Interaction between double helix DNA fragments and the new antitumor agent sabarubicin,Men10755

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials.The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4′ of the proximal sugar) with the hexanucleotides d(CGTACG)₂ and d(CGATCG)₂ was studied by a combined use of 2D-¹H and ³¹P NMR techniques. Both ¹H and ³¹P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (k_off) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of k_off , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)₂ sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues.A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.     

Intercalation complex of imidazoacridinone C-1311, a potential anticancer drug,with DNA helix d(CGATCG)2: stereostructural studies by 2D NMR spectroscopy

Imidazoacridinone C-1311 (Symadex®) is a powerful antitumor agent, which successfully made its way through the Phase I clinical trials and has been recommended for Phase II few a years ago. It has been shown experimentally that during the initial stage of its action C-1311 forms a relatively stable intercalation complex with DNA, yet it has shown no base-sequence specificity while binding to DNA. In this paper, the d(CGATCG)₂:C-1311 intercalation complex has been studied by means of two-dimensional NMR spectroscopy, yielding a full assignment of the resonance lines observed in ¹H NMR spectra. The observation of the intermolecular NOE contacts between C-1311 and DNA allowed locating the ligand between the guanine and adenine moieties. Formation of a symmetric complex was pointed out on the basis of the lack of a second set of the ¹H resonances. The resulting stereostructure of the complex was then improved by means of molecular dynamics, using the CHARMM force field and GROMACS software. To this end, distance restraints derived from the NOESY cross-peak volumes were applied to the atomistic model of the d(CGATCG)₂:C-1311 complex. Obtained results are in full agreement with biochemical data on the mechanism of action of C-1311, in particular with the previously postulated post-intercalation enzymatic activation of the studied drug.     

DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.     

Crystal Structures of Four Morpholino-Doxorubicin Anticancer Drugs Complexed with d(CGTACG) and d(CGATCG): Implications in Drug-DNA Crosslink

Abstract

Among the new generations of anthracycline drugs, morpholino-doxorubicin (MDox) and its derivative have unusually potent activity when compared with the parent doxorubicin. 3″- Cyano-morpholino-doxorubicin (CN-MDox) has been suggested to form a covalent crosslink to DNA, although the exact mode of interactions remains unclear. To establish the structural basis of this crosslink, we carried out X-ray diffraction analyses of the complexes between four different morpholino-doxorubicins (i.e., MDox, CN-MDox, (R)- and (S)-2″-methoxy- morpholino-Dox (MMDox)) and two DNA hexamers CGTACG and CGATCG. Their crystal data are similar to other Dau/Dox complexes with space group P4₁2₁2,a=b ～28 Å, c～ 53 Å. The refined structures at ～1.8 Å resolution revealed that two drug molecules bind to the duplex with the aglycons intercalated between the CpG steps with their N^3′ -morpholino- daunosamines in the minor groove. The morpholino moiety is flexible and may adopt different conformations dependent on the sequence context The O¹ atoms of the two morpholino groups in the drug-DNA complexes are in van der Waals contact The structural results suggest possible crosslinking mechanism of CN-MDox. It is worth pointing out that by linking two piperazinyl- or piperidinyl-doxorubicins at the 1″ positions a new type of bis-doxorubicin derivatives may be synthesized which may bind to a hexanucleotide sequence with some specificity.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

A Theoretical Investigation on the Sequence Selective Binding of Daunomycin to Double-Stranded Polynucleotides

Abstract

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self- complementary double-stranded hexanucleotides: d(CGTACG)₂, d(CGATCG)₂, d(CITACI)₂, d(TATATA)₂, d(CGCGCG)₂ and d(TACGTA)₂. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)₂- DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)₂ and d(CGCGCG)₂, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT)-poly(dA-dT) and poly(dG-dC)<<poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)₂ and d(CGTACG)₂, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)₂ complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent. Altogether, it is seen that the selective base pair recognition by daunomycin cannot, in general, be defined in terms of the two base pairs implicated in the intercalation site alone (with the exception of homogeneous AT or GC base sequences) but must be expressed in terms of a triplet of base pairs.     

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.     

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2)

The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG^5BrUACG)₂ and d(CGTACG)₂ have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

A theoretical investigation on the sequence selective binding of daunomycin to double-stranded polynucleotides

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self-complementary double-stranded hexanucleotides: d(CGTACG)2,d(CGATCG)2,d(CITACI)2, d(TATATA)2, d(CGCGCG)2 and d(TACGTA)2. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)2-DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)2 and d(CGCGCG)2, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)2 and d(CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)2 complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tetraplex Formation of d(GGGGGTTTTT): 1H NMR Study in Solution

Abstract

The oligonucleotide d(G₅T₅) can in principle form a fully matched duplex with G · T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A₅C₅) at low temperature, d(G₅T₅) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G₅T₅) and d(A₅C₅) are mixed together in equimolar amounts and heated for several minutes at 85°C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz ¹H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.     

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.     

Tin-119 NMR Studies on adduct formation and stereochemistry of organoyltin(IV)trihalides

qazTin-119 and phosphorus-31 NMR spectra have been recorded for a series of adducts of RSnX₃ (R  Me, Ph; X  Cl, Br) with halide, tributylphosphine (P) and tributylphosphine oxide (L). The adducts were either 1:1 five coordinate or 1:2 six coordinate complexes. The tin-ll9 NMR spectra of mixtures of corresponding chloro and bromo complexes reveal, in most cases, all possible mixed halide species but much additional structural information is obtained from these spectra which could not be extracted from the spectra of individual compounds themselves. Thus in some cases, in the five coordinate species the Berry pseudorotation between isomers within a particular stoichiometry could be slowed on the NMR timescale which allowed a determination of the molecular structure. An equimolar mixture of [PhSnCl₅]²⁻ and [PhSnBr₅]²⁻ shows eleven of the twelve geometries possible for [PhSnCl_xBr_5−x]²⁻. In the six coordinate series [RSnX₄P]⁻ the tin-119 NMR spectra of the mixtures of [RSnCl₄P]⁻ and [RSnBr₄P]⁻ allow the geometry to be determined as trans. Application of the pairwise additivity model for calculation of the tin-119 chemical shift positions for the mixed halide systems are discussed.     

Complexes of d(+)-biotin with Pd(II) and Pt(II)

The reactions of d(+)-biotin with K₂MX₄, where M = Pd(II) or Pt(II) and X = Cl or Br have been studied in acidic, neutral or alkaline aqueous solutions. Complexes of the type trans-M(Bio)₂X₂ have been isolated for both metals and characterized with elemental analyses, conductivity measurments, ir spectra, ¹Hnmr and ¹³Cnmr spectra. The complex of the type [Pd(Bio)Cl₂]₂ has also been isolated from DMF solutions. The results indicate that d(+)-biotin coordinates exclusively through its sulfur atom with these metals in all the complexes in the present study, in the solid state or in solution.     

Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC)

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)]₂, the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the ¹H and ³¹P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)]₂ is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C⁺ pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9⁺/5mC1•5mC1⁺/C8•C8⁺/C2•C2⁺/T7.T7/C6•C6⁺/C4•C4⁺. The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8⁺) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)]₂ were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10^−pH.     

Acetyl pyridine-based palladium(II) compounds as an artificial metallonucleases

Palladium(II) complexes of type [Pd(Lⁿ)Cl₂] and [Pd(bdt)(Lⁿ)] have been synthesized using 2-acetyl pyridine derivatives(Lⁿ) and benzene-12-dithiol(bdt). The synthesized complexes have been characterized by various analytical techniques like thermo gravimetric analysis, elemental analysis, conductance measurement, and spectroscopic techniques like elemental analysis, mass spectra, absorption spectra, IR, ¹H NMR, energy-dispersive X-ray spectroscopy. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption titration, viscosity measurement methods. Based on the observations, an intercalative binding mode of DNA has been proposed. In order to provide additional evidence for the intercalation mode of binding between the complex and CT-DNA, fluorescence titration experiment was performed. In addition, molecular modeling study has been carried out with the aim of establishing the complex’s binding mode. Antibacterial activity study of the complexes have been screened against pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Serratia marcescens, and Pseudomonas aeruginosa. Gel electrophoresis assay demonstrates that all the complexes can cleave the pUC19 plasmid DNA.     

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 °C. Structural models of the complex with d(CGTACG)₂ were developed on the basis of DOSY, 2D NOESY and ³¹P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.     

Structural characterization of the head-to-head isomers of the [Pd2(Ph2Ppy)2Cl2] and [PtPd(Ph2Ppy)2I2] complexes (Ph2Ppy = 2-(diphenylphosphino)pyridine)

The molecular structures of the thermodynamically unstable head-to-head isomers, HH-[Pd₂(Ph₂Ppy)₂Cl₂] and HH-[PtPd(Ph₂Ppy)₂I₂], have been determined by single crystal X-ray diffraction. The two complexes have proved to be isostructural. The severe distortions of the bond angles from the ideal square planar geometry around the metal centers ligating the trans phosphorus donor atoms are indicative of a more pronounced internal strain in the HH isomers as compared to the HT counterparts. The enhanced internal strain is thought to be the major driving force responsible for the spontaneous conversion of the head-to-head isomers to their head-to-tail congeners. ¹³C NMR spectra in solution phase as well as solid-state ³¹P MAS NMR spectra have proved to be informative regarding the orientation of the asymmetric Ph₂Ppy ligands.