Molecular modelling of the interactions of tetra-(4-N-methylpyridyl) porphin with TA and CG sites on DNA.

The molecular structure of the DNA-intercalating ligand tetra-(4-N-methylpyridyl) porphin has been determined by X-ray crystallography. The porphyrin has a precise centre of symmetry; the central core is planar, with the N-methylpyridyl groups inclined to it at angles of 66-72 degrees. Molecular modelling of this structure into TpA and CpG sites of intercalated DNA, has been performed, and approximate energetics calculated. It has been shown that only the CpG site can have full ligand intercalation, since the thymine methyl group sterically hinders such geometry at TpA sites. Modelling indicates the importance of electrostatic effects in the low-energy forms of intercalated and part-intercalated complexes at both sequences.     

Theoretical Design, Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)(2) and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)(2)and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN(6) and N(7) (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N(7) of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N(7) and O(6) of the 3' guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

Theoretical Design,Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract

In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)₂ and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)₂and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN₆ and N₇ (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N₇ of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N₇ and O₆ of the 3′ guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

Predicted mode of intercalation of doxorubicin with dinucleotide dimers

Intermolecular molecular mechanics energy calculations have been carried out for doxorubicin interacting with two dinucleotide dimer sequences. The preferred mode of intercalation is in the minor groove with the anthraquinone ring of the drug nearly perpendicular to the base pairs for the (CpG) sequence having alternate C3′ endo-C2′ endo sugar ring puckering. The preferred intercalation conformation of the drug is nearly identical to the N-bromacetyldaunomycin crystal structure. This prediction is qualitatively consistent with the recently reported crystal structure of a d(CpGpCpGpCpG) dimer-daunomycin complex. For the other dinucleotide sequence, (TpC-ApG), minor groove intercalation is also preferred, but the drug conformation can be changed.     

Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)₂ also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.     

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.     

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.     

A theoretical investigation of the intercalative binding of 7-H pyrido[4.3C]carbazole chromophore into a d(CpG)2 minihelix

Theoretical computations are performed of the intercalative binding to a model d(CpG)2 minihelix of 7-H pyrido[4.3C]carbazole, the precursor of the antitumor bisintercalating drug ditercalinium. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structures) of Miller and co-workers. The ligand-nucleotide interactions and the nucleotide conformational energies are computed with the SIBFA procedures (sum of interactions between fragments ab initio computed), which use formulas of empirical origin that reproduce ab initio SCF (self-consistent field) computations. Among the candidate intercalation sites most favored energetically, one has a pattern of conformational angles related to the one determined crystallographically by Sobell et al. in a series of x-ray structural studies of small intercalator-dinucleotide monophosphate complexes. Optimal values of the unwinding angle, found in the range of -12 degrees to -14 degrees, are consistent with available experimental data on DNA.     

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.     

The molecular structure of a 4'-epiadriamycin complex with d(TGATCA) at 1.7A resolution: comparison with the structure of 4'-epiadriamycin d(TGTACA) and d(CGATCG) complexes.

The structure of the complex between d(TGATCA) and the anthracycline 4'-epiadriamycin has been determined by crystallographic methods. The crystals are tetragonal, space group P4(1)2(1)2 with unit cell dimensions of a = 28.01, c = 52.95A. The asymmetric unit consists of one strand of hexanucleotide, one molecule of 4'-epiadriamycin and 34 waters. The R-factor is 20.2% for 1694 reflections with F greater than or equal to 2 sigma F to 1.7A. Two asymmetric units associate to generate a duplex complexed with two drug molecules at the d(TpG) steps of the duplex. The chromophore intercalates between these base pairs with the anthracycline amino-sugar positioned in the minor groove. The double helix is a distorted B-DNA type structure. Our structure determination of d(TGATCA) complexed to 4'-epiadriamycin allows for comparison with the previously reported structures of 4'-epiadriamycin bound to d(TGTACA) and to d(CGATCG). The three complexes are similar in gross features and the intercalation geometry is the same irrespective of whether a d(CpG) or d(TpG) sequence is involved. However, the orientation of the amino-sugar displays a dependence on the sequence adjacent to the intercalation site. The flexibility of this amino-sugar may help explain why this class of antibiotics displays a relative insensitivity to base sequence when they bind to DNA.     

Site specificity of binding of antitumor antibiotics to DNA

The site specificities for intercalation of steffimycin B, adriamycin, echinomycin, and ethidium bromide with DNA have been determined by CD first-neighbor analysis. The first three, which are antineoplastic antibiotics, all exhibit preferential binding to sites comprised of guanine and cytosine (GpC, CpG, and CpC or its complement GpG). Ethidium bromide displays nonspecific intercalation. The results are compared with findings from “footprinting” studies.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.     

A theoretical investigation of the base sequence preferences of monointercalating polymethylene carboxamide derivatives 9-aminoacridine.

Theoretical computations are performed of the comparative binding affinities of five polymethylene carboxamide derivatives of 9-aminoacridine to a series of double-stranded hexanucleotides. The purpose of this investigation is to ascertain whether minor groove recognition of a guanine base adjacent to the intercalation site can occur, and be preferentially stabilized, for a given length of the polymethylene side chain, encompassing from n = 2 up to n = 6 methylene groups. For that purpose, several representative sequences were investigated, in which intercalation of the 9-aminoacridine chromophore occurred at a central d(CpG) or d(TpA) step. Investigated were the self-complementary sequences d(CGCGCG)2, d(GCCGGC)2, d(TATATA)2 and d(ATTAAT)2, as well as the 'mixed' sequences d(ACTAAT) .d(ATTAGT) and d(TGTATA). d(TATACA). For n = 3 up to n = 6, such a recognition was enabled only when the guanine base was located downstream of the intercalation site, i.e. with steps d(CGG) and d(TAG). It occurred by means of a bidentate interaction involving, on the one hand, H(N2) and N3 of the base, and, on the other hand, the carbonyl oxygen and the cis amino hydrogen of the terminal formamide moiety of the ligand. Because of the flexibility of the side chain, however, alternative binding modes were also found to occur competitively, involving backbone-only interactions of the side chain. On the basis of the present computations, upon binding to the sequence d(GCCGGC)2, an optimal value of n = 5 could be derived, with the corresponding acridine derivative eliciting both a significant prevalence of the bidentate over backbone only binding mode, and the most favourable energy balance within the investigated series. This privileged value of n = 5 is fully consistent with the experimental results of Markovits et al. and Gaugain et al. The very flexibility of the side chain, however, hampered any preferential recognition of a triplet sequence with a downstream guanine, such as d(CGG) or d(TAG), to be elicited over sequences such as d(TAA), d(TAT) or d(TAC).     

DNA-drug interactions. The crystal structure of d(CGATCG) complexed with daunomycin

The structure of a d(CGATCG)-daunomycin complex has been determined by single crystal X-ray diffraction techniques. Refinement, with the location of 40 solvent molecules, using data up to 1.5 A, converged with a final crystallographic residual, R = 0.25 (RW = 0.22). The tetragonal crystals are in space group P4(1)2(1)2, with cell dimensions of a = 27.98 A and c = 52.87 A. The self-complementary d(CGATCG) forms a distorted right-handed helix with a daunomycin molecule intercalated at each d(CpG) step. The daunomycin aglycon chromophore is oriented at right-angles to the long axis of the DNA base-pairs. This head-on intercalation is stabilized by direct hydrogen bonds and indirectly via solvent-mediated, hydrogen-bonding interactions between the chromophore and its intercalation site base-pairs. The cyclohexene ring and amino sugar substituent lie in the minor groove. The amino sugar N-3' forms a hydrogen bond with O-2 of the next neighbouring thymine. This electrostatic interaction helps position the sugar in a way that results in extensive van der Waals contacts between the drug and the DNA. There is no interaction between daunosamine and the DNA sugar-phosphate backbone. We present full experimental details and all relevant conformational parameters, and use the comparison with a d(CGTACG)-daunomycin complex to rationalize some neighbouring sequence effects involved in daunomycin binding.     

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.     

Cell Cycle Arrest and Induction of Apoptosis in Colon Adenocarcinoma Cells by a DNA Intercalative Quinoline Derivative, 4-Morpholinopyrimido [4′,5′:4,5] Selenolo (2,3-b) Quinoline

Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of 4-morpholinopyrimido [4′,5′:4,5] selenolo (2,3-b) quinoline (MPSQ) with DNA. The association constants of MPSQ–DNA interactions were of the order of 10⁴ M^?1. Melting temperature, topological, and docking studies confirmed that the mode of interaction was by intercalation with preference to d(GpC)–d(CpG) site of DNA. Cytotoxicity studies showed the MPSQ-induced dose-dependent inhibitory effect on the proliferation of different cancer cells. Colon adenocarcinoma (COLO 205) cells are more sensitive among the cell lines tested, with an IC₅₀ value of 15 μM. Flow cytometry revealed that MPSQ affects the cell cycle progression by arresting at G2M phase. Further, Annexin V staining, mitochondrial membrane potential assay, and caspase-3 activity assay confirmed that MPSQ leads to mitochondria-mediated apoptotic cell death in COLO 205 cells.     

Solution structure of 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol intercalated in the DNA duplex d(CGATCG)2

The solution structure of the complex formed between d(CGATCG)(2) and 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol, a new antitumor drug under design, has been resolved using NMR spectroscopy and restrained molecular dynamic simulations. The drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the minor groove. Analysis of NMR data establishes a weak stacking interaction between the intercalated ligand and the DNA bases; however, the drug/DNA affinity is enhanced by a hydrogen bond between the hydroxyl group of the end of the intercalant side chain and the amide group of guanine G6. Unrestrained molecular dynamic simulations performed in a water box confirm the stability of the intercalation model. The structure of the intercalated complex enables insight into the structure-activity relationship, allowing rationalization of the design of new antineoplasic agents.     

Molecular cloning and analysis of the fragile X region in man.

The fragile X syndrome (FraX), the most common inherited form of mental retardation, has been located to Xq27.3. As a step in the molecular analysis of this mutation, we have cloned a contiguous 1.8 Mb region containing the entire fragile X region in YAC and cosmid clones. The cloned area defines a region of 50 kb containing a CpG island, found to be selectively methylated in patients expressing the fragile X phenotype. In this 50kb area we have localised the breakpoints of four somatic cell hybrids selected to break at the position of the fragile site. Fluorescence in-situ hybridisation of cosmids flanking this area shows that the breakpoints, the CpG island and the fragile site coincide.