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Analysis of cross-linking between DNA and anthracyclines
Full text linkAnthracyclines are a group of chemotherapeutics that include adriamycin (doxorubicin), daunorubicin, idarubicin, and epirubicin. Anthracyclines are active against a wide range of tumours, in particular, adriamycin is used in the treatment of breast cancer, Hodgkin’s lymphoma, lung cancer, multiple myeloma and re-occurring ovarian cancer. Despite the broad spectrum of actions, resistance or severe cardio-toxicity limits the use of these important anticancer-drugs. The search for a “better anthracycline” has resulted in more than 2000 analogs, but only a few more anthracyclines have attained clinical approval. Although the exact mechanism by which adriamycin exerts its anti-tumour activity is uncertain, the dominant mechanism appears to involve impairment of topoisomerase IIα activity consistent with observed DNA intercalation and nuclear localization. The search for less toxic and more effective anthracyclines has led to the discovery of nemorubicin, a doxorubicin derivative in which the amino nitrogen of the daunosamine unit is incorporated into a methoxymorpholinyl ring. Preclinical investigations showed that nemorubicin, unlike classic anthracyclines, is not cardiotoxic and retains antitumour activity in various multidrug-resistant tumor models. Encouraging results have been obtained in phase I/II clinical trials in which the drug was administered by the intra-hepatic artery route. Nemorubicin is 80-120 times more potent than doxorubicin in vivo; in contrast, its in-vitro activity is only eight times greater than doxorubicin’s toward cultured drug sensitive tumour cells. A recent study established that nemorubicin is converted by enzyme CYP3A in a more cytotoxic metabolite PNU-159682, which was found to be 700-2400 times more potent that its parent drug toward cultured human cancer cells and which exhibits significant efficacy in in vivo tumor models. Ongoing studies aimed at exploring the molecular mechanism of action of PNU-159682 indicate that it has different effects on cell cycle progression and different DNA interacting properties, compared to both MMDX and doxorubicin. Moreover, further recent data suggest that PNU-159682 retains its activity against tumor cell lines with mechanisms of resistance different from those classical anticancer agents including MDR-1 gene overexpression, reduced topoisomerase II activity, and mutations in the topoisomerase I gene, these latter genetic alterations conferring resistance in vitro to the parent drug, MMDX [1]. We used different experimental approaches aimed to rationalizing the high activity of this metabolite. Test in vitro performed in our laboratory with kinetoplast DNA confirmed the inactivity of the metabolite against topoisomerase IIα. The absence of activity toward topoisomerase suggests that the high cytotoxicity of this compound had to be searched elsewhere. Anthracyclines such as doxorubicin and daunorubicin can bind covalently the DNA when activated with formaldehyde. Moreover, anthracyclines that have intrinsic ability to form cross-links to the DNA, such as cyanomorpholinyl-doxorubicin or barminomycin, were found to exhibit high cytotoxicity comparable with the PNU. Then we considered the possibility that PNU interacts with the DNA as a preactivated anthracycline. Our work evidenced that PNU behaves similarly to the activated doxorubicin (doxorubicin mixed with H2CO) in DNA melting analyses. PNU quickly reacts with double-strand oligonucleotides to form adducts detectable by DPAGE. These adducts are sufficiently stable to be isolated by HPLC. Mass characterization confirmed that these complexes are formed by duplex DNA bound to the anthracycline. These investigations suggest that the reaction between PNU and DNA does not involve the formation of a classical cross-link, but in relation to the electrophoretic, chromatographic and mass spectrometry results these adducts can be ascribed to the family of “virtual cross-link” (VXL). Anthracyclines-formaldehyde conjugate or anthracyclines in formaldehyde buffer have the specific ability to intercalate into DNA, forming covalent bonding; a methylene bridge links the amino group of the anthracycline to the 2-amino group of a G-base in the minor groove, while the other strand of DNA is stabilized by hydrogen bonds. Such unusual combination of intercalation, covalent bonding and hydrogen bonding is referred to as the virtual cross-linkink [2], that leads to the formation of more stable complexes between the anthracyclines and the DNA, improving the drugs’ cell killing ability. Among the different mechanisms of anticancer activity of anthracyclines, anthracycline-DNA adducts formation elicited interest related to the possibility to find safer and more efficacious anticancer drugs. Anthracycline-formaldehyde conjugates and cross-linking anthracyclines exhibit high cytotoxicity comparable to classical cross-linking drugs. We used different anthracyclines aimed to rationalize the structure activity relationship for the formation of VXL. We confirmed by electrophoretic and chromatographic analyses that aminosugar and its amino nitrogen is absolutely necessary for the formation of “VXL” and we discussed the role of the 4' position of the daunosamine in modulation of this activity. The presence of methylene bridge and its relationship with guanine was confirmed by mass spectrometry.Le antracicline sono un’importante famiglia di chemioterapici, tra queste sono usate prevalentemente l’adriamicina (doxorubicina), la daunorubicina, l’idarubicina e l’epirubicina. Sono farmaci con un ampio spettro d’azione, in particolare l’adriamicina è usata nel trattamento del cancro al seno, del linfoma di Hodgkin, del cancro al polmone, del mieloma multiplo e del cancro ovarico recidivo. Nonostante l’ampio spettro d’azione la resistenza e la severa cardiotossicità limitano l'uso di questi importanti farmaci anticancro. La ricerca di migliori derivati ha dato luogo a più di 2000 analoghi dei quali solamente pochi di essi hanno raggiunto l’approvazione clinica. Anche se il meccanismo esatto con il quale l’adriamicina esercita l’attività anticancro è incerta, il meccanismo principale coinvolge un danno nei confronti dell’enzima topoisomerasi II, un meccanismo supportato dall’intercalazione nel DNA e dalla localizzazione nucleare. La ricerca di antracicline meno tossiche e più efficaci ha portato alla scoperta della nemorubicina, un derivato della doxorubicina in cui l'azoto amminico della daunosamina è incorporato in un anello metossimorfolinico. Le indagini precliniche hanno mostrato che la nemorubicina, diversamente dalle antracicline classiche non è cardiotossica e l'attività antitumorale è mantenuta nei vari modelli di tumore resistenti alla terapia. Risultati incoraggianti sono stati ottenuti in fase I/II dove il composto è stato somministrato attraverso l’arteria intraepatica. La nemorubicina è 80-120 più potente della doxorubicina in vivo, diversamente la sua attività in vitro è solamente otto volte rispetto alla doxorubicina verso culture cellulari tumorali sensibili alle antracicline. Un recente studio ha stabilito che la nemorubicina è convertita dall’enzima CYP3A in un metabolita estremamente più citotossico, il PNU-159682. Questo metabolita è risultato dalle 700 alle 2400 volte più potente rispetto al progenitore nemorubicina verso cellule cancerose umane in coltura e ha mostrato un’efficacia significativa in diversi modelli di tumore in vivo. Studi in corso finalizzati a definire il meccanismo molecolare di azione di PNU-159682 indicano differenti effetti sul ciclo cellulare e una differente interazione col DNA rispetto al progenitore nemorubicina e alla doxorubicina. Inoltre, dati recenti indicano che PNU-159682 mantiene la sua attività anche verso cellule aventi diversi meccanismi di resistenza rispetto a diversi agenti anticancro classici, inclusa la sovraespressione del gene MDR-1, la riduzione dell’attività di topoisomerasi II e mutazioni nel gene codificante per la topoisomerasi I: quest’ultima modifica genetica conferisce resistenza in vitro alla nemorubicina [1]. Noi abbiamo usato diversi approcci sperimentali per razionalizzare l’elevata attività di questo metabolita. Test condotti in vitro nel nostro laboratorio con kinetoplast DNA hanno confermato l'inattività del metabolita nei confronti della topoisomerasi II. L'assenza dell'attività verso la topoisomerasi ci suggerisce che l’alta citotossicità di questo metabolita è da ricercarsi altrove. Antracicline come doxorubicina e daunorubicina possono legare covalentemente il DNA quando attivate con formaldeide. Inoltre è stato trovato che antracicline che hanno un’intrinseca attività a formare cross-link col DNA come la cianomorfolino-doxorubicina o la barminomicina posseggono un’alta citotossicità comparabile col PNU. Quindi abbiamo considerato la possibilità che il PNU interagisca col DNA come una antraciclina preattivata. Il nostro lavoro ha evidenziato che il PNU si comporta in modo analogo alla doxorubicina attivata (doxorubicina con formaldeide) nelle analisi di melting del DNA. PNU reagisce velocemente con oligonucleotidi a doppio filamento per formare addotti visualizzati in DPAGE. Questi addotti sono sufficientemente stabili per essere isolati tramite HPLC. La caratterizzazione ottenuta tramite spettrometria di massa ha confermato che questi complessi sono formati da DNA a doppio filamento legato all’antraciclina. Questi studi suggeriscono che la reazione tra PNU e DNA non coinvolge la formazione di un classico cross-link, ma in relazione ai risultati elettroforetici, cromatografici e di spettrometria di massa questi addotti possono essere annoverati nella famiglia dei “virtual cross-link” (VXL). I coniugati antracicline-formaldeide o le antracicline in tampone contenente formaldeide hanno la specifica abilità di intercalarsi nel DNA formando legami covalenti; un ponte etilenico lega l’ammino gruppo dell’antraciclina col 2-amino gruppo della base guaninica nel solco minore, mentre l’ altra catena del DNA è stabilizzata tramite legami idrogeno. Questa particolare combinazione di intercalazione, legame covalente e legame ad idrogeno è chiamata virtual cross-link (VXL) [2], che porta alla formazione di complessi più stabili tra le antracicline e il DNA aumentando la tossicità cellulare delle antracicline. Tra i diversi meccanismi anticancro delle antracicline, la formazione di addotti DNA-antracicline ha suscitato notevole interesse riferito alla possibilità di trovare nuovi farmaci anticancro più sicuri e più efficaci. I coniugati antraciclina-formaldeide e le antracicline cross-linkanti esibiscono un’elevata citotossicità comparabile con i classici agenti cross-linkanti. Abbiamo usato differenti antracicline con lo scopo di razionalizzare il rapporto struttura attività nella formazione del VXL. Abbiamo confermato attraverso l’analisi elettroforetica e cromatografica che l’amminozucchero e l’azoto amminico sono assolutamente necessari per la formazione del “VXL” e abbiamo discusso il ruolo della posizione 4' nella daunosamina nella modulazione di questa attività. La presenza del ponte metilenico e la sua relazione con la guanina è stata confermata mediante spettrometria di massa
Characterization of the interaction with nucleic acids of the highly cytotoxic oxidative metabolite of the anthracycline nemorubicin by fluorescence quenching assays
No full textHighly Improved Electrospray Ionization-Mass Spectrometry Detection of G-Quadruplex-Folded Oligonucleotides and Their Complexes with Small Molecules
Full text linkG-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication
Full text linkG-quadruplexes (G4s) are nucleic acids secondary structures, epigenetic regulators in cells and viruses.
In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We
here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene
diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the
HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially
recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less
abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low
nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of
action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment
of viral genes transcription. Our data suggest that the observed potent antiviral activity and low
cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive
presence during infection. HSV-1 G4s may thus represent new effective antiviral targets: the fact that
no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both
active and latent HSV infections, makes them particularly attracting
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