Three putative DNA replication/repair elements encoding genes confer self-resistance to distamycin in Streptomyces netropsis.

Distamycin (DST) is a well-characterized DNA minor groove binder with antivirus activity and antitumor potency. Two separate gene clusters (a 28-kb cluster and a 7-kb cluster) have recently been identified to coordinately encode the biosynthetic machinery of DST in Streptomyces netropsis. Here we report a gene cassette, which is linked to the aforementioned smaller dst gene cluster and plays an important role in the self-resistance to DST in S. netropsis. This cassette consists of three uncharacterized genes that might be implicated in DNA replication/repair. Knockout of the cassette led to the decrease in the production of DST, while heterologous expression of part of the cassette in S. lividans made it become resistant to both DST and mitomycin C, another DNA-binding agent. More interestingly, homologs of these three genes were found in genomes of other actinomyces that produce DNA-binding antibiotics, suggesting that a novel common mechanism in addition to pumping may enable these strains to resist the cytotoxic metabolites they produced.

Differential scanning calorimetric study of antibiotic distamycin A binding with chromatin within isolated rat liver nuclei.

CONTEXT: Natural oligopeptide antibiotic distamycin A (Dst) biosynthesized by Streptomyces distallicus is traditionally used in medical practice as an anti-inflammatory and antitumour drug. OBJECTIVE: Dst was investigated for its effect on the structural components of native chromatin directly within isolated rat liver nuclei in the presence of physiologically significant cations (magnesium or spermine and spermidine). MATERIALS AND METHODS: Differential scanning calorimetry (DSC) was used to study the Dst action at molar ratio Dst/DNA = 0.1 and 0.15 mM Dst on the melting profile of nuclei suspension in different conditions. RESULTS: Results showed that the thermodynamic parameters of control nuclei in the presence of polyamines or Mg2+ were different. The incubation of nuclei with Dst raised transition temperatures of relaxed (peak II) and topologically constrained DNA (peak III) by 6-8 °C and decreased by 2-4 °C that of core-histones (peak I). The total excess transition enthalpy (ΔHexc) in buffer with polyamines (24.7 kJ/mol DNA nucleotides) increased by1.5 times versus control but in buffer with Mg2+, the value of ΔHexc (35.8 kJ/mol DNA nucleotides) remained unchanged. CONCLUSIONS: The association of Dst with chromatin in the nucleus weakens histone-DNA contacts and causes additional strengthening of interaction between two complementary DNA chains. Our results contribute towards validation of DSC to test drug ability to modulate chromatin structure in the physiological environment and to clarify the mechanism of these modulations.

An evaluation of Minor Groove Binders as anti-lung cancer therapeutics.

A series of 47 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for anti-lung cancer activity by screening against the melanoma cancer cell line B16-F10. Five compounds have been found to possess significant activity, more so than a standard therapy, Gemcitabine. Moreover, one compound has been found to have an activity around 70-fold that of Gemcitabine and has a favourable selectivity index of greater than 125. Furthermore, initial studies have revealed this compound to be metabolically stable and thus it represents a lead for further optimisation towards a novel treatment for lung cancer.

FeON-FeOFF: the Helicobacter pylori Fur regulator commutates iron-responsive transcription by discriminative readout of opposed DNA grooves.

Most transcriptional regulators bind nucleotide motifs in the major groove, although some are able to recognize molecular determinants conferred by the minor groove of DNA. Here we report a transcriptional commutator switch that exploits the alternative readout of grooves to mediate opposite output regulation for the same input signal. This mechanism accounts for the ability of the Helicobacter pylori Fur regulator to repress the expression of both iron-inducible and iron-repressible genes. When iron is scarce, Fur binds to DNA as a dimer, through the readout of thymine pairs in the major groove, repressing iron-inducible transcription (FeON). Conversely, on iron-repressible elements the metal ion acts as corepressor, inducing Fur multimerization with consequent minor groove readout of AT-rich inverted repeats (FeOFF). Our results provide first evidence for a novel regulatory paradigm, in which the discriminative readout of DNA grooves enables to toggle between the repression of genes in a mutually exclusive manner.

DNA Minor Groove Binders-Inspired by Nature.

The synthesis and biological activity of a variety of analogues to the naturally occurring antibacterial and antifungal Distamycin A were explored by a number of authors. These compounds were subject to a large array of assays. Some of these compounds showed high activity against a range of Gram-positive, Gram-negative bacteria as well as fungi. To explore the anti-parasitic activity of this class of compounds, specific modifications had to be made. A number of these compounds proved to be active against Trypanosoma brucei. The binding of a number of these compounds to short sequences of DNA were also examined using footprinting assays as well as NMR spectroscopy. Computer modelling was employed on selected compounds to understand the way these compounds bind to specific DNA sequences. A large number of variations were made to the standard structure of Distamycin. These changes involved the replacement of the pyrrole moieties as well as the head and tail groups with a number of heterocyclic compounds. Some of these minor groove binders (MGBs) were also investigated for their capability for the treatment of cancer and in particular lung cancer.

ANTIPROLIFERATIVE EFFECTS ON BREAST CANCER CELLS AND SOME INTERACTIONS OF NEW DISTAMYCIN ANALOGUES WITH DNA, ENDONUCLEASES AND DNA TOPOISOMERASES.

The evaluation of a new group of distamycin analogues 1-6 as potential minor groove binders for the treatment of cancer were investigated. The activity of the new compounds against several restriction enzymes was examined. The studied compounds did not block GC-rich sequences regions of DNA but inhibited catalytic action of endonucleases in AA, AT, TT and AG restriction sites. Determination of association constants using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 have confirmed that the tested compounds bind within minor groove of B-DNA. All of the compounds demonstrated activity against DNA topoisomerases II at the concentration 10 µM, but they did not inhibit activity of topoisomerase I. The studied derivatives were evaluated in human MCF-7 breast cancer cells and showed antiproliferative and cytotoxic effects in the range of 81.70 µM and 200.00 µM.

Synthesis, anti-mycobacterial activity and DNA sequence-selectivity of a library of biaryl-motifs containing polyamides.

The alarming rise of extensively drug-resistant tuberculosis (XDR-TB) strains, compel the development of new molecules with novel modes of action to control this world health emergency. Distamycin analogues containing N-terminal biaryl-motifs 2(1-5)(1-7) were synthesised using a solution-phase approach and evaluated for their anti-mycobacterial activity and DNA-sequence selectivity. Thiophene dimer motif-containing polyamide 2(2,6) exhibited 10-fold higher inhibitory activity against Mycobacterium tuberculosis compared to distamycin and library member 2(5,7) showed high binding affinity for the 5'-ACATAT-3' sequence.

Extraction of histone H1 and decondensation of nuclear chromatin with various Mg-dependent organization levels under treatment with polyglutamic acid and distamycin.

Chromatin in rat liver nuclei under conditions of low ionic strength (20-25 mM) and [Mg2+] from 2 to 5 mM has a condensed structure (100-200 nm globules) and gives the same CD signal (320-340 nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1 mM leads to chromatin decondensation to 30 nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA = 6 and in the presence of 5 mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3 mM Mg2+ leads to chromatin decondensation to 30 nm fibrils. Extraction of about half of histone H1 at [Mg2+] ≤ 2 mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1-2 min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5-7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA = 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA = 30 and DM/DNA = 0.1, about 5-10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging.

Energetics of ligand binding to G-quadruplexes.

G-quadruplex ligands are potential anticancer agents as telomerase inhibitors and potential transcriptional regulators of oncogenes. The search for best-in-class drugs is addressed to identify small molecules able to promote and stabilize G-quadruplex structures. What features should the G-quadruplex ligands possess? They should have selective antiproliferative effects on cancer cells and induce telomerase inhibition or oncogene suppression. One of the main challenges in their design and synthesis is to make the ligands selective for G-quadruplex DNA. These features should be amplified by careful analyses of physico-chemical aspects of G-quadruplex-drug interactions. In particular, the study of the energetics of G-quadruplex-drug interactions can enhance drug design by providing thermodynamic parameters that give quantitative information on the biomolecular interactions important for binding. The main methodologies used to gain information on energetics of binding are based on spectroscopic or calorimetric principles. Spectroscopic techniques such as fluorescence and circular dichroism are rapid and cheap methods, but are not sufficient to characterize completely the thermodynamics of interaction. Calorimetric techniques such as isothermal titration calorimetry offer a direct measure of binding enthalpy, in addition to the stoichiometry and affinity constants. With the complete thermodynamic signature of drug-target interaction, dissecting the enthalpic and entropic components of binding is possible, which can be a useful aid to decision-making during drug optimization.

Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells.

Acquired resistance to cisplatin (cDDP) is a multifactorial process that represents one of the main problems in ovarian cancer therapy. Distamycin A is a minor groove DNA binder whose toxicity has limited its use and prompted the synthesis of derivatives such as NAX001 and NAX002, which have a carbamoyl moiety and different numbers of pyrrolamidine groups. Their interaction with a B-DNA model and with an extended-TATA box model, [Polyd(AT)], was investigated using isothermal titration calorimetry (ITC) to better understand their mechanism of interaction with DNA and therefore better explain their cellular effects. Distamycin A interactions with Dickerson and Poly[d(AT)(6)] oligonucleotides show a different thermodynamic with respect to NAX002. The bulkier distamycin A analogue shows a non optimal binding to DNA due to its additional pyrrolamidine group. Cellular assays performed on cDDP-sensitive and -resistant cells showed that these compounds, distamycin A in particular, affect the expression of folate cycle enzymes even at cellular level. The optimal interaction of distamycin A with DNA may account for the down-regulation of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and the up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) caused by this compound. These effects seem differently modulated by the cDDP-resistance phenotype. NAX002 which presents a lower affinity to DNA and slightly affected these enzymes, showed a synergic inhibition profile in combination with cDDP. In addition, their combination with cDDP or polyamine analogues increased cell sensitivity to the drugs suggesting that these interactions may have potential for development in the treatment of ovarian carcinoma.

Molecular basis for the inhibition of HMGA1 proteins by distamycin A.

The molecular mechanism for the displacement of HMGA1 proteins from DNA is integral to disrupting their cellular function, which is linked to many metastatic cancers. Chemical shift and NOESY NMR experiments provide structural evidence for the displacement of an AT hook peptide (DNA binding motif of HMGA1 proteins) by both monomeric and dimeric distamycin. However, the displaced AT hook alters distamycin binding by weakening the distamycin:DNA complex, while slowing monomeric distamycin dissociation when AT hook is in excess. The central role of the AT hook was evaluated by monitoring full-length HMGA1a protein binding using fluorescence anisotropy. HMGA1a was effectively displaced by distamycin, but the cooperative binding exhibited by distamycin was eliminated by displaced HMGA1a. Additionally, these studies indicate that HMGA1a is displaced from the DNA by 1 equiv of distamycin, suggesting the ability to develop therapeutics that take advantage of the positively cooperative nature of HMGA1a binding.

Influence of distamycin, chromomycin, and UV-irradiation on extraction of histone H1 from rat liver nuclei by polyglutamic acid.

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.

[Effect of of distamycin A on histone H1 methylation, extraction and formation of UV-inducible crosslinks with DNA in the interphase rat liver nucleus].

Incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-(3)H]methionine ([(3)H] SAM) leads to incorporation of the radioactive label not only into core-histones H3 and H4, but also into linker histone H1. Addition of distamycine A to the incubation medium stimulates label incorporation into histone H1 ~ in 6 times and into histone H3 ~ in 2 times. The presence of distamycine facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases of UV-induced DNA-histone cross-links formation. These effects give evidence of weakening of H1-chromatin interaction by distamycin to be results of histone H1 position change relative to nucleosome and(or) disturbance of histones H1-H3 interactions so as these histones are exposed to additional methylation.

[Antiviral properties of the derivatives of netropsin and distamycin against herpes simplex viruses type 1 and variolovaccine].

The antiherpesvirus activity of newly synthesized DNA-binding compounds for cultured Vero E6 cells was examined. The compounds were found to have selective antiherpesviral activity. Their antiviral activity was shown against the virus strains isolated from clinical specimens. The test compounds were ascertained to have also a high activity against herpes simplex virus type 1 (HSV-1/L2 TC-) that was very resistant to acyclovir. The authors' data demonstrated an obvious dose-dependent antiviral effect, which was representatively seen when Pt-bis-Dst and Lys-bis-Nt were used. The findings have offered the challenge to test some of these compounds in experiments on laboratory animals.

Specific stabilization of promoter G-Quadruplex DNA by 2,6-disubstituted amidoanthracene-9,10-dione based dimeric distamycin analogues and their selective cancer cell cytotoxicity.

We have designed and synthesized anthraquinone containing compounds which have oligopyrrole side chains of varying lengths. These compounds stabilized the G-quadruplex DNA formed in the promoter regions of c-MYC oncogenes selectively over the duplex DNA. These observations were recorded using UV-vis spectroscopic titrations, fluorescence measurements and circular dichroism (CD) spectral titrations. The potency of the compounds to stabilize the G4 DNA has been shown from the thermal denaturation experiments. The compound interacts with c-MYC G-quadruplex DNA through stacking mode as obtained from ethidium bromide displacement assay, cyclic voltammetric titration, and docking experiments. Molecular modeling studies suggested that the stacking of the anthraquinone moiety over the G-tetrad of the G4 structures are responsible for the stability of such quadruplex secondary structure. Furthermore, polymerase stop assay also supported the formation of stable G4 structures in the presence of the above-mentioned compounds. The compounds have shown selective cancer cell (HeLa and HEK293T) cytotoxicity over normal cells (NIH3T3 and HDFa) under in vitro conditions as determined from MTT based cell viability assay. Apoptosis was found to be the mechanistic pathway underlying the cancer cell cytotoxicity as obtained from Annexin V-FITC and PI dual staining assay which was further substantiated by nuclear morphological changes as observed by AO/EB dual staining assay. Cellular morphological changes, as well as nuclear condensation and fragmentation upon treatment with these compounds, were observed under bright field and confocal microscopy.

Human chromosome fragility.

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.

EhMLBP is an essential constituent of the Entamoeba histolytica epigenetic machinery and a potential drug target.

EhMLBP has been identified as a protein that specifically binds to methylated long interspersed element (LINE) retrotransposons and rDNA in Entamoeba histolytica. EhMLBP is unique to Entamoeba parasites, which makes this protein a possible drug target for treating amebiasis. In the work described here, we evaluated this potential. Downregulation of EhMLBP using antisense technology resulted in trophozoites with impaired growth and cytopathic activity. This indicated that EhMLBP is an essential protein. With a view to identifying new antiamebic agents, we tested the effect of distamycin A, a drug with known antimalarial activity, on the growth of the parasite and on the ability of EhMLBP to bind to DNA. Distamycin A (IC(50) = 13 microM) efficiently inhibited the growth of E. histolytica. Indeed, distamycin A at a concentration of 5-20 microM inhibited the binding of EhMLBP to methylated LINE DNA in vitro. As an additional approach to identify molecules that inhibit EhMLBP activity, a selective biopanning assay was performed using the DNA-binding domain of EhMLBP and the Ph.D.-12 phage display peptide library. Remarkably, four out of the 11 phages selected after three rounds of biopanning expressed the peptide 'SYFDQNERWGAP' (Pept3) at their surface. The binding of EhMLBP to Pept3 was confirmed by ELISA. Phage expressing Pept3 inhibited the binding of EhMLBP to RT LINE DNA. The growth of E. histolytica transfectants expressing Pept3 was significantly impaired compared with that of trophozoites expressing a scrambled version of Pept3. These results highlight EhMLBP as an essential constituent of the parasite E. histolytica and a novel target for antiamebic chemotherapy.

ABCG2-associated resistance to Hoechst 33342 and topotecan in a murine cell model with constitutive expression of side population characteristics.

Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations.

alpha-halogenoacrylic derivatives of antitumor agents.

In this review article we have reported a series of hybrid compounds characterized by the presence of a alpha-halogenocryloyl alkylating moiety of low chemical reactivity, linked to known antitumor agents or their active moieties. Among them, brostallicin (PNU-166196), was selected for clinical development and is now undergoing Phase II studies in patients with advanced or metastatic soft tissue sarcoma.

Bis-epoxyethyl derivatives of distamycin A modified on the amidino moiety: induction of production of fetal hemoglobin in human erythroid precursor cells.

Derivatives of distamycin A modified at the C-terminal amidine moiety and tethered to bis-epoxyethyl moieties at the N-terminal position were tested for their ability to induce erythroid differentiation in the human erythroleukemic cell line K562. None of the compounds without bis-epoxyethyl moiety were active. A comparison of the biological activity of diepoxy compounds containing different non-basic amidine-modified moieties, showed low activity of amidoxime, carbamoyl and N-methyl carbamoyl derivatives as differentiation agents. In contrast, a cyanamidine derivative, compound 3, was able to induce erythroid differentiation of K562 cells. In addition, the cyanamidine derivative 3 was able to induce HbF accumulation following treatment of cultures of erythroid precursor cells isolated from the peripheral blood of normal subjects.