Aryl ethynyl anthraquinones: a useful platform for targeting telomeric G-quadruplex structures

2,7-Diaryl ethynyl anthraquinones have been synthesized by Sonogashira cross-coupling and evaluated as telomeric G-quadruplex ligands, with good G-quadruplex/duplex selectivity

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an \u201cin house\u201d library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets. \ua9 2012 Elsevier Masson SAS. All rights reserved

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.This research was supported by a grant from MIUR, Rome (PRIN), University of Bologna (RFO) and Polo Scientifico-Didattico di Rimini (to V.T.). We thank the National Cancer Institute (Bethesda, MD) for the anticancer assays. MICINN (Spanish Government) is acknowledged for grant BIO2010-16351 (to J.F.D.). Lizzia Raffaghello is a recipient of MFAG Grant. Giovanna Bianchi is a recipient of a FIRC fellowship

SELECTIVE TARGETING OF NUCLEIC ACIDS BY SMALL MOLECULES: A DNA STRUCTURE RECOGNITION APPROACH

The discovery of new anticancer targets is the key factor for the development of more efficacious therapies. Sequence selective binding of double stranded DNA in the classical B form has been extensively employed to target small molecules to defined polynucleotide portions. More recently, ligand recognition of non canonical DNA foldings has been additionally considered a useful approach to selectively target distinct genomic regions. In this connection, G-quadruplexes represent an interesting system since they are believed to be physiologically significant arrangements. These non-canonical DNA structures are found at the ends of the human chromosomes (telomeres) as well as at promoter regions of several oncogenes where there is a cluster of guanine-rich sequences and they are likely to play important roles in the regulation of biological events. The induction and stabilization of the G-quadruplex arrangement by small molecules can lead to the inhibition of the telomerase activity by interfering with the interaction of the enzyme and its single stranded template. A similar molecular mechanism is likely involved in the transcriptional control that leads to the suppression of the oncogene transcription and, ultimately, in the regulation of the gene expression. As a result, the quadruplex topic is very attractive for the development of a specific anticancer strategy defined by a dramatic reduction of side effects, typical of chemotherapy. The purpose of this work is to investigate the interactions between novel classes of small molecules and different quadruplex DNA sequences and conformations. These new molecules were properly designed providing systematic atom-wise substitutions based on rational evaluations of previous studied compounds in order to increase their selectivity for G-quadruplex structures and to reduce toxic effects. Biophysical and biological properties of all new derivatives are herein evaluated at molecular and cellular level. The thesis work is divided into three main sections based on the structural features of the compounds object of study. The first part focuses on heterocyclic dications: upon changing their molecular binding shape, a correlation with G-quadruplex binding have been drawn. In particular it was possible to rationalize a shift in the binding modes, in particular between end stacking and groove recognition. Nevertheless a correlation between biophysical (G-quadruplex affinity) and biological (telomerase inhibition and cytotoxicity) results was not always clear. This feature may suggest the involvement of cellular targets different from the telomere and that are now under investigation. In Chapter 3, the DNA binding properties of some phenantroline derivatives in presence and in absence of Ni(II) and Cu(II) are investigated. We confirmed that different complex geometries involving one, two or three ligands per metal ion can affect the pattern of DNA recognition by driving nucleic acid conformational changes. Finally, in Chapter 4 some transplatin derivatives are evaluated. We focused our attention on defining the compounds capability to form adducts, with the nucleic acids, the nature of adducts and the kinetics of adduct formation not only on double strand DNA but also using single strand as well as G-quadruplex as targets. The results showed how different structural modifications can cooperate to greatly affect the potential interaction of the compounds. Interestingly it turned out their preference to react on single stranded DNA portions than to double stranded ones. This is probably due to an unfavourable orientation of the reactive groups when the molecule interacts with the DNA substrate. As a result, they appear to crosslink unpaired strands. By extending these results at cellular level they can reflect distinct distribution of platination site along the genome in comparison to cisplatin and even transplatin. The results obtained increment the available knowledge of DNA-small molecules interaction. In particular it emerged that a conserved interaction mode is consistent with biological effects. On the other hand, a shift in the binding mode can drive to different cytotoxic effects. This can provide a rationale for subsequent drug structure optimization leading to the development of new efficient and selective anticancer agents.La scoperta di nuovi target anticancro è il fattore chiave per lo sviluppo di terapie sempre più efficaci. Lo studio del legame selettivo a sequenze di DNA a doppia elica nella classica forma B è stato largamente impiegato al fine di direzionare piccole molecole verso porzioni polinucleotidiche definite. Più recentemente, il riconoscimento (da parte di ligandi) di porzioni non canoniche di DNA si può tradurre in un metodo vantaggioso per indirizzare questi composti verso regioni distinte del genoma. A tale proposito, le strutture G-quadruplex rappresentano un sistema interessante poiché sono ritenute fisiologicamente significative. Queste strutture “non-canoniche” di DNA si trovano alle estremità del cromosoma (telomeri) così come in varie regioni promotrici di oncogeni in cui vi è un’abbondante presenza di residui guaninici e sembrano coinvolte nella regolazione di importanti eventi biologici. Pare infatti che l'induzione e la stabilizzazione di strutture G-quadruplex dalle parte di piccole molecole porti all'inibizione dell'attività della telomerasi interferendo con l'interazione tra l’enzima e il suo substrato a singola catena. Un simile meccanismo molecolare è probabilmente coinvolto anche nel controllo della regolazione dell'espressione genica e può portare alla soppressione della trascrizione di un oncogene. Di conseguenza, “l’approccio G-quadruplex” si rivela molto interessante per lo sviluppo di una strategia anticancro specifica caratterizzata anche da una riduzione drammatica degli effetti collaterali, tipici della chemioterapia. Lo scopo di questo lavoro è lo studio delle interazioni tra nuove famiglie di piccole molecole e diverse conformazioni di DNA G-quadruplex. Queste nuove molecole sono state opportunamente progettate apportando sostituzioni di atomi o gruppi funzionali basate sulla valutazione di composti precedentemente studiati al fine di aumentare la loro selettività per strutture G-quadruplex e di ridurre gli effetti tossici. Le proprietà biofisiche e biologiche di tutti i nuovi derivati sono state valutate al livello molecolare e cellulare. Il lavoro di tesi si divide in tre parti in base alle caratteristiche strutturali dei composti. La prima parte è dedicata alla studio di dicationi eterociclici: si è cercato correlare modifiche nella conformazione molecolare con l’affinita’ verso strutture G-quadruplex. In particolare è stato possibile razionalizzare cambiamenti della modalità di legame in base alla struttura dei composti esaminati. Tuttavia una correlazione fra i risultati biofisici (affinità G-quadruplex) e biologici (inibizione della telomerasi e citotossicità) non è risultata sempre definita. Ciò può suggerire il coinvolgimento di bersagli cellulari diversi dal telomero umano. Nel capitolo 3, sono state studiate le proprietà di legame al DNA di alcuni derivati fenantrolinici in presenza ed in assenza di Ni (II) e Cu (II). Abbiamo confermato che complessi caratterizzati da diverse geometrie che coinvolgono una, due o tre molecole per ione possono compromettere o meno il riconoscimento del DNA o determinare cambiamenti conformazionali dell'acido nucleico. Per concludere, il capitolo 4 è dedicato allo studio di derivati del transplatino. In particolare ci siamo focalizzati nel definire la capacità dei composti di formare addotti, la natura dei complessi e la cinetica di formazione del complesso non solo con DNA a doppio filamento ma utilizzando anche substrati a singola catena come il G-quadruplex. I risultati hanno dimostrato come diverse modifiche strutturali possano avere un ruolo importante nell’interazione dei composti con gli acidi nucleici. E’ risultata interessante la loro preferenzialità a reagire con porzioni di DNA a singolo filamento rispetto a sequenze a doppia elica. Ciò è probabilmente dovuto ad uno sfavorevole orientamento dei gruppi reattivi quando la molecola interagisce con il substrato di DNA. Di conseguenza, i composti sembrano formare un cross-link tra due filamenti non appaiati. A livello cellulare, questi risultati riflettono una distinta distribuzione del sito di platinazione all’interno del genoma rispetto al cisplatino e perfino rispetto al transplatino. I risultati ottenuti incrementano la conoscenza disponibile sull’interazione tra DNA e piccole molecole. In particolare è emerso che la conservazione della modalità di interazione si correla con effetti biologici definiti. Al contrario, una variazione della modalità di legame può portare a effetti citotossici differenti. Ciò può fornire una spiegazione razionale per una successiva ottimizzazione della struttura dei composti finalizzata allo sviluppo di nuovi agenti antitumorali efficaci e selettivi

Heterocyclic Cations as Potential Anticancer Agents: An Approach that Targets G-quadruplex with Different Binding Modes

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity

Cationic porphyrin-\u200bruthenium(II) conjugates as potential selective stabilizers of G-\u200bquadruplex

Porphyrins and metalloporphyrins have been extensively investigated as potential anticancer drugs working as DNA binding agents as well as photosensitizer in the photodynamic therapy (PDT)\u200b. Conjugation of the macrocycle with organo-\u200bmetallic complexes have been considered a powerful tool to implement the soly. of the porphyrin and to provide addnl. cytotoxic properties. Here, we prepd. six four-\u200bfold sym. cationic porphyrin-\u200bruthenium(II) conjugates which differ one from each other's for the nature of the linker, the substituents on the Ru(II) fragments and the total pos. charge and we investigated their interaction with DNA templates different from the B form. Our data suggested that the tested conjugated are not selective toward the G-\u200bquadruplex form of the human telomeric sequence. Indeed, unfolded DNA efficiently works as matrix for the cationic conjugates. Nevertheless, they are extremely efficient in cleaving the macromol. upon irradn., irresp. of its structural arrangement

Molecular Basis for Differential Recognition of G-Quadruplex versus Double-Helix DNA by Bis-Phenanthroline Metal Complexes

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\u2013DNA ionic pair has ever been identified. These results provide a rationale for the selective recognition of distinct DNA arrangements in view of targeted pharmacological applications

Ni2+ and Cu2+ complexes of a phenanthroline-based ligand bind to G-quadruplexes at non-overlapping sites

Transition metal complexes allow fine tuning of DNA binding affinity and selectivity. Here we report on the nucleic acid recognition properties of a phenanthroline-based ligand coordinated to Ni(2+) or Cu(2+). The resulting complexes clearly bind to telomeric G-quadruplexes at different sites according to the nature of the bound metal ion