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.

Proximicins F and G and Diproximicin A: Aminofurans from the Marine-Derived Verrucosispora sp. SCSIO 40062 by Overexpression of PPtase Genes.

Overexpression of phosphopantetheinyl transferase (PPtase)-encoding genes sfp and svp in the marine-derived Verrucosispora sp. SCSIO 40062 led to the production of two new aminofuran monomers, proximicin F (1) and proximicin G (3) and a new dimer diproximicin A (2), along with two known compounds, proximicins B (4) and C (5). Their structures were unambiguously elucidated on the basis of detailed NMR spectroscopic analysis and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Proximicin B (4) showed moderate antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus, and Bacillus subtilis.

Monitoring DNA-Ligand Interactions in Living Human Cells Using NMR Spectroscopy.

Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

Efficacy of netropsin dihydrochloride against the viability, cytopathogenicity and hemolytic activity of Trichomonas vaginalis clinical isolates.

Trichomonas vaginalis (T. vaginalis) is a common sexually transmitted infection, affecting the urogenital tract. Trichomoniasis is customarily treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects and there is several reports on MTZ resistant T. vaginalis. Thus, the present study aimed to in-vitro evaluate the activity of DNA minor groove binder drug ''Netropsin dihydrochloride'' against metronidazole-sensitive T. vaginalis isolates (G and U isolates) and resistant T. vaginalis isolate (ATCC50138) (R isolate). Netropsin was tested at concentrations ranging from 3.5 to 200 µg/ml. It showed effectiveness against all isolates with MLC of 12.5 µg/ml for G and U isolates and of 25 µg/ml for R isolate. Cytotoxicity assay of isolates exposed to the respective MLC of netropsin for 42 h showed a highly significant reduction in the death percentage of MCDK cell line as compared to the effect elicited by drug free controls. The hemolytic activity was evaluated by hemolytic assay and by monitoring the interaction of T. vaginalis isolates with human erythrocytes by inverted microscopy and scanning electron microscopy. The hemolytic assay showed (0%) hemolysis of RBCs incubated with T. vaginalis isolates treated with the corresponding MLC of netropsin for 24 h. Scanning electron microscopy revealed cytoskeletal deformities of netropsin treated isolates. Taken together, these observations suggest that netropsin is a promising therapy for T. vaginalis infection affecting its viability, virulence, cytopathogenic and hemolytic activity with a mechanism of action that might overcome T. vaginalis resistance to metronidazole.

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.

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.

Anti-MRSA and anti-TB metabolites from marine-derived Verrucosispora sp. MS100047.

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.

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.

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.

DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

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.

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.

Complex of the herpes simplex virus type 1 origin binding protein UL9 with DNA as a platform for the design of a new type of antiviral drugs.

The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80 bp). The protein also binds to a single-stranded DNA (OriS*) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3'-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5'- and 3'- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А + Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.

[Estimation of activity of bis-netropsin derivatives based on a model of an experimental cutaneous herpes simplex virus disease of guinea pigs].

Using the model of an experimental cutaneous infection of guinea pig males caused by herpes simple virus type 1, it is shown that application of dimerico derivatives of netropsin Lys-bis Nt and 15Lys-bis Nt in the form of polietilenglicol-based ointment suppresses viral infection more effectively than acyclovir.

[Antiherpetic activity of netropsin derivatives as tested in experiments in laboratory animals].

Two dimeric netropsin derivatives (Lys-bis-Nt 15Lys-bis-Nt) were comprehensively tested for antiviral and toxic activity in cell cultures and laboratory animals. The two compounds were found to provide effective and selective inhibition of reproduction of herpes simplex I both in cell culture Vero E6 and in brain of infected white mice, thereby increasing the survival rate and mean life expectation of treated animals as compared to control.

[Inhibition of herpes simplex virus helicase UL9 by netropsin derivatives and antiviral activities of bis-netropsins].

Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies bis-netropsins get bound selectively to an A+T-cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA bis-netropsins stabilize a structure of the A+T-cluster and inhibit thermal fluctuation-induced opening of AT- base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3?- and 5?-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT- rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.

Efficient synthesis and biological evaluation of proximicins A, B and C.

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.

Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma.

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.

DNA site-specific N3-adenine methylation targeted to estrogen receptor-positive cells.

A compound that can target cells expressing the estrogen receptor (ER), and produce predominantly 3-MeA adducts in those cells has been designed and synthesized. This compound produces mainly the 3-MeA adduct upon reaction with calf thymus DNA, and binds to the ER with a relative binding affinity of 51% (estradiol = 100%). The compound is toxic to ER-expressing MCF-7 breast cancer cells, and pre-treatment with the ER antagonist fulvestrant abrogates the toxicity. Pre-treatment of MCF-7 cells with netropsin, which inhibits N3-adenine methylation by the compound, resulted in a threefold decrease in the toxicity. These results demonstrate the feasibility of this strategy for producing 3-MeA adducts in targeted cells.