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Computational Approaches for the Rational Design of Novel Topoisomerase I Poisons as Potential Anticancer Drugs.
No full textTopoisomerase I (TopoI) is an essential DNA-targeting enzyme which alters the supercoiling of DNA through a concerted process of breaking and rejoining of a DNA strand, thereby controlling the DNA topology required for replication and transcription. TopoI mediates relaxation of supercoiled DNA by creating a transient single-strand break in the DNA duplex that originates from a transesterification reaction involving a nucleophilic attack by the active-site tyrosine (Tyr723) hydroxyl group on a DNA phosphodiester bond situated at the site of cleavage. The resulting 3'-phosphotyrosine enzyme-DNA complex (“covalent binary complex” or “cleavable complex”) is then reversed, after DNA relaxation through strand passage, when the released 5'-OH of the nicked strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. These events result in the relaxation of the DNA structure, which is required during transcription or replication.
TopoI is a specific target for the pentacyclic alkaloid Camptothecin (CPT), which was first isolated in 1966 from extracts of Camptotheca Acuminata, and its derivatives, known as TopoI poisons.
These molecules block DNA religation, thus converting TopoI into a DNA-damaging agent. In the presence of a TopoI poison a ternary complex between DNA, an intercalator and topoisomerase is formed. Such a ternary complex is more stable than the DNA-TopoI associate, which may lead to an enhanced lifetime of the initially cleaved DNA. As a consequence of the misalignment of the free 5-hydroxyl group and the scissile tyrosine-DNA bond due to the presence of the drug, the religation of the broken strand cannot take place, i.e. the strand break persists. This activates a complex sequence of intacellular responses, that ultimately lead to cell death by apoptosis. Therefore, molecules which form such stabilized ternary complexes with DNA and TopoI exhibit a high potential as DNA-targeting anticancer drugs.
Camptothecin was early shown to be clinically problematic because, in addition to its negligible water solubility, its active “ring-closed” ?-hydroxylactone form is rapidly converted under physiological conditions to the open carboxylate form, which is inactive and readily binds to human serum albumin, making it inaccessible for cellular uptake.
To date, only two semisynthetic analogs of Camptothecin (Topotecan and Irinotecan) have been approved by FDA for the clinical treatment of the ovarian, small cell-lung and colon cancers.
Solving crystal structures of Top1 in complex both with Camptothecin and Topotecan and with structurally different molecules (indolocarbazoles and indoloquinolines) has significantly increased the amount of structural information about the interaction between the Top1-DNA binary complex and the poison molecule. This encouraged the application of structure-based drug design to investigate
and rationalize the activity of Top1 poisons and to rationally design new potential anticancer drugs. Thus, we considered the possibility to find a new pharmacophore exploiting all the available crystal structures to find the set of structural features which are in common to all of the five TopoI poisons.
In order to obtain new derivatives easier to be synthesized while maintaining the pharmacophoric features required for the formation of a stable TopoI-DNA-poison ternary complex, the synthesis of simplified CPT derivatives was proposed. It is possible to suppose that a pentacyclic scaffold is not necessarily required for a molecule to effectively bind the TopoI-DNA binary complex, providing that it maintains an aromatic planar system for the intercalation and appropriate functional groups to interact with specific residues of TopoI. On the basis of this assumption, several scaffolds were designed and docking studies with different search algorithms were performed to predict the ability of simplified analogs to form stable ternary complex with TopoI and DNA.
Moreover, we performed a molecular docking analysis on a series of new 5-substituted CPT derivatives to achieve more insight into the interactions of the new compounds with the binary TopoI-DNA complex. The introduction of substituents in position 5 of the CPT scaffold results in derivatives with reduced cytotoxic potency compared with the reference drugs. In general, the presence of a small lipophilic substituent is well tolerated while the modification with hydrophilic groups results in reduced affinity for the binding site. The presence of bulky groups is detrimental for cytotoxic potency because of the loss of important stabilizing interactions. Computational results indicated as a general feature that the 5-?-epimer is better tolerated in the binding site compared to the 5-?-epimer, resulting in higher biological activity.
Recently, a new series of 16a-thio-CPT derivatives has been synthesized. Biological assays for this class of compounds revealed that 16a-thio-CPT derivatives have higher anticancer activity compared to their oxo-analogs, both in vitro and in vivo, and are potent TopoI inhibitors. In order to find a rational explanation to their remarkable activity, a computational analysis was performed, both for predicting elements of their pharmacokinetic behavior and for describing their binding mode through a docking analysis. Docking results did not reveal striking differences between thio- and oxo-derivatives in terms of binding mode and docking score. On the contrary, the predicted values for features such as lipophilicity, water solubility and cell permeability indicated that these properties may be responsible for a significantly different pharmacokinetic profile for the two classes of compounds.
For the docking analysis of 5-substituted and 16a-thio CPT derivatives we used the quantum mechanics (QM)-polarized ligand docking (QPLD) protocol (Schrödinger software suite) with an aim to improve accuracy in docking calculations. Traditional docking methods employ an approximated physical chemistry-based representation of protein-ligand interactions, obtaining charges from a molecular mechanics force-field. The QPLD protocol uses an ab initio methodology to calculate ligand charges within the protein environment, thus taking into account the charge polarization induced by the protein environment.
The work on 5-substituted and 16a-thio CPT derivative was sponsored by the pharmaceutical company Indena S.p.A., Milano, Italy. The work was carried out in collaboration with the group of Professor Arturo Battaglia (Centro Nazionale Richerche Bologna, Italy) for the synthesis of the compounds, and with the group of Professor Franco Zunino (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy), that was concerned with biological assays.La Topoisomerasi I eucariotica (TopoI) è un enzima essenziale che modifica il grado di superavvolgimento del DNA mediante un processo di rottura e successiva ricongiunzione di uno dei due filamenti del DNA, regolandone lo stato topologico richiesto nei processi di replicazione e di trascrizione.
Il rilassamento del DNA superavvolto indotto dalla TopoI avviene attraverso la rottura transitoria di un filamento per mezzo di una reazione di transesterificazione. Quest'ultima coinvolge l'attacco nucleo lo da parte del gruppo ossidrilico di un residuo di tirosina (Tyr723) del sito attivo dell'enzima ad un legame fosfodiesterico del DNA a livello del sito di taglio, con formazione di un complesso covalente 3'-fosfotirosina TopoI-DNA (noto anche come cleavable complex). La rotazione del filamento tagliato intorno a quello intatto rende possibile il rilassamento della molecola di DNA. Il complesso binario covalente subisce in seguito un attacco nucleofilo a livello della fosfotirosina da parte dell'estremità libera 5'-OH del lamento rotto, che permette la riformazione del legame fosfodiesterico.
La TopoI è il target specifico dell'alcaloide pentaciclico Camptotecina (CPT), isolato per la prima volta nel 1966 dalla corteccia di Camptotheca Acuminata, e dei suoi derivati, noti come veleni di TopoI.
Queste molecole bloccano il processo di ricongiunzione del filamento di DNA tagliato, convertendo la TopoI in un agente dannoso per il DNA stesso. Queste molecole si intercalano tra le basi del DNA a livello del sito di taglio dando luogo alla formazione di un complesso ternario con la TopoI e il DNA. La maggiore stabilità del complesso ternario rispetto al complesso binario TopoI-DNA risulta in un aumento del tempo di vita del DNA tagliato. Poiché la presenza del veleno induce il disallineamento dell'estremità libera 5'-OH del filamento rotto con il legame fosfotirosinico, il processo di ricongiunzione non può avvenire. Questo porta all'attivazione di una serie complessa di segnali intracellulari, il cui risultato ultimo è la morte della cellula per apoptosi. Di conseguenza, le molecole in grado di formare complessi ternari stabili hanno attività antitumorale.
L'utilizzo clinico della CPT è ostacolato sia dalla sua scarsissima idrosolubilità, sia perchè la sua forma attiva lattonica “chiusa” è rapidamente convertita, in condizioni fisiologiche, nella forma carbossilica “aperta”, che è inattiva e si lega in elevata percentuale all'albumina serica.
Attualmente solo due derivati semisintetici della CPT (Topotecan ed Irinotecan) sono utilizzati nella pratica clinica per il trattamento di cancro ovarico, polmonare e colo-rettale.
La recente risoluzione, mediante cristallografia a raggi X, della struttura tridimensionale di TopoI in complesso sia con la Camptotecina e il Topotecan che con derivati strutturalmente diversi (indolocarbazolici ed isochinolinici), ha fornito chiarimenti sulle caratteristiche dell'interazione enzima-inibitore. Tali informazioni costituiscono elementi molto utili nello studio dei veleni di TopoI con un approccio di tipo computazionale il cui scopo è comprendere in che modo queste molecole interagiscono con il loro target e progettare nuovi nuovi derivati come potenziali farmaci ad attività antitumorale. È stato quindi possibile individuare un nuovo farmacoforo sfruttando le cinque strutture cristallogra che disponibili, al fine di rintracciare un set di caratteristiche strutturali comuni a tutti i veleni di TopoI.
Con lo scopo di disporre di nuovi derivati più semplici da ottenere dal punto di vista della sintesi ma che mantengano tutte le caratteristiche farmacoforiche richieste per la formazione di complessi ternari stabili con TopoI e DNA, è stata proposta la sintesi di analoghi “semplificati” della CPT. Si può infatti ipotizzare che una molecola non debba necessariamente possedere uno scaffold
pentaciclico per legarsi al complesso binario TopoI-DNA, purchè mantenga un sistema planare aromatico per intercalare il DNA e opportuni gruppi funzionali in grado di interagire con specifici aminoacidi dell'enzima. Sulla base di questa assunzione, diversi possibili scaffold sono stati disegnati e sottoposti a studi di docking con diversi protocolli di ricerca per predire la loro capacità di formare stabili complessi ternari con TopoI e DNA.
È stato inoltre condotto uno studio di docking molecolare per una serie di nuovi derivati della CPT sostituiti in posizione 5 (5-derivati) per descrivere la loro modalità di interazione con il complesso TopoI-DNA. In generale, l'introduzione di sostituenti in posizione 5 ha come risultato una diminuzione dell'attività citotossica rispetto alla CPT. La presenza di sostituenti lipofili di piccole dimensioni sembra essere tollerata all'interno del sito di binding, mentre l'introduzione di gruppi idro lici dà luogo ad una diminuzione dell'affinità. L'introduzione di gruppi stericamente ingombranti provoca la perdita di importanti interazioni al'interno del sito di legame. I risultati computazionali indicano come caratteristica generale che l'epimero ? dei 5-derivati ha un'affinità maggiore per il sito di binding rispetto all'epimero ?.
Recentemente sono stati sintetizzati una serie di 16a-tio-CPT analoghi. I saggi biologici condotti su questi composti hanno rivelato che i tio-derivati sono potenti inibitori della TopoI ed hanno attività antitumorale maggiore rispetto ai loro oxo-analoghi, sia in vitro che in vivo. Al fine di razionalizzare la maggiore attività dei tio-analoghi rispetto ai derivati tradizionali, è stato eseguito
uno studio computazionale volto sia a predire proprietá chimico-fisiche rilevanti per la descrizione del possibile profilo farmacocinetico delle nuove molecole, sia a descriverne la modalità di legame
nel sito del complesso binario TopoI-DNA tramite studi di docking.
I risultati di docking non hanno messo in evidenza differenze di rilievo tra i tio- e gli oxo-derivati in termini di modalità di binding e di docking score. Al contrario, i valori predetti per proprietà come la lipofilicità, la solubilità in acqua e la permeabilità cellulare indicano che il profilo farmacocinetico delle due classi di composti potrebbe essere significativamente diverso.
Per le simulazioni di docking condotte sui 5-CPT-derivati e sui tio-CPT-derivati è stato utilizzato il protocollo quantum mechanics (QM)-polarized ligand docking (QPLD) (implementato nella suite Schrödinger) con lo scopo di aumentare l'accuratezza dei calcoli di docking. I metodi tradizionali utilizzati per il docking molecolare utilizzano una rappresentazione chimico-fisica approssimata delle interazioni proteina-ligando, dove le cariche atomiche sono calcolate in base al campo di forza. Il protocollo QPLD utilizza invece una metodologia ab initio per il calcolo delle cariche del ligando all'interno della cavità della proteina, tenendo in considerazione la polarizzazione di carica indotta dagli aminoacidi del sito di legame sul ligando.
Il lavoro relativo ai 5-CPT-derivati e ai tio-CPT-derivati à stato supportato dall'azienda farmaceutica Indena S.p.A., Milano, Italia. Il lavoro è stato condotto in collaborazione con il gruppo del Professor Arturo Battaglia (Centro Nazionale Richerche Bologna) per la sintesi dei composti e con il gruppo del Professor Franco Zunino (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano) per i saggi biologici
Novel camptothecin derivatives as topoisomerase I inhibitors
No full textBACKGROUND:
Camptothecin (CPT), a pentacyclic alkaloid isolated by Wall et al. in 1958 from the Chinese tree Camptotheca acuminata, was reported to possess an interesting antitumor activity. Late in 1985, it was reported by Liu et al. that the cytotoxic activity of CPT was attributed to a novel mechanism of action involving the nuclear enzyme classified as type I DNA topoisomerase. Since the explanation of the unique mechanism of action, many derivatives have been synthesized and some of them are in various stages of preclinical and clinical development. Among them, two derivatives, topotecan and irinotecan, have successfully entered into the market and are used as topoisomerase I poisons in clinical practice.
OBJECTIVE:
The main focus of the present review is to describe the development of CPT derivatives over the years dividing them by decades according to the date of discovery and focusing attention on molecules that were published in patents.
RESULTS/CONCLUSION:
To summarize, > 150 patents have been deposited over the past 30 years. Structure-activity relationship studies suggest that substitutions at the 7-, 9- or 10-positions of most CPT derivatives enhance their antitumor activity, but at the 11- or 5-position usually lead to activity decrease
A Novel Generalized 3D-QSAR Model of Camptothecin Analogs
No full textIn the present paper, we are interested to explore if the application of docking-driven conformational analysis could increase the goodness of 3D-QSAR statistical models, as alternative approach to a conventional ligand-based conformer generation. In particular, we have selected as peculiar key-study an ensemble of Camptothecin (CPT) analogs classified as human DNA Topoisomerase I (Top1) selective inhibitors. The CPT analogs dataset has been recently analyzed by Hansch and Verma using a classical 2D-QSAR study
Relationship between the structure and the DNA binding properties of diazoniapolycyclic duplex- and triplex-DNA binders: Efficiency, selectivity, and binding mode
No full textThe association of dicationic polycyclic ligands, namely, four diazoniapentaphene derivatives, three diazoniaanthra[1,2-a]anthracenes, diazoniahexaphene, and a partly saturated hydroxy-substituted diazoniapentaphene with double-stranded and triple-helical DNA, was investigated by spectrophotometric and viscosimetric titrations, CD and LD spectroscopy, DNA melting experiments, and molecular modeling studies. All experimental and theoretical data reveal an intercalative DNA-binding mode of the diazoniapentaphenes and diazoniaanthra[1,2-a]anthracenes; the latter have approximately 10-fold higher affinity for the DNA duplex. CD spectroscopic investigations and molecular modeling studies show that only one azonianaphthalene part of the ligand is intercalated between the DNA base pairs, whereas the remaining part of the ligand points outside the intercalation pocket. In contrast, the diazoniahexaphene is a DNA groove binder, which binds selectively to [poly(dAdT)](2). At low ligand-to-DNA ratios (r 0.2. Studies of the interaction of diazoniapolycyclic ions with triplex DNA reveal a preferential binding of both diazoniapentaphenes and diazoniaanthra[1,2-a]anthracenes to the triplex and stabilization thereof. These properties are more pronounced in the case of the hexacyclic diazoniaanthra[1,2-a]anthracenes; however, the diazoniahexaphene shows no preferential binding to the triplex. The DNA binding properties of the diazoniapentaphene derivatives remain essentially the same upon variation of the positions of nitrogen atoms or substitution with methyl groups. In contrast, the interactions of the diazoniaanthra[1,2-a]anthracence isomers with triplex DNA are slightly different. Notably, the 14a,16a-diazoniaanthra[1,2-a]anthracene is among the most efficient triplex stabilizers, with a 9-fold larger binding affinity for the triplex than for the DNA duplex. Moreover, the diazoniapentaphene and diazoniaanthra[1,2-a]anthracene derivatives represent the first examples of triplex-DNA binders that do not require additional aminoalkyl side chains for efficient triplex stabilization
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
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