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PubMed1987

Inhibition of estrous behavior in rats by intrahypothalamic application of agents that disrupt nuclear binding of estrogen-receptor complexes.

This study tested the hypothesis that estrogen facilitation of reproductive behavior in female rats requires the binding of estrogen-receptor complexes to the genomic components of hypothalamic cell nuclei. Female rats were implanted stereotaxically with bilateral guide cannulae aimed at the ventromedial nucleus of the hypothalamus (VMH). Animals were ovariectomized following recovery from the implant surgery and randomly assigned to receive one of four drug treatments: actinomycin-D, ethidium bromide, netropsin, or 4', 6-diamidino-2-phenylindole. Each female received at least two tests for estrous behavior 48 hr after estrogen priming. On one test, drug-filled cannulae were lowered into the VMH 1 hr prior to a subcutaneous injection of 2-3 micrograms of estradiol benzoate (EB); on the other test blank cannulae were inserted 1 hr prior to EB treatment. Intracranial administration of all four compounds, which disrupt estrogen-receptor binding to hypothalamic nuclei, inhibited both the quantity and the quality of lordosis responses to systemic injections of EB. The results support the hypothesis that specific receptor interactions with the genome of hypothalamic cells mediate estrogen facilitation of estrous behavior in female rats.

Animals↗
PubMed1987

Binding of Hoechst 33258 to the minor groove of B-DNA.

An X-ray crystallographic structure analysis has been carried out on the complex between the antibiotic and DNA fluorochrome Hoechst 33258 and a synthetic B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G. The drug molecule, which can be schematized as: phenol-benzimidazole-benzimidazole-piperazine, sits within the minor groove in the A-T-T-C region of the DNA double helix, displacing the spine of hydration that is found in drug-free DNA. The NH groups of the benzimidazoles make bridging three-center hydrogen bonds between adenine N-3 and thymine O-2 atoms on the edges of base-pairs, in a manner both mimicking the spine of hydration and calling to mind the binding of the auti-tumor drug netropsin. Two conformers of Hoechst are seen in roughly equal populations, related by 180 degrees rotation about the central benzimidazole-benzimidazole bond: one form in which the piperazine ring extends out from the surface of the double helix, and another in which it is buried deep within the minor groove. Steric clash between the drug and DNA dictates that the phenol-benzimidazole-benzimidazole portion of Hoechst 33258 binds only to A.T regions of DNA, whereas the piperazine ring demands the wider groove characteristic of G.C regions. Hence, the piperazine ring suggests a possible G.C-reading element for synthetic DNA sequence-reading drug analogs.

Base Sequence↗
PubMed1992

Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon.

The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions -84.5, -74 and -63, with respect ot the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.

Base Sequence↗
PubMed1979

Interaction of phenosafranine with nucleic acids and model polyphosphates. III. Heterogeneity in phenosafranine interactions with DNA base pairs.

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20

Bacillus cereus↗
PubMed1994

The purine 2-amino group as a critical recognition element for binding of small molecules to DNA.

The expedient of preparing homologous DNA samples substituted with I for G, DAP for A, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for antibiotics on DNA. The selectivity of echinomycin for CpG steps, of actinomycin for GpC steps, and of netropsin for A + T-rich tracts, is seen to be radically altered in the substituted DNA molecules.

2-Aminopurine↗
PubMed2004

Aromatic analogues of DNA minor groove binders--synthesis and biological evaluation.

Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in MCF-7 breast cancer cells.

Antineoplastic Agents↗
PubMed2004

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems.

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.

Antiviral Agents↗
PubMed2001

Significance of ligand tails for interaction with the minor groove of B-DNA.

Minor groove binding ligands are of great interest due to their extraordinary importance as transcription controlling drugs. We performed three molecular dynamics simulations of the unbound d(CGCGAATTCGCG)(2) dodecamer and its complexes with Hoechst33258 and Netropsin. The structural behavior of the piperazine tail of Hoechst33258, which has already been shown to be a contributor in sequence-specific recognition, was analyzed. The simulations also reveal that the tails of the ligands are able to influence the width of the minor groove. The groove width is even sensitive for conformational transitions of these tails, indicating a high adaptability of the minor groove. Furthermore, the ligands also exert an influence on the B(I)/B(II) backbone conformational substate behavior. All together these results are important for the understanding of the binding process of sequence-specific ligands.

Base Sequence↗
PubMed1998

Electrostatic contributions to heat capacity changes of DNA-ligand binding.

Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain.

Antineoplastic Agents↗
PubMed1997

Small ligands that neither bind to nor alter the structure of d(GA x TC)n sequences in DNA.

Three minor-groove binding ligands have been used to study the characteristics of two d(GA x CT)n DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia-Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA x CT)5 nor to d(GA x CT)22 extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA x CT)n flanking sequences located near DNA regions where the drug was bound. The d(GA x CT)n sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.

Base Sequence↗
PubMed2003

New propylamine oligopyrrole carboxamides linked to a heterocyclic or anthraquinone system: synthesis, DNA binding, topoisomerase I inhibition and cytotoxicity.

Continuing our studies on combilexines, compounds consisting of a DNA intercalator linked to a minor groove ligand, new results are presented. The synthesis of a series of new propylamine oligopyrrole carboxamides closely related to netropsin and distamycin A, linked to a heterocyclic or anthraquinone system is reported. The cytotoxic activity in vitro, the DNA binding characteristics and the inhibition of the topoisomerase I of the compounds were studied in order to explain the biological mechanism of action of these new potential combilexines. Some of the synthesised compounds showed cytotoxic activity against human tumour cell lines, as well as DNA binding and topoisomerase I inhibiting properties.

Animals↗
PubMed2001

Thiazole orange as the fluorescent intercalator in a high resolution fid assay for determining DNA binding affinity and sequence selectivity of small molecules.

The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.

Anti-Bacterial Agents↗
PubMed1997

Defining GC-specificity in the minor groove: side-by-side binding of the di-imidazole lexitropsin to C-A-T-G-G-C-C-A-T-G.

BACKGROUND: Polyamide drugs, such as netropsin, distamycin and their lexitropsin derivatives, can be inserted into a narrow B-DNA minor groove to form 1:1 complexes that can distinguish AT base pairs from GC, but cannot detect end-for-end base-pair reversals such as TA for AT. In contrast, 2:1 side-by-side polyamide drug complexes potentially are capable of such discrimination. Imidazole (Im) and pyrrole (Py) rings side-by-side read a GC base pair with the Im ring recognizing the guanine side. But the reason for this specific G-Im association is unclear because the guanine NH2 group sits in the center of the groove. A 2:1 drug:DNA complex that presents Im at both ends of a GC base pair should help unscramble the issue of imidazole reading specificity. RESULTS: We have determined the crystal structure of a 2:1 complex of a di-imidazole lexitropsin (DIM), an analogue of distamycin, and a DNA decamer with the sequence C-A-T-G-G-C-C-A-T-G. The two DIM molecules sit antiparallel to one another in a broad minor groove, with their cationic tails widely separated. Im rings of one drug molecule stack against amide groups of the other. DIM1 rests against nucleotides C7A8T9G10 of strand 1 of the helix, whereas DIM2 rests against G14G15C16C17 on strand 2. All DIM amide nitrogens donate hydrogen bonds to N and O atoms on the floor of the DNA groove and, in addition, the two Im rings on DIM2 accept hydrogen bonds from guanine N2 amines, thereby providing specific reading. The guanine N2 amine can bond to Im on its own side of the groove, but not on the cytosine side, because of limits on close approach of the two Im rings and the geometry of sp2 hybridization about the amide nitrogen. CONCLUSIONS: Im and Py rings distinguish AT from GC base pairs because of steric factors involving the bulk of the guanine amine, and the ability of Im to form a hydrogen bond with the amine. Side-by-side Im and Py rings differentiate GC from CG base pairs because of tight steric contacts and sp2 hybridization at the amine nitrogen atom, with the favored conformations being G/Im,Py/C and C/Py,Im/G. Discrimination between AT and TA base pairs may be possible using bulkier rings, such as thiazole to select the A end of the base pair.

Binding Sites↗
PubMed1993

Binding affinities of synthetic peptides, pyridine-2-carboxamidonetropsin and 1-methylimidazole-2-carboxamidonetropsin, that form 2:1 complexes in the minor groove of double-helical DNA.

The designed peptides pyridine-2-carboxamidonetropsin (2-PyN) and 1-methylimidazole-2-carboxamidonetropsin (2-ImN) are crescent-shaped analogs of the natural products netropsin and distamycin A. 2-PyN and 2-ImN bind the 5'-TGTCA-3' sequence as antiparallel side-by-side dimers in the minor groove of DNA. The binding affinities of 2-PyN and 2-ImN to four different 5-bp sites on DNA were determined by quantitative MPE-Fe(II) footprint titration and compared with the tripeptide D from distamycin. The binding affinities of D to the sites 5'-TTTTT-3' and 5'-TGTCA-3' are 2.6 x 10(7) and < 1 x 10(5) M-1, respectively (pH 7.0, 100 mM NaCl). 2-PyN binds these sites with similar affinities, 2.3 x 10(5) and 2.7 x 10(5) M-1, respectively. The affinities of 2-ImN to the same two sites are < 5 x 10(4) and 1.4 x 10(5) M-1, respectively. Substitution of an N-methylpyrrole-2-carboxamide of the distamycin tripeptide by 1-methylimidazole-2-carboxamide has changed the specificities for the two binding sites by a factor of 10(3). The data for 2-PyN and 2-ImN binding the 5'-TGTCA-3' site are best fit by a cooperative binding curve consistent with 2:1 peptide-DNA complexes.

Base Composition↗
PubMed1994

Short peptide fragments derived from HMG-I/Y proteins bind specifically to the minor groove of DNA.

Short peptides derived from chromosomal proteins have previously been proposed to bind specifically to the minor groove of A,T-rich DNA [for a review, see M. E. A. Churchill and A. A. Travers (1991) Trends Biochem. Sci. 16, 92-97]. Using NMR spectroscopy, we investigated the DNA binding of SPRKSPRK, which is one such A,T-specific motif. Under the conditions studied SPRKSPRK interacts only nonspecifically with d(CGCAAAAAAGGC).d(GCCTTTTTTGCG). The peptides TPKRPRGRPKK, PRGRPKK, and PRGRP derived from the non-histone chromosomal protein HMG-I/Y, however, bind specifically to the central A,T sites of d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2. 2D NOE measurements show that the RGR segment of each peptide is in contact with the minor groove. The arginine side chains and the peptide backbone are buried deep in the minor groove, in a fashion generally similar to the antibiotic netropsin. Under the same conditions the peptide PKGKP does not interact with the same oligonucleotide duplexes, indicating that the arginine guanidinium groups are major determinants of the A,T specificity.

Amino Acid Sequence↗
PubMed1994

Effects of minor groove binding drugs on the interaction of TATA box binding protein and TFIIA with DNA.

TBP (TATA box binding protein), a general transcription factor required for proper initiation of gene expression by RNA polymerase II, and minor groove binding drugs (MGBs) both interact with DNA within the minor groove at AT sites. This study has evaluated MGBs as inhibitors of DNA/TBP complex formation by gel mobility shift assays. Our results demonstrate that reversible MGBs (DAPI, distamycin A, Hoechst 33258, and netropsin) are effective inhibitors of the formation of DNA/TBP complex and that distamycin A is the most potent (0.16 microM inhibits TBP complex formation by 50%). CC-1065, a drug that covalently binds to DNA in the minor groove, is even more active than distamycin A (0.00085 microM inhibits TBP complex formation by 50%). Significantly more CC-1065 (0.009 microM) is required to break up preformed DNA/TBP complex compared to the drug concentration needed to prevent complex formation. In comparison, the order of drug addition has little influence on the ability of reversible MGBs to disrupt DNA/TBP complex. In the presence of TFIIA, a factor that enhances TBP association with DNA, greater drug concentrations (distamycin A and CC-1065, respectively) are needed to disrupt a preformed complex of DNA/TBP/TFIIA. In comparison to MGBs, drugs capable of binding to DNA by intercalation are generally weaker at blocking TBP complex formation except for hedamycin, which can intercalate and irreversibly bind to DNA and is as effective as reversible MGBs.

Base Sequence↗
PubMed1993

Site-specific cleavage at a DNA bulge by neocarzinostatin chromophore via a novel mechanism.

The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) oxidatively cleaves single-stranded or duplex DNA site-specifically in the absence of activating thiol provided that the DNA contains a bulged structure. Point mutations, deletions, and insertions in the DNA analogue and its complement of the 3'-terminus of yeast tRNA(Phe) show that for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must generate a hairpin structure with an apical loop and at least a two-base-pair stem hinged to a region of duplex structure via a bulge containing a target nucleotide at its 3' side. The size of the loop is not critical so long as it contains at least three nucleotides; the bulge requires a minimum of two nucleotides but must have fewer than five. With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom. Maintenance of the bulged structure requires stable duplex regions on each side of the bulge. A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cleavage in the thiol-independent reaction. Drugs such as netropsin, which sequester the DNA into nonbulge containing structures inhibit the reaction. In the absence of O2 strand cleavage is blocked and quantitatively replaced by a presumed drug-DNA covalent adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗
PubMed1989

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment.

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.

Base Sequence↗