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PubMed1995

Structure of a dicationic monoimidazole lexitropsin bound to DNA.

An X-ray crystal structure has been solved of the complex of a dicationic lexitropsin with a B-DNA duplex of sequence CGCGAATTCGCG. The lexitropsin is identical to netropsin except for replacement of the first methylpyrrole ring by methylimidazole, converting a =CH- to =N-. Crystals are isomorphous with those of the DNA dodecamer in the absence of drug. Although the =N- for =CH- substitution was intended to make that locus on the drug molecule compatible with a G.C base pair, electrostatic attraction for the two cationic ends of the drug predominates, and this lexitropsin binds to the same central AATT site as does the parent netropsin. But unlike netropsin, this lexitropsin exhibits end-for-end disorder in the crystal. Both orientations were refined separately to completion. Final residual errors at 2.25 A resolution for the 2358 reflections above 2 sigma in F are R = 0.165 for one orientation (LexA) with 37 water molecules and 0.164 for the inverted drug orientation (LexB) with 40 water molecules. This molecular disorder is probably attributable to a weakening of binding to the AATT site occasioned by the imidazole-for-pyrrole substitution.

Antibiotics, Antineoplastic↗
PubMed1991

Bis(pyrrolecarboxamide) linked to intercalating chromophore oxazolopyridocarbazole (OPC): selective binding to DNA and polynucleotides.

We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator. The hybrid molecule Net-OPC binds to poly[d(A-T)] at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0). Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly[d(A-T)] than does netropsin. On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly[d(A-T)] is suggested to involve both intercalation and external binding of the OPC chromophore. In contrast, on poly[d(G-C)], Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated. The binding constant of Net-OPC to poly[d(G-C)] was found to be about 350-fold lower than that of the high-affinity binding site in poly[d(A-T)]. As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site. The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate. The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.

Base Sequence↗
PubMed1987

Molecular recognition between oligopeptides and nucleic acids. Monocationic imidazole lexitropsins that display enhanced GC sequence dependent DNA binding.

A series of monocationic lexitropsins, or information-reading oligopeptides, were synthesized to minimize and offset the AT bias for doubly cationic ligands bound in the minor groove of DNA. The compounds possess an N-formyl group in place of the guanidinium moiety normally present in netropsin. By systematic replacement of the N-methylpyrrole groups of the dipeptide with N-methylimidazole, a remarkably high degree of sequence specificity was obtained. One of the compounds having two N-methylimidazole residues was found to exhibit dramatically altered specificity when compared with netropsin and preferred to bind to the sequence 5'-CCGT-3' 3'-GGCA-5'. The structural elements underlying sequence recognition in terms of the model for the netropsin-DNA interaction are presented and discussed.

Base Sequence↗
PubMed2001

Allosteric interaction of minor groove binding ligands with UL9-DNA complexes.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

Allosteric Regulation↗
PubMed2006

DNA intramolecular triplexes containing dT --> dU substitutions: unfolding energetics and ligand binding.

We used a combination of optical and calorimetric techniques to investigate the incorporation of deoxythymidine --> deoxyuridine (dT --> dU) substitutions in the duplex and third strand of the parallel intramolecular triplex d(A(7)C(5)T(7)C(5)T(7)) (ATT). UV and differential scanning calorimetry melting experiments show that the incorporation of two substitutions yielded triplexes with lower thermal stability and lower unfolding enthalpies. The enthalpies decrease with an increase in salt concentration, indirectly yielding a heat capacity effect, and the magnitude of this effect was lower for the substituted triplexes. The combined results indicate that the destabilizing effect is due to a decrease in the level of stacking interactions. Furthermore, the minor groove ligand netropsin binds to the minor groove and to the hydrophobic groove, created by the double chain of thymine methyl groups in the major groove of these triplexes. Binding of netropsin to the minor groove yielded thermodynamic profiles similar to that of a DNA duplex with a similar sequence. However, and relative to ATT, binding of netropsin to the hydrophobic groove has a decreased binding affinity and lower binding enthalpy. This shows that the presence of uridine bases disrupts the hydrophobic groove and lowers its cooperativity toward ligand binding. The overall results suggest that the stabilizing effect of methyl groups may arise from the combination of both hydrophobic and electronic effects.

Binding Sites↗
PubMed1997

A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: a potential mechanism for topoisomerase I poisoning.

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A.T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated "A-tract" conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as "bent." By contrast, the d(GT4A4C)2 duplex contains two 4-bp A.T tracts separated by a TpA dinucleotide step, which should induce a less hydrated "B-like" conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20 degrees C, 5PTB exhibits an approximately 2. 3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4',6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

Base Sequence↗
PubMed1987

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity.

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.

Antineoplastic Agents↗
PubMed2001

Mixed mode of ligand-DNA binding results in S-shaped binding curves.

S-shaped binding curves often characterize interactions of ligands with nucleic acid molecules as analyzed by different physico-chemical and biophysical techniques. S-shaped experimental binding curves are usually interpreted as indicative of the positive cooperative interactions between the bound ligand molecules. This paper demonstrates that S-shaped binding curves may occur as a result of the "mixed mode" of DNA binding by the same ligand molecule. Mixed mode of the ligand-DNA binding can occur, for example, due to 1) isomerization or dimerization of the ligands in solution or on the DNA lattice, 2) their ability to intercalate the DNA and to bind it within the minor groove in different orientations. DNA-ligand complexes are characterized by the length of the ligand binding site on the DNA lattice (so-called "multiple-contact" model). We show here that if two or more complexes with different lengths of the ligand binding sites could be produced by the same ligand, the dependence of the concentration of the complex with the shorter length of binding site on the total concentration of ligand should be S-shaped. Our theoretical model is confirmed by comparison of the calculated and experimental CD binding curves for bis-netropsin binding to poly(dA-dT) poly(dA-dT). Bis-netropsin forms two types of DNA complexes due to its ability to interact with the DNA as monomers and trimers. Experimental S-shaped bis-netropsin-DNA binding curve is shown to be in good correlation with those calculated on the basis of our theoretical model. The present work provides new insight into the analysis of ligand-DNA binding curves.

DNA↗
PubMed2005

Sequence-specific minor groove binding ligands as potential regulators of gene expression in Xenopus laevis oocytes.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent gene activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the DNA-protein interactions following hormone activation. The strategy of artificial regulating of gene activity by sequence-specific minor groove binding ligands is very attractive. We have synthesized and studied the interaction with DNA of bis-linked netropsin derivatives in which two monomers are attached via short linkers in head-to-head and tail-to-tail manners. We have found that cis-diammine-platinum bridged bis-netropsin added to Xenopus oocytes media penetrates cellular and nuclear membrane and binds selectively to the MMTV promoter at the DNA segment that partly overlaps with the site recognized by glucocorticoid receptor. DNase I footprinting studies demonstrate that there are more stronger binding sites for cis-diammine-platinum bridged bis-netropsin on the naked MMTV DNA which are found to be inaccessible for its binding in oocytes.

Animals↗
PubMed1997

Sequence-specific interactions of minor groove binders with the 154 base pair HindIII-RsaI restriction fragment of cDNA of the human Tau 40 protein involved in pathology of Alzheimer's disease.

The DNA minor groove binders netropsin, distamycin and four structurally related bisquaternary ammonium heterocycles (BQA), SN 6999, SN 6570, SN 6132 and SN 6131, were investigated for sequence-specific interactions with the 154 base pair fragment of cDNA of the human Tau 40 protein (h Tau 40 protein), involved in pathology of Alzheimer's disease. The base sequences 5' AATCTT 3', 5' AATATT 3' and 5' TTTCAATCTTTTTATTT 3' were identified as ligand specific binding sites and demonstrate the obvious dA.dT binding preference. Footprinting titration experiments were performed to estimate sequence-specific binding constants (KA). The KA-values were in the order of 10(6)M-1 and dependent on DNA base sequence as well as ligands used. The highest values estimated were for netropsin (KA = 5.0 x 10(6)M-1) and the quinoline derivative SN 6999 (KA = 6.2 x 10(6)M-1) binding to the sequence 5' ATAAT 3'. Microscopic binding constants are determined by the base sequence rather than by the length of dA.dT stretches. In the extended dA.dT run, 5' TTTCAATCTTTTTATTT 3', netropsin and distamycin binding tolerates the presence of two dG.dC base pairs, as indicated by nearly unaffected footprints. In contrast, the failure of BQAs to form footprints demonstrates their significantly decreased binding selectivity.

Alzheimer Disease↗
PubMed1984

Infrared linear dichroism of oriented DNA-ligand complexes prepared with the wet-spinning method.

Oriented DNA films prepared by the wet-spinning technique have been complexed with several ligands: the anthracycline antibiotic violamycin BI, the dipeptide L-carnosine, and the oligopeptide antibiotic netropsin. The formation of the DNA-ligand complexes is accompanied by dramatic changes of the conformational flexibility of DNA. The B-A transition which occurs usually between 80% and 70% relative humidity (RH) is more or less suppressed by the ligands. Violamycin BI at a total ligand per DNA base pair ratio, rt, of approximately 0.03 and L-carnosine at rt approximately 1.5 inhibit the B-A transition of approximately 18 and approximately 0.25 base pairs per ligand molecule, respectively. Netropsin at rt = 0.2 induces a very stable B-DNA even at rather low RH (23%). The total hydration of this complex is significantly higher than for a drug-free DNA film. Netropsin-DNA complexes at rt of 0.02 and 0.01 result in an inhibition of approximately 45 base pairs per drug molecule with respect to the B-A transition.

Aminoglycosides↗
PubMed1995

The binding modes of a rationally designed photoactivated DNA nuclease determined by NMR.

The complex between the rationally designed synthetic DNA cleaving agent netropsin-diazene and the double-stranded DNA oligomer 5'-CGCAAAGGC-3'.5'-GCCTTTTGCG-3' was characterized by two-dimensional NMR spectroscopy in solution. Photolysis of netropsin-diazene bound to DNA generates a trimethylenemethane diradical intermediate that induces single-strand breaks in the DNA. The pi-diyl trimethylenemethane based compounds are a new class of DNA nucleases. We tested the following design criteria: (i) binding of the diazene and subsequent reactive diyl to the DNA, (ii) sequence selectivity in the ligand binding and (iii) prevention of diyl dimerization. Sixteen NOE derived ligand-DNA distance restraints were used to obtain the energy minimized model of the complex. The ligand is bound to the minor groove of the oligomer with the diazene at the 5' end of the A-tract in the predominant conformation of the complex. This form of the complex exchanges with a minor conformation in which the ligand is in the opposite orientation. The DNA maintains a B-form structure. Netropsin-diazene has fulfilled all of the design criteria, binding to the DNA duplex studied in the minor groove of the central AAAA tract in a 1:1 mode, preventing diyl dimerization and other side reactions from occurring.

Base Sequence↗
PubMed1996

The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism.

For the first time, the interaction between a series of small molecules and the TAR RNA of HIV-1 has been investigated by electric linear dichroism (ELD). The compounds tested include the DNA intercalating drugs proflavine and ethidium bromide and an amsacrine-4-carboxamide DNA-threading intercalator as well as the AT-specific DNA minor groove binders netropsin, Hoechst 33258, berenil and DAPI. In all cases except for netropsin, negative reduced dichroism signals were measured in the drug absorption band. In agreement with previous studies, the results indicate that both classical and threading intercalation can occur with the TAR RNA. The ELD data show that the mode of binding of the drugs Hoechst 33258, berenil and DAPI to the TAR RNA is similar to their binding mode in GC-rich regions of DNA and likely involves intercalation into the A-form TAR RNA helix. The wide and shallow minor groove of the TAR RNA is apparently not accessible to DNA minor groove binding drugs such as netropsin. The ELD technique appears uniquely valuable as a means of investigating the interaction of drugs with the TAR RNA.

Antiviral Agents↗
PubMed1998

Use of capillary electrophoresis in the study of ligand-DNA interactions.

Free solution capillary electrophoresis (FSCE) has been used to separate two non-self-complementary 12mer oligonucleotide duplexes: d(AAATTATATTAT).d(ATAA-TATAATTT) and d(GGGCCGCGCCGC).d(GCGGCGCGGCCC). Titration of mixtures of the two oligonucleotides with model intercalators (ethidium bromide andactinomycin D) and minor groove binders (netropsin, Hoechst 33258 and distamycin) has shown the suitability of FSCE as a method to study the sequence selectivity of DNA binding agents. Binding data have shown cooperativity of binding for netropsin and Hoechst 33258 and have provided ligand:DNA binding ratios for all five compounds. Cooperativity of netropsin binding to a 12mer with two potential sites has been demonstrated for the first time. Ligands binding in the minor groove caused changes in migration time and peak shape which were significantly different from those caused by intercalators.

Binding, Competitive↗
PubMed1979

The effect of antibiotics on the T4 polynucleotide ligase catalyzed template dependent polymerization of oligodeoxythymidylates.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.

Anti-Bacterial Agents↗
PubMed1994

Synthesis and antiviral activity of three pyrazole analogues of distamycin A.

The synthesis of three new monopyrazole analogues of the antiviral compound distamycin A is reported. Suitably protected 4-amino-1-methylpyrrole-2-carboxylic acid and 3-amino-1-methylpyrazole-5-carboxylic acid derivatives were chosen as starting materials. The construction of the trimeric polyamide framework was accomplished by assembly of the monomeric precursors under condensing conditions by analogy with our previous methodology, although with significant improvements in some pivotal steps. After chromatographic purification and spectroscopic characterisation, the analogues were assayed for antiviral activity. Compounds 7a-c inhibited vaccinia virus at a concentration similar to or lower than distamycin A and the related antibiotic netropsin. Analogues 7b and 7c exhibited an antiviral effect comparable to those of distamycin A and netropsin against HSV-1 and HSV-2, whereas their antiviral activity against several other viruses including HIV-1 and HIV-2 was somewhat lower. The cellular toxicity of 7a-c toward different host cell types proved to be of similar magnitude or lower than those of distamycin A and netropsin.

Animals↗
PubMed2000

Design and synthesis of novel thiazole-containing cross-linked polyamides related to the antiviral antibiotic distamycin.

A family of naturally occurring oligopeptides includes netropsin, distamycin, anthelvencin, kikumycin B, amidinomycin, and norformycin. Netropsin (I) and distamycin (II) express their biological activities by targeting specific sequences of chemical functionalities in the minor groove of DNA. Both netropsin and distamycin can be regarded as polyamide chains in which each alpha-carbon has been replaced by a five-membered pyrrole ring. The repeat distance in such an augmented polyamide chain is almost the same as the distance from one base pair to the next along the floor of a minor groove within beta-DNA. In this paper we report the synthesis of 16-21 cross-linked polyamides containing a thiazole heterocyclic ring bearing the active functionalites NH(2), NHCHO, or H. 16 and 17 were synthesized by DCC and HOBt catalyzed reaction of 5 with 14 and 15, while the formylation products 18 and 19 were obtained by coupling the formylated 4-methyl-thiazolated acid 6 with 14 and 15. The deaminated compounds 20 and 21 were obtained by the coupling of 5-trichloroacetyl-4-methylthiazole 7 synthesized from 4-methylthiazole. All the six cross-linked polyamides 16-21 were tested for their DNA gyrase inhibition. The studies have shown these polyamides have better sequence recognition and a greater percentage of inhibition than the corresponding monomers. The compound 17 shows complete inhibition of gyrase at 0.5 microM concentration as compared to the naturally occurring distamycin at 1.0 microM.

Antiviral Agents↗
PubMed1995

A camptothecin-resistant DNA topoisomerase I mutant exhibits altered sensitivities to other DNA topoisomerase poisons.

The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.

Alanine↗