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M J Waring

Publications and source records attributed to M J Waring.

At least 37 records · Page 2Linked to original sources
PubMed1998

DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM.

The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.

Anti-Bacterial Agents↗
PubMed1998

Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5'(A/T)GC and 5'(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5'IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5'(A/T)GC and 5'(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.

Adenosine↗
PubMed1998

Recognition of specific sequences in DNA by a topoisomerase I inhibitor derived from the antitumor drug rebeccamycin.

We investigated the interaction with DNA of two synthetic derivatives of the antitumor antibiotic rebeccamycin: R-3, which is a potent topoisomerase I inhibitor and contains a methoxyglucose moiety appended to the indolocarbazole chromophore, and its aglycone, R-4. Spectroscopic measurements indicate that R-3 intercalates into DNA and that its carbohydrate domain contributes significantly to reinforce the affinity for DNA. Two complementary ligation assays concur that R-3, but not its aglycone counterpart, exerts a significant effect on the curvature and/or the flexibility of DNA. The sugar moiety may be responsible for preferential binding of R-3 to circular (or bent) DNA molecules as opposed to linear DNA fragments. The sequence selectivity of binding to DNA has been studied thoroughly by footprinting with DNase I and two other nucleases. The glycosylated compound is highly selective for nucleotide sequences containing GpT (ApC) and TpG (CpA) steps. The derivative lacking the sugar moiety on the indolocarbazole chromophore binds at essentially identical sites but with considerably lower affinity, so it seems that the chromophore rather than the carbohydrate is responsible for the preferential binding to sequences surrounding GpT and TpG steps. The influence of the exocyclic substituents present on the bases at the recognition sites (i.e., the 2-amino group of guanine and the 5-methyl group of thymine) was evaluated using two series of modified DNA molecules prepared by polymerase chain reaction containing inosine and/or 2,6-diaminopurine and uridine and/or 5-methylcytosine residues. The introduction of the amino group onto purine residues or the addition of a methyl group to pyrimidine residues suffices to create new drug binding sites. Therefore, unlike most DNA-binding small molecules, the rebeccamycin analogue seems to be highly sensitive to any modification of the exocyclic substituents on the bases in both the major and minor grooves of the double helix. The footprinting profiles with the different DNA fragments bear a remarkable resemblance to those determined for nogalamycin and bisnaphthalimide compounds known to recognize their preferred GpT and TpG sites via intercalation from the major groove. The unique DNA binding characteristics of the rebeccamycin analogue correlate well with its inhibitory effects on topoisomerase I.

Aminoglycosides↗
PubMed1998

Odour-absorbing dressings.

This study uses an objective test system that compares the ability of different dressings to prevent the passage of a volatile amine when applied to a wound model under simulated 'in-use' conditions. Five dressings containing activated charcoal were tested by this method, and a low-adherent absorbent dressing was used as a control. Results demonstrate clear differences in the ability of the products to contain the test solution and prevent the loss of the volatile diethylamine into the surrounding air. Products which combine a physical absorbent with a charcoal component show enhanced performance.

Administration, Cutaneous↗
PubMed1997

Effects of diaminopurine and inosine substitutions on A-tract induced DNA curvature. Importance of the 3'-A-tract junction.

Gel migration and uranyl photoprobing have been used to study the effects of inosine and 2,6-diaminopurine (2,6-DAP) substitution on adenine-tract (A-tract) induced DNA curvature. Using a 10mer repeated sequence including five inosines we show by uranyl photoprobing that a narrow minor groove varying in phase with the helix repeat is not the cause of DNA curvature. Further, we have systematically studied by gel migration the effects on A-tract induced curvature of either single or full substitution with inosine and/or 2,6-DAP in a 5'-AAAAAGCCGC-3'sequence. DNA curvature is shown to increase when inosines are substituted for the guanosines in the sequence between the A-tracts. By comparing the effects of each monosubstitution it can be seen that when the G closest to the 3'-end of the A-tract is substituted the effect on DNA curvature is much stronger than when substitution is made at any other position. By contrast, curvature is abolished when 2,6-DAP residues are substituted for all adenines, and monosubstitution reveals that the effect of substituting a single adenine is strongest at the 3'-end of the A-tract. These results favor a model in which the curvature induced by an A-tract in DNA molecules is primarily located at the junction with the 3'-end of the A-tract, and this peculiar junction is created because the A-tract has a preference to form a non-B-DNA structure which builds up from the 5'-end.

2-Aminopurine↗
PubMed1997

Footprinting of echinomycin and actinomycin D on DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine.

In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.

2-Aminopurine↗
PubMed1997

Altered cleavage of DNA sequences by bleomycin and its deglycosylated derivative in the presence of actinomycin.

The antitumour antibiotics bleomycin and actinomycin are commonly used therapeutically in combination. One causes metal ion- and oxygen-dependent oxidative damage to DNA, while the other acts at the level of DNA via intercalation of its phenoxazone chromophore and probable inhibition of topoisomerases. Both drugs bind and/or cleave DNA primarily at guanine-containing sequences, which could lead to mutual interference. Using three different restriction fragments we show that binding of actinomycin to DNA causes major alterations in the sequence specificity of bleomycin.Fe-mediated cleavage, including the appearance of new cleavage sites and the suppression of others. The subtle sequence-dependence of the interference is illustrated by the different effects of actinomycin on DNA cleavage by the deglycobleomycin.Fe complex. Actinomycin sharply decreases the extent of cleavage at GpC sites by both bleomycin and deglycobleomycin whereas cleavage at GpT sites is much less affected, while novel cleavage sites are generated at GpA, ApT and, to a lesser extent, TpT steps. A dramatic increase in bleomycin.Fe cutting at GpA is barely detectable with deglycobleomycin.Fe, confirming that the carbohydrate moiety of bleomycin is important for DNA recognition. The results contribute to a better understanding of how two individually well-characterized small molecules interact simultaneoulsy with specific sequences in DNA and as such assist clarification of the principles governing drug-DNA recognition.

Antibiotics, Antineoplastic↗
PubMed1997

Sequence-selective binding to DNA of bis(amidinophenoxy)alkanes related to propamidine and pentamidine.

The DNA sequences targeted by a complete homologous series of aromatic diamidines have been determined at single-nucleotide resolution via protection from cutting by the endonucleases DNase I, DNase II and micrococcal nuclease. Propamidine, pentamidine and to a lesser extent hexamidine bind selectively to nucleotide sequences composed of at least four consecutive A-T base pairs. In contrast, the binding to DNA of butamidine, heptamidine, octamidine and nonamidine is poorly sequence-selective. Sequences composed of only three consecutive A-T base pairs do not afford a potential binding site for propamidine or the longer homologues, and none of the drugs tolerate the presence of a G-C base pair within the binding site. Experiments with DNA molecules containing inosine in place of guanosine and 2,6-diaminopurine in place of adenine reveal that the lack of binding of propamidine to GC-containing sites is attributable to an obstructive effect of the exocyclic 2-amino group of guanosine. The present data support the view that the local conformation of the double helix (in particular the width of the minor groove) plays a dominant role in the binding reaction and that the capacity of diamidines to recognize AT-rich sequences selectively varies considerably depending on the length of the alkyl chain. The evidence indicates that binding to AT-tracts in DNA must play a role in the biological activity of these diamidines, but there is no simple correlation between binding and pharmacological efficacy.

Alkanes↗
PubMed1997

Copper-dependent oxidative and topoisomerase II-mediated DNA cleavage by a netropsin/4'-(9-acridinylamino)methanesulfon-m-anisidide combilexin.

A conjugate molecule was synthesized by linking the DNA-intercalative antitumor drug 4'-(9-acridinylamino)methanesulfon-manisidide (mAMSA) via a 4-carboxamide side chain to a dipyrrolecarboxamide moiety structurally related to the minor groove-binding antibiotic netropsin. The molecule (netropsin/ mAMSA) behaves as a threading intercalator. Its netropsin-like tail becomes located in the minor groove of the double helix and serves to drive the hybrid molecule preferentially to AT-rich sites on various DNA fragments as revealed by DNase I footprinting. The hybrid retains the susceptibility to copper-dependent oxidation characteristic of the parent mAMSA moiety as well as its ability to generate oxygen radicals, which can mediate DNA damage, mainly at cytidine and guanosine nucleotides. It also retains the property of stimulating the formation of cleavable complexes with DNA in the presence of topoisomerase II, but its netropsin-like moiety confers little or no influence on the reaction with topoisomerase I. Although netropsin/mAMSA is less potent than mAMSA at producing cleavable complexes with topoisomerase II, it promotes the appearance of cleavage sites at much the same nucleotide sequences as does the parent compound. The dipyrrolecarboxamide tail is not silent, however, since it modifies the concentration-dependence of cleavable complex formation.

Amsacrine↗
PubMed1997

Synthesis, DNA-binding and cytotoxic properties of a bis(netropsin)-anthracenedione conjugate.

A combilexin molecule containing two netropsin moieties attached to the aminoalkyl side chains of mitoxantrone has been synthesized and evaluated for cytotoxic activity towards murine L1210 leukaemia and human MCF7 carcinoma cells in vitro. It is marginally less cytotoxic than mitoxantrone but much more growth-inhibitory than netropsin. Various spectroscopic and biochemical techniques have been employed to characterize the interaction of the drug, NetMitox, with DNA. Circular dichroism (CD) and electric linear dichroism (ELD) data indicate that binding of the netropsin moiety or moieties within the minor groove of the double helix impedes the intercalation of the adjacent anthracenedione ring. ELD and footprinting experiments reveal a certain amount of mutual interference between the two functionalities of the conjugate molecule but the selective recognition of AT-rich sequences by netropsin largely dominates the recognition pattern. The lack of interaction with GC-rich sequences is attributable to steric hindrance occasioned by the 2-amino group of guanine which impedes access of the netropsin moiety into the minor groove, as is evident by the good binding of the hybrid to poly(dI-dC) x poly(dI-dC) as well as by the redistribution of its binding sites on DNA molecules substituted with inosine and/or 2,6-diaminopurine. The difficulty of the anthracenedione system in intercalating correlates with the lack of effect of the drug on cleavable complex formation with topoisomerase II as well as its diminished cytotoxicity compared to mitoxantrone. However, the finding that the drug retains significant toxicity towards leukaemia cells may suggest that DNA is perhaps not the unique molecular target.

Animals↗
PubMed1996

PCR-based development of DNA substrates containing modified bases: an efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins.

DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of sequence-selective ligands, both small and large. Footprinting patterns reveal that the binding sites for AT- or GC-specific antibiotics (distamycin or mithramycin, respectively) are completely changed in the modified DNAs, as expected for direct sequence readout involving contact with the purine 2-amino group. However, we also find large changes in the binding of HMG-D, a member of the HMG-1 family of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformability of the double helix. This interpretation is confirmed by the finding that deoxyuridine-containing poly- and oligonucleotides, which lack the exocyclic methyl group of thymidine in the major groove, interact 5-10 times more strongly with HMG-D than do their counterparts containing natural nucleotides.

2-Aminopurine↗
PubMed1996

A simple ligation assay to detect effects of drugs on the curvature/flexibility of DNA.

Circular DNA molecules can readily be formed from the 169 bp tyrT fragment in the presence of T4 DNA ligase. We have analyzed the formation of DNA circles in the presence of the clinically important antitumour drugs amsacrine, mitoxantrone and daunomycin. All three are intercalating agents but they affect the closure reaction differently: daunomycin and mitoxantrone progressively inhibit the formation of circles whereas at low concentrations amsacrine strongly enhances the yield of circles suggesting that this drug can increase the flexibility and/or curvature of DNA. The ligation assay described here may prove useful and widely applicable for investigating the effects of small molecules on the secondary structure of DNA.

Amsacrine↗
PubMed1996

Sequence-selective intercalation of antitumour bis-naphthalimides into DNA. Evidence for an approach via the major groove.

LU 79553, a bis-naphthalimide drug highly active against human solid tumour xenografts, has been shown to bis-intercalate into DNA with a helix-unwinding angle of 37 degrees. Footprinting experiments with DNase I reveal that the drug is selective for mixed nucleotide sequences characterised by an alternating purine-pyrimidine motif, particularly those containing GpT (ApC) and TpG (CpA) steps. Derivatives bearing nitro or amino substituents on the naphthalimide chromophores bind at essentially identical sites. The footprinting profiles on tyrT DNA and on two fragments from pBS bear a remarkable resemblance to those determined for nogalamycin, an antibiotic which binds intercalatively leaving bulky carbohydrate substituents blocking both the major and minor grooves of the helix. Several lines of evidence indicate that the bis-naphthalimides recognise their preferred binding sites via the unusual expedient of intercalating from the major groove. Footprints on the complementary DNA strands sometimes appear staggered in the 5'direction. Repositioning the 2-amino group of G.C base pairs, which serves as a critical minor-groove marker, by substitution with inosine and/or 2,6-diaminopurine has little effect on the distribution of binding sites for LU 79553. The bis-naphthalimides affect the guanine-specific reaction with dimethyl sulfate (which reacts with the N7 position of the base located in the major groove) but not reactions with tetrachloropalladinate or methylene blue. Photoactivation of LU 79553-DNA complexes leads to a small amount of strand scission mainly at guanine residues. These observations make a strong case for binding via the major groove of the double helix, in contrast to nearly all common intercalating drugs, which could be important in explaining the unique biological selectivity of bis-naphthalimides.

Amides↗
PubMed1996

Stabilization of triple helical DNA by a benzopyridoquinoxaline intercalator.

Biophysical, footprinting, and chemical probing experiments are described which characterize the triple helix-stabilizing effects of a benzo[f]pyridoquinoxaline derivative BfPQ-4,3 structurally related to the previously reported benzo[f]pyridoindole compound BePI [Mergny et al. (1992) Science 256, 1681-1684]. Two parallel triple helix model systems have been investigated; one in which the third strand matched perfectly a 27 base pair purine-pyrimidine motif in target DNA and another in which the third strand was one nucleotide longer, i.e., a 28-mer. In the latter system, the pairing of the (Y)28 third strand to the (Y.R)27 target induces the formation of a bulge containing at least one unpaired base, which can be evidenced by chemical probing experiments with osmium tetroxide. BPQ, which uinwinds a duplex DNA by 17 degrees as judged by viscometric experiments and otherwise behaves as a typical nonspecific intercalculating drug, promotes the formation of Y.R.Y parallel triple helix containing both T.A.T and C.G.C+ triplets. Both DNase I and MPE.FeII footprinting experiments concur that triplex formation with the target (Y.R)27 sequence can be detected in the presence of BPQ at about 10-fold lower oligonucleotide concentrations than are required to produce an equivalent footprint in the absence of the drug. In addition, BPQ will promote binding to the polypurine-polypyrimidine target sequence by the longer mismatched oligonucleotide, providing significant stabilization of the parallel bulge-containing(Y.R)27,(Y)28 triplex with nearly the same efficiency as the bulge-free (Y.R)27.(Y)28 triplex. Thus in vivo BPQ might enhance the formation of both undesired and desired DNA triplexes. By performing an MPE*FeII probing reaction with a 5'-32 P-labeled oligonucleotide third strand, we have obtained evidence that BPQ is actually bound to the triplex region and may distort in a sequence-specific fashion.

Base Sequence↗
PubMed1996

Interaction of a DNA-threading netropsin-amsacrine combilexin with DNA and chromatin.

Combilexins are a group of DNA ligands having a sequence-specific minor groove binding element combined with an intercalating chromophore which stabilizes the DNA complex and can interfere with topoisomerases. In this study, complementary methods of spectroscopy (absorption, circular dichroism, electric linear dichroism) and biochemistry (viscometry, footprinting) have been applied to explore the nature of the complex formed between a new amsacrine-4-carboxamide-netropsin combilexin and DNA or chromatin. Collectively, the structural and kinetic data concur that the conjugate threads through the DNA double helix so as to intercalate its acridine chromophore, leaving the netropsin moiety and the methanesulfonanilino group positioned within the minor and major grooves of the double helix, respectively. The hybrid retains the AT selectivity conferred by the netropsin moiety. The threading-type intercalation process, evidenced by stopped-flow measurements, is affected when the DNA is wrapped around histones. The composite drug can bind to both the DNA linker segments and the nucleosomal cores in chromatin though, unlike its constituents, it antagonizes the salt-induced condensation of chromatin. As far as its mode of binding to DNA is concerned, the netropsin-amsacrine hybrid molecule exhibits structural features reminiscent of the antitumor antibiotics nogalamycin and pluramycin. The design of DNA-threading combilexins provides an original route for the development of sequence-specific ligands capable of forming stable complexes with DNA.

Amsacrine↗
PubMed1996

DNA recognition by two mitoxantrone analogues: influence of the hydroxyl groups.

The clinically useful anticancer drug mitoxantrone intercalates preferentially into 5'-(A/T)CG and 5'-(A/T)CA sites on DNA. The 5,8 hydroxyl substituents on its anthracenedione chromophore are available to interact with the double helix. Footprinting experiments with two anthraquinone derivatives structurally related to mitoxantrone and ametantrone have been undertaken to assess the influence of the hydroxyl groups on the DNA recognition process. The results confirm that they do play a role in the recognition of preferred nucleotide sequences and suggest that the binding of anthraquinones to a 5'-(A/T)CG site is dependent on the presence of the 5,8 hydroxyl substitutes whereas binding to 5'-(A/T)CA sites appears to proceed just as well without them.

Antineoplastic Agents↗
PubMed1996

Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

Antibiotics, Antineoplastic↗