1,721,023 research outputs found
Meccanismi d'azione di farmaci antitumorali in uso e in sperimentazione: attività a livello di topoisomerasi e telomerasi
Full text linkThe mechanism of action of antitumor drugs, both used in clinical practice and investigated for preclinical development, has been studied.
The first part of this thesis focused on the interactions between anticancer drugs currently used for treatment of solid malignancies. The study was aimed at optimizing drug combination and administration protocols, by analyzing the rational basis for their interaction both in vivo and in vitro. In particular, the effect of the interaction between alkylating agents, antimetabolites and antitopoisomerasic drugs on DNA topoisomerase expression was studied, based on the observation that topological enzymes are essential enzymes involved in most of DNA repair/replication processes. Indeed, the current study has been able to show that both antimetabolites and alkylating agents affect topoisomerases expression, and that higher cytotoxic effects are obtained when the topoisomerase poison is administered (at high doses) before the alkylating or antimetabolite drugs (at doses lower than their IC50).
In the second part, new synthetic quinone methide derivates have been studied. These display alkylating properties upon selective and controlled bioactivation. The aim of this thesis was to define the tested compound mechanism of action and intracellular molecular targets, for potential development of telomerase-targeted anticancer drugs.
Some of these innovative compounds have demonstrated to selectively recognize and alkylate telomeric sequences in their folded G-quadruplex conformation. Compounds presenting the naphtho diimidic nucleus and conformationally free reactive aromatic arms were the best stabilizers and binders of the G-quadruplex folded sequence. Moreover, preliminary studies highlighted an antiviral activity toward HSV-1, not directly dependent on the compound covalent reactivity
Clerocidin-mediated DNA Footprinting Discriminates among Different G-Quadruplex Conformations and Detects Tetraplex Folding in a Duplex Environment
No full textG-Quadruplex Visualization in Cells via Antibody and Fluorescence Probe
Full text linkG-quadruplexes (G4s) are noncanonical nucleic acids structures involved in key regulatory and pathological roles in eukaryotes, prokaryotes, and viruses: the development of specific antibodies and fluorescent probes represent an invaluable tool to understand their biological relevance. We here present three protocols for the visualization of G4s in cells, both uninfected and HSV-1 infected, using a specific antibody and a fluorescent G4 ligand, and the effect of the fluorescent ligand on a G4 binding protein, nucleolin, upon binding of the molecule to the nucleic acids structure
Reactivity of Clerocidin towards adenine: implications for base modulated DNA damage
No full textClerocidin is a complex natural molecule which induces DNA damage both directly and through irreversible/reversible poisoning of prokaryotic/eukaryotic topoisomerases II. By analysis of clerocidin reactivity towards adenine and thymine bases, we were able to fully characterize and compare the unique direct reactivity of clerocidin towards the four DNA bases, both in solution and in the DNA context. We showed that thymine was not reactive, while adenine gave a single stable covalent adduct, which was unambiguously identified as the 1,6-dialkylated species by means of modified clerocidin derivatives, modified adenine nucleotides, ESI-MS and multinuclear NMR spectroscopy. The mechanism of formation of the clerocidin adenosine adduct was similar to that occurring with cytosine, while being substantially different from that with guanine. An electrophoresis-based assay was able to highlight the unique ability of clerocidin to chemically discriminate among DNA nucleotides within a nucleic acid sequence. Finally, molecular modelling analysis gave useful indications to solve the apparent contradiction between direct and topoisomerase II-mediated covalent clerocidin reactivity with deoxyadenosine
Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment
Full text linkBACKGROUND: G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.
METHODS:
Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.
RESULTS:
The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlight stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.
CONCLUSIONS:
CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.
GENERAL SIGNIFICANCE:
Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions
Topoisomerase I involvement in schedule-dependent interaction between 5-fluoro-uracil and irinotecan in the treatment of colorectal cancer.
No full text5-Fluoro-uracil (5FU), an antimetabolite drug, stimulates expression of topoisomerase I (tpI) in adenocarcinoma cancer cells. When 5FU is given in combination with Irinotecan (IR), a tpI poison, the most effective regimen is represented by IR given before low doses of 5FU. Hence, despite their distinct mechanisms of action, the molecular basis for successful combination and schedule of 5FU and IR in the treatment of colorectal cancer rests on the opposing drug effects on the expression and poisoning of the tpI enzyme
The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids
Full text linkBACKGROUND:
G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants.
METHODS:
Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding.
RESULTS:
The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding.
CONCLUSIONS:
Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies.
GENERAL SIGNIFICANCE:
The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis
Synthesis and G-Quadruplex Binding and Selectivity of Novel Tri- and Tetra-Substituted Ethynyl Naphthalene Diimides
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