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PubMedDate not supplied

Molecular recognition between oligopeptides and nucleic acids: DNA binding specificity of a series of bis netropsin analogues deduced from footprinting analysis.

A series of tether-linked bis netropsins have been synthesized in order to assess the phasing problem, which arises because of the lack of dimensional correspondence between oligopeptides and oligonucleotides in DNA binding characteristics. The consequences of incorporating variable-length flexible and rigid tethers [poly(methylene), Z and E ethylene, m- and p-phenylene] between the two netropsin-like moieties on the DNA binding properties were assessed by DNase I footprinting. The conformational freedom associated with two netropsins linked by a flexible methylene tether allows ligand binding in both a mono- and bidentate fashion, with bidentate binding requiring a minimum linker length of (CH2)3. For compounds possessing rigid tethers, for example, cis and trans ethylene moieties, the cis geometry excludes bidentate ligation while the trans structure favors it. Bis netropsins possessing aryl linking groups have reduced DNA binding affinities. This is most plausibly due to the aryl groups, which are not coplanar with the netropsin moieties, thus blocking the ligand from penetrating deeply into the minor groove of DNA.

Base Sequence↗

PubMed1985

The molecular origin of DNA-drug specificity in netropsin and distamycin.

X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.

Base Sequence↗

PubMed1990

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.

Base Sequence↗

PubMed1992

The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).

Animals↗

PubMedDate not supplied

Effect of netropsin on one-electron oxidation of duplex DNA.

The effect of netropsin on the oxidative reactions of duplex DNA was examined. One-electron oxidation of DNA creates a radical cation that migrates through duplex DNA and reacts primarily at GG steps. Netropsin is a dication that specifically binds primarily by hydrogen bonding in the minor groove at sites that have four or more contiguous A.T base pairs. We showed that the oxidation potential of netropsin is less than that of any of the four nucleobases. We find that netropsin quenches the oxidative reactions of DNA independent of whether it is specifically bound. Within the Perrin model of static quenching, a netropsin within a rather large fixed volume around the DNA is an effective quencher.

DNA↗

PubMed1986

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution.

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.

Base Sequence↗

PubMed1995

Design of composite drug molecules: mutual effects on binding to DNA of an intercalator, amsacrine, and a minor groove binder, netropsin.

A variety of spectroscopic and biochemical techniques have been employed to investigate the extent to which binding to DNA of an intercalator (amsacrine or its 4-carboxamide derivative SN16713) affects the binding of netropsin, a minor groove-targeted ligand, and vice versa. In general, rather little mutual interference has been found and the binding of one drug is compatible with binding of the other. The anilinoacridines exert little or no effect on the positioning of netropsin in the minor groove, judged by circular dichroism spectroscopy and electric linear dichroism, whereas netropsin has a perceptible effect on the intercalative binding of amsacrine, but not that of SN16713. Neither acridine drug prevents the netropsin-induced Z-->B structure reversion observed with poly(dG-dC).poly(dG-dC) in buffer containing 60% ethanol. The kinetics of dissociation of any one drug from its DNA complex are affected little, if at all, by the simultaneous presence of the other. Footprinting experiments with the several drugs singly or in combination reveal a certain amount of mutual interference, but the selective recognition of AT-rich sequences by netropsin tends to dominate the recognition pattern and is largely maintained in the presence of a considerable excess of amsacrine or its 4-carboxamide derivative.

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↗

PubMed1975

The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid.

The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other. Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites. Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs. The binding of actinomycin to DNA was decreased by the presence of netropsin. Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding. Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain. Supercoiled DNA gave the same result as linear DNA. These results imply that DNA can absorb alterations in conformation within a short distance.

Anti-Bacterial Agents↗

PubMed2005

Cloning of the netropsin resistance genes from Streptomyces flavopersicus NRRL 2820.

Streptomyces flavopersicus NRRL 2820 (synonym: Streptomyces netropsis DSM40093) is resistant to the N-methylpyrrole-containing oligopeptide antibiotic netropsin. A 9.38 kb DNA-fragment was isolated from a genomic library of Streptomyces flavopersicus using an Escherichia coli-Streptomyces lividans shuttle vector which enables S. lividans to grow on netropsin-containing agar plates. By subcloning, the resistance was conferred to a 5.9 kb Eco RV fragment. DNA sequence analysis of this Eco RV fragment revealed two open reading frames (netP1 , 1556 bp and netP2 , 1773 bp). The deduced proteins share significant similarity to each other (27% identity) and to the large family to ABC-type multidrug resistance proteins. In each protein a conserved transmembrane and ATP binding domain was identified. Deletion analysis showed that both proteins are necessary for netropsin resistance indicating that the proteins form a heterodimeric ABC-transporter exporting netropsin.

Adenosine Triphosphate↗

PubMed1985

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G.

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.

Base Sequence↗

PubMed1995

Refinement of netropsin bound to DNA: bias and feedback in electron density map interpretation.

The X-ray crystal structure of the complex of the B-DNA dodecamer CGCGAATTCGCG with the antitumor drug netropsin has been reexamined to locate the drug accurately for computer-based drug design. The optimum solution is with the drug centered in the AATT region of the minor groove, making three good bifurcated hydrogen bonds with adenine N3 and thymine O2 atoms along the floor of the groove. Pyrrole rings of netropsin are packed against the C2 positions of adenines, leaving no room for the amine group of guanine and, hence, providing a structural rationale for the A.T specificity of netropsin. An alternative positioning in which the drug is shifted along the minor groove by ca. one-half base pair step is rejected on the basis of free R factor calculations and the appearance of the original drug-free difference maps. Final omit maps, although of more pleasing appearance, are not a dependable means of discriminating between right and wrong structures. The shifted alternative drug position ignores potential hydrogen bonding along the floor of the groove, provides no explanation for netropsin's observed A.T specificity, and is contradicted by NMR results [Patel, D. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6424].

Base Sequence↗

PubMed1989

Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment.

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

Base Composition↗

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↗

PubMedDate not supplied

Development and validation of empirical force field parameters for netropsin.

The netropsin molecule preferentially binds to the four consecutive A.T base pairs of the DNA minor groove and could therefore inhibit the expression of specific genes. The understanding of its binding on a molecular level is indispensable for computer-aided design of new antitumor agents. This knowledge could be obtained via molecular dynamics (MD) and docking simulations, but in this case appropriate force field parameters for the netropsin molecule should be explicitly defined. Our parametrization was based on the results of quantum chemical calculations. The resulting set of parameters was able to reproduce bond lengths, bond angles, torsional angles of the ab initio minimized geometry within 0.03 A, 3 deg and 5 deg, respectively, and its vibrational frequencies with a relative error of 4.3% for low and 2.8% for high energy modes. To show the accuracy of the developed parameters we calculated an IR spectrum of the netropsin molecule using MD simulation and found it to be in good agreement with the experimental one. Finally, we performed a 10 ns long MD simulation of the netropsin-DNA complex immersed in explicit water. The overall complex conformation remained stable at all times, and its secondary structure was well retained.

Algorithms↗

PubMed1985

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers.

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.

Binding Sites↗

PubMed2002

Experimental studies on the nature of bonding of DNAbipyridyl-(ethylenediamine)platinum(II) and DNAnetropsin complexes in solution and oriented wet-spun films.

The stability of complexes of NaDNA with bipyridyl- (ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)](2+)) and with netropsin has been studied using two techniques: (i) ultraviolet (UV) melting experiments were done on NaDNA* [(bipy)Pt(en)](2+), showing that the [(bipy)Pt(en)](2+) ligand stabilizes the DNA double helix structure; and (ii) swelling measurements (via optical microscopy) as a function of relative humidity were done on wet-spun oriented films of NaDNA*[(bipy)Pt(en)](2+) and of NaDNA*netropsin. The swelling data shows that an irreversible transition of the films occurs at high relative humidity, first for the NaDNA*netropsin, then for pure NaDNA, and lastly for the NaDNA*[(bipy)Pt(en)](2+). These results are indicative that the [(bipy)Pt(en)](2+) complex stabilizes the intermolecular bonds which mediate the film swelling characteristics. A model is suggested for the binding of [(bipy)Pt(en)](2+) to DNA to explain why the swelling experiments show this ligand as increasing the intermolecular bond strength between the DNA double helices, while netropsin decreases this degree of stabilization.

2,2'-Dipyridyl↗

PubMed1974

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers.

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) . poly (dT), poly (dA-dT) . poly(dA-dT) and poly (dI-dC) . poly (dI-dC) but poorly, if at all, to poly (dG) . poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K(a)=2.9 . 10(5) M(-1)); corresponding values for distamycin A are one site per 6.1 nucleotides with K(a)= 11.6 . 10(5) M(-1). Binding sites apparently involve predominantly A.T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3 degrees per bound antibiotic molecule.

Animals↗