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.

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.

Carbocyclic Analogues of Distamycin and Netropsin.

The DNA as the depository of genetic information is a natural target for chemotherapy. A lot of anticancer and antimicrobial agents derive their biological activity from their selective interaction with DNA in the minor groove and from their ability to interfere with biological processes such as enzyme catalysis, replication and transcription. The discovery of the details of minor groove binding drugs, such as netropsin and distamycin A, oligoamides built of 4-amino-1-methylpyrrole-2-carboxylic acid residues, allowed to develop various DNA sequence-reading molecules, named lexitropsins, capable of interacting with DNA precisely, strongly and with a high specificity, and at the same time exhibiting significant cytotoxic potential. Among such compounds, lexitropsins built of carbocyclic sixmembered aromatic rings occupy a quite prominent place in drug research. This work is an attempt to present current findings in the study of carbocyclic lexitropins, their structures, syntheses and biological investigations such as DNA-binding and antiproliferative activity.

An evaluation of Minor Groove Binders as anti-fungal and anti-mycobacterial therapeutics.

This study details the synthesis and biological evaluation of a collection of 19 structurally related Minor Groove Binders (MGBs), derived from the natural product distamycin, which were designed to probe antifungal and antimycobacterial activity. From this initial set, we report several MGBs that are worth more detailed investigation and optimisation. MGB-4, MGB-317 and MGB-325 have promising MIC80s of 2, 4 and 0.25 µg/mL, respectively, against the fungus C. neoformans.MGB-353 and MGB-354 have MIC99s of 3.1 µM against the mycobacterium M. tuberculosis. The selectivity and activity of these compounds is related to their physicochemical properties and the cell wall/membrane characteristics of the infective agents.

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.

In vitro activity and mode of action of distamycin analogues against African trypanosomes.

Distamycin, a natural polyamide containing three heterocycle rings with a polar end, has inspired several groups to prepare synthetic analogues, which proved to have anti-trypanosomal and anti-tumoral activity. We describe the synthesis of bi and tri thiazoles amides that harbor different substitutions at their ends and the evaluation of their anti-Trypanosoma brucei activity. The most active compound 10b showed better biological activity (EC50 310 nM and selectivity index 16) than the control drug nifurtimox (EC50 15 µM and selectivity index 10). Studies on the mode of action show that the parasiticidal activity of 10b originates from disruption of lysosomal homeostasis, which is followed by release of redox active iron, an increase in oxidizing species and collapse of cell membrane integrity. In this respect, our study suggests that non-charged lipophylic distamycins destabilize cell membranes.

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.

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.

New Fe(iii) and Co(ii) salen complexes with pendant distamycins: selective targeting of cancer cells by DNA damage and mitochondrial pathways.

Minor groove binding distamycin like moieties were conjugated with core salens and the corresponding Fe(iii) and Co(ii) complexes were synthesized. Herein, we have shown efficient DNA minor groove binding specificities along with excellent DNA cleavage capacities with metallosalen conjugates. The metal complexes showed toxicity toward various cancer cells over normal cells with high specificity. Interestingly, the Co(ii) complexes exhibited greater activity than the Fe(iii) complexes in accordance with the stronger affinity of the former in the biophysical studies. Active DNA damage, and prominent nuclear condensation along with the release of cytochrome-c from the mitochondria unanimously showed that the metal complexes followed apoptotic pathways to induce cell death.

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.

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.

Minor groove binder distamycin remodels chromatin but inhibits transcription.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.

Probing the electrostatics and pharmacological modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1: a binding affinity and kinetics investigation.

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

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.

Effect of DNA groove binder distamycin A upon chromatin structure.

BACKGROUND: Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods--dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. CONCLUSIONS/SIGNIFICANCE: We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration.

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.

HPV episome levels are potently decreased by pyrrole-imidazole polyamides.

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s≤150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.