Exploring the interaction of biologically active compounds with DNA through the application of the SwitchSense technique, UV-Vis spectroscopy, and computational methods.

DNA is a key target for anticancer and antimicrobial drugs. Assessing the bioactivity of compounds involves in silico and instrumental studies to determine their affinity for biomolecules like DNA. This study explores the potential of the switchSense technique in rapidly evaluating compound bioactivity towards DNA. By combining switchSense with computational methods and UV-Vis spectrophotometry, various bioactive compounds' interactions with DNA were analyzed. The objects of the study were: netropsin (as a model compound that binds in the helical groove), as well as derivatives of pyrazine (PTCA), sulfonamide (NbutylS), and anthraquinone (AQ-NetOH). Though no direct correlation was found between switchSense kinetics and binding modes, this research suggests the technique's broader utility in assessing new compounds' interactions with DNA. used as analytes whose interactions with DNA have not been yet fully described in the literature.

Disruption of nucleosomes by DNA groove binders of clinical significance and implications for chromatin remodeling.

Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.

Transcriptional Regulation of Congocidine (Netropsin) Biosynthesis and Resistance.

The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.

Nonhost Disease Resistance in Pea: Chitosan's Suggested Role in DNA Minor Groove Actions Relative to Phytoalexin-Eliciting Anti-Cancer Compounds.

A stable intense resistance called "nonhost resistance" generates a complete multiple-gene resistance against plant pathogenic species that are not pathogens of pea such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph). Chitosan is a natural nonhost resistance response gene activator of defense responses in peas. Chitosan may share with cancer-treatment compounds, netropsin and some anti-cancer drugs, a DNA minor groove target in plant host tissue. The chitosan heptamer and netropsin have the appropriate size and charge to reside in the DNA minor groove. The localization of a percentage of administered radio-labeled chitosan in the nucleus of plant tissue in vivo indicates its potential to transport to site(s) within the nuclear chromatin (1,2). Other minor groove-localizing compounds administered to pea tissue activate the same secondary plant pathway that terminates in the production of the anti-fungal isoflavonoid, pisatin an indicator of the generated resistance response. Some DNA minor groove compounds also induce defense genes designated as "pathogenesis-related" (PR) genes. Hypothetically, DNA targeting components alter host DNA in a manner enabling the transcription of defense genes previously silenced or minimally expressed. Defense-response-elicitors can directly (a) target host DNA at the site of transcription or (b) act by a series of cascading events beginning at the cell membrane and indirectly influence transcription. A single defense response, pisatin induction, induced by chitosan and compounds with known DNA minor groove attachment potential was followed herein. A hypothesis is formulated suggesting that this DNA target may be accountable for a portion of the defense response generated in nonhost resistance.

Mechanism for the Binding of Netropsin to Hairpin DNA Revealed Using Nanoscale Ion Emitters in Native Mass Spectrometry.

Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand-DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand-DNA and DNA-DNA interactions can be rapidly obtained simultaneously. This research indicates a "simultaneous binding mechanism" for the interactions of netropsin with DNA.

Enzyme-free optical DNA mapping of the human genome using competitive binding.

Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.

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.

Towards the Shell Biorefinery: Sustainable Synthesis of the Anticancer Alkaloid Proximicin†A from Chitin.

A shell biorefinery would involve fractionation of crustacean shells and incorporation of the components into value-added products, particularly those that contain nitrogen. In a proof-of-concept study that validates this concept, the anticancer alkaloid proximicin A has been synthesized from the chitin-derived platform chemical 3-acetamido-5-acetylfuran (3A5AF). This study accentuates the leading role chitin is likely to play in the sustainable production of nitrogen-containing fine chemicals that are not directly attainable from lignocellulose.

ATPase activity of Plasmodium falciparum MLH is inhibited by DNA-interacting ligands and dsRNAs of MLH along with UvrD curtail malaria parasite growth.

Malaria caused by Plasmodium falciparum is the major disease burden all over the world. Recently, the situation has deteriorated because the malarial parasites are becoming progressively more resistant to numerous commonly used antimalarial drugs. Thus, there is a critical requirement to find other means to restrict and eliminate malaria. The mismatch repair (MMR) machinery of parasite is quite unique in several ways, and it can be exploited for finding new drug targets. MutL homolog (MLH) is one of the major components of MMR machinery, and along with UvrD, it helps in unwinding the DNA. We have screened several DNA-interacting ligands for their effect on intrinsic ATPase activity of PfMLH protein. This screening suggested that several ligands such as daunorubicin, etoposide, ethidium bromide, netropsin, and nogalamycin are inhibitors of the ATPase activity of PfMLH, and their apparent IC50 values range from 2.1 to 9.35 µM. In the presence of nogalamycin and netropsin, the effect was significant because in their presence, the V max value dropped from 1.024 µM of hydrolyzed ATP/min to 0.596 and 0.643 µM of hydrolyzed ATP/min, respectively. The effect of double-stranded RNAs of PfMLH and PfUvrD on growth of P. falciparum 3D7 strain was studied. The parasite growth was significantly inhibited suggesting that these components belonging to MMR pathway are crucial for the survival of the parasite.

Cell-based high-throughput compound screening reveals functional interaction between oncofetal HMGA2 and topoisomerase I.

HMGA2 is an important chromatin factor that interacts with DNA via three AT-hook domains, thereby regulating chromatin architecture and transcription during embryonic and fetal development. The protein is absent from differentiated somatic cells, but aberrantly re-expressed in most aggressive human neoplasias where it is causally linked to cell transformation and metastasis. DNA-binding also enables HMGA2 to protect cancer cells from DNA-damaging agents. HMGA2 therefore is considered to be a prime drug target for many aggressive malignancies. Here, we have developed a broadly applicable cell-based reporter system which can identify HMGA2 antagonists targeting functionally important protein domains, as validated with the known AT-hook competitor netropsin. In addition, high-throughput screening can uncover functional links between HMGA2 and cellular factors important for cell transformation. This is demonstrated with the discovery that HMGA2 potentiates the clinically important topoisomerase I inhibitor irinotecan/SN-38 in trapping the enzyme in covalent DNA-complexes, thereby attenuating transcription.

HMGA2 as a potential molecular target in KMT2A-AFF1-positive infant acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) in infants is an intractable cancer in childhood. Although recent intensive chemotherapy progress has considerably improved ALL treatment outcome, disease cure is often accompanied by undesirable long-term side effects, and efficient, less toxic molecular targeting therapies have been anticipated. In infant ALL cells with KMT2A (MLL) fusion, the microRNA let-7b (MIRLET7B) is significantly downregulated by DNA hypermethylation of its promoter region. We show here that the expression of HMGA2, one of the oncogenes repressed by MIRLET7B, is reversely upregulated in infant ALL leukaemic cells, particularly in KMT2A-AFF1 (MLL-AF4) positive ALL. In addition to the suppression of MIRLET7B, KMT2A fusion proteins positively regulate the expression of HMGA2. HMGA2 is one of the negative regulators of CDKN2A gene, which encodes the cyclin-dependent kinase inhibitor p16(INK4A) . The HMGA2 inhibitor netropsin, when combined with demethylating agent 5-azacytidine, upregulated and sustained the expression of CDKN2A, which resulted in growth suppression of KMT2A-AFF1-expressing cell lines. This effect was more apparent compared to treatment with 5-azacytidine alone. These results indicate that the MIRLET7B-HMGA2-CDKN2A axis plays an important role in cell proliferation of leukaemic cells and could be a possible molecular target for the therapy of infant ALL with KMT2A-AFF1.

The AT-hook DNA binding ability of the Epstein Barr virus EBNA1 protein is necessary for the maintenance of viral genomes in latently infected cells.

Epstein Barr Virus (EBV) is a human tumor virus that is causally linked to malignancies such as Burkitt׳s lymphoma, and gastric and nasopharyngeal carcinomas. Tethering of EBV genomes to cellular chromosomes is required for the synthesis and persistence of viral plasmids in tumor cells. However, it is not established how EBV genomes are tethered to cellular chromosomes. We test the hypothesis that the viral protein EBNA1 tethers EBV genomes to chromosomes specifically through its N-terminal AT-hook DNA-binding domains by using a small molecule, netropsin, that has been shown to inhibit the AT-hook DNA-binding of EBNA1 in vitro. We show that netropsin forces the loss of EBV genomes from epithelial and lymphoid cells in an AT-hook dependent manner and that EBV-positive lymphoma cells are significantly more inhibited in their growth by netropsin than are corresponding EBV-negative cells.

Semi-automatic synthesis, antiproliferative activity and DNA-binding properties of new netropsin and bis-netropsin analogues.

A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the all new substances prepared in this investigation were fully characterized by NMR ((1)H, (13)C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322 confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.

Compounds from marine-derived Verrucosispora sp. FIM06054 and their potential antitumour activities.

Strain FIM06054 was isolated from a marine sponge sample collected from the East China Sea and was characterised as a strain of Verrucosispora genus on the basis of its 16S rRNA gene sequence. One new compound, FW054-1 (1), together with a known aminofuran compound proximicin A (2), was isolated from the culture broth of Verrucosispora sp. FIM06054. Their structures were elucidated on the basis of spectral analysis. 1 and 2 showed antiproliferative activity against several human tumour cell lines.

Mining of the pyrrolamide antibiotics analogs in Streptomyces netropsis reveals the amidohydrolase-dependent "iterative strategy" underlying the pyrrole polymerization.

In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual "iterative strategy" underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.

Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth.

BACKGROUND: Human malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host. RESULTS: Recently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3' to 5' direction and interacts with MLH to modulate each other's activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 µM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 µM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo. CONCLUSIONS: As PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Conformational investigation of antibiotic proximicin by X-ray structure analysis and quantum studies suggest a stretched conformation of this type of γ-peptide.

The proximicins A-C are naturally occurring cytotoxic γ-peptides that contain the unique 4-amino-furan-carboxylic acid. In contrast to the structurally related cytotoxic natural DNA binder netropsin and distamycin, both exhibiting as core building block N-methyl-4-amino-pyrrol-carboxylic acid, no DNA binding was observed for the procimicins. X-ray analysis of crystals of a protected 4-amino-furan-2-carboxylic acid dipeptide revealed a stretched conformation. In contrast, for netropsin and distamycin, sickle-shaped crystal conformations were observed. DFT-calculations elegantly confirm these conformational arrangements. The most stable conformers of the proximicins are linear whereas sickle-shaped conformations are less stable, having higher Gibbs energies. For netropsin, distamycin and the netropsin-proximicin-hybrid a sickle shaped conformation appears energetically favored. The reported results are consistent with the observations that the proximicins A-C do not bind to the DNA and have a different mode of action concerning their cytotoxic activity with respect to netropsin and distamycin.

Interaction of marine Streptomyces compounds with selected cancer drug target proteins by in silico molecular docking studies.

The criteria currently followed for selecting antitumor compounds include agents that can target apoptosis inhibitor proteins and cancer cell markers. In silico studies are often used to identify suitable antitumor compounds for the cancer targets. The aim of the present study is to evaluate the interactions of some antitumor compounds reported from marine Streptomyces with cancer target proteins. Nine compounds were selected from marine Streptomyces based on previous reports and evaluated for their interactions with cancer target proteins by in silico molecular docking approach. Interactions of the selected ligand with target proteins were studied by PatchDock bioinformatics docking tool. Among the compounds tested marmycin A was interacted very effectively with human epidermal growth factor receptor 2 (HER2) and showed a least binding energy of -472.92 kcal/mol. The compound altemicidin showed a least binding energy of -415.66 kcal/mol with cyclin dependent kinase 4 (CDK4). The ligands resistoflavine and resistomycin also interacted with HER2 and showed the binding energy of -402.10 kcal/mol and -377.78 kcal/mol respectively. Other ligands proximycin A, chandrananimycin C, echinosporin, streptochlorin and streptokordin also showed the binding energy of -341.11 kcal/mol, -313.31 kcal/mol, -305.64 kcal/mol, -291.91 kcal/mol and 222.34 kcal/mol respectively with CDK4 protein. These results of our study suggest that HER2 and CDK4 are better cancer drug targets for therapy.

Cooperative stabilization of Zn(2+):DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA.

The simultaneous binding of netropsin in the minor groove and Zn(2+) in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3'-terminus (3'FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn(2+)/netropsin: 3'FdU complex had very high thermal stability with aspects of the complex intact at 85 °C, conditions that result in complete dissociation of Mg(2+) complexes. CD and (19)F NMR spectroscopy were consistent with Zn(2+) binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 (1)H and netropsin (1)H resonances. The Zn(2+)/netropsin: 3'FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn(2+):3'FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:32.

Structural studies on ligand-DNA systems: A robust approach in drug design.

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.