... Abstract #4994. G-quadruplex stabilization by anthraquinone-peptide conjugates. Claudia Sissi... more ... Abstract #4994. G-quadruplex stabilization by anthraquinone-peptide conjugates. Claudia Sissi,Lorena Lucatello, Sergio Alfredo Cadamuro, Giuseppe Zagotto, Manlio Palumbo and Keith Fox University, Padova, Italy and Unversity, Southampton, United Kingdom. ...
The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) ... more The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.
G-quadruplex structures at telomeric region and in oncogene promotorial sequences represent promi... more G-quadruplex structures at telomeric region and in oncogene promotorial sequences represent promising chemotherapeutic targets. Among these latter ones, an interesting site of intervention is represented by the promotorial sequence of KIT, a proto-oncogene whose overexpression or mutations characterize several human cancers. This region contains two G-rich sequences, kit1 and kit2, whose G-quadruplex structures have been extensively studied by using X-ray crystallography and NMR. The stabilization of these secondary structures by small molecules has been shown to result in a suppression of gene transcription. The peculiarity of these G-quadruplex structures rest in the presence of unprecedented structural domains which foresees the possibility to design small molecules able to recognize them, with a higher selectivity in comparison to other nucleic acid tetra-helixes. In order to identify novel ligands for these sequences, we started a screening program by using an available “in house” library of compounds. Each member was previously confirmed to provide a basal level of nucleic acid recognition. Ligand selection was performed by applying two validated different screening assays (FRET and FID); they were properly designed to identify the best binders and to preliminary exclude compounds with low selectivity among different G-quadruplexes. For selected compounds, the DNA binding properties were fully characterized in terms of binding affinity, binding mode and ability to convert unfolded sequences into a G-quadruplex structure. On parallel, the ligands cellular effects were explored in two human cancer cell lines (MCF7 and HCG27). At first, we evaluated their cytotoxic potential; then, we examined the extent of potential c-kit down-regulation following the exposure to candidates at both mRNA and protein level. By merging these results we were able to highlight the relevance of one pharmacophore as potential selective KIT targeting agent. Citation Format: Claudia Sissi, Silvia Da Ros, Eleonora Zorzan, Caterina Musetti, Lara Zorro Shahidian, Manlio Palumbo, Mery Giantin, Mauro Dacasto. Impairment of c-kit expression in human cancer cell lines by a novel pharmacophoric unit selected for the recognition of the proto-oncogene KIT promotorial region. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2015-4551
Selective recognition of DNA folding is central to multiple biological and pharmacological applic... more Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.
DNA sequences rich in guanines can fold up to form peculiar structures, known as a G-quadruplex, ... more DNA sequences rich in guanines can fold up to form peculiar structures, known as a G-quadruplex, deriving from pairing of four guanines by Hoogsteen-like hydrogen bonds. Nowadays, these structure(s) are being exploited as suitable targets for anti-cancer drug design. Indeed, G-quadruplexes can be formed at the telomers, the ends of human chromosomes as well as in the promoters of several important oncogenes such as c-myc, bcl-2, c-kit and VEGF. At physiological conditions an equilibrium between different conformations builds up. It has been shown that the interconversion between folded-unfolded conformation can be used by the cell to modulate the oncogene transcriptional state. Up-to-date, several classes of compounds have been tested and identified as efficient G-quadruplex binders. DNA recognition occurs mainly through stacking on a terminal G-tetrad, thus, these ligands share a general consensus structural motif based on a large flat aromatic surface. Several natural compounds characterized by cytotoxic activity present this structural motif. Here, we selected several structurally related natural derivatives of plant origin characterized by four fused rings with different degree of π electron delocalization. Among them, Berberine, Sanguinarine and Cheleritrine were identified as the most efficient stabilizers for G-quadruplex structures. Their interaction with various nucleic acid conformations was examined in detail in terms of affinity constant and mode of binding by NMR studies, spectroscopic (fluorometric and CD) and ITC titrations. In particular, we focussed our attention on the role of DNA sequence and structure on binding geometry and affinity. Biophysical data were then related to the cytotoxic response of each derivative. From these data we could asses the potential of the tested ligands as G-quadruplex binders (and prospective drug response) at physiological conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2674.
Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisom... more Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisomerase II function through stimulation of enzyme-mediated DNA cleavage. To clarify the drug structural requirements for stimulation of topoisomerase II DNA cleavage, the cytotoxic activity and molecular effects of mitoxantrone, ametantrone, and a new derivative (BBR2577), bearing a modification on one of the side chains, were examined in relation to their DNA binding affinities and modes of drug-DNA interaction. The results showed a good correlation between cytotoxicity and topoisomerase II DNA cleavage. The modification of one side chain did not influence the cytotoxic potency or the ability of the drug to stimulate DNA cleavage. In contrast, removal of the hydroxyl substituents in the planar aromatic moiety (ametantrone) markedly affected the efficacy of the drug. Ametantrone showed a markedly lower capacity, compared with the other two compounds, to induce cleavable complexes both in intact cells and in SV40 DNA, which suggests a critical role of these substituents in the formation of the ternary topoisomerase II-DNA-drug complex. The poor efficacy of ametantrone is likely due to low stability of the ternary complex. This is possibly related to a different orientation of the drug chromophore intercalated into DNA, compared with those of mitoxantrone and BBR2577. The DNA cleavage efficiencies of the tested drugs at low concentrations correlated with the DNA binding affinity. Identical DNA cleavage patterns were observed with the three compounds, which suggests that all tested drugs share a similar specificity for interaction with sites recognized by the enzyme.
The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl) amine (BAPA) metal binding u... more The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl) amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn (II) complexes of BAPA are known to ...
Proceedings of 2nd International Online-Conference on Nanomaterials, 2020
Phosphate diesters and plasmid DNA are cleaved by gold nanoparticles functionalized with Zn(II)-t... more Phosphate diesters and plasmid DNA are cleaved by gold nanoparticles functionalized with Zn(II)-triazacyclonononane complexes with different mechanisms, dinuclear and mononuclear, respectively, with impressive rate accelerations with respect to the uncatalyzed processes.
1,10‐Phenanthroline (Phen) derivatives are attractive ligands to provide metal complexes that are... more 1,10‐Phenanthroline (Phen) derivatives are attractive ligands to provide metal complexes that are selective for different DNA secondary structures. Herein, we analyze the binding processes of two bis‐Phen analogues and their NiII complexes toward double‐stranded DNA and telomeric G‐quadruplex DNA by calorimetric and spectroscopic techniques. The free ligands can adapt to both DNA arrangements. Conversely, metal ion coordination produces an increase in ligand affinity for the tetrahelical structure, whereas it dramatically decreases binding to double‐stranded DNA as a result of distinct binding modes on the two templates. In fact, NiII complexes effectively stack on the G‐quadruplex terminals, with an entropic loss counterbalanced by favorable enthalpy changes, whereas they cause a conformational reshaping of the double‐helix form with a substantial decrease in the binding free energy. Consistently, no NiII–DNA ionic pair has ever been identified. These results provide a rationale fo...
RNA G-quadruplexes have been recently proposed as physiologically relevant structures in cellular... more RNA G-quadruplexes have been recently proposed as physiologically relevant structures in cellular processes which suggests they represent attractive novel drug targets. Up-to-date, limited information of their interactions with small molecules is available. In particular, no systematic comparative analysis of the DNA vs RNA recognition processes has been performed. Here, we investigated the RNA G-quadruplex binding properties of a phenanthroline-based metal complex, (P115)Ni(II), known to efficiently recognize the DNA telomeric sequence. We showed that one ligand molecule can be sandwiched between two RNA G-quadruplex units. Conversely, this binding mode is not occurring using the DNA counterpart as the target sequence.
... Abstract #4994. G-quadruplex stabilization by anthraquinone-peptide conjugates. Claudia Sissi... more ... Abstract #4994. G-quadruplex stabilization by anthraquinone-peptide conjugates. Claudia Sissi,Lorena Lucatello, Sergio Alfredo Cadamuro, Giuseppe Zagotto, Manlio Palumbo and Keith Fox University, Padova, Italy and Unversity, Southampton, United Kingdom. ...
The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) ... more The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.
G-quadruplex structures at telomeric region and in oncogene promotorial sequences represent promi... more G-quadruplex structures at telomeric region and in oncogene promotorial sequences represent promising chemotherapeutic targets. Among these latter ones, an interesting site of intervention is represented by the promotorial sequence of KIT, a proto-oncogene whose overexpression or mutations characterize several human cancers. This region contains two G-rich sequences, kit1 and kit2, whose G-quadruplex structures have been extensively studied by using X-ray crystallography and NMR. The stabilization of these secondary structures by small molecules has been shown to result in a suppression of gene transcription. The peculiarity of these G-quadruplex structures rest in the presence of unprecedented structural domains which foresees the possibility to design small molecules able to recognize them, with a higher selectivity in comparison to other nucleic acid tetra-helixes. In order to identify novel ligands for these sequences, we started a screening program by using an available “in house” library of compounds. Each member was previously confirmed to provide a basal level of nucleic acid recognition. Ligand selection was performed by applying two validated different screening assays (FRET and FID); they were properly designed to identify the best binders and to preliminary exclude compounds with low selectivity among different G-quadruplexes. For selected compounds, the DNA binding properties were fully characterized in terms of binding affinity, binding mode and ability to convert unfolded sequences into a G-quadruplex structure. On parallel, the ligands cellular effects were explored in two human cancer cell lines (MCF7 and HCG27). At first, we evaluated their cytotoxic potential; then, we examined the extent of potential c-kit down-regulation following the exposure to candidates at both mRNA and protein level. By merging these results we were able to highlight the relevance of one pharmacophore as potential selective KIT targeting agent. Citation Format: Claudia Sissi, Silvia Da Ros, Eleonora Zorzan, Caterina Musetti, Lara Zorro Shahidian, Manlio Palumbo, Mery Giantin, Mauro Dacasto. Impairment of c-kit expression in human cancer cell lines by a novel pharmacophoric unit selected for the recognition of the proto-oncogene KIT promotorial region. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2015-4551
Selective recognition of DNA folding is central to multiple biological and pharmacological applic... more Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.
DNA sequences rich in guanines can fold up to form peculiar structures, known as a G-quadruplex, ... more DNA sequences rich in guanines can fold up to form peculiar structures, known as a G-quadruplex, deriving from pairing of four guanines by Hoogsteen-like hydrogen bonds. Nowadays, these structure(s) are being exploited as suitable targets for anti-cancer drug design. Indeed, G-quadruplexes can be formed at the telomers, the ends of human chromosomes as well as in the promoters of several important oncogenes such as c-myc, bcl-2, c-kit and VEGF. At physiological conditions an equilibrium between different conformations builds up. It has been shown that the interconversion between folded-unfolded conformation can be used by the cell to modulate the oncogene transcriptional state. Up-to-date, several classes of compounds have been tested and identified as efficient G-quadruplex binders. DNA recognition occurs mainly through stacking on a terminal G-tetrad, thus, these ligands share a general consensus structural motif based on a large flat aromatic surface. Several natural compounds characterized by cytotoxic activity present this structural motif. Here, we selected several structurally related natural derivatives of plant origin characterized by four fused rings with different degree of π electron delocalization. Among them, Berberine, Sanguinarine and Cheleritrine were identified as the most efficient stabilizers for G-quadruplex structures. Their interaction with various nucleic acid conformations was examined in detail in terms of affinity constant and mode of binding by NMR studies, spectroscopic (fluorometric and CD) and ITC titrations. In particular, we focussed our attention on the role of DNA sequence and structure on binding geometry and affinity. Biophysical data were then related to the cytotoxic response of each derivative. From these data we could asses the potential of the tested ligands as G-quadruplex binders (and prospective drug response) at physiological conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2674.
Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisom... more Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisomerase II function through stimulation of enzyme-mediated DNA cleavage. To clarify the drug structural requirements for stimulation of topoisomerase II DNA cleavage, the cytotoxic activity and molecular effects of mitoxantrone, ametantrone, and a new derivative (BBR2577), bearing a modification on one of the side chains, were examined in relation to their DNA binding affinities and modes of drug-DNA interaction. The results showed a good correlation between cytotoxicity and topoisomerase II DNA cleavage. The modification of one side chain did not influence the cytotoxic potency or the ability of the drug to stimulate DNA cleavage. In contrast, removal of the hydroxyl substituents in the planar aromatic moiety (ametantrone) markedly affected the efficacy of the drug. Ametantrone showed a markedly lower capacity, compared with the other two compounds, to induce cleavable complexes both in intact cells and in SV40 DNA, which suggests a critical role of these substituents in the formation of the ternary topoisomerase II-DNA-drug complex. The poor efficacy of ametantrone is likely due to low stability of the ternary complex. This is possibly related to a different orientation of the drug chromophore intercalated into DNA, compared with those of mitoxantrone and BBR2577. The DNA cleavage efficiencies of the tested drugs at low concentrations correlated with the DNA binding affinity. Identical DNA cleavage patterns were observed with the three compounds, which suggests that all tested drugs share a similar specificity for interaction with sites recognized by the enzyme.
The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl) amine (BAPA) metal binding u... more The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl) amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn (II) complexes of BAPA are known to ...
Proceedings of 2nd International Online-Conference on Nanomaterials, 2020
Phosphate diesters and plasmid DNA are cleaved by gold nanoparticles functionalized with Zn(II)-t... more Phosphate diesters and plasmid DNA are cleaved by gold nanoparticles functionalized with Zn(II)-triazacyclonononane complexes with different mechanisms, dinuclear and mononuclear, respectively, with impressive rate accelerations with respect to the uncatalyzed processes.
1,10‐Phenanthroline (Phen) derivatives are attractive ligands to provide metal complexes that are... more 1,10‐Phenanthroline (Phen) derivatives are attractive ligands to provide metal complexes that are selective for different DNA secondary structures. Herein, we analyze the binding processes of two bis‐Phen analogues and their NiII complexes toward double‐stranded DNA and telomeric G‐quadruplex DNA by calorimetric and spectroscopic techniques. The free ligands can adapt to both DNA arrangements. Conversely, metal ion coordination produces an increase in ligand affinity for the tetrahelical structure, whereas it dramatically decreases binding to double‐stranded DNA as a result of distinct binding modes on the two templates. In fact, NiII complexes effectively stack on the G‐quadruplex terminals, with an entropic loss counterbalanced by favorable enthalpy changes, whereas they cause a conformational reshaping of the double‐helix form with a substantial decrease in the binding free energy. Consistently, no NiII–DNA ionic pair has ever been identified. These results provide a rationale fo...
RNA G-quadruplexes have been recently proposed as physiologically relevant structures in cellular... more RNA G-quadruplexes have been recently proposed as physiologically relevant structures in cellular processes which suggests they represent attractive novel drug targets. Up-to-date, limited information of their interactions with small molecules is available. In particular, no systematic comparative analysis of the DNA vs RNA recognition processes has been performed. Here, we investigated the RNA G-quadruplex binding properties of a phenanthroline-based metal complex, (P115)Ni(II), known to efficiently recognize the DNA telomeric sequence. We showed that one ligand molecule can be sandwiched between two RNA G-quadruplex units. Conversely, this binding mode is not occurring using the DNA counterpart as the target sequence.
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