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The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) – K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258.

DNA condensation is a ubiquitous phenomenon in biology, yet the physical basis for it has remained elusive. Here, we have explored the mechanism of DNA condensation through the protamine-DNA interaction, and by examining on it the influence of DNA binding drugs. We observed that the DNA condensation is accompanied by B to Ψ-DNA transition as a result of DNA base pair distortions due to protamine binding, bringing about the formation of toroidal structure through coil-globule transition. The binding energetics suggested that electrostatic energy, bending energy and hydration energy must play crucial roles in DNA condensation. EtBr intercalation interferes with the protamine-DNA interaction, challenging the distortion of the DNA helix and separation of DNA base pairs by protamine. Thus, EtBr, by competing directly with protamine, resists the phenomenon of DNA condensation. On the contrary, netropsin impedes the DNA condensation by an allosteric mechanism, by resisting the probable DNA major groove bending by protamine. In summary, we demonstrate that drugs with distinct binding modes use different mechanism to interfere with DNA condensation.

Microbes belonging to the genus Verrucosispora possess significant chemical diversity and biological properties. They have attracted the interests of many researchers and are becoming promising resources in the marine natural product research field. A bioassay-guided isolation from the crude extract of Verrucosispora sp. strain MS100047, isolated from sediments collected from the South China Sea, has led to the identification of a new salicylic derivative, glycerol 1-hydroxy-2,5-dimethyl benzoate (1), along with three known compounds, brevianamide F (2), abyssomicin B (3), and proximicin B (4). Compound 1 showed selective activity against methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) value of 12.5 μg/mL. Brevianamide F (2), which was isolated from actinomycete for the first time, showed a good anti-BCG activity with a MIC value of 12.5 μg/mL that has not been reported previously in literatures. Proximicin B (4) showed significant anti-MRSA (MIC = 3.125 μg/mL), anti-BCG (MIC = 6.25 μg/mL), and anti-tuberculosis (TB) (MIC = 25 μg/mL) activities. This is the first report on the anti-tubercular activities of proximicins. In addition, Verrucosispora sp. strain MS100047 was found to harbor 18 putative secondary metabolite gene clusters based on genomic sequence analysis. These include the biosynthetic loci encoding polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) consistent with abyssomicins and proximicins, respectively. The biosynthetic pathways of these isolated compounds have been proposed. These results indicate that MS100047 possesses a great potential as a source of active secondary metabolites.

The synthesis and the antitumor potential of 2-substituted aminothiazole-4-carboxylate, structurally-related to the antitumor antibiotic netropsin ( NT ) were reported. However, the exact mode of action and SAR of these compounds remained undefined. Currently, an energy-based molecular modeling approach has been utilized to highlight the mode of interaction of these compounds with β -DNA and characterize the structural requirements of biologically active aminothiazoles. Employing netropsin ( NT ) as a template and nine different 2-aminothiazole analogues have been examined for their capacity to interact with the DNA structure based on the number and pattern of H-bonding, energy of binding, conformational changes of compounds due to docking, and the width change of DNA minor groove. Positive correlation (R 2 = 0.94) was found between (E binding ) of the used 2-aminothiazoles and their antitumor activity, thus substantiating the utility of this modeling approach in future design of similar compounds.

Minor groove binding compounds have been shown to induce changes in global DNA conformation, allosterically inhibiting DNA-protein interactions necessary for transcriptional processes. Many minor groove binders are specific for AT-base pairs but have little preference over alternating AT or A-tract sequences. Few compounds, other than polyamides, show selectivity for mixed sequences with AT and GC base pairs. Electrospray ionization mass spectrometry (ESI-MS) can provide insight on the stoichiometry and relative affinities in minor groove recognition of different DNA sequences with a library of minor groove binders. A goal in our current research is to develop new compounds that recognize mixed sequences of DNA. In an effort to optimize screening for compounds that target mixed AT and GC base pair sequences of DNA, ESI-MS was used to study the competitive binding of compounds with a mixed set of DNA sequences. The method identified preferred binding sites, relative affinities, and concentration-dependent binding stoichiometry for the minor groove binding compounds netropsin and DB75 with AT-rich sequences, and DB293 with ATGA and AT-sites.

Polymyxins are last-resort antibiotics for treating infections of Gram-negative bacteria. The recent emergence of polymyxin-resistant bacteria, however, urgently demands clinical optimisation of polymyxin use to minimise further evolution of resistance. In this study we developed a novel combination therapy using minimal concentrations of polymyxin B. After large-scale screening of Streptomyces secondary metabolites, we identified a reliable polymixin synergist and confirmed as netropsin using high-pressure liquid chromatography, nuclear magnetic resonance and mass spectrometry followed by in vitro assays using various Gram-negative pathogenic bacteria. To evaluate the effectiveness of combining polymixin B and netropsin in vivo, we performed survival analysis on greater wax moth Galleria mellonella infected with colistin-resistant clinical Acinetobacter baumannii isolates as well as Escherichia coli, Shigella flexineri, Salmonella typhimuruim, and Pseudomonas aeruginosa. The survival of infected G. mellonella was significantly higher when treated with polymyxin B and netropsin in combination than when treated with polymyxin B or netropsin alone. We propose a netropsin combination therapy that minimises the use of polymyxin B when treating infections with multidrug resistant Gram-negative bacteria.

Although the molecular mechanism and thermodynamic profile of a wide variety of chemical agents have been examined intensively in the past decades in terms of specific recognition of their protein receptors, to date the physicochemical nature of DNA-drug recognition and association still remains largely unexplored. The present study focused on understanding the structural basis, energetic landscape, and biological implications underlying the binding of small-molecule ligands to their cognate or non-cognate DNA receptors. First, a new method to capture the structural features of DNA-drug complex architecture was proposed and then used to correlate the extracted features with binding affinity of the complexes. By employing this method, a statistical regression-based predictor was developed to quantitatively evaluate the interaction potency of drug compounds with DNA in a fast and reliable manner. Subsequently, we use the predictor to examine the binding behavior of a number of structure-available, affinity-known DNA-drug complexes as well as a large pool of randomly generated DNA decoys in complex with the same drugs. It was found that (1) as compared with protein-DNA recognition, small-molecule agents have relatively low specificity in selecting their cognate DNA targets from the background of numerous random decoys; (2) the abundance of A-T base pairs in the DNA core motif exhibits a significant positive correlation with the affinity of drug ligand binding to the DNA receptor; and (3) high affinity seems not to be closely related to high selectivity for a DNA-targeting drug, although high-affinity drug entities have a greater possibility of being ranked computationally as top binders. We hope that this work will provide a preliminary insight into the molecular origin of sequence-specific interactions in DNA-drug recognition.

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.

Overexpression of high mobility group AT-hook 1 (HMGA1) is common in human cancers. Little is known about the mechanisms underlying its deregulation and downstream targets, and information about its clinical and biological significance in medulloblastoma (MB) is lacking. Here, we demonstrated frequent genomic gain at 6p21.33-6p21.31 with copy number increase leading to overexpression of HMGA1 in MB. The overexpression correlated with a high proliferation index and poor prognosis. Moreover, we found that hsa-miR-124a targeted 3'UTR of HMGA1 and negatively modulated the expression in MB cells, indicating that loss/downregulation of hsa-miR-124a reported in our previous study could contribute to the overexpression. Regarding the biological significance of HMGA1, siRNA knockdown and ectopic expression studies revealed the crucial roles of HMGA1 in controlling MB cell growth and migration/invasion through modulation of apoptosis and formation of filopodia and stress fibers, respectively. Furthermore, we identified cdc25A as a target of HMGA1 and showed that physical interaction between HMGA1 and the cdc25A promoter is required for transcriptional upregulation. In clinical samples, HMGA1 and cdc25A were concordantly overexpressed. Functionally, cdc25A is involved in the HMGA1-mediated control of MB cell growth. Finally, netropsin, which competes with HMGA1 in DNA binding, reduced the expression of cdc25A by suppression of its promoter activity and inhibited in vitro and in vivo intracranial MB cell growth. In conclusion, our results delineate the mechanisms underlying the deregulation and reveal the functional significance of HMGA1 in controlling MB cell growth and migration/invasion. Importantly, the results highlight the therapeutic potential of targeting HMGA1 in MB patients.

Binding of ligands to DNA gives rise to several relevant biological and biomedical effects. Here, through the use of atomic force microscopy (AFM), we studied the consequences of drug binding on the morphology of single DNA molecules. In particular, we quantitatively analyzed the effects of three different DNA-binding molecules (doxorubicin, ethidium bromide, and netropsin) that exert various pharmacologic and therapeutic effects. The results of this study show the consequences of intercalation and groove molecular binding on DNA conformation. These single-molecule measurements demonstrate morphological features that reflect the specific modes of drug-DNA interaction. This experimental approach may have implications in the design of therapeutically effective agents.

New structurally diverse natural products are discovered when novel screening procedures are introduced or when high quality biological materials from new sources are examined in existing screens, hence it is important to foster these two aspects of novelty in drug discovery programmes. Amongst prokaryotes, actinomycetes, notably streptomycetes, remain a rich source of new natural products though it has become increasingly difficult to find such metabolites from common actinomycetes as screening 'old friends' leads to the costly rediscovery of known compounds. The bioprospecting strategy which is the subject of this review is based upon the premise that new secondary metabolites can be found by screening relatively small numbers of dereplicated, novel actinomycetes isolated from marine sediments. The success of the strategy is exemplified by the discovery of a range of novel bioactive compounds, notably atrop-abyssomicin C and proximicins A, B and C from Verrucosispora strains isolated from sediment samples taken from the Sea of Japan and the Raune Fjord, respectively, and the dermacozines derived from Dermacoccus strains isolated from the Challenger Deep of the Mariana Trench in the Pacific Ocean. The importance of current advances in prokaryotic systematics in work of this nature is stressed and a plea made that resources be sought to train, support and employ the next generation of actinobacterial systematists.

Prefixation treatment of Chinese hamster V79-E cells with the AT-specific agents netropsin, distamycin A and Hoechst 33258 produced a dose- and time-dependent alteration of chromosome morphology. Compared to control cultures the chromosomes appear less condensed and revealed, after Giemsa staining, a non-random pattern of light and dark segments. It was found that the induced banding pattern corresponded for the three agents tested. Some of the darkly stained regions coincided with the extracentromeric C-bands seen in the karyotype of this cell strain, suggesting these areas to be GC-rich heterochromatin. The binding specifity and the mode of action of netropsin, distamycin A and Hoechst 33258 in DNA and chromatin suggest that the structural modifications induced depend on the amount of AT-base clusters in specific regions. Differential deprivation of chromosomal proteins might also contribute to the observed differences in stainability of chromosome segments. Attempts to induce elongations or banding patterns by prefixation treatment with the GC-specific agents actinomycin D and oligomycin were unsuccessful.

The localization of SCEs was studied by simultaneous treatment of V79-E cells with netropsin-HCl and BUdR. The SCEs occurred in a non-random pattern, but no clear-cut correlation of SCEs and C-band heterochromatin or dAdT-clusters, resp., was observed. BUdR substitution completely abolishes the differential elongation pattern of chromosomes obtained after in vitro treatment with netropsin alone, but does not prevent an overall chromosome elongation. Netropsin enforces the unequal stretching of sister chromatids produced by BUdR.

In vitro exposure of mammalian cells to dA-dT probes partially inhibits chromosome contraction in mitosis. Especially dA-dT rich regions are affected. Combined with BUdR incorporation in late S-phase this treatment induces banding which depends on both replication pattern and location of dA-dT rich regions. This method was used in normal mouse fibroblasts and in Chinese hamster V79-E cells, with netropsin as the dA-dT specific agent. Banding was observed both after conventional Giemsa and after FPG-staining. In those cases where the G bands in general are dA-dT rich and late replicating, the banding obtained with this technique corresponds to R-bands.

The photophysical properties of the polyamide PyPyPyβDp (PPP) were investigated by means of steady-state absorption and fluorescence spectroscopies, as well as time-resolved fluorescence spectroscopy. It was found that the excited-state properties of PPP are very sensitive to solvents. In TKMC buffer PPP exhibited weak fluorescence with a decay time constant of 16 ps, while with the decrease of the solvent polarity PPP showed the blue-shifted peak position, increased intensity and lengthened life-time for its fluorescence behavior. In the presence of calf thymus DNA, it was observed that the fluorescence intensity was enhanced and the fluorescence lifetime increased from 16 to 32 ps for PPP, which verified that PPP bound into the minor groove of DNA duplex.

Combilexeins are high-affinity DNA ligands with a structure comprising both recognition (lexitropsin) and functional (intercalator, alkylating fragment, etc.) groups. These molecules have drawn the attention of researchers due to a unique combination of properties, whereby highly specific binding of lexitropsin is retained together with a high biological activity of the functional fragment [1]. Combilexeins with functional fragments possessing intercalation properties are of special interest because their binding to DNA allows two main types of ligand – DNA interaction, minor groove binding (MGB) and intercalation, to take place simultaneously [2]. A series of acridine-containing combilexeins synthesized on the basis of distamycin and netropsin exhibit such a bifunctional binding to DNA [2]. At the same time, Bailly and Chaires [1] quite reasonable classify some “usual” intercalators (e.g., actinomycin D) as combilexeins because the side chains of these agents are capable of performing recognition functions. Combilexeins have not been considered as potential antiviral agents. Previously [3], we suggested that biodegradable bisintercalators can be promising antiviral agents. Indeed, bisacridinylaminoacyldiamines have proved to be highly effective antiviral agents [4] and interferon inductors [5], being superior in these respects to amyxin (I), a drug used in clinical practice as an antiviral drug and interferon inductor. The development of this approach has led us to a hypothesis that bisinterlexeins (lexitropsins combined with two intercalators) can be promising antiviral agents [6]. To our knowledge, only one trifunctional DNA ligand containing two different intercalators (ethidium and anilinoacridine fragments) possessing cytotoxic properties (topoisomerase II inhibition) have been described so far [7]. The need to introduce two intercalating fragments into a molecule implies the presence of two functional groups in the connecting lexitropsin fragment (linker). Lexitropsin-like properties have been reported for symmetric polyamides based on pyridine-2,5-dicarboxylic acid (II), which exhibit a good isohelical fit to the minor groove of DNA (coincidence of the curvature radii of the ligand and groove) [8].

Selective N3-adenine methylation represents a novel strategy for tumors with a phenotype of poor responsiveness to a number of anticancer agents currently used in the clinic. Resistance to N3-methyl-adenine-inducing agents, such as MeOSO2(CH2)(2)-lexitropsin (Me-Lex), is due to high levels of N-methylpurine glycosylase (MPG). However, tumor cells with high MPG activity can be rendered susceptible to Me-Lex using poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. Purpose: To evaluate the potential toxicity of Me-Lex, used as single agent or combined with PARP-1 inhibitors, in normal peripheral blood lymphocytes (PBL). Methods: PBL either resting or activated with phytohemagglutinin (PHA), obtained from healthy donors, were treated with graded concentrations of Me-Lex with or without PARP-1 inhibitor (3-aminobenzamide, AB, or NU1025, NU). MPG activity, apoptosis and sister chromatid exchanges (SCE) were evaluated. Results: (a) Me-Lex was cytotoxic mainly in PHA-activated PBL with low MPG activity; (b) combincd treatment with Me-Lex and AB induced apoptotic effects as early as 24 h after drug exposure both in non-stimulated and PHA-activated PBL. When concentrations of PARP-I inhibitors (25 muM NU and 4 mM AB) that produced a twofold increase in Me-Lex cytotoxicity in tumor cells were compared, NU induced a less-pronounced increase in apoptosis in PBL treated with Me-Lex; (c) Me-Lex at concentrations that allowed cytogenetic analysis did not induce a significant number of SCE; (d) PARP-1 inhibitors provoked a dose-dependent increase in SCE, but 25 muM NU was devoid of genotoxic effects and did not significantly increase SCE in PBL treated with Me-Lex. Conclusions: Me-Lex showed preferential cytotoxicity against mitogen-activated PBL. Our results also indicated that for each PARP-1 inhibitor it is necessary to define the concentration devoid of genotoxic effects in normal cells, but still capable of enhancing the efficacy of DNA-damaging agents in tumor cells.

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group ( ) or aliphatic pentamethylene chain ( ), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for and binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A 4 T 4-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T 4 A 4-3" sequences.

The poly(ADP-ribose) polymerase (PARP) is involved in cell recovery from DNA damage, such as methylation of N3-adenine, that activates the base excision repair process. In the present study we demonstrated that MeOSO2(CH2)2-lexitropsin (Me-Lex), a methylating agent that almost exclusively produces N3-methyladenine, induced different modalities of cell death in human leukemic cell lines, depending on the presence of PARP inhibitor. Growth inhibition, provoked by the combination of Me-Lex and PARP inhibitor, was associated with a marked down-regulation of c-myc, increased generation of single strand breaks and apoptosis. When used as single agent, at concentrations that saturated cell repair ability, Me-Lex induced mainly cell death by necrosis. Surprisingly, addition of a PARP inhibitor enhanced apoptosis and reduced the early appearance of necrosis. Telomerase activity was completely suppressed in cells exposed to Me-Lex alone, by 24 h after treatment, whereas it did not change when Me-Lex was combined with PARP inhibitor. Thereafter, inhibition of telomerase was observed with both treatments. The results suggest new insights on different modalities of cell death induced by high levels of N3-methyladenine per se, or by the methylated base in the presence of PARP inhibitor. Cell Death and Differentiation (2001) 8, 817–828

Two-dimensional 1H NMR (NOESY, COSY) spectroscopy was used to study the spatial structure of a DNA complex CGTTTATTp-Net:AATAAACG with a netropsin analog bound at the terminal 3′-phosphate group in aqueous solution. The positions of the proton NMR signals of the duplex are compared with those for the unmodified duplex and the free ligand. A mean conformation of the duplex is constructed by the molecular mechanics technique using the obtained NMR limitations on the interproton distances. The data obtained indicate that the aromatic pyrrole rings of the netropsin analog are involved in a stacking interaction with the terminal AT base pair of the duplex; this is generally not typical of ordinary minor-groove netropsin type ligands.

Mismatch repair deficiency contributes to tumor cell resistance to O6-guanine methylating compounds and to other antineoplastic agents. Here we demonstrate that MeOSO2(CH2)2- lexitropsin (Me-Lex), a DNA minor groove alkylating compound which generates mainly N3-methyladenine, has cytotoxic and clastogenic effects in mismatch repair-deficient leukemic cells. Moreover, MT-1 cells, which express p53 upon drug treatment and possess low levels of 3-methylpurine DNA glycosylase activity, are more susceptible to cytotoxicity induced by Me-Lex, with respect to p53-null and 3-methylpurine DNA glycosylase-proficient Jurkat cells. In both cell lines, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, which inhibits base excision repair capable of removing N-methylpurines, increases cytotoxicity and clastogenicity induced by Me-Lex or by temozolomide, which generates low levels of N3-methyl adducts. The enhancing effect is more evident at low Me-Lex concentrations, which induce a level of DNA damage that presumably does not saturate the repair ability of the cells. Nuclear fragmentation induced by Me-Lex + 3-aminobenzamide occurs earlier than in cells treated with the single agent. Treatment with Me-Lex and 3-aminobenzamide results in augmented expression of p53 protein and of the X-ray repair cross- complementing 1 transcript (a component of base excision repair). These results indicate that N3-methyladenine inducing agents, alone or combined with poly(ADP-ribose) polymerase inhibitors, could open up novel chemotherapeutic strategies to overcome drug resistance in mismatch repair-deficient leukemic cells.

Zimm and Bragg theory for helix to coil transition has been suitably amended and applied to explain the two step transition in DNA triplex. The experimental measurements reported by G. E. Plum et al. and Y. W. Park et al. have been interpreted. The order-order and order-disorder transitions associated with the melting of DNA triple helix, with and without netropsin binding, have been characterized by the nucleation parameter, enthalpy change, sharpness of transition, and heat capacity. The destabilization of the triplex and stabilization of duplex on netropsin binding are reflected in these characteristic parameters.

We report the design of optimal linker geometries for the synthesis of stapled DNA-minor-groove-binding molecules. Netropsin, distamycin, and lexitropsins bind side-by-side to mixed-sequence DNA and offer an opportunity for the design of sequence-reading molecules. Stapled molecules, with two molecules covalently linked side-by-side, provide entropic gains and restrain the position of one molecule relative to its neighbor. Using a free-atom simulated annealing technique combined with a discrete mutable atom definition, optimal lengths and atomic composition for covalent linkages are determined, and a novel hydrogen bond 'zipper' is proposed to phase two molecules accurately side-by-side.

This report deals with the cleavage reaction of calf thymus (CT) topoisomerase II with oligonucleotides containing one main cleavage site and adjacent binding sites for minor groove binders. The sequences of the oligonucleotides were derived from a pBR 322 sequence, which contains one main topoisomerase II cleavage site. The cleavage reaction was performed under increasing concentrations of minor groove binders and it showed characteristic inhibition dependences of topoisomerase II to the binding sites and to the binding length of the minor groove binders. The extension of the minor groove binder length on DNA from 4 to 10 base pairs (bp) by netropsin and bis-netropsin, respectively, causes a strong increase of the topoisomerase II cleavage inhibition. The same is observed by the introduction of a second minor groove binder sequence symmetrically positioned around the topoisomerase II main cleavage site. The combination of two different minor groove binders can lead to an increased topoisomerase II inhibition but also to a prevention of total inhibition as shown with chromomycin A3 and distamycin A at concentrations of 0.1 and 0.25 microM, respectively.

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca2+-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.

The antibiotic netropsin decreased only slightly the exponential growth rate in a medium with mineral salts and glucose but its effect was increased during postexponential growth. Protein turnover and the basal intracellular serine proteinase (ISP) activity in the crude cytoplasmic fraction were not significantly affected by netropsin during exponential growth. However, ISP activity increased during postexponential growth and its rise was inhibited by the antibiotic. The population in the postexponential phase was not committed to sporulation.

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.

A method of indirect mutant selection, analogous to the bacterial penicillin technique, has been developed for Physarum polycephalum. The antibiotic netropsin was found to be lethal to growing but not to nongrowing (cycloheximide inhibited) myxamoebae. In reconstruction experiments, a 200 fold enrichment of cycloheximide sensitive over cycloheximide resistant cells was obtained. This technique was used to isolate temperature sensitive mutants. The mutant frequency among survivors was increased at least 40 fold by netropsin selection.

Distamycin and netropsin are two oligopeptides which bind to DNA in a nonintercalative manner. Analogues of distamycin have been synthesized and their binding with poly d(A-T) studied using ultraviolet absorption spectroscopy. Preliminary biological activity tests on a gram positive bacteria using these analogues have also been carried out.

This paper presents results of theoretical computations on the interaction energies and geometries for the binding to nucleic acids of a number of representative groove binding non intercalating drugs: netropsin, distamycin A, SN 18071, etc. The computations account for the specificity of binding in all cases and demonstrate that the formation of hydrogen bonds is not necessary neither for binding nor for the preference for the minor groove of AT sequences of B-DNA. It appears that if a relatively good steric fit can be obtained in the minor groove, the interaction will be preferentially stabilized there by the favorable electrostatic potential generated in this groove by the AT sequences. The computation of the interaction energies in free space does not reproduce, however, the order of affinities of the ligands studied and yields too great values of the binding energies. The introduction of the solvent effect, through the computation of the hydration and cavitation effects, confirms the specificity, improves the ordering and brings the values of the energies close to the experimental ones. The theoretical account of the "surprising" effect of netrospin binding to the major groove of the TψC stem of tRNA Phe confirms the decisive significance of the distribution of the molecular electrostatic potential for the selection of the binding site. The inclusion in the computations of the flexibility of DNA enables to predict correctly the main features of the macromolecular deformation upon the binding of the ligand.

The interaction of 4′-6-diamidino-2-phenylindole · 2 HCl with natural and synthetic polydeoxy- and polyribonucleotides of different base content and sequences was studied with circular dichroism, ultracentrifugation, viscosity and calorimetry. All the polymers show two types of binding. The strength of interaction and its resistence to ionic strength are related to the content of AT clusters in the chain. On the other hand, sedimentation measurements rule out an intercalation mechanism. A model of 4'-6-diamidino-2-phenylindole · 2 HCl interaction with DNA and double stranded RNA, similar to that displayed by distamycin and netropsin, is proposed.

The appearance of poly(A)RNA species at the onset of sporulation has been demonstrated in B. polymyxa. These RNA species, characterized by short oligo(A) stretches, are synthesized at the end of exponential growth (t0) and their level reaches a maximum two hours later (t2 of sporulation). This new feature of sporulation appears relevant to this process as indicated by the lack of formation of poly(A)RNA in mutants blocked at the early stages of sporulation. In addition, the antibiotic netropsin, which is known to inhibit sporulation without affecting the vegetative growth of B. subtilis, also inhibits poly(A) RNA synthesis and sporulation in B. polymyxa. The selective inhibitory effect of netropsin on the poly(A)RNA formation, as reflected in the reduced incorporation of [3H]-adenosine into RNA by sporulating cells, is traced to the inhibition of sporulation-specific RNA polymerase under in vitro conditions. The inhibition of transcription concerns mainly the DNA regions that are firmly associated with membranes. Moreover, [32P]-labeled poly(A)RNA isolated from sporulating cells at t2 hybridizes preferentially with membrane bound DNA (mDNA). Further evidence that the mDNA is enriched in d(AT) sequences was obtained by demonstration of the resistance of the mDNA-netropsin complex to DNase action. Based on the property of netropsin to bind to AT sequences in DNA, it is proposed that early spore genes might be enriched in d(AT) sequences and are localized in mDNA.

The ability of electronic energy transfer and direct binding competition between pairs of dyes to enhance contrast in human or bovine metaphase chromosome staining patterns is illustrated, and the relative effectiveness of these two mechanism compared. The existence of energy transfer between quinacrine or 33258 Hoechst and 7-amino-actinomycin D in doubly stained chromosomes is demonstrated directly by microfluorometry. The ability of the dyes 7-amino-actinomycin D, methyl green, or netropsin, acting as counterstains, to displace quinacrine, 33258 Hoechst, or chromomycin A3 from chromosomes, is estimated by quantitative analysis of energy transfer data, by photobleaching of the counterstains, or by selective removal of counterstains by appropriate synthetic polynucleotides. Effects on the fluorescence of soluble 33258 Hoechst-DNA complexes due to energy transfer or binding displacement, by actinomycin D or netropsin, respectively, are further differentiated by nanosecond fluorescence decay measurements. Examples are presented of dye combinations for which (a) energy transfer is the primary mechanism operative, (b) binding competition exists, with consequences reinforcing those due to energy transfer, or (c) binding competition is the most important interaction. These analyses of mechanisms responsible for contrast enhancement in doubly stained chromosomes are used to derive information about the relationship between chromosome composition and banding patterns.