THE DISCOVERY AND CHARACTERIZATION OF NOVEL POTENT 5-SUBSTITUTED 3, 3’, 4’, 7-TETRAMETHOXYFLAVONOID DNA TRIPLEX SPECIFIC BINDING LIGANDS

Chemotherapy works by killing fast dividing cells. Unfortunately, these drugs are not specific to cancer tissue and can damage normal cells. Chemotherapy is like taking poison and hoping it kills the cancer cells before it kills you. As an alternative, many researchers have investigated the use of antigene therapy to selectively target cancer causing genes to avoid off target effects. Although promising, the theory is limited by the stability of the triplex structure. Here, we report the discovery of potent triplex binding ligands derived from the natural product quercetin. Chemical derivatives of 5-substituted 3, 3’, 4’, 7-tetramethoxyquercetin derivatives were characterized using several biophysical methods: thermal denaturation monitored by UV, circular dichroism, viscometry, differential scanning calorimetry, and isothermal titration calorimetry. The data revealed that these derivatives specifically stabilize triplex DNA and do not influence the stability of duplex DNA, triple RNA, or duplex RNA. Structurally, the amino containing side chains at the 5-position and the linker length are critical for the observed binding affinity and specificity. Two derivatives, 5 and 7, are comparable (if not better) to the triplex groove binder Neomycin. Our data confirm the binding mode as enthalpically driven intercalation. Piperidine or pyrrolidine 5-substituted 3, 3’, 4’, 7-tetramethoxyquercetin derivatives with a three-carbon linker are the lead compounds for development as a potential antigene enhancer

Interactions of flavones and related compounds with nucleic acids.

This project focused on a group of active ingredients isolated from herbal medicines, most of them flavonoids, as a foundation for the development of new anticancer drugs. Interactions between these compounds and genomic DNA, synthetic polynucleotides, or higher order DNA isoforms (triple and quadruple helical forms) have been studied using a range of physicochemical and biological techniques. The studies demonstrated that flavonoids and isoflavonoids bind to the various nucleic acid forms with weak or moderate affinities and no significant specificity, apart from quercetin that demonstrated a differential binding for G-quadruplex structures. Rational drug design has been subsequently employed to elucidate the mode of binding and novel compounds have been synthesized in order to provide precise structure-activity related studies and result in a second generation of chemotherapeutic anticancer agents with improved properties. Interaction with DNA is thought to involve the planar ring structures. The double bond of the O in C4 in a flavone scaffold gives planarity to the molecule, and this allows the molecule to intercalate between the bases in the different nucleic acid structures. The comparison between flavone, flavanone and isoflavonoid indicates that the position of the B ring and the double bond in the C ring are important for the interactions with DNA. Introduction of nitrogen in the ring did not improve the binding but tertiary amines improved binding 100 fold. Introduction of sulfur produced two binding constants. Position 7 in the A ring of the flavones is extremely relevant for the binding to the nucleic acids, but substitutions in the B ring did not improve the binding. Methoxylation or acetoxylation in positions 5 and 7 decreased the affinity for DNA. Novel compounds were tested for their specificity against different DNA isoforms and their anticancer activity in various tumour cell lines

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future

DNA related enzymes as molecular targets for antiviral and antitumoral chemotherapy. A natural overview of the current perspectives

Background: The discovery of new chemotherapeutic agents still remains a continuous goal to achieve. DNA polymerases and topoisomerases act in nucleic acids metabolism modulating different processes like replication, mitosis, damage repair, DNA topology and transcription. It has been widely documented that Polymerases serve as molecular targets for antiviral and antitumoral chemotherapy. Furthermore, telomerase is a ribonucleoprotein with exacerbated activity in most of the tumor cell lines, becoming as an emergent target in Cancer treatment. Methods: We undertook an exhaustive search of bibliographic databases for peer-reviewed research literature related to the last decade. The characteristics of screened bibliography describe structure activity relationships and show the principal moieties involved. This work tries to summarize the investigation about natural and semi-synthetic products with natural origin with the faculty to inhibit key enzymes that play a crucial role in DNA metabolism. Results: Eighty-five data references were included in this review, showing natural products widely distributed throughout the plant kingdom and their bioactive properties such as tumor growing inhibitory effects, and anti-AIDS activity. Conclusion: The findings of this review confirm the importance to find new drugs and biologically active natural products, and their potential medicinally useful benefits.Fil: Garro, Hugo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Pungitore, Carlos Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; Argentin

Exploring the stabilizing effect on the i-motif of neighboring structural motifs and drugs

Cytosine-rich DNA sequences may fold into a structure known as i-motif, with potential in vivo modulation of gene expression. The stability of the i-motif is residual at neutral pH values. To increase it, the addition of neighboring moieties, such as Watson-Crick stabilized loops, tetrads, or non-canonical base pairs have been proposed. Taking a recently described i-motif structure as a model, the relative effect of these structural moieties, as well as several DNA ligands, on the stabilization of the i-motif has been studied. To this end, not only the original sequence but different mutants were considered. Spectroscopic techniques, PAGE, and multivariate data analysis methods have been used to model the folding/unfolding equilibria induced by changes of pH, temperature, and the presence of ligands. The results have shown that the duplex is the moiety that is responsible of the stabilization of the i-motif structure at neutral pH. The T:T base pair, on the contrary, shows little stabilization of the i-motif. From several selected DNA-binding ligands, the G-quadruplex ligand BA41 is shown to interact with the duplex moiety, whereas non-specific interaction and little stabilization has been observed within the i-motif

The role of miRNAs in regulating the expression of flavonol pathway genes and its possible impact on the crosstalk between UV-B and flg22 signal cascades in Arabidopsis thaliana

In cell culture, we have shown that flavonol metabolite accumulation depends on expression of the Arabidopsis flavonol pathway genes (FPGs), which are upregulated by UV-B irradiation but repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (MTI). The suppression of flavonoid production during MTI is believed to allow the plant focusing its metabolism on the pathogen defense by directing phenylalanine resources from UV-B protective flavonol production towards production of phytoalexins and cell wall fortification by lignin incorporation during MTI. Here we extend our observations made initially in cell cultures by describing that this kind of signal crosstalk between UV-B and flg22 is also functional in planta. We demonstrate that such signal crosstalk is fully functional in Arabidopsis in planta. However, we observed some differences in the expression patterns of MYB transcriptions factors (TFs) as compared to data from the cell culture system. Our data suggest that in planta the TF MYB111 might play a more dominant role than the TF MYB12, which was strongly regulated in cell cultures. Thus we can present an updated working model how this crosstalk might function. We believe that this system based on seedlings of the model plant Arabidopsis thaliana constitutes a valuable platform for further dissection of the underlying molecular mechanism, e.g. by deploying gain/loss-of-function of candidate genes. Plants are confronted with various abiotic and biotic stress factors in their natural habitats, and have evolved sophisticated and multifaced mechanisms to defense themselves. In our previous works, we have demonstrated the crosstalk between flg22 and UV-B-induced signal cascades in plants, in which the expression of Arabidopsis flavonol pathway genes (FPGs) are upregulated by UV-B irradiation but simultaneously repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (PTI). Although several transcription factors proved to be involved in the crosstalk, the underlying regulatory mechanism remains largely unsolved. By deep sequencing we identified 217 miRNAs representing 204 conserved and 13 novel miRNAs involved in the crosstalk. Among them, e.g. 106 miRNAs were upregulated by flg22 and downregulated by UV-B. Furthermore, a set of specific interactions between miRNAs and their targets were confirmed showing reciprocal changes in their expression levels, simultaneously. As revealed by GO and KEGG analysis in silico, predicted miRNA target genes participate in a series of plant biological and molecular processes as well secondary metabolism pathways. Two modulations of miRNA-target interactions were obtained in the crosstalk. The first one consisting of miR158, miR165, miR166, miR167, miR168, miR172, miR391, miR393, miR447, miR824, miR828, miR846 and miR858, which were all repressed by UV-B irradiation, while upregulated by flg22, and the second one constitute miR159, miR164, miR171 and miR822, being repressed by flg22 and upregulated by UV-B. Both miRNA sets display converse regulation with the change in transcript levels of their targets. Furthermore, we demonstrated that knockdown of miR858 (Group I) in Arabidopsis goes along with increased the chalcone synthase (CHS) expression while its overexpression results in its depression. Vice versa, knockout of miR164b (Group II) depresses the CHS gene expression while its overexpression strongly elevated the CHS gene expression, providing the first genetic evidence that miRNAs identified in this study constitute an additional layer in regulating the crosstalk between flg22 and UV-B induced signaling cascades. In nature plants are often simultaneously challenged by different stress factors. The abiotic stress UV-B irradiation induces the production of UV-protective flavonols, but their accumulation is attenuated by biotic stress, e.g. by treatment with pathogen elicitors (flg22). This suppression has been shown to occur via suppression of flavonol pathway genes (FPGs) enabling the plant to direct its secondary metabolism to a more efficient pathogen defense response. Identification of two highly conserved miRNAs (miR858 and miR828) being involved in the crosstalk and their targets MYB111 and MYB75 that proved to play an important role in regulation of FPGs and the flavonoid accumulation provoked us to assume that miR858-MYB111 and miR828-MYB75 interactions play an important role in the crosstalk. Here, we demonstrate that both miR858 and miR828 are regulated by UV-B and flg22 in a UVR8 and FLS2 receptors-dependent manner and relying on the respective signaling pathways. Comparison between miR858/MYB111-promoter-GUS and their transcript levels evidences that the MYB111 is regulated not only at the transcriptional level, but also suffered from post-transcriptional modification in response to the flg22 and UV-B challenges, in which miR858 acts as a determinant regulator. Following this, we conclude that the post- transcriptional regulation mediated by plant-derived miRNAs constitutes the crosstalk between the flg22 and UV-B induced signal cascades in Arabidopsis. This allows an extension of the crosstalk model between plant responses to biotic (flg22) and abiotic (UV-B) stresses in Arabidopsis

Flavone: the Molecular and Mechanistic Study of How a Simple Flavonoid Protects DNA from Oxidative Damage.

Dietary flavonoids are ubiquitous and are marketed as supplements. Characterized as antioxidants, they offer protection against a number of degenerative diseases. Flavonoid mechanics involve free radical scavenging, metal chelation, and substrate association. The skeletal structure of flavonoids is a fused ring system modified by hydroxyl, sugar, and carbohydrate additions. Flavone is a structurally simple flavonoid. Quercetin and its glycosidic analog rutin are complex structures. Using a DNA oxidation/cleavage assay, flavone reduces DNA nicking by 91%. Depending on the solvent system used, quercetin can either increase or decrease DNA oxidation. Rutin exhibits neither pro- nor antioxidant activity. The molecular interactions responsible for these results are defined for flavone. 1) Flavone intercalates into DNA and saturates DNA at a 1/3.5 flavone:DNA molar ratio. 3) Flavone reduces iron-dependent DNA oxidation. 4) Flavone interacts with quercetin to enhance DNA protection. These results characterize the primary activities of a simple flavonoid

Anti- Japanese-Encephalitis-Viral Effects of Kaempferol and Daidzin and Their RNA-Binding Characteristics

Background: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an a-1 translational frameshift to synthesize the NS1 ’ protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described. Methodology/Principal Findings: The 50 % cytotoxic concentration (CC50) and 50 % effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC 50 values of Kae and Dai were 12.6 and 25.9 mM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 mM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes wit

Development And Study Of Inhibitors Of Heat Shock Protein 70 Induction

Tumor and cancer cells that over express heat shock protein 70: HSP70) are found to be multidrug resistant and thermo tolerant, creating a hurdle to existing therapy. Although HSP70 is recognized as an increasingly important drug target, the protein structure of this highly conserved chaperone remains challenging for direct targeting. An alternative strategy is to inhibit the transcription of HSP70. Among known small molecule inhibitors of HSP70 induction, quercetin has very low toxicity and has the advantage of being easily modified for structure-activity studies. One part of the dissertation focuses on the identification of quercetin derivatives with improved specificity and activity, and to determine the protein targets of quercetin responsible for inhibition of heat shock induction of HSP70. A library of quercetin derivatives was synthesized and screened for their ability to inhibit HSP70 induction and at the same time not enhance HSP27 phosphorylation. The derivatives that inhibit HSP70 induction were also found to be inhibitors of both Ck2 kinase and CaMKII kinase that are known to activate heat shock transcription factor 1 that leads to HSP70 induction. A biotinylated quercetin affinity agent was also developed that was able to pull down the CK2 kinase target in vitro and several other proteins in vivo under UVA irradiation. In collaboration with mass spectrometry center, these unknown protein targets were identified by proteomic studies, and found to be previously identified chemotherapeutic targets. Another goal of this dissertation was to develop polyamide-based gene inhibitors of heat shock induction that interfere with the binding of the heat shock transcription factor. A series of polyamides that targeted the heat shock elements were designed and synthesized and demonstrated to bind the target DNA by DNase I footprinting. These polyamides were evaluated by gel shift assay for their ability to block binding of the heat shock factor and the most effective polyamide was shown to decrease HSP70 expression in Jurkat cells by western blot assay