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PubMed2000

Synthesis of a netropsin conjugate of a water-soluble epi-quinocarcin analogue: the importance of stereochemistry at nitrogen.

The efficient synthesis of a water-soluble C11a-epi-analogue (6b) of quinocarcin is described. This substance, and a netropsin amide conjugate (8) lack the capacity to inflict oxidative damage on DNA due to the stereoelectronic geometry of their oxazolidine nitrogen atoms. The capacity of these substances to alkylate DNA through the generation of an iminium species has been examined. Both compounds were found to be unreactive as DNA alkylating agents. The results of this study are discussed in the context of previous proposals on the mode of action of this family of antitumor alkaloids.

Alkylating Agents↗
PubMed2000

Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs.

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.

Base Composition↗
PubMed1993

Molecular structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor groove binding drug netropsin.

The molecular structure of the complex between the minor groove binding drug netropsin and the dodecamer d(CGCAAATTTGCG) has been solved and refined by X-ray diffraction analysis to an R-factor of 19.8% and 2.2-A resolution. The drug lies in the narrow minor groove of the B-DNA fragment, covering five of the six A.T base pairs (from A5.T20 to T9.A16). The long six A.T base pair tract allows the drug to bind in a position that optimizes its contacts with the DNA, establishing hydrogen bonds with O2 of thymines and N3 of adenines. The DNA molecule shows a high propeller twist only at the A6.T19 step of the A-tract. Two three-centered hydrogen bonds are observed in the major groove at half of the A-tract.

Base Sequence↗
PubMed1987

Specific activation of transcription initiation by the sequence-specific DNA-binding agents distamycin A and netropsin.

A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted at an early step in the RNA polymerase-promoter interaction. We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.

Base Sequence↗
PubMed1988

Interaction of synthetic analogues of distamycin and netropsin with nucleic acids. Does curvature of ligand play a role in distamycin-DNA interactions?

Distamycin and netropsin, a class of minor groove binding nonintercalating agents, are characterized by their B-DNA and A-T base-specific interactions. To understand the conformational and chemical basis of the above specificities, the DNA-binding characteristics of a novel synthetic analogue of distamycin have been studied. The analogue, mPD derivative, has the requisite charged end groups and a number of potential hydrogen-bonding loci equal to those of distamycin. The difference in the backbone curvatures of the ligands, distamycin, the mPD derivative, and NSC 101327 (another structurally analogous compound), is a major difference between these ligands. UV and CD spectroscopic studies reported here show the following salient features: The mPD derivative recognizes only B-DNA, to which it binds via the minor groove. On the other hand, unlike distamycin, it binds with comparable affinities to A-T and G-C base pairs in a natural DNA. These DNA-binding properties are compared with those reported earlier for distamycin and NSC 101327 [Zimmer, Ch., & Wahnert, U. (1986) Prog. Biophys. Mol. Biol. 47, 31-112]. The backbone structures of these three ligands were compared to show the progressive decrease in curvatures in the order distamycin, mPD derivative, and NSC 101327. The plausible significance of the backbone curvature vis-à-vis the characteristic B-DNA and AT-specific binding of distamycin is discussed. To our knowledge, this is the first attempt (with a model synthetic analogue) to probe the possible influence of backbone curvature upon the specificity of interactions of the distamycin class of groove-binding ligands with DNA.

Circular Dichroism↗
PubMed1980

Interaction of netropsin and distamycin with deoxyribonucleic acid: electric dichroism study.

We report dichroism and equilibrium binding studies of netropsin (Net) and distamycin A3 (Dist) binding to deoxyribonucleic acid (DNA). We show that at low degrees of binding (r) to calf thymus DNA, Net induces a considerable increase in the apparent DNA length (14 A/drug molecule bound), closely analogous to the results reported earlier for Dist. In addition, we show that chicken erythrocyte DNA shows length changes similar to those of calf thymus DNA upon distamycin binding. DNA length reaches a maximum at 1 bound drug/20-30 base pairs and then decreases to its initial value by r = 0.1. This effect is not seen for two other DNAs with nearly identical A + T base pair content and may therefore arise from the details of base sequence or base modification in eukaryotic DNA. We also show that Dist binding to calf thymus DNA at low r values is positively cooperative and shows a DNA affinity which is primarily nonionic. We demonstrate that independent of the DNA to which they are bound, the Net and Dist transition moments are inclined by 43 +/- 3 degrees from the helix axis, consistent with the idea that both drugs bind inside and parallel to the DNA small groove. From dichroism measurements, we show that the conformational change induced in calf thymus DNA by Dist does not kink or bend the helix and does not substantially alter the average inclination of the bases. Finally, we outline a statistical mechanical theory for calculation of binding isotherms when binding is coupled to a DNA structural change.

Animals↗
PubMed1998

Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin.

We have investigated the thermodynamic aspects of the ligand binding to chromatin, using isothermal titration calorimetry. Two classical DNA ligands were used: an intercalator, ethidium bromide, and a minor groove binder, netropsin. Stoichiometry, affinity constant, and thermodynamic parameters were determined at various salt concentrations and different temperatures. The effect of ionic strength was analyzed according to the Record theory applied to chromatin. We also compared the binding parameters on naked DNA, H1/H5-depleted chromatin, and chromatin. We demonstrated that the presence of histones on DNA still allows the ligand binding that takes place according to a simple one single-site model. For both ligand types, the thermodynamic driving force is enthalpic and the association is characterized by a somewhat weaker affinity and more scattered ligand distribution than on naked DNA. The ligand affinity is weakly altered by the salt-induced compaction of the chromatin and the binding is accompanied by a release of one counterion per ligand molecule. The temperature-dependent studies revealed the existence of a small heat capacity change associated with ligand binding to chromatin, together with an enthalpy-entropy compensation that maintains the free energy constant over the investigated temperature range.

Animals↗
PubMed1989

Synthesis, DNA binding, and biological evaluation of synthetic precursors and novel analogues of netropsin.

A series of oligopeptides have been synthesized that are structurally related to the natural agent netropsin. The binding constants to double-stranded polynucleotides as well as the cytostatic activity against both murine human tumor cell lines and the in vitro activity against a range of DNA and RNA viruses have been determined for these novel compounds and some of their synthetic precursors. 1-Methyl-5-nitropyrrole-2-carboxylic acid methyl ester (4), N-[[1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrol-2- yl]carbonyl]-L-alanine tert-butyl ester (28), and N-[[1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrol-2- yl]carbonyl]-L-alanyl-L-alanine tert-butyl ester (29) showed modest inhibitory effect on tumor cell proliferation (CD50 = 26-85 micrograms/mL). Of all the compounds that were evaluated, 28 proved the most potent antiviral agent. It was inhibitory to parainfluenza-3 virus and Coxsackie virus B4 in Vero cells at a concentration of 20 micrograms/mL.

Animals↗
PubMed1986

Structure-activity relationship of novel oligopeptide antiviral and antitumor agents related to netropsin and distamycin.

A group of oligopeptides have been synthesized that are structurally related to the natural antiviral antitumor agents netropsin and distamycin. Cytostatic activity against both human and murine tumor cell lines as well as their in vitro activity against a range of viruses is reported. The biological activity of these agents is discussed both in terms of their structural differences and, in particular, in relation to their observed base- and sequence-dependent minor-groove binding to duplex oligonucleotides.

Animals↗
PubMed1978

Conformational features of distamycin-DNA and netropsin-DNA complexes by Raman spectroscopy.

The binding of distamycin and netropsin to duplex DNA has been studied by Raman spectroscopy. Several changes occur in the Raman spectra of these drugs upon binding DNA. These changes were analyzed by assigning specific motions to the observed Raman bands through the use of molecular subunits of the drugs and normal mode calculations. Our analysis indicates that pyrrole ring and peptide group vibrations are altered upon binding to DNA. The environments of the pyrrole ring methyl groups are not affected by the binding. These data provide physical evidence consistent with a binding model in which the methyl groups on the pyrroles project away from the DNA and the peptide N-H groups form hydrogen bonds with the DNA.

Animals↗
PubMed1983

Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.

Animals↗
PubMed1992

Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin.

The designed peptide 1-methylimidazole-2-carboxamide netropsin (2-ImN) binds specifically to the sequence 5'-TGACT-3'. Direct evidence from NMR spectroscopy is presented that this synthetic ligand binds DNA as a 2:1 complex, which reveals that the structure is an antiparallel dimer in the minor groove of DNA. This is in contrast to the 1:1 complexes usually seen with most crescent-shaped minor groove binding molecules targeted toward A+T-rich tracts but reminiscent of a dimeric motif found for distamycin at high concentrations. These results suggest that sequence-dependent groove width may play an important role in allowing an expanded set of DNA binding motifs for synthetic peptides.

Base Sequence↗
PubMed1985

Structure of poly(dA).poly(dT) is not identical to the AT rich regions of the single crystal structure of CGCGAATTBrCGCG. The consequence of this to netropsin binding to poly(dA).poly(dT).

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA).poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA).poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA).poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations-dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA).poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).

Molecular Structure↗
PubMed1993

Abolition of intrinsically bent DNA structure components in AT clusters by netropsin interaction; titration viscometric investigations.

It is argued that the enhancement of the apparent DNA contour length by the specifically binding non-intercalating drug netropsin (Nt) (Reinert et al., NAR 9, 2335, 1981) at very low Nt/DNA-phosphate ratios essentially is the result of an abolition of periodically arranged intrinsic helix bends in A.T rich tracts of base pairs. In the preceding paper the existence of pronounced DNA tertiary structure components has been postulated for (two species of) natural eukaryotic DNA. The resulting model suggests local apparent solenoid-related DNA tertiary structure components at high sodium ion concentration cs, partly/totally molten out at 45/60 C. With decreasing cs the tertiary structure components have been found to be gradually reduced, at least below cs = 0.010 M, as titration viscometrically revealed by a gradual rise of the apparent DNA contour length (Reinert et al., JBSD 9, 537, 1991). Hence, we performed titration viscometric analyses about Nt interaction with calf thymus DNA (ctDNA) at cs = 0.075 M, 0.010 M and 0.004 M Na+. The concomitant DNA conformational changes are quantitatively described in terms of the relative changes of both DNA persistence length and hydrodynamically operative apparent DNA contour length for the three first resolved interaction modes below a Nt/DNA-P ratio of 0.03. These experiments, together with previous respective analyses at cs = 0.20 M Na+ and different temperatures (l.c.), suggest that those DNA sites binding Nt most strongly predominantly are responsible for the formation of solenoid-related DNA tertiary structure components. Most probably these are A tract-containing sequences. As the essential factor for their apparent elongation effect at low Na+ concentrations, a gradual alteration of the number of base pairs per helix turn seems to occur below cs = 0.010 M Na+ and, concomitantly, a change in phasing between intrinsic helix bends and helix screw.

Adenine↗
PubMed1994

Modulation of DNA supercoiling by interaction with netropsin and other minor groove binders.

The assay of DNA unwinding by ethidium, followed by sedimentation velocity techniques, was applied to complexes of supercoiled plasmid DNA with different non-intercalating drugs which strongly and sequence-specifically bind to DNA. Compared with the behaviour of naked DNA, most of the complexes exhibit an increase in the critical EB/nucleotide binding ratio associated with the principal minimum in the sedimentation profile. Using netropsin (Nt) as the paradigm of the minor groove binders investigated, the drug-induced alterations in various structural parameters of both the relaxed and supercoiled form of DNA are described. Whereas winding number, helical repeat (both being defined with reference to a surface normal), and linking number of the superhelical DNA remain constant in our experiments, its twist number, surface twist, number of superhelical turns as well as the absolute values of linking number difference, superhelix density, and writhing number increase on binding of Nt. Correspondingly, compared with the naked relaxed DNA a higher linking number (or twist number, or winding number), a higher average duplex winding angle and a lower helical repeat have to be assigned to the relaxed Nt-DNA complex. The various minor groove binders investigated were found to differ considerably in their efficiency to alter the structure of supercoiled DNA.

Base Sequence↗
PubMed1995

DNA interaction of the imidazole-containing lexitropsin ImPy: titration viscometric study in comparison to netropsin.

The imidazole (Im) containing lexitropsin ImPy related to netropsin (Nt) is a sequence reading DNA ligand which, in contrast to Nt, permits binding to a GC base pair. The ImPy induced DNA conformational changes differ significantly from those induced by Nt as monitored by titration viscometry, although interaction modes have also been resolved with boundaries at the same ligand to DNA phosphate ratio, r. Evidently ImPy covers similar binding sites (in the same sequence) as Nt for natural calf thymus DNA at r < 0.023. This result suggests that the preferred binding sites of ImPy are A tracts (cf. K.E.R. JBSD 9(1993) 973), in agreement with previous data. The respective DNA coil expansion, most probably caused by unbending (l.c.), is similar but smaller compared to the Nt-DNA interaction. These results again suggest that, at low r values, the van der Waals interaction in the narrowed minor groove of AT clusters provides a dominating energy contribution to ImPy binding. At r > 0.03 the DNA coil expansion increases to extremely high values in that r range where Nt binding (to mixed AT/GC sequences) induces no effect at all owing to steric hindrance with the amino group of guanine. On the basis of many quantitative results for the Nt-DNA systems these effects can be understood in terms of an unbending of intrinsic helix bends (l.c.). They are of considerable interest in connection with the ability of such compounds to influence the direction of the local regulatory relevant DNA curvature.

Animals↗
PubMed1996

Reversal of the Z- to B-conformation of poly(dA-dT) center dot poly(dA-dT) induced by netropsin and distamycin A.

Poly(dA-dT) center dot poly(dA-dT) which adopts the Z-form at 5 M NaCl in presence of 95 mM Ni2+ions is reversed to the B-conformation by the nonintercalating drugs netropsin (Nt) and distamycin A (Dst). The drug-induced reversal from the Z-to B-form of poly(dA-dT) center dot poly(dA-dT) is evidenced by CD spectral changes at characteristic wavelengths around 295 nm and 248 nm. The drug-induced conformational transition is accompanied by a slow kinetic process. The results suggest the preference of these AT-specific drugs for the B-form and the inability of Nt and Dst to form a stable complex with the Z-form of poly(dA-dT) center dot poly(dA-dT).

Bacterial Proteins↗
PubMed1996

Binding of Net-Fla, a netropsin-flavin hybrid molecule, to DNA: molecular mechanics and dynamics studies in vacuo and in water solution.

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A. T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the drug-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.

Binding Sites↗