Computational studies on effects of MDMA as an anticancer drug on DNA

This research is designed to further understand the effects of the novel drug MDMA on biologic receptor of DNA. The ultimate goal is to design drugs that have higher affinity with DNA. Understanding the physicochemical properties of the drug as well as the mechanism by which it interacts with DNA should ultimately enable the rational design of novel anticancer or antiviral drugs. Molecular modeling on the complex formed between MDMA and DNA presented this complex to be fully capable of participating in the formation of a stable intercalation site. Furthermore, the molecular geometries of MDMA and DNA bases (Adenine, Guanine, Cytosine, and Thymine) were optimized with the aid of the B3LYP/6-31G* method. The properties of the isolated intercalator and its stacking interactions with adenine···thymine (AT) and guanine···cytosine (GC) nucleic acid base pairs were studied with the DFTB method. DFTB method is an approximate version of the DFT method that was extended to cover the London dispersion energy. The B3LYP/6-31G* stabilization energies of the intercalator···base pair complexes were found to be −9.40 and −12.57 kcal/mol for AT···MDMA and GC···MDMA, respectively. Results from comparison of the DFTB method and HF method conclude close results and support each other.     

Novel computational study on π-stacking to understand mechanistic interactions of Tryptanthrin analogues with DNA

Based on recently published initial experimental results on the intercalation of a class of broad spectrum antiparasitic compounds, we present a purely theoretical approach for determining if these compounds may preferentially intercalate with guanosine/cytosine (GC)-rich or adenosine/thymidine (TA)-rich regions of DNA. The predictive model presented herein is based upon utilization of density functional theory (DFT) to determine a priori how the best intercalator may energetically and sterically interact with each of the nucleoside base pairs. A potential new method using electrostatic potential maps (EPMs) to visually select the best poses is introduced and compared to the existing brute-force center of mass (COM) approach. The EPM and COM predictions are in agreement with each other, but the EPM method is potentially much more efficient. We report that 4-azatryptantrin, the best intercalator, is predicted to favor π-stacking with GC over that of TA by approximately 2–4?kcal/mol. This represents a significant difference if one takes into account the Boltzmann distribution at physiological temperature. This theoretical method will be utilized to guide future experimental studies on the elucidation of possible mechanism(s) for the action of these antiparasitic compounds at the molecular level.     

Structural studies on bio-active compounds. Part XIV. Molecular modelling of the interactions between pentamidine and DNA

Molecular mechanics modelling was carried out on the antimicrobial aromatic diamidine, pentamidine, bound to the minor groove of several AT-rich DNA octamers. The pentamidine molecule was found to span four base pairs, with its highly charged amidinium groups forming hydrogen bonds to the O2 of thymine or the N3 of adenine, but not to the backbone phosphate groups. The mean binding energy of the pentamidine-DNA complexes was -52 kcal/mol, of the same order as that of berenil-DNA complexes. There was no significant energy difference between those models which contained GC base pairs and those which did not. The presence of bound pentamidine resulted in some distortion of helix geometry involving helix opening towards the minor groove in most cases and a decrease in the number of residues per turn.     

DNA sequence selectivity of three biosynthetic analogues of the quinoxaline antibiotics

Mono- and bis-quinoline analogues of echinomycin, and a bis-3-amino-quinoxaline analogue of triostin A, have been prepared by directed biosynthesis and investigated for sequence selectivity in binding to DNA. Binding isotherms for bis-3-amino triostin A interacting with four natural and two synthetic DNA species have been determined by using solvent-partition analysis with radiolabelled antibiotic. They reveal a similar pattern of preferences to that seen with the parent compound: tighter binding occurs with the more guanine and cytosine (GC)-rich DNAs and in every case the association constant is increased two- to five-fold over the value recorded for triostin A. Deoxyribonuclease I footprinting patterns measured for the quinoline analogues of echinomycin differ from those observed with the parent antibiotic in that an additional strong site of protection occurs around the CpG sequence at position 35 in the tyrT fragment. Footprints for bis-3-amino triostin A reveal a substantially more selective pattern of cleavage inhibition than seen with the natural antibiotics: only two or three distinct binding sites are identified in tyrT DNA and four in pTyr2 DNA. Each is centred around one or more CpG steps, but many more CpG-containing sequences are unprotected. The analogue seems to prefer CpG steps flanked by at least one adenine and thymine (AT) pair, optimally ACGN. Enhancement of cutting at AT-rich sequences surrounding their binding sites is seen with all three of the new antibiotics. The results lend weight to the idea that novel sequence selectivity can be attained by making appropriate substitution on the chromophores of quinoxaline antibiotics.     

In vitro DNA deamination by alpha-nitrosaminoaldehydes determined by GC/MS-SIM quantitation

The deamination of DNA bases by three alpha-nitrosaminoaldehydes, butylethanalnitrosamine, methylethanalnitrosamine, and N-nitroso-2-hydroxymorpholine (NHMOR), the direct metabolite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides, oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively. The order of relative reactivities of the bases was as listed above. Deamination of cytosine to uracil was detected by the reaction of (32)P-labeled oligonucleotide ([5'-(32)P]CGAT) followed by enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf thymus DNA was performed by a gas chromatography/mass spectrometry-selected ion monitoring method. Both the extent and the rate of the deamination reactions which occur by transnitrosation from the alpha-nitrosaminoaldehyde to the base were determined for formation of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant at low substrate levels.     

Base specificity for DNA interstrand cross-linking induced by anticancer agent bizelesin

Bizelesin is a promising novel anticancer agent which is known to alkylate N3 of adenine to induce DNA interstrand cross-links (ISC) within 5′-TAATTA and 5′-TAAAAAA. We have investigated the base specificity for DNA ISC induced by bizelesin using oligomers containing the cross-linkable sequence 5′-TAATTN, in which “N” was either A, C, G, or T. An analysis of denaturing polyacrylamide gel showed that bizelesin is able to induce DNA ISC in the duplex oligomer containing sequences 5′-TAATTA and 5′-TAATTG. The formation of interstrand cross-linking did not occur in the sequences 5′-TAATTC and 5′-TAATTT. DNA strand cleavage assay to determine the cross-linking site within 5′-TAATTG sequence showed that bizelesin alkylates guanine. These results demonstrate that bizelesin is able to induce DNA ISC at guanine but not at cytosine or thymine. In addition, guanine adducts have been found to be susceptible to DNA strand cleavage by exposure to hot piperidine. The extent of DNA strand cleavage, however, was not 100% efficient in either neutral pH buffer or hot piperidine.     

Isolation and frontier molecular orbital investigation of bioactive quinone-methide triterpenoids from the bark of Salacia petenensis

The crude dichloromethane bark extract of Salacia petenensis (Hippocrateaceae) from Monteverde, Costa Rica, shows antibacterial and cytotoxic activity. Bioactivity-directed separation led to the isolation of tingenone and netzahualcoyonol as the biologically active materials. Also isolated from the extract were 3-methoxyfriedel-2-en-1-one (a new natural product) and 29-hydroxyfriedelan-3-one. The structures of these compounds were elucidated on the basis of NMR spectral analysis. Molecular orbital calculations have been carried out using the semi-empirical PM3 and Hartee-Fock 3-21G ab initio techniques on the quinone-methide nortriterpenoids tingenone and netzahualcoyonol, as well as on the nucleotide bases adenine, guanine, cytosine, and thymine. The molecular orbital calculations suggest that a possible mode of cytotoxic action of quinone-methide triterpenoids involves quasi-intercalative interaction of the compounds with DNA followed by nucleophilic addition of the DNA base to carbon-6 of the triterpenoid.     

Separation of purine and pyrimidine bases by capillary electrophoresis using beta-cyclodextrin as an additive

Capillary electrophoresis was applied to separate purine and pyrimidine bases in the basis of their partial ionization in the alkaline buffer. The effects of buffer pH, buffer and beta-cylclodextrin concentration were systematically investigated using a commercial capillary electrophoresis instrument with UV detector at 254nm. We found that the resolutions of bases (especially for adenine and thymine) were significantly improved in the presence of beta-cylclodextrin. The satisfactory separation of five bases such as cytosine, thymine, adenine, guanine and uracil were achieved by capillary electrophoresis using beta-cylclodextrin as an additive. Under the optimal conditions, the linear range was from 2 to 200microg/ml for bases (R= 0,991-0,9977 ) and the detection limits were from 0.8 to 1.8microg/ml (S/N = 2). The detection limit of 0.05microg/ml ( S/N=2 ) for uracil was obtained by stacking injection mode. The assay was used to determine the deamination of cytosine to uracil by heating in the presence of sodium hydroxide. Our primarily results show that capillary electrophoresis is a very useful tool for determination of purine and pyrimidine bases and study on nucleic acids.     

小肽对Hoechst33258与DNA特异部位结合的竞争性作用

为了了解小肽如何识别ＤＮＡ特异的结合位点，我们研究了两个小肽ＹＲ和ＧＧＨ对ＤＮＡ与Ｈｏｅｃｈｓｔ３３２５８特异结合的竞争性抑制作用。对Ｌｉｎｅｗｅａｖｅｒ－Ｂｕｒｋ图的分析显示当Ｈｏｅｃｈｓｔ３３２５８的浓度在３．６６（１０－９ｍｏｌ／Ｌ（１．０９（１０－８ｍｏｌ／Ｌ范围内，这种作用是典型的竞争性抑制作用，ＹＲ结合在ＤＮＡ小沟区（ＡＴ富集区），结合常数为１．０２（１０８（ｍｏｌ／Ｌ）－１，而ＧＧＨ的ＡＴ富集区结合特异性在浓度高时明显减弱，因为它也能与ＧＣ碱基结合     

Fluorine‐18 labeling of peptide nucleic acids

Peptide nucleic acids (PNAs) are a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double stranded DNA. From a chemical point of view, the deoxyribose phosphate backbone of DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while the nucleobases of DNA (adenine, guanine, cytosine and thymine) are retained. Due to the increasing interest in the labeling of peptide nucleic acids (PNAs) as potent diagnostic agents in nuclear medicine, we have used and adapted the reliable methodology developed for the fluorine‐18 labeling of oligonucleotides and have now demonstrated that it is possible to label PNAs in sufficient quantity and with high specific radioactivity for PET studies in a time compatible with the half life of fluorine‐18. Copyright © 2002 John Wiley & Sons, Ltd.     

DNA structural perturbation induced by the CPI-derived DNA interstrand cross-linker: molecular mechanisms for the sequence specific recognition

The highly potent cytotoxic DNA-DNA cross-linker consists of two cyclopropa[c]pyrrolo[3, 4-3]indol-4(5H)-ones indoles [(+)-CPI-I] joined by a bisamido pyrrole (abbreviated to "Pyrrole"). The Pyrrole is a synthetic analog of Bizelesin, which is currently in phase II clinical trials due to its excellent in vivo antitumor activity. The Pyrrole has 10 times more potent cytotoxicity than Bizelesin and mostly form DNA-DNA interstrand cross-links through the N3 of adenines spaced 7 bp apart. The Pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., 5'-T(A/T)2G(A/T)2A*-3'), while Bizelesin prefers purely AT-rich sequences (e.g., 5'-T(A/T)4 or 5A*-3', where T represents the cross-strand adenine alkylation and A* represents an adenine alkylation) (Park et al., 1996). In this study, the high-field 1H-NMR and rMD studies are conducted on the 11-mer DNA duplex adduct of the Pyrrole where the 5'-TTAGTTA*-3' sequence is cross-linked by the drug. A severe structural perturbation is observed in the intervening sequences of cross-linking site, while a normal B-DNA structure is maintained in the region next to the drug-modified adenines. Based upon these observations, we propose that the interplay between the bisamido pyrrole unit of the drug and central G/C base pair (hydrogen-bonding interactions) is involved in the process of cross-linking reaction, and sequence specificity is the outcome of those interactions. This study suggests a mechanism for the sequence specific cross-linking reaction of the Pyrrole, and provides a further insight to develop new DNA sequence selective and distortive cross-linking agents.     

Binding of cis(1,2-diaminocyclohexane)platinum(II) and its derivatives to duplex DNA

A theoretical study is presented for the binding of RR, SS, SR, and RS isomers 1,2-diaminocyclohexane (DAC) or cis-PtII(DAC) to DNA. cis-PtII(DAC) is ligated to N7(G) on two adjacent intrastrand guanine bases in a kinked pentamer duplex of DNA (AT, CG*, CG*, GC, AT). The relative stability of the complexes is determined by calculating the relative conformational energy of the cis-PtII(DAC)(DNA) complexes with molecular mechanics (MM) and the intrinsic binding or ligation energy with quantum mechanics (QM). The results suggest that the RR and SS isomers of PtII(DAC) adducts with DNA are more stable than the SR/RS isomer by 1.7 kcal/mol relative to the cis-PtII(DAC(H2O)2 aquated species. Calculations on the overall stability of these isomers show that the SS and RR isomers are 6.5-8.2 kcal/mol more stable than the SR/RS isomers when bound to DNA, and this is attributed to differences in the strain energy in the DAC rings. The theoretical analyses of these compounds correlate a small differential activity with the trend in intrinsic binding energies. The RR isomer is more active in B16 melanoma cells, and the SS is most active in L1210 leukemia, and in general the RR and SS isomers are more active than the SR and RS in most cell types. The fact that the activity is DNA dependent suggests that excision or repair mechanisms may be taking place and that additional mechanistic steps beyond molecular modeling and quantum mechanical calculations are required to fully understand the activity. These studies of molecular fit of cis-PtII(DAC) to DNA are used to suggest substituted DAC compounds that may yield similar binding characteristics. Modifications to yield DAC derivatives are recommended in anticipation that they may also exhibit activity.     

P-postlabeling analysis of DNA adducts formed in vitro and in rat skin by methylenediphenyl-4,4'-diisocyanate (MDI)

The ³²P-postlabeling method was adapted for the detection of DNA adducts formed by methylenediphenyl-4,4'-diisocyanate (MDI). Incubation of the 3'-phosphates of the deoxyribosides of cytosine (C), adenine (A), guanine (G) and thymine (T) with MDI in Tris buffer resulted in the formation of 5, 7, 8, and 2 reaction products, respectively. Incubation of DNA with MDI resulted in detectable levels of 5, 2, and 1 adducts attributable to C, A, and G. Analysis of DNA isolated from the epidermis of rats treated dermally with 9 mg MDI showed an adduct pattern similar to the one seen in the in vitro DNA incubation. A total adduct level of 7 per 10⁸ nucleotides was measured, the limit of detection was 2 adducts per 10¹⁰ nucleotides. The data indicate that a minute fraction of MDI can reach DNA in vivo in a chemically reactive form. In comparison with the genotoxic skin carcinogen 7,12-dimethylbenz[a]anthracene on the other hand, the DNA-binding potency of MDI was more than 1000-fold lower.     

Structural elucidation of a novel DNA-DNA cross-link of 1,2,3,4-diepoxybutane

DNA-DNA cross-linking by 1,2,3,4-diepoxybutane (DEB) is considered the molecular basis for its potent cytotoxic and genotoxic effects. DEB reactions with DNA initially lead to N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then alkylate neighboring DNA bases to form bifunctional lesions. We recently reported the structures of four regioisomeric guanine-adenine adducts of DEB involving the N7 position of guanine and the N1, N3, N6, and N7 positions of adenine (Park, S., et al. (2004) Chemical Research in Toxicology 17, 1638-1651). In the present work, a novel bifunctional DNA lesion of DEB was identified as 1-(hypoxanth-1-yl)-4-(guan-7-yl)-2,3-butanediol (N1HX-N7G-BD). An authentic standard of N1HX-N7G-BD was prepared and structurally characterized by proton NMR, UV, and mass spectrometry. HPLC-ESI-MS/MS analyses of acid hydrolysates of DEB-treated calf thymus DNA revealed a peak that had the same retention time, MS/MS fragmentation, and UV spectrum as the authentic standard of N1HX-N7G-BD. We propose that N1HX-N7G-BD is formed by the hydrolytic deamination of previously reported 1-(aden-1-yl)-4-(guan-7-yl)-2,3-butanediol. Although N1HX-N7G-BD adducts are less abundant in DEB-treated DNA than the corresponding guanine-guanine cross-links, they may play a role in the induction of both AT and GC base pair mutations.     

DNA Sequence Preference and Adduct Orientation of Pyrrolo[2,1-c][1,4]benzodiazepine Antitumor Agents

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DNA binding properties of the indolocarbazole antitumor drug NB-506

Indolocarbazoles derived from the antibiotic rebeccamycin represent an important group of antitumor agents. Several indolocarbazoles are currently undergoing clinical trials. These compounds inhibit topoisomerase 1 to produce DNA breaks that are responsible for cell death. Unlike classical topoisomerase I poisons like camptothecin, glycosyl indolocarbazoles can form stable complexes with DNA even in the absence of topoisomerase I. At least in part, their mode of action is reminiscent of that of the anthracyclines, which also bind to nucleic acids and interfere with topoisomerase II. The lead synthetic compound in the series is the uncharged drug NB-506, which bears a glucose residue attached to the indolocarbazole chromophore substituted with two hydroxyl groups at positions 1 and 11. Here we report a detailed biophysical study aimed at characterizing the DNA binding properties of NB-506. Molecular modeling was used to compare the conformation and electronic properties of NB-506 and its analogue ED-571 bearing the two hydroxyl groups at positions 2 and 10. Surface plasmon resonance experiments, performed with DNA hairpin oligomers, indicate that NB-506 binds almost equally well to both AT and GC base pairs, and the binding affinity (K = 10(5) M(-1)) is similar to that of certain classical intercalators such as amsacrine and bisantrene. Isothermal titration calorimetry experiments show that the binding of NB-506 is enthalpy-driven (deltaH = -7.2 kcal/mol). The binding enthalpy measured for NB-506 is similar to that obtained with doxorubicin but the DNA interaction processes for the two drugs differ markedly in terms of entropy and deltaG. The free energy of NB-506 binding to DNA is considerably less favorable than that of doxorubicin. These biophysical data help us to understand further how rebeccamycin-type anticancer drugs interact with DNA.     

The interaction of unfused polyaromatic heterocycles with DNA: intercalation, groove-binding and bleomycin amplification.

A number of unfused-aromatic cations have been found to bind to DNA by intercalation and to amplify the bleomycin catalysed cleavage of DNA. These molecules are more similar in structure to unfused minor-groove binding compounds such as netropsin and DAPI than to fused-ring intercalators such as proflavine. An analysis of DAPI interactions with specific sequence DNA polymers has indicated that the binding modes for the molecule are sequence dependent: minor groove binding in sequences of three or more AT base pairs and intercalation in mixed or pure GC base pair sequences. As with other unfused intercalators which bind with their cationic side chains in the major groove, the amidinium groups of DAPI are in the major groove in the GC intercalation complex. DAPI is, thus, a good bleomycin amplifier in GC sequences but its minor-groove binding mode in AT sequences leads to bleomycin inhibition.     

Cross-linked thymine-purine base tandem lesions: synthesis, characterization, and measurement in gamma-irradiated isolated DNA

5-(Phenylthiomethyl)-2'-deoxyuridine has been recently shown to be a specific photolabile precursor of 5-(2'-deoxyuridilyl)methyl radical that is involved in the formation of tandem base lesion with vicinal guanine in oxygen-free aqueous solution. The thionucleoside was incorporated by either liquid or solid-phase phosphoramidite synthesis into dinucleoside monophosphates with a 2'-deoxyadenosine residue as the vicinal nucleoside located either at the 3' or 5'-extremity. UV-C irradiation of the modified dinucleoside monophosphate under anaerobic conditions gives rise to cross-linked thymine(CH2-C8)adenine tandem base lesions which were isolated and characterized by (1)H NMR and mass spectrometry analyses. The formation of the latter tandem lesions involved an intramolecular addition of the 5-(2'-deoxyuridilyl)methyl radical to the C8 of the adenine moiety. A sensitive and specific assay aimed at monitoring the formation of the four thymine(CH2-C8)purine adducts, namely d(T Delta G), d(G Delta T), d(T Delta A), d(A Delta T), within DNA, was designed. This was based on a liquid chromatography analysis coupled to tandem mass spectrometry (HPLC-MS/MS) detection of the dinucleoside monophosphates which were quantitatively released from gamma-irradiated DNA and oligodeoxyribonucleotides by enzymatic hydrolysis. The four lesions were detected in both single-stranded oligodeoxyribonucleotide and isolated DNA upon exposure to gamma-radiation in oxygen-free aqueous solution. It was found that the tandem guanine-thymine lesions were produced more efficiently than the adenine-thymine cross-links. Moreover, a significant sequence effect was observed. Thus, the yield of formation of the tandem lesions is higher when the purine base is located at the 5' position of the 5-(2'-deoxyuridilyl)methyl radical.     

Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase

The interaction of dihydrofolate reductase (DHFR) from Escherichia coli with drugs such as methotrexate (MTX) and 2,4-diamino-6,7-dimethylpteridine (DAM) has been studied by means of site-directed mutagenesis, fluorescence spectroscopy, and steady-state as well as transient kinetics. A strictly conserved residue at the dihydrofolate binding site of DHFR, phenylalanine-31, has been replaced with tyrosine or valine to ascertain the importance for binding of this hydrophobic amino acid, which interacts with both the pteridine ring and the p-aminobenzoyl moiety. The first mutation (Phe-31----Tyr) has a minimal effect on the binding of the classical inhibitor, DAM. On the other hand, the second mutation (Phe-31----Val) has increased the dissociation constant of DAM from the DHFR.NADPH.DAM ternary complex over 150-fold (greater than 3 kcal/mol). The dissociation constant of DAM from the (Val31-DHFR).DAM binary complex was too large to be measured fluorometrically. More importantly, these mutations have decreased the overall tight binding of MTX, from 100- to 140-fold (corresponding to a loss of binding energy of 2.2-2.4 kcal/mol) for the Tyr-31 and Val-31 mutants, respectively. These results indicate that hydrophobic interactions between MTX and DHFR are at least as important as formation of the MTX.DHFR salt bridge in the tight binding of MTX.