98G and Panc 1 Cells. DNA repairing enzyme PARP was cleaved in mahanine (25 μM) treated T98G and Panc 1 cells after 24 h (Figure 3E), which is known to induce nucleosomal condensation and apoptosis.14 The PARP cleavage was completely rescued by O-Me-mahanine treatment under identical conditions. However, dehydroxy-mahanine even at a 50 μM concentration cleaved little PARP whereas N-Medehydroxy-mahanine was unable to show any PARP cleavage. Mahanine Decreases the UV Absorption upon DNA Binding. DNA shows an intense absorption band at 260 nm, and this band intensity reduced significantly in the presence of mahanine or its derivatives (Figure 4A) when we plotted the wavelength vs normalized absorbance. This decrease in absorption was maximum in presence of mahanine, while other derivatives also showed reduced-interaction with DNA in the following order mahanine > dehydroxy-mahanine > O-Memahanine > N-Me-dehydroxy-mahanine. All the above experiments revealed that mahanine is the most active compound among all the derivatives with C-7-OH and N-9 imino groups and also shows the greater interaction with DNA. So, we then focused on the binding efficacy, mode of binding, and involvement of DNA molecules toward the mahanine−DNA complex. The values of the cooperativity of binding (i.e., n = 2.214) and binding constant (Kb) of mahanine were obtained from the DNA absorption at 260 nm from Hill plot.15 From the Hill plot analysis, we calculated the binding constant of mahanine as Kb = 11.9 μM using the equation: log {v/(2 − v)} = αH·log(Dfree) − αH·log(Kb). Here, Dfree = concentration of free mahanine, v = absorbance, αH = slope of the hill plot curve, Kb = binding constant of mahanine. The cooperativity of binding of DNA− mahanine complex was calculated using the following equation:

Figure 4. Effect of mahanine and its derivatives in DNA binding. (A) Absorption spectra of DNA (100 μg/mL) was taken after six times dilution with the same buffer in the absence and presence of mahanine and its three derivatives (100 μM) in Tris-HCl and 50 mM NaCl buffer after 2 h of incubation as described in Materials and Methods. (B) Absorption spectra of DNA (100 μg/mL) after 2 h of incubation with the various dose of mahanine (a = 0, b = 20, c = 40, d = 60, e = 80, f = 100, g = 120 μM) was taken after six times dilution with the same buffer. The Hill plot was drawn with the help of free mahanine present after addition of each concentration at the similar time point. (C) Fourier transform infrared spectroscopy of DNA (250 μg/mL) the absence and presence of mahanine (1 mM) and dehydroxymahanine (1 mM) as described in Materials and Methods. (D)

log(Y /2 − Y ) = n log([L]) − log(2Kb)

Here, n = Hill coefficient, Y = (ODmax − ODmax(1)/(max (ODmax) − ODmax(1)), L is the concentration of mahanine, i.e. 3.33, 6.66, 9.99, 13.32, 16.65, and 20 μM, respectively. ODmax = maximum intensity of DNA−mahanine complex which might be at 260 nm or slight red- or blue-shifted for a particular concentration; ODmax (1) = maximum intensity of DNA when E

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

used to generate the graph represented in the Figure 4D. With the help of Origin 7 software, enthalpy change (ΔH) was calculated to be −3943 ± 455 cal/mol and the entropy change (TΔS) was 2686.81 cal/mol. The free energy change (ΔG) for mahanine−DNA complex was −6.6 ± 0.45 kcal/mol and the KD value was about 11 μM. Because the entropy change was more than 40% of the total free energy, it might be concluded that mahanine was a minor groove binding agent. Mahanine Is Unable to Induce Conformational Changes in DNA. To identify the conformational changes in DNA duplex as a result of its interaction with mahanine, the CD spectral studies were carried out (Figure 4E). It is noted that the CD band at 275 nm, due to base stacking and at 245 nm due to the B-conformation of DNA did not change significantly even at a concentration of 100 μM of mahanine in comparison with a known intercalator (EtBr, 300 μM). As reported earlier, simple electrostatic interaction or groove binding of small molecules with DNA did not affect the intensity of the band at 275 nm, whereas a change in intensity of the band at 245 nm indicated the change of B-DNA conformer, i.e., formation of a different conformation of DNA.20 From this spectral analysis, it might be concluded that mahanine did not act as an intercalator. Molecular Modeling. A. Mahanine Binds to a Minor Groove of DNA by Molecular Docking. Out of the 100 docking runs performed for each DNA−ligand complexes, favorable binding poses were obtained with those presenting the most negative binding free energies (Table 1). Molecular docking results showed that mahanine had the best binding energy of −8.4 kcal/mol when bound between the gap of DNA bases (DNA1) as an intercalator. Interestingly, mahanine when bound in minor groove with DNA2 and DNA3, providing more favorable binding energy of −8.8 and −9.1 kcal/mol, respectively. However, from molecular docking results, it was very uncertain to distinguish whether mahanine was a minor groove binder or an intercalator. From the two controls, it could be stated that netropsin, a known minor groove binder, when bound as an intercalated complex, showed less stable energy (−6.9 kcal/mol) in comparison to its minor groove bound complex with both DNA2 (−10.0 kcal/mol) and DNA3 (−11.4 kcal/mol). On the contrary, ellipticine, an intercalator, showed a significantly higher binding energy in intercalated form (−8.7 kcal/mol) rather than minor groove form (−7.4 kcal/mol). From these results, it could be stated that the general trend of mahanine is being that of a minor groove binder. The minor groove bound state of mahanine resulted in a close fit of the molecule along the wall of the minor groove (Figure 5A). The three fused aromatic carbon rings, the carbazole moiety, is placed in the middle of the minor groove and the nonpolar tail portion spreads toward the side wall of minor groove. The functional C-7-OH group forms a bifurcated hydrogen bond interaction with the oxygen atom of the backbone deoxyribose sugar of G9 (2.11 Å) and amino

Figure 4. continued Isothermal calorimeter titrations of the DNA against mahanine The symbol filled square indicated, data points taken after deduction of the dilution effect, and the line is calculated from the average of the best fit parameters shown for the DNA (10 μM). All titration were conducted in 50 mM tris-HCl buffer, pH = 7.5. (E) CD spectra of DNA (1 mg/ mL) in the presence and absence of mahanine (100 μM). EtBr (300 μM) was used as a positive control.

there was no mahanine; max (ODmax) = among all different concentration of mahanine used the one which showed maximum intensity. Strong Involvement of the Phosphate Backbone during Mahanine−DNA Interaction. DNA contains two kinds of nucleophilic sites, exocyclic nitrogen of the bases and carbonyl oxygen of phosphate group, and these can function as hydrogen bond acceptors.16 The molecular vibrations arising from these different moieties of DNA are observed in specific regions of the IR spectrum. The most significant changes in the FTIR spectra of DNA were observed in the region between 1531 and 950 cm−1 after binding with mahanine (Figure 4C). The FTIR signal at 968 cm−1 was due to the sugar−phosphate stretching frequency and showed strong spectral intensity changes upon mahanine complexation. This intensity change indicated that mahanine binds to the phosphate backbone of DNA. The change in intensity of the PO2− symmetric stretch at 1082 cm−1 indicated the restriction in the mobility of the functional group, thus implying the binding of the mahanine to DNA. The shift in frequency and change in intensity of the PO2− asymmetric stretch at 1219 cm−1 suggested that the phosphate group was an important site with which mahanine interacted with DNA and the binding site was close to the phosphate groups. It was reported that the change in intensity at 1531 cm−1 is due to the involvement of cytosine.17,18 It might be pointed out that the change in intensity or shift at 1531 cm−1 indicated that mahanine must have some interaction with cytosine. It was also known that the alteration in IR spectra at 1643 cm−1 indicates the possibility of a minor groove binder.19 Alteration in the spectra of the DNA bases at 1643 cm−1 (cytosine, C2O) suggested the interaction between DNA and mahanine occurs in the minor groove. Hence, the changes observed in the infrared spectra of DNA due to binding of mahanine indicated the strong involvement of phosphate backbone and weak interaction with cytosine in the minor groove of DNA. Mahanine a Minor Groove Binder: A Thermodynamic Signature of Mahanine−DNA Binding by ITC. ITC is a well established technique for analysis of enthalpy and equilibrium constant for DNA−ligand binding. Groove binding is principally entropic motivated, while intercalation is enthalpic motivated.17 The data sets from the DNA titration with the mahanine independently, and the average fit parameters were

Table 1. Binding Modes and Free Energy Values of Mahanine−DNA Complexes Achieved by Docking Netropsin (Known Minor Groove Binder) and Ellipticine (Known Intercalator) Were Used As Separate Controls DNA1

DNA2

DNA3

ligand

binding mode

ΔGbinding (kcal/mol)

binding mode

ΔGbinding (kcal/mol)

binding mode

ΔGbinding (kcal/mol)

mahanine netropsin ellipticine

intercalation intercalation intercalation

−8.4 −6.9 −8.7

minor groove minor groove minor groove

−8.8 −10.0 −7.4

minor groove minor groove minor groove

−9.1 −11.4 −8.5

F

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 5. continued of mahanine forms bifurcated hydrogen bond with G9 and G5. N9 atom forms another hydrogen bond with T7. (C) Closed proximity of neighboring residues A6, C8, G5 T7, and G9 in the minor groove region of the DNA with the ligand imply van der Waals interactions. (D) Regression analysis between ΔGexp and ΔGcalcd values is shown for predicting the binding free energies of mahanine and its derivatives after MD simulation (number of sample was 8 with mean x as 36.6 and mean y as 10.35, the slope and intercept were 0.165 and 4.32 respectively).

nitrogen atom of G5 (2.16 Å). The N-9 imino group formed hydrogen bond with the carbonyl oxygen of base T7 (2.28 Å) (Figure 5B). However, other significant close electrostatic and van der Waals interactions were with neighboring bases G5, A6, T7, C8, G9, and G10 in the minor groove region of the DNA (Figure 5C). B. Mahanine Stays in the Stable Conformation in the Minor Groove of DNA during Molecular Dynamic Simulation. It is well documented in the case of protein−ligand interactions, the free energies calculated using perturbation method with Gromacs comes much higher than the experimental values.21 However, Gromacs has not been used earlier for DNA−ligand interactions. Our observation in the case of DNA−ligand interactions also followed the similar trend, requiring the correlation procedure in order to predict any reasonable free energy values. Hence, we selected a data set of eight DNA−ligand complexes for which both crystal structures and the experimental free energies are available in the literature22 and statistical regression analysis was performed. Table 2 showed the calculated and experimental free energies of Table 2. Standard Free Energy (ΔG) Data for a Set of DNA Complexes with Ligand for Which the Experimental ΔG Values As Well As X-Ray Structures Are Availablea known ligands

PDB ID

ΔGexpb (kcal/mol)

ΔGcalcdc (kcal/mol)

netropsin distamycin berenil Hoechst33258 benzamidazole bisfuramidine symmetric benzamidazole asymmetric benzamidazole

121D 1JTL 2GVR 8BNA 2IKJ 227D 2GXV 2GYM

−12.0 −12.0 −8.0 −11.0 −9.0 −9.3 −11.0 −12.0

−43.8 −42.7 −24.3 −45.5 −27.2 −31.1 −36.8 −41.7

a

These values have been used to develop the regression equation between the experimental and calculated values fitting a statistical model. bExperimental ΔG values taken from literatures. cCalculated ΔG values obtained from thermodynamic integration method.

binding of this set of molecules. The results of simple linear regression analysis were shown in Figure 5D. A value of R2 (0.96) close to 1 indicated a very good positive correlation between ΔGexp and ΔGcalcd values. The regression equation was y = 4.32 + 0.165x and was used for predicting the binding free energies of mahanine and its derivatives after MD simulation. The free energy changes of ligand in water and in the complex are given in Table 2. The total free energy change of mahanine bound as a minor groove to DNA2 is −9.5 and DNA3 is −9.7 kcal/mol (Table 3). On the contrary, the free energy of binding of mahanine in intercalated conformation

Figure 5. Molecular docking and dynamics simulation of the binding of mahanine and its derivatives to DNA (A) Structure of minor groove-bound state of mahanine with DNA. DNA was shown in a surface representation in atom-based color (white for C, cyan for H, red for O, blue for N, and yellow for P), whereas mahanine is represented in sticks model with atom-based color (green for C, red for O, blue for N, and white for H). (B) The functional C-7-OH group G

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

kcal/mol revealed from ITC studies confirmed that mahanine acts as a groove binder to DNA. When we inspected the CD data then we might note the minor change in the CD pattern in presence of EtOH mimics the change occurring in presence of mahanine. Such insignificant conformational change of the double helix from in its usual B-form suggested a nonintercalative nature of mahanine binding to the DNA duplex compared to substantial change induced by a known intercalator, EtBr. If we now compared this by the cooperativity of binding, i.e., a positive n value (n = 2.214), it can only be interpreted as a nonintercalating binding occurring in a region which may be palandromic in nature. Such sites are normally targeted by various DNA-binding proteins.20 So obviously in the future the possible sequence specificity of the binding might be validated. If we use only alternative argument, absence of an intercalation like effect and presence of cooperativity can hardly be explained. This observation was further supported by molecular docking studies. It was established that mahanine did occupy a stable position at the minor groove of DNA showing a higher binding affinity than its intercalated state. This observation was in line with the binding of netropsin, a well-known minor groove binder, which also showed a higher strength of binding to the minor groove of DNA rather than an intercalated state. Similarly, ellipticine, an intercalator, showed higher binding strength upon intercalation in comparison to its complex docked in the minor groove. Consequently, simulation of the mahanine−DNA docked complex showed its flexibility to remain in a minor groove bound state with DNA. However, on modifying the −OH and −NH group of mahanine, the binding strength with the surrounding nucleotides got reduced. This depicted that these functional groups played a significant role in modulating the interaction of mahanine with nucleotide oligomers. Hence, we could conclude that mahanine interacts with DNA as a minor groove binder. Mahanine-induced growth inhibition led to apoptosis through mitochondrial membrane depolarization followed by activation of several apoptotic-related molecules. Establishment of the effective functional group(s) of an agent enhances the possibility for further development.17 The potential contribution of C-7-OH group in mahanine was confirmed by the presence of a significant percent of dUTP-FITC and 7-ADD positive cells, suggesting cancer cell growth inhibition was due to apoptosis, which was drastically reduced in dehydroxymahanine. Previously, we had shown that mahanine is a potential pro-oxidant agent both in the leukemic and the pancreatic cancer cells.4,6 Mahanine could produce a huge amount of ROS within very early hours of treatment. Mitochondrial depolarization is a major alteration in inner mitochondrial membrane mainly triggered by Ca2+ ion channel alteration and production of ROS in mitochondrial lumen. Here we had identified that modification of the C-7-OH or 9NH group(s) and the presence or absence of a −OH group contribute significantly toward variable cellular fate in an array of cancer cells. Among all the derivatives tested, mahanine showed the highest efficacy to trigger cell death and mitochondrial membrane depolarization in cancer cells. Therefore it might be envisaged that mahanine is capable of producing the highest amount of ROS among all the derivatives. So, it could be hypothesized that blockage of the −OH and/or −NH group might reduce the pro-oxidant property of these of compounds. Hence mitochondrial

Table 3. Calculated Free Energy (ΔG) Values of Mahanine and Its Analogues with DNA Duplex with Respective Binding Modes ligand mahanine

dehydroxy mahanine Me-dehydroxy mahanine Me-mahanine

receptor

binding mode

ΔGcalcd (kcal/mol)

DNA1 DNA2 DNA3 DNA2 DNA2 DNA2

intercalation minor groove minor groove minor groove minor groove minor groove

−8.8 −9.5 −9.7 −8.3 −8.6 −8.8

was −8.8 kcal/mol, substantiating the minor groove binding of mahanine. Further, on modification of the functional hydroxyl (C-7-OH) group (dehydroxy mahanine) and both the hydroxyl and amine group of mahanine (N-Me-dehydroxy mahanine), the binding free energy turned out to be less favorable. This suggested that hydroxyl group at C-7 position and the secondary amine group in mahanine plays a very important role in its interaction with DNA. On the other hand, absence of the functional hydroxyl group and secondary amine group in the derivatives weakens its interaction.

■

DISCUSSION The evaluation of structure−activity correlation facilitates the determination of the chemical groups responsible for biological effect.17 The present investigation was initiated mainly to address two problems: (i) identification of functional groups of a nontoxic novel carbazole alkaloid, mahanine, purified from an edible plant, responsible for its cytotoxicity against various cancer cells and (ii) examination the prospect and nature of their interaction with DNA. The present study is the first of its kind to provide evidence that C-7-OH group of mahanine was the main functional group, modification of which drastically reduced cytotoxicity toward different types of cancer cells. Additionally, the imino group at the N-9 position (−NH) also play an additional important role in exhibiting its biological effect. Several biophysical investigations were in good agreement with molecular dynamic studies, establishing that mahanine occupied a stable position at the minor groove of DNA showing higher binding affinity than its intercalated state. It is recommended that absorption spectroscopy studies provide a more complete description of the interaction between biomolecules.20 UV−visible spectral data analysis suggested that the interaction of mahanine with DNA resulted in maximum decrease in absorption intensity as a consequence of masking of base pairs due to interaction with nucleotides. It was also clear that the lesser interactive molecule being the NMe-dehydroxy-mahanine, and the intermediate interactive molecules were dehydroxy-mahanine and O-Me-mahanine further supported the major contribution of C-7-OH of mahanine. From high positive value of cooperativity of binding in the Hill plot analysis, it might be concluded that the strong complexation between DNA and mahanine. It was of interest to address the above query by examining the mahanine binding mode to DNA, using FTIR spectroscopy and ITC.18−20 The changes observed in the infrared spectra of DNA by FTIR due to binding of mahanine indicated a strong involvement of phosphate backbone and weak participation of the bases of DNA. Groove binding is in general entropically favored.17 TΔS value 2.7 kcal/mol and ΔG value −6.6 ± 0.45 H

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

and 1.3 equiv of methyl iodide (16.72 mg, 117 mmol) for 3 h. The excess NaH was deactivated by saturated NH4Cl solution. The mixture was treated with saturated brine solution to isolate the N-methylateddehydroxy-mahanine (Figure 1A). After each modification, the desired products were identified by IH and 13C NMR data analysis (Supporting Information Figure S2), and purity of the compounds were determined by HPLC, which was ≥95%. The details of NMR spectral data for each the synthesized and isolated compounds are listed here. Mahanine. 1H NMR (CDCl3, 300 MHz): δ 1.42 (s, 3H), 1.57 (s, 3H), 1.65 (s, 3H), 1.73 (t, 2H, J = 7.8 Hz), 2.15 (m, 3H), 2.31 (s, 3H), 5.10 (brs, 1H), 5.59 (d, 1H, J = 9.6 Hz), 6.51 (d, 1H, J = 9.3 Hz), 6.68 (d, 1H, J = 6.9 Hz), 6.74 (merged s with the doublet, 1H), 7.52 (s, 1H), 7.69 (d, 1H, J = 7.8 Hz), 7.75 (merged s with the doublet, 1H). 13 C NMR (CDCl3, 75 MHz): δ 16.28 (CH3), 17.56 (CH3), 22.72 (CH2), 25.10 (CH3), 25.66 (2-CH3), 32.91 (CH), 40.13 (CH2), 77.42 (C), 105.73 (C), 108.13 (CH), 116.88 (C), 118.25 (C), 118.89 (CH), 119.07 (CH), 121.24 (CH), 122.99 (CH), 124.07 (CH), 124.21 (CH), 127.58 (CH), 131.64 (C), 136.05 (C), 141.58 (C), 150.67 (C). MS (ESI+) m/z = 370 (M + Na+) for C23H25O2NNa. Dehydroxy-mahanine. 1H NMR (DMSO-d6, 300 MHz): δ 1.40 (s, 3H), 1.52 (s, 3H), 1.60 (s, 3H), 1.69 (t-like, 2H, J = 7.2, 7.8 Hz), 2.10 (brd, 2H, J = 6.6 Hz), 2.26 (s, 3H), 5.09 (brs, 1H), 5.75 (d, 1H, J = 9.6 Hz), 6.96 (d, 1H, J = 9.6 Hz), 7.08 (t, 1H, J = 7.2 Hz), 7.27 (t, 1H, J = 7.5 Hz), 7.41 (d, 1H, J = 7.8 Hz), 7.70 (s, 1H), 7.92 (d, 1H, J = 7.5 Hz), 11.17 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): δ 16.2 (CH3), 17.7 (CH3), 22.6 (CH2), 25.7 (CH3), 26.0 (CH3), 40.6 (CH2), 78.2 (C), 77.42 (C), 104.4 (C), 110.9 (CH), 116.3 (C), 116.8 (C), 118.6 (CH), 118.9 (CH), 119.4 (CH), 121.3 (CH), 123.2 (CH), 124.3 (CH), 124.5 (CH), 128.4 (CH), 131.2 (C), 135.5 (C), 140.1 (C), 149.4 (C). MS (ESI+) m/z = 354 (M + Na+) for C23H25ONNa. Methylated Dehydroxy-mahanine. 1H NMR (CDCl3, 300 MHz): δ 1.45 (s, 3H), 1.58 (s, 3H), 1.66 (s, 3H), 1.72−1.83 (m, 2H), 2.15 (d, 1H, J = 10.4 Hz), 2.20 (d, 1H, J = 10.4 Hz), 2.34 (s, 3H), 4.00 (s, 3H), 5.12 (dt, 1H, J = 1.5, 6.5 Hz), 5.63 (d, 1H, J = 9.9 Hz), 7.15 (d, 1H, J = 5.7), 7.17 (d, 1H, J = 9.9 Hz), 7.25−7.38 (m, 2H), 7.69 (s, 1H), 7.91 (d, 1H, J = 7.5 Hz). 13C NMR (CDCl3, 75 MHz): δ 16.28 (CH3), 17.56 (CH3), 22.72 (CH2), 25.10 (CH3), 25.66 (CH3), 32.91 (CH), 40.13 (CH2), 77.42 (C), 105.73 (C), 108.13 (CH), 116.88 (C), 118.25 (C), 118.89 (CH), 119.07 (CH), 121.24 (CH), 122.99 (C), 124.07 (CH), 124.21 (CH), 127.58 (CH), 131.64 (C), 136.05 (C), 141.58 (C), 150.67 (C). MS (ESI+) m/z = 368 (M + Na+) for C24H26ONNa. Methylated Mahanine. 1H NMR (CDCl3, 300 MHz): δ 1.44 (s, 3H), 1.58 (s, 3H), 1.65 (s, 3H), 1.75 (t, 2H, J = 7.8 Hz), 2.15 (brd, 2H, J = 5.7 Hz), 2.32 (s, 3H), 3.87 (s, 3H), 5.11 (brs, 1H), 5.65 (d, 1H, J = 9.63 Hz), 6.62 (d, 1H, J = 9.6 Hz), 6.79 (d, 1H, J = 7.8 Hz), 7.88 (s, 1H), 7.55 (s, 1H), 7.76 (d, 1H, J = 7.8 Hz). 13C NMR (CDCl3, 75 MHz): δ 16.28 (CH3), 17.55 (CH3), 22.72 (CH2), 25.73 (CH3), 40.67 (CH2), 55.62 (CH3), 77.95 (C), 95.14 (CH), 104.27 (C), 107.59 (CH), 116.70 (C), 117.51 (CH), 117.86 (C), 118.19 (C), 119.85 (CH), 120.40 (CH), 124.20 (CH), 128.62 (CH), 131.62 (C), 134.73 (C), 140.66 (C), 148.88 (C), 157.86 (C). MS (ESI+) m/z = 384 (M + Na+) for C24H27O2NNa. Acetylated Mahanine. 1H NMR (CDCl3, 300 MHz): δ 1.45 (s, 3H), 1.59 (s, 3H), 1.66 (s, 3H), 1.76 (t-like, 2H, J = 7.5, 8.7 Hz), 2.17 (t, 2H, J = 6.0 Hz), 2.30 (s, 3H), 2.36 (s, 3H), 5.12 (brs, 1H), 5.61 (d, 1H, J = 9.9 Hz), 6.55 (d, 1H, J = 9.9 Hz), 6.80 (d, 1H, J = 8.1 Hz), 6.99 (s, 1H), 7.43 (s, 1H), 7.66 (d, 1H, J = 8.4 Hz), 8.04 (s, 1H). 13C NMR (CDCl3, 75 MHz): δ 16.0 (CH3), 17.6 (CH3), 21.3 (CH3), 22.7 (CH2), 25.7 (CH3), 25.8 (CH3), 40.7 (CH2), 78.1 (C), 103.6 (CH), 104.1 (C), 112.5 (CH), 115.9 (C), 117.7 (CH), 118.1 (C), 119.5 (CH), 120.9 (CH), 121.7 (CH), 124.2 (CH), 128.1 (CH), 131.6 (C), 135.5 (C), 139.7 (C), 147.6 (C), 149.6 (C), 170.8 (C). MS (ESI+) m/ z = 412 (M + Na+) for C25H27O4NNa. Biotinylated Mahanine. 1H NMR (CDCl3, 600 MHz): δ 1.43 (s, 3H), 1.57 (s, 3H), 1.65 (s, 3H), 1.75 (t, 3H, J = 8.4 Hz), 1.85 (t, 2H, J = 6.6 Hz), 2.13−2.20 (m, 3H), 2.31 (s, 3H), 2.72 (d, 1H, J = 13.2), 2.89 (dd, 1H, J = 4.2, 13.2), 3.13 (brs, 1H), 4.26 (brs, 1H), 4.44 (brs, 1H), 4.96 (d, 1H, J = 2.4), 5.11 (t, 1H, J = 5.4), 5.4 (d, 1H, J = 1.8),

depolarization and also substantially decrease in cell death were observed both in C-7-OH blocked mahanine-treated and Nmethylated derivatives of dehydroxy-mahanine-treated cells. Additionally, decrease in the survival rate of these mahanineinduced cells was noted to be associated with a decrease in the Bcl-xl and Bid expression, strongly recommending the importance of the C-7-OH group in mitochondrial damage by affecting those Bcl-2 family proteins. Methylation of C-7OH of mahanine led to the restoration of those protein levels with O-Me-mahanine-treated cells supported again its significance. A similar kind of degradation of Bcl-xl and Bid proteins were observed only at much higher concentrations, suggesting at least some contribution of −NH group present in dehydroxy-mahanine-induced apoptosis of cancer cells. Taken together, our results confirmed that C-7-OH and to some extent imino group at N-9 position of mahanine, simultaneously enhance the effectiveness of anti-proliferative activity of cancer cells towards apoptosis through the mitochondrial pathway. To the best of our knowledge, this is the first report on the identification of functional group of a potentially important nontoxic anticancer herbal compound mahanine and to understand the prospect and nature of their interaction with DNA. These findings will help for the discovery of a potent anticancer agent that binds with DNA minor groove.

■

MATERIALS AND METHODS

Reagents. Primary antibodies (Bcl-xl, Bid, caspase-9, caspase-7, caspase-8, and PARP), secondary HRP conjugated antibodies, all flow cytometry compatible fluorescence conjugated antibodies, 7-AAD, and dUTP-FITC were purchased from BD Bioscience (USA). JC-1 was purchased from Invitrogen (USA). Bromophenol blue, RPMI-1640, antibiotic−antimycotic, molecular grade BSA, Tween-20, Tris-HCl, and DMSO were from Sigma-Aldrich (USA). The CT-DNA was purchased from Bangalore Genie. The Tris buffer, sodium chloride, pyridine, dimethyl formamide, methyl iodide, sodium hydride, acetic anhydride, dimethyl amino-pyridine, potassium carbonate, and hydrochloric acid (AR) were from Merck, and all the other reagents used elsewhere from Sigma-Aldrich (USA). Purification and Characterization of Two Carbazole Alkaloids (Mahanine and Dehydroxy-mahanine). Mahanine and dehydroxy-mahanine were isolated and purified from fresh leaves of an Indian plant Murraya koenigii, which belongs to the family of Rutaceae (Supporting Information Table S1). The purity was confirmed by HPLC which was ≥95% pure (Supporting Information Figure S1). MS and NMR (1H and 13C) spectral analysis (Supporting Information Figure S2) established their structures as mahanine and dehydroxy-mahanine (Figure 1B).23 Chemical Modification of Functional Groups of Mahanine. Mahanine (30 mg, 86 mmol) was dissolved in anhydrous DMF (5 mL) and stirred with methyl iodide (12.1 mg, 86 mmol) and K2CO3 (12 mg, 86 mmol) (1:1) for 3 h. The product was isolated using silica gel column chromatography and confirmed by mass spectrometry. 1H and 13C NMR spectral analyses established it as C-7-OH methylated mahanine (Figure 1A). The acetylated derivative at C-7 hydroxyl group of mahanine was prepared by stirring mahanine (30 mg, 86 mmol) in anhydrous pyridine (∼7 mL) with acetic anhydride (10 mL, 100 mmol) for 6 h (Figure 1A). Additionally, to check the stability of acetylated mahanine, we incubated this derivative with culture medium for 48 h at physiological condition. After incubation, the solution was lyophilized and analyzed by ESI-MS. Biotinylated mahanine was synthesized by dissolving mahanine (30 mg) in DMSO (10 mL), followed by adding 1 equiv each of DMAP (12 mg, 87 mmol) and PFP−biotin (35.47 mg, 86 mmol) and stirring at 37 °C for 3 h. The product was purified by silica gel column chromatography (Figure 1A). Dehydroxy-mahanine (30 mg, 90 mmol) was dissolved in anhydrous DMF (2.0 mL) and stirred with 3 equiv of NaH (6.6 mg, 275 mmol) I

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

dehydroxy-mahanine at concentrations 0 and 30 μM for 24 h. Quantitative flow cytometric evaluation of the apoptotic cells was assessed by the FITC conjugated dUTP according to the manufacturer’s protocol (ApoDirect kit). At least 20000 cells were acquired26 and analyzed by CellQuest Pro software. Measurement of Mitochondrial Membrane Depolarization. Cells (1 × 106) were exposed without or with mahanine (0 30 μM) for 24 h, washed with PBS, and probed with JC-1 (a potentiometric probe for mitochondria, 25 μM) in the dark for 30 min at 37 °C, and 10000 cells analyzed by flow cytometer.27 In parallel, cells were similarly exposed with dehydroxy-mahanine (0 and 30 μM) under identical conditions for comparison. Immunoblot Analysis. Cells (1.5 × 106) were exposed to mahanine, O-Me-mahanine (25 μM), dehydroxy-mahanine (50 μM), and N-Me-dehydroxy-mahanine (50 μM) separately for 24 h. An equivalent amount of protein (50 μg/well) from each sample was resolved in SDS-PAGE (10%) and electrotransferred into a nitrocellulose membrane.28 The membrane was blocked by 2% TBS-BSA (for 5−30 min at 25 °C) and probed with primary antibody for overnight at 4 °C, washed with TBS containing 0.1% Tween-20, and incubated with appropriate HRP conjugated secondary antibody. Subsequently, the membrane was washed and immune-reactive protein was identified by the West-pico ECL (Pierce) system. A control set experiments were also carried without the test molecules (mahanine and its derivatives) under the same conditions. Absorption Spectroscopy Study. A. Interaction of Mahanine and Its Derivatives with DNA. Interaction of mahanine and its different derivatives with DNA were studied by UV−vis absorption spectroscopy. The spectra were scanned in the wavelength range of 220−350 nm with a Thermo-Scientific spectrophotometer (model Evolution 300, USA) with 1 cm quartz cell at 25 °C.29,30 The stock DNA (1.0 mg/mL) was prepared in 50 mM Tris-HCl buffer of pH 7.5. Mahanine and its derivative were fixed at 100 μM concentration. Mahanine−DNA complex was incubated at 37 °C for 1 h, and then the absorbance measurements were recorded after appropriate dilution. B. Binding Constant and the Cooperativity of Binding of Mahanine−DNA Complex. For determining the cooperativity of binding, DNA concentration was fixed (100 μg/mL) and the concentration of mahanine was varied from 0 to 120 μM. This mahanine−DNA complex was incubated for 2 h at 37 °C, and the absorbance spectra were taken in the range 220−350 nm. From the absorbance spectra, the binding constant (Kb) and the cooperativity of binding for the complex formed between mahanine and DNA were determined according to the methods published in the literature where the bindings of proteins to DNA were described.15 Infrared Spectroscopy Study. Infrared spectra were recorded on a Nicolet FT-IR (model 6700, USA) spectrometer equipped with DTGS KBr detector and KBr beam splitter by attenuated total reflection (ATR) mode. The spectra of the DNA (250 μg/mL) in the presence and absence of mahanine (1 mM) was recorded by forming thin films by air drying the samples on the ATR plate to avoid the water interference at the primary amine signature region. Each spectrum was taken after 256 scans and 4 cm−1 wavenumber resolution at 25 °C in the wavenumber region of 1800−900 cm−1 which is the characteristic of the vibrational frequency of DNA bases (A, T, G, and C).31 CD Spectroscopy Study. CD spectra were recorded on a Jasco J815 CD spectrometer, in the 50 mM Tris-HCl buffer of pH 7.5, at room 25 °C. DNA (1000 μg/mL) was used for recording spectra in the presence and absence of mahanine (100 μM). EtBr (300 μM) was used as a positive control. Spectra were recorded after 2 h of incubation at 37 °C. Isothermal Titration Calorimetry (ITC) Study. ITC experiment was carried out using a Microcal VP-ITC (Northhampton, MA, USA) at 20 °C. Mahanine was dissolved in minimum volume of DMSO and diluted in 50 mM tris buffer, pH 7.5. In control experiments, an equal amount of DMSO was used to nullify the extra heat change. Each of the titration was conducted by filling the ITC cell with DNA (10 μM, 1.5 mL) solution and adding up to 28 injections (in each time 10 μL)

5.61 (d, 1H, J = 10.2 Hz), 6.68 (d, 1H, J = 10.2 Hz), 6.81 (d, 1H, J = 8.4 Hz), 7.13 (s, 1H), 7.53 (s, 1H), 7.77 (d, 1H, J = 8.4 Hz), 9.14 (brs, 1H), 4-H are not detected. 13C NMR (CDCl3, 150 MHz): δ 16.1 (CH3), 17.6 (CH3), 22.7 (CH2), 24.7 (CH2), 25.8 (CH3), 28.50 (CH2), 28.84 (CH2), 34.1 (CH2), 40.4 (CH2), 40.8 (CH2), 41.0 (CH3), 55.7 (CH), 60.1 (CH), 62.2 (CH), 76.8 (C), 103.8 (CH), 104.4 (C), 112.6 (CH), 116.0 (C), 117.9 (CH), 118.2 (C), 119.47 (CH), 120.9 (CH), 121.8 (CH), 124.2 (C), 128.2 (CH), 131.6 (C), 135.8 (C), 140.1 (C), 147.7 (C), 149.7 (C), 163.4 (C), 173.5 (C). MS (ESI+) m/z = 596 (M + Na+) for C33H39O4N3SNa. Cell Lines. Human glioblastoma (U87MG, U373MG, LN229, T98G, and A172), nonsmall cell lung carcinoma (A549, NCI H23), colorectal carcinoma (SW480, HCT116), pancreatic ductal carcinoma (MIAPaCa2, AsPC1), T-cell lymphocytic leukemia (MOLT-3, MOLT-4, and CEMC-7), B-cell lymphocytic leukemia (REH), chronic myeloid leukemic (K562), normal monkey kidney (Vero), and human embryonic kidney (HEK-293T) cells were purchased from ATCC (USA). Pancreatic ductal carcinoma (Panc 10.05 and Panc1) cell lines were a kind gift by Dr. Kaustubh Dutta from Mayo Clinic, Rochester, Minnesota. Cells were grown in RPMI-1640 medium or IMDM supplemented with 10% heat inactivated FCS and 1% antibiotic− antimycotic solution in a humidified atmosphere at 37 °C and with 5% CO2. Cell Viability Analysis Using 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl Tetrazolium Bromide (MTT). Cells (1 × 104) in the log phase were incubated separately with the various concentrations (0− 60 μM) of mahanine in 96-well tissue culture plate in triplicate at 37 °C in a humidified atmosphere containing 5% CO2 for different times as indicated. MTT (10 μL, 10 μg/μL) was then added to each well and incubated for 3 h at 37 °C. The viability of treated and untreated cells was checked by MTT assay as described elsewhere.24 In parallel, cells were exposed with five different derivatives of mahanine and processed similarly. In each treatment, cells without compound (control cells) were exposed to the highest amount of vehicles (0.15% absolute ethanol). All of the experiments were done in triplicate on three different days to determine the IC50 values. Results are presented as means ± SE. DNA Damage Was Determined by Comet Assay. Mahanine and its derivative-induced DNA damage in the cell were determined using the comet assay.12 Mahanine and its derivative (0, 20 μM), pretreated T 98G cells (1 × 105 cells) for 24 h, were used for this assay. After treatment with the said compounds, cells were tripsinized and resuspended in ice-cold PBS. Approximately 20000 cells in a volume of 50 μL of 0.5% (w/v) low melting point agarose were pipetted onto a frosted glass slide coated with a thin layer of 1.0% (w/ v) agarose, covered with a coverslip, and allowed to set on ice for 10 min. Following removal of the coverslip, the slides were immersed in ice-cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2-EDTA, 1% (w/v) N-lauroyl-sarcosine, adjusted to pH 10.0, and 1.0% Triton X-100 was added immediately before use). After overnight incubation at 4 °C, the slides were placed into a horizontal electrophoresis tank filled with electrophoresis buffer (0.3 M NaOH, 1 mM EDTA (pH 13) and subjected to electrophoresis for 30 min at 300 mA at 4 °C. Slides were transferred to neutralization solution (0.4 M Tris-HCl) for 3 × 5 min washes and stained with ethidium bromide solution for 5 min. Slides were viewed using the 20× objective of a Zeiss Axioskop microscope equipped with epifluorescence optics. For each sample, the tail lengths (μm) of a minimum of 20 comets were analyzed. The length of the comet was quantified as the distance from the centrum of the cell nucleus to the tip of the tail in pixel units and the tail length was expressed as a mean ± SD from 20 comets. Flow Cytometric Detection of 7-Amino-actinomycin D (7AAD) Positive Cells. Cells (1 × 106) were exposed to mahanine and dehydroxy-mahanine at concentrations 0 and 30 μM for 24 h. Evaluation of apoptosis was performed using the 7-AAD according to the manufacturer’s instructions. At least 10000 cells were acquired and data were analyzed by CellQuest Pro software (BD FACS Calibur).25 Analysis of DNA Fragmentation by Terminal Deoxyribonucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labeling (TUNEL) Assay. Cells (1 × 106) were treated with mahanine and J

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

of mahanine solution (250 μM) at 240 s intervals. Three replicate titrations were performed. The integrated heat/injection data obtained in ITC titration were fitted to a oneSites model and a nonlinear regression algorithm developed with Origin-7 software. Statistical Analysis. Data were from at least three independent experiments. Statistical analysis of data was performed using two-tail student t test. Error bars represent standard deviation of mean (±SD). Significant differences (P < 0.05) between the means of control and compound-treated cells or two test groups were analyzed by Microsoft Excel and Graph pad Prism. Molecular Modeling Studies. A. Preparation of Receptor and Ligand Models. To examine whether mahanine binds to DNA as an intercalator or a groove binder, two forms of crystallographic DNA structures were taken. A crystallographic DNA with gap (PDB ID 1Z3F, resolution 1.5 Å) was taken so that it was capable of accommodating an intercalator. This crystal structure was of a six base pair d(CGATCG)2 oligo nucleotide duplex (DNA1) complexed with ellipticine, a known intercalator, and contains two ellipticine molecules, intercalated between two separate CG base pairs.32 To simulate the minor groove bound state, three-dimensional structure of a DNA duplex having a dodecameric sequence of d(ATGCGATCGACG)2 (DNA2), in an ideal canonical B-DNA conformation was modeled using Builder module of InsightII software package (Accelrys Inc., San Diego, CA). Additionally, a crystallographic DNA with no gap (PDB ID 1DNE, resolution 2.4 Å) having the sequence d(CGCGATATCGCG)2 (DNA3) was also used (a crystal structure of a minor groove binder netropsin−DNA complex).33 Water molecules were removed from all the structures. The structure of mahanine was modeled using the Builder module of Insight II. The structure was energy minimized for 200 steps each of steepest descent and conjugate gradient using cff91 force field followed by MD simulation to find the optimized modeled structure.6 B. Molecular Docking. Cross docking of mahanine and its derivatives were performed with the nucleotide oligomers from crystallographic complexes 1Z3F, 1DNE, and the modeled dodecamer B-DNA using Autodock4.2.34 In parallel, self-docking and cross docking of two DNA ligands netropsin, a minor groove binder, and ellipticine, an intercalator, were also performed as controls with DNA as a receptor.35 The rigid docking protocol was used, and the best binding mode of interaction was taken for MD simulation. Polar and aromatic hydrogens were added, and Gastegier charges were computed by Autodock tool on each atom of the ligand. The AutoTors utility was used to define torsional degrees of freedom for the ligand. The grid box was centered in the macromolecule and the dimension of the grid was 82 × 92 × 100 Å3 with the spacing between the grid points at 0.403 Å in order to include the entire DNA fragment. Grid potential maps were calculated using the module AutoGrid 4.0. The Lamarckian genetic algorithm was used to perform docking simulation, with an initial population of 150 randomly placed individuals with a maximum number of 250000 energy evaluations, 150000 generations, mutation rate of 0.02, a crossover rate of 0.8, and an elitism value of 1, were used. Then 100 docking runs were performed. Pseudo-Solis and Wets algorithm was used for local search method. Finally, the resulting docked conformations were clustered together on the basis of rootmean-square deviation (RMSD) tolerance of 2.0 Å and represented by most favorable free energy of binding. C. Molecular Dynamics Simulation. All MD simulations were carried out, one for the solvated ligand−DNA complex and the other for the free ligand in solution, using Gromacs 4.5.3.36 Amber 94 force field was applied to the DNA fragment. The ligand force field parameters were acquired using Amber 94 and GAFF.37 The partial charges of the ligand were calculated with the Gasteiger charge method, incorporated in Open Babel program (www.openbabel.org). The initial configuration of receptor−ligand complex was placed in a cubic box with solute-wall cutoff distance of 15 Å. The system was solvated using TIP3P water molecules, and the appropriate number of counterions was added in order to neutralize. After initial configurations, a standard equilibration protocol was followed for drug DNA simulation.38 In the first step, the starting structure was restrained with 25 kcal/mol residue harmonic restraint and 1000

iterations of potential energy minimization, followed by MD at 300 K for about 3 ps. The restraints were then released on the solute in five steps by reducing the restraint by 5 kcal/mol residue per step followed by 600 iterations of conjugate gradient minimization. Finally, in the fifth step, the whole system was minimized without harmonic restraints. The system was then heated to 300 K with a temperature coupling time of 0.2 ps. All the covalent bonds to the hydrogen were held constant to a tolerance of 0.0005 Å by applying the SHAKE routine. We used a 1 fs time step in all of our MD simulations. The temperature of the system was allowed to fluctuate around 300 K with a temperature coupling time of 0.2 ps, and the pressure was allowed to fluctuate around 1 bar with a pressure coupling time of 0.6 ps. The simulations were performed at constant temperature and pressure conditions using the Berendsen algorithm. Periodic boundary conditions with a 12 Å uniform cutoff for Lennard-Jones potential and for electrostatic contribution, particle mesh wald (PME) were used. The stochastic dynamic (SD) integrator was applied to the MD simulation. Trajectories and simulated structures were analyzed using VMD software.39 D. Free Energy Calculation. To compute the binding free energy of the complexes, we have used the double annihilation free energy method proposed by Jorgensen.38 The free energy of binding of ligand to the DNA was calculated using the thermodynamic windows method with fixed widths for the electrostatic portion and the slow growth method for the van der Waals portion.40 This was carried out by mutating the ligand in the solvated complex and free in solution in both forward (λ 1 → 0) and backward directions (λ 0 → 1). With electrostatic coupling, the electrostatic and van der Waals contributions to the free energy change of ligand → 0 in the complex and in solution were calculated. The electrostatic free energy change was computed at an interval of Δλ = 0.00002 and van der Waals free energy change at Δλ = 0.0000025. The electrostatic and van der Waals component of the isolated ligand in solution and in complex were calculated from both forward and backward simulations.

■

ASSOCIATED CONTENT

S Supporting Information *

Structural characterizations of mahanine and dehydroxymahanine. The NMR (1H and 13C) spectra of all the isolated and synthesized compounds. Effect of mahanine and dehydroxy-mahanine on inhibition of cancer cell proliferation in a time dependent. Concentrations of mahanine and dehydroxy-mahanine were determined in MeOH, and EtOAc extracts from Murraya Koenigii leaf extract. Name, source and structural identification of mahanine and its derivatives. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +91-33-2429-8861. Fax: 91-33-2473-5197. E mail: [email protected] or [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We sincerely acknowledge Prof. Siddhartha Roy, Director, CSIR-IICB, for his helpful comments and criticism. CSIR-IICB, CSIR under IAP-0001, HCP004, NMITLI, TLP-004 and DBT under GAP 235, ICMR, Government of India, supported this work. Financial support from J.C. Bose Fellowship, DST of Government of India, and mutual grant from ICMR and German Cancer Research Centre are also acknowledged. We sincerely convey our thanks to Dr. G. Suresh Kumar, CSIRIICB for providing CD and ITC instruments and Jyoti Shaw for her valuable suggestion about comet assay. K

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

■

Article

O-rutinoside from Barringtonia racemosa, targets mitochondrial apoptotic pathway in acute lymphoblastic leukemia. J. Asian Nat. Prod. Res. 2010, 12, 639−48. (15) Karsten, R. Analysis of protein−DNA binding at equilibrium. B. I. F. Futura 1997, 12, 20−26. (16) Mao, Y.; Daniel, L. N.; Whittaker, N.; Saffiotti, U. DNA binding to crystalline silica characterized by Fourier-transform infrared spectroscopy. Environ. Health Perspect. 1994, 102, 165−171. (17) Rahul, P.; Paul, J. H. DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Curr. Opin. Biotechnol. 2007, 18, 497−503. (18) Roy, S.; Basak, S.; Dasgupta, A. K. Nanoparticle Induced Conformational Change in DNA and Chirality of Silver Nanoclusters. J. Nanosci. Nanotechnol. 2010, 10 (2), 819−825. (19) Alexander, P.; Helmut, W. Structural organization of DNA− protein complexes of chromatin studied by vibrational and electronic circular dichrosim. Spectroscopy 2010, 3−4, 239−244. (20) Kashanian, S.; Ghobadi, A. T. DNA Interaction Studies of Ethylene Diamine Tetraacetic Acid Food Additive and Selenium Effect in DNA Cleavage Inhibition. DNA Cell Biol. 2011, 30 (12), 1085− 1090. (21) Aqvist, J.; Luzhkov, V. B.; Brandsdal, B. O. Ligand binding affinities from MD simulations. Acc. Chem. Res. 2002, 35, 358−365. (22) Shaikh, S. A.; Ahmed, S. R.; Jayaram, B. A molecular thermodynamic view of DNA−drug interactions: a case study of 25 minor-groove binders. Arch. Biochem. Biophys. 2004, 429 (1), 81−99. (23) Joshi, B. S.; Kamat, V. N.; Gawad, D. H. On the structures of girinimbine, mahanimbine, isomahanimbine, koenimbidine and murrayacine. Tetrahedron 1970, 26 (5), 1475−82. (24) Liu, T.; Liu, D.; Liu, J.; Song, J. T.; Gao, S. L.; Li, H.; Hu, L. H.; Liu, B. R. Effect of NF-κB inhibitors on the chemotherapy-induced apoptosis of the colon cancer cell line HT-29. Exp. Ther. Med. 2012, 4 (4), 716−722. (25) Lai, R. C.; Choo, A.; Lim, S. K. Derivation and Characterization of Human ESC-Derived Mesenchymal Stem Cells. Methods Mol. Biol. 2011, 698, 141−150. (26) Lange, T.; Schilling, A. F.; Peters, F.; Mujas, J.; Wicklein, D.l; Amling, M. Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro. Biomaterials 2011, 32 (17), 4067−4075. (27) Mandal, C.; Dutta, A.; Mallick, A.; Chandra, S.; Misra, L.; Sangwan, R. S.; Mandal, C. Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade. Apoptosis 2008, 13 (12), 1450−1464. (28) Nakanishi, Y.; Shiraha, H.; Nishina, S.; Tanaka, S.; Matsubara, M.; Horiguchi, S.; Yamamoto, K. Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis. BMC Cancer 2011, 4 (11), 3. (29) Banerjee, S.; Mondal, S.; Sen, S.; Das, S.; David, L.; Hughes, Corrado, R.; Cédric, D.; Mandal, C.; Mitra, S. Four new dinuclear Cu(II) hydrazone complexes using various organic spacers: syntheses, crystal structures, DNA binding and cleavage studies and selective cell inhibitory effect towards leukemic and normal lymphocytes. Dalton Trans. 2009, 14 (34), 6849−6860. (30) Wahl, P.; Paoletti, J.; Le Pecq, J. B. Decay of fluorescence emission anisotropy of the ethidium bromide−DNA complex. Evidence for an internal motion in DNA. Proc. Natl. Acad. Sci. U. S. A. 1970, 65 (2), 417−421. (31) Fredericks, J. D.; Bennett, P.; Williams, A.; Rogers, K. D. FTIR spectroscopy: a new diagnostic tool to aid DNA analysis from heated bone. Forensic Sci. Int.: Genet. 2012, 6 (3), 375−380. (32) Canals, A.; Purciolas, M.; Aymamí, J.; Coll, M. The anticancer agent ellipticine unwinds DNA by intercalative binding in an orientation parallel to base pairs. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61, 1009−1012. (33) Coll, M.; Aymami, J.; van der Marel, G. A.; van Boom, J. H.; Rich, A.; Wang, A. H. Molecular structure of the netropsin−

ABBREVIATIONS USED 7-AAD, 7-amino actinomycin D; BSA, bovine serum albumin; EtOH, ethanol; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; ITC, isothermal titration calorimetry; FTIR, Fourier transform infrared spectroscopy; H2DCFDA, 5-(and6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester; HPLC, high performance liquid chromatography; JC1, 5,5′-6,6′-tetracholoro-1,1′-3,3′-tetraethylbenzimidazolylcarbocyanine iodide; DMAP, 4-dimethylaminopyridine; PFPbiotin, pentafluorophenyl-ester biotin; MS, mass spectroscopy; NMR, nuclear magnetic resonance; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; TDT, terminal deoxy transferase; TUNEL, terminal deoxyneuleotidyltransferase enzyme-mediated dUTP end labeling; UV, ultraviolet

■

REFERENCES

(1) Mondal, S.; Bandyopadhyay, S.; Ghosh, M. K.; Mukhopadhyay, S.; Roy, S.; Mandal, C. Natural products: promising resources for cancer drug discovery. Anti-Cancer Agents Med. Chem. 2012, 12, 49− 75. (2) Roy, M. K.; Thalang, V. N.; Trakoontivakorn, G.; Nakahara, K. Mahanine, a carbazole alkaloid from Micromelum minutum, inhibits cell growth and induces apoptosis in U937 cells through a mitochondrial dependent pathway. Br. J. Pharmacol. 2005, 145 (2), 145−155. (3) Nakahara, K.; Trakoontivakorn, G.; Alzoreky, N. S.; Ono, H.; Onishi-Kameyama, M.; Yoshida, M. Antimutagenicity of some edible Thai plants, and a bioactive carbazole alkaloid, mahanine, isolated from Micromelum minutum. J. Agric. Food Chem. 2002, 50 (17), 4796−4802. (4) Bhattacharya, K.; Samanta, S. K.; Tripathi, R.; Mallick, A.; Chandra, S.; Pal, B. C.; Shaha, C.; Mandal, C. Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways. Biochem. Pharmacol. 2010, 79 (3), 361−372. (5) Sinha, S.; Pal, B. C.; Jagadeesh, S.; Banerjee, P. P.; Bandyopadhaya, A.; Bhattacharya, S. Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases. Prostate 2006, 66 (12), 1257−1265. (6) Sarkar, S.; Dutta, D.; Samanta, S. K.; Bhattacharya, K.; Pal, B. C.; Li, J.; Datta, K.; Mandal, C.; Mandal, C. Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo. Int. J. Cancer 2012, 132 (3), 695−706. (7) Rahul, P.; Paul, J. H. DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Curr. Op. Biotechnol. 2007, 18, 497−503. (8) Hegde, S.; Schmidt, M.; Doherty, A. To market, to market 2004. Annu. Rep. Med. Chem. 2005, 40, 443−473. (9) Jagadeesh, S.; Sinha, S.; Pal, B. C.; Bhattacharya, S.; Banerjee, P. P. Mahanine reverses an epigenetically silenced tumor suppressor gene RASSF1A in human prostate cancer cells. Biochem. Biophys. Res. Commun. 2007, 362 (1), 212−217. (10) Paz, M. M.; Das, T. A.; Tomasz, M. Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity. Bioorg. Med. Chem. 1999, 7 (12), 2713−2726. (11) Mahato, R.; Tai, W.; Cheng, K. Prodrugs for improving tumor targetability and efficiency. Adv. Drug Delivery Rev. 2011, 63 (8), 659− 670. (12) Lorenzo, Y.; Costa, S.; Collins, A. R.; Azqueta, A. The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead. Mutagenesis 2013, 28, 427−432. (13) Mondal, S.; Bhattacharya, K.; Mallick, A.; Sangwan, R.; Mandal, C. Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide Dinduced apoptosis. PLoS One 2012, 7 (3), e34277. (14) Samanta, S. K.; Bhattacharya, K.; Mandal, C.; Pal, B. C. Identification and quantification of the active component quercetin 3L

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment. Biochemistry 1989, 28 (1), 310−320. (34) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785−2791. (35) Ricci, C. G.; Netz, P. A. Docking studies on DNA−ligand interactions: building and application of a protocol to identify the binding mode. J. Chem. Inf. Model. 2009, 49 (8), 1925−1935. (36) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. GROMACS: fast, flexible, and free. J. Comput. Chem. 2005, 26, 1701−1718. (37) Mukherjee, A.; Lavery, R.; Bagchi, B.; Hynes, J. T. On the molecular mechanism of drug intercalation into DNA: a simulation study of the intercalation pathway, free energy, and DNA structural changes. J. Am. Chem. Soc. 2008, 130 (30), 9747−9755. (38) Suresh, S. B.; Peter, A. K. Calculating the Absolute Free Energy of Association of Netropsin and DNA. J. Am. Chem. Soc. 1999, 121, 3267−3271. (39) Humphrey, W.; Dalke, A.; Schulten, K. VMDvisual molecular dynamics. J. Mol. Graphics 1996, 14, 33−38. (40) Singh, U. C; Brown, F. K.; Bash, P. A.; Kollman, P. A. An approach to the application of free energy perturbation methods using molecular dynamics: applications to the transformations of methanol to ethane, oxonium to ammonium, glycine to alanine, and alanine to phenylalanine in aqueous solution and to H 3 O + (H 2 O) 3 to NH4+(H2O)3 in the gas phase. J. Am. Chem. Soc. 1987, 109, 1607− 1614.

M

dx.doi.org/10.1021/jm400290q | J. Med. Chem. XXXX, XXX, XXX−XXX