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PubMed1995

X-ray structures of the B-DNA dodecamer d(CGCGTTAACGCG) with an inverted central tetranucleotide and its netropsin complex.

The crystal structures of the B-DNA dodecamer d(CGCGTTAACGCG) duplex (T2A2), with the inverted tetranucleotide core from the duplex d(CGCGAATTCGCG) [A2T2, Dickerson & Drew (1981). J. Mol. Biol. 149, 761-768], and its netropsin complex (T2A2-N) have been determined at 2.3 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), unit-cell dimensions of a = 25.7, b = 40.5 and c = 67.0 A, for T2A2 and a = 25.49, b = 40.87, c = 67.02 A for T2A2-N and are isomorphous with A2T2. The native T2A2 structure, with 70 water molecules had a final R value of 0.15 for 1522 reflections (F > 2sigma), while for the netropsin complex, with 87 water molecules, the R value was 0.16 for 2420 reflections. In T2A2, a discontinuous string of zig-zagging water molecules hydrate the narrow A.T minor groove. In T2A2-N, netropsin binds in one orientation in the minor groove, covering the TTAA central region, by displacing the string of waters, forming the majority of hydrogen bonds with DNA atoms in one strand, and causing very little perturbation of the native structure. The helical twist angle in T2A2 is largest at the duplex center, corresponding to the cleavage site by the restriction enzymes HpaI and HincII. The sequence inversion AATT-->TTAA of the tetranucleotide at the center of the molecule results in a different path for the local helix axis in T2A2 and A2T2 but the overall bending is similar in both cases.

Journal Article↗
PubMed2002

Carbocyclic analogues of netropsin and distamycin: DNA-binding properties and inhibition of DNA topoisomerases.

Inhibition of DNA topoisomerase and DNA-binding properties of a series of benzene-containing and C-terminus-modified analogues of distamycin and netropsin are described. These analogues contain two or three benzene units, respectively. Dibenzene analogues did not inhibit the topoisomerases, I and II. In this case, relaxation of DNA was inhibited with tribenzene analogues. Data from the ethidium displacement assay showed that these compounds were able to bind in the minorgroove binding mode in AT sequences of DNA. Molecular modelling experiments were performed to rationalize the lower binding affinity of tribenzene analogues of distamycin and netropsin, 3 and 4, compared to dibenzene analogues, 1 and 2. The superior DNA-binding afforded by 1 and 2 in comparison to 3 and 4 results from their more effective penetration into the minor groove of DNA and smaller perturbation of molecular structure upon complex formation.

Animals↗
PubMed1995

The osmotic sensitivity of netropsin analogue binding to DNA.

The binding of a netropsin analogue to random sequence DNA, monitored by CD, is seen dependent on the concentration of neutral solutes. The binding free energy decreases linearly with solute osmolal concentration and the magnitude of the effect is insensitive to the chemical identity of the solute for betaine, sorbitol, and triethylene glycol. These solutes appear to modulate binding through their effect on water activity and changes in the hydration of the drug and DNA in the complex reaction, not through a direct interaction with the reactants or the product. The dependence of binding constant on solute concentration can be interpreted as an additional binding of some 50-60 extra solute excluding water molecules by the complex. A water sensitivity of drug binding is further seen from the dependence of binding constants on the type of anion in solution. Anions in the Hofmeister series strongly affect bulk water free energies and entropies. The differences in netropsin analogue binding to DNA with Cl-, F-, and ClO4- are consistent with the effect observed with neutral solutes. The ability to measure changes in water binding associated with a specific DNA interaction is a first step toward correlating changes in hydration with the strength and specificity of binding.

Binding Sites↗
PubMed1987

The superstructure of chromatin and its condensation mechanism. IV. Enzymatic digestion, thermal denaturation, effect of netropsin and distamycin.

Changes in the structure of chicken erythrocyte chromatin fibres at low ionic strength resulting from enzymatic digestion, thermal denaturation and binding of Netropsin and Distamycin were monitored by synchrotron X-ray solution scattering. Digestion with micrococcal nuclease confirms the previous assignment of the 0.05 nm-1 band to an interference between nucleosomes with an average distance of 23 nm. The results of thermal denaturation indicate that above 40 degrees C there is a progressive increase of the internucleosomal distance and that above 60 degrees C the characteristic structure of the chromatin fibre is destroyed. Binding of Netropsin and Distamycin also results in an increase of the internucleosomal distance which can be estimated to correspond to about 0.2 nm/mol.

Animals↗
PubMed1977

Inhibition of the development of Q-bands on human chromosomes by netropsin.

Netropsin, an oligopeptide-type basic antibiotic, having exclusively A-T-specific DNA-binding affinity and situating itself into the minor groove of the double helix, represses the development of Q-bands if human chromosome preparations are treated with it before quinacrine mustard staining. The most probable interpretation of this effect is that netropsin interferes with the intercalation of the dye molecules. It is assumed this phenomenon supports the hypothesis that quinacrine mustard binds preferentially to A-T-rich sequences of DNA in the metaphase chromosomes.

Adenosine↗
PubMed1993

Netropsin inhibits the increase of intracellular Ca(2+)-dependent serine proteinase activity in sporulating Bacillus megaterium.

Netropsin suppressed the increase of intracellular proteolytic activity when added to B. megaterium incubated in a sporulation medium. The inhibited enzyme was a Ca(2+)-dependent serine proteinase. Sporulation and protein turnover in later sporulation phases were inhibited as well. Different concentrations of netropsin affected various aspects of protein catabolism differently.

Bacillus megaterium↗
PubMed1979

Netropsin, a DNA-binding oligopeptide structural and binding studies.

The crystal structure of netropsin, an oligopeptide which binds to DNA, has been determined. The molecule is bowed with the amide groups on the concave side, and the carbonyl and methyl groups on the convex side. The amide groups participate in extensive hydrogen bonding with water molecules; the charged amino end groups interact with the sulfate anions. Binding of netropsin to poly(dA) . poly(dT) under conditions of different ionic strength was also studied. Utilizing the crystallographic as well as the binding data, it is possible to build a model which explains the specificity of this antibiotic.

Circular Dichroism↗
PubMed1984

Enhancement of S1 nuclease-susceptibility of negatively superhelical DNA by netropsin.

It was evidenced that the antibiotic netropsin enhances the single-strand-specific nuclease S1-susceptibility of negatively superhelical DNA. In contrast, an intercalating drug inhibited S1 action on the superhelical DNA. Negatively superhelical DNA is known to possess several (or a number of) unbasepaired sites sensitive to S1 cleavage. S1 cleaves generally the DNA once at these sites to result in production of the full-length linear form. However netropsin-bound DNA had a tendency to be cleaved by S1 simultaneously at plural sites producing several species of linear DNAs smaller than full-length size.

DNA, Viral↗
PubMed1992

Biological activity and molecular interaction of a netropsin-acridine hybrid ligand with chromatin and topoisomerase II.

A hybrid molecule, which combines an anilinoacridine chromophore related to the antitumour drug amsacrine (m-AMSA) and a bispyrrole moiety analogous to the antiviral agent netropsin, has been examined for its ability to bind chromatin and to modulate the activity of topoisomerase II. The results show that the presence of histones does not alter the bimodal DNA binding process. Intercalation of the acridine and groove binding of the netropsin part of the drug are both observed with chromatin preparations. Moreover, the hybrid has a clear topoisomerase II-DNA cleavable complex-inducing activity close to that of m-AMSA. The role of the two parts of the hybrid ligand is discussed in relation to ternary complex formation. Two cell lines (L1210 leukemia and MCF7 mammary carcinoma) were compared in their sensitivity to the tested ligand. The drug, which appears to be an efficient growth inhibitor of leukemic cells in vitro, reveals moderate activity against P388 leukemia in vivo. The biological activity of the hybrid may derive from a mechanism that involves DNA binding and topoisomerase II inhibition. This study demonstrates that agents which intercalate and bind to the minor groove of DNA simultaneously represent a new class of drugs interfering with topoisomerase II and provide opportunities for the development of new antitumour agents.

Amsacrine↗
PubMed1989

Variation of DNA sequence specificity of DNA-oligopeptide binding ligands related to netropsin: imidazole-containing lexitropsins.

CD binding studies of nonintercalative oligopeptides related to netropsin, named lexitropsins, have been carried out with synthetic duplex DNAs and natural DNA. While netropsin possesses a high dA.dT sequence specificity, these ligands show a progressive lowering of the ability to bind to dA.dT basepairs in DNA and a dramatic reduction of the sequence specificity seen at high salt concentration due to a replacement of pyrrole moieties by imidazoles. This variation in DNA sequence specificity of lexitropsins is mirrored in corresponding large differences in the template inactivation of poly(dA-dT).poly(dA-dT) in the RNA polymerase reaction by these drugs. The presence of imidazole permits binding of the oligopeptide to dG.dC pairs, which is most effective for the triimidazole peptide. Results at increasing salt concentration reveal, however, that a tight binding to pure dG.dC sequences does not occur. A proper sequence containing dG.dC and dA.dT pairs is supposed to be required for a higher specificity. The CD data accord well with previously reported melting studies and are in favor of recent theoretical results suggesting that the diminished AT preference may be due to an increase in the complexation energy with the dG.dC pairs.

Base Composition↗
PubMed1995

Netropsin and spermine conjugates of a water-soluble quinocarcin analog: analysis of sequence-specific DNA interactions.

BACKGROUND: Quinocarcin is the simplest of the bioxalmycin/naphthyridinomycin/tetrazomine/saframycin class of anti-tumor antibiotics, which damage DNA in a process that is inhibited by superoxide dismutase (SOD). The oxazolidine moiety of this class of anti-tumor antibiotics undergoes a redox self-disproportionation reaction of the Cannizzaro type. The reaction is proposed to proceed via an intermediate carbon-centered radical, which then reduces molecular oxygen to give superoxide. We set out to determine whether the DNA-cleavage properties of these anti-tumor antibiotics could be retained in less complex analogs of quinocarcin. RESULTS: A totally synthetic, water-soluble analog of quinocarcin has been prepared. This analog produced superoxide, but had considerably reduced ability to cleave supercoiled circular DNA compared to quinocarcin or tetrazomine. When conjugated to the DNA-binding molecule spermine, however, it cleaved DNA as effectively as quinocarcin at less than 1/10 the concentration. A conjugate with netropsin displayed selective cleavage around the sequence 5'-d(ATTT)-3'. Molecular modeling of the interaction between the conjugate and DNA, together with the pattern of cleavage, indicates that a non-diffusable oxidant is involved in sequence-selective DNA cleavage. The spermine conjugate displayed weak antimicrobial activity. CONCLUSIONS: Knowledge of the stereoelectronic requirements for superoxide production by quinocarcin has allowed us to design a structurally less complex analog which has many of the same physical properties, including water solubility, the ability to produce superoxide and the ability to cleave DNA. Covalently attaching known DNA-binding molecules to this analog gave a compound that produced sequence-specific DNA damage. Our results suggest that a mechanism other than superoxide production can mediate DNA damage by the netropsin conjugate.

Aerobiosis↗
PubMedDate not supplied

Sequence-recognition and cleavage of DNA by a netropsin-phenazine-di-N-oxide conjugate.

We report the synthesis, DNA-binding and cleaving properties, and cytotoxic activities of R-128, a hybrid molecule in which a bis-pyrrolecarboxamide-amidine element related to the antibiotic netropsin is covalently tethered to a phenazine-di-N-oxide chromophore. The affinity and mode of interaction of the conjugate with DNA were investigated by a combination of absorption spectroscopy, circular dichroism, and electric linear dichroism. This hybrid molecule binds to AT-rich sequences of DNA via a bimodal process involving minor groove binding of the netropsin moiety and intercalation of the phenazine moiety. The bidentate mode of binding was evidenced by linear dichroism using calf thymus DNA and poly(dA-dT).(dA-dT). In contrast, the drug fails to bind to poly(dG-dC).poly(dG-dC), because of the obstructive effect of the guanine 2-amino group exposed in the minor groove of this polynucleotide. DNase I footprinting studies indicated that the conjugate interacts preferentially with AT-rich sequences, but the cleavage of DNA in the presence of a reducing agent can occur at different sequences not restricted to the AT sites. The main cleavage sites were detected with a periodicity of about 10 base pairs corresponding to approximately one turn of the double helix. This suggests that the cleavage may be dictated by the structure of the double helix rather than the primary nucleotide sequence. The conjugate which is moderately toxic to cancer cells complements the tool box of reagents which can be utilized to produce DNA strand scission. The DNA cleaving properties of R-128 entreat further exploration into the use of phenazine-di-N-oxides as tools for investigating DNA structure.

Animals↗
PubMed1988

Determination of netropsin-DNA binding constants from footprinting data.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

Base Composition↗
PubMed1988

Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting.

A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka (25 degrees C) = 1.0 X 10(5) and 2.0 X 10(5) M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.

Binding Sites↗
PubMed2003

Evidence in Escherichia coli that N3-methyladenine lesions and cytotoxicity induced by a minor groove binding methyl sulfonate ester can be modulated in vivo by netropsin.

The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate. We report herein the relationship between the in vivo formation of N3-methyladenine and toxicity in wild-type and base excision repair defective mutant Escherichia coli. In addition, it is demonstrated that both N3-methyladenine adduction and cytotoxicity can be inhibited in vivo with netropsin, a potent competitive inhibitor of binding of lex to DNA. The results show a clear relationship between the levels of N3-methyladenine and toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome. For methyl methanesulfonate, which does not sequence selectively methylate DNA, a relationship between the formation of N3-methyladenine and toxicity is also observed. However, netropsin affects neither the level of N3-methyladenine nor the toxicity of methyl methanesulfonate in E. coli.

Adenine↗
PubMed1989

Novel linked antiviral and antitumor agents related to netropsin and distamycin: synthesis and biological evaluation.

A group of oligopeptides have been synthesized which are structurally related to the natural antiviral antitumor antibiotics netropsin and distamycin bearing two such moieties linked by polymethylene bridges. Cytostatic activity against both human and murine tumor cell lines and their in vitro activity against a range of viruses are reported. Enhanced antiviral activity was obtained against vaccinia virus. As a result of the introduction of the polymethylene linkers [( CH2)n, with n = 1, 2, and 6-8], both the antitumor and antivaccinia virus activity was markedly enhanced, relative to that of the parent compounds netropsin and distamycin. The biological activity of these agents is discussed both in terms of their structural differences and in relation to their minor groove binding to duplex DNA.

Animals↗
PubMed2004

Synthesis and antimicrobial activity of some netropsin analogues.

Nine novel lexitropsins were synthesized by linking two netropsin-like moieties through three different dicarboxylic acids; 9,10-dihydro-2,7-phenanthrenedicarboxylic acid; [(3-[[(carboxymethyl)amino]carbonyl]benzoyl)amino]acetic acid and indole-2,5-dicarboxylic acid. The netropsin residues were modified by the use of N-isopentylpyrrole, 5-methylthiophene or 5-isopropylthiazole heterocyclic building blocks in place of the usual N-methylpyrrole. The compounds were tested against five gram-positive bacteria: Staphylococcus aureus, Streptomyces faecalis, methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Mycobacterium fortuitum, three gram-negative bacteria: Klebsiella aerogenes, Proteus vulgaris, Escherichia coli and three fungi: Aspergillus niger, Candida albicans and Aspergillus nidulans. Some of the compounds showed significant inhibitory effects on the growth of the microorganisms.

Anti-Infective Agents↗
PubMed1992

Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.

Base Sequence↗