NONO è una proteina dimerica coinvolta in diversi tipologie di cancro. Essa è implicata sia nel controllo del ciclo cellulare che nella trascrizione genetica. E’ inoltre presente nei meccanismi di riparazione del DNA di tipo non omologo.
NONO appartiene alla famiglia delle proteine DBHS e per questo motivo è caratterizzata dalla presenza di due motivi di riconoscimento in tandem dell’RNA altamente conservati (RRM1, RRM2), che assumono un ruolo fondamentale nel legame con il ligando naturale, e di un dominio NONa/paraspeckle (NOPS), rappresentato dalla regione a spirale. Tutti questi domini rappresentano l’interfaccia d’interazione che è di tipo idrofobico. Lavorando sotto forma di dimeri, saranno presenti ben otto domini: due domini RRM2 che si dispongono in modo pseudosimmetrico andando a circoscrivere un canale di 20Å a diretto contatto con il solvente. I due domini RRM1 si dispongono invece esternamente a questo canale. Il dominio NOPS si lega al dominio RRM2 determinando in quest’ultimo la formazione di loop a cappio all’ estremità 3 e 5 che presentano dei legami ad idrogeno. Il loop numero 3 si impegna nel legame con l’acido nucleico.
Ad oggi in letteratura non sono presenti ligandi conosciuti e per questo motivo, visto l’importanza della NONO e la sua implicazione in diverse patologie, si è ritenuto importante effettuare delle ricerche per la scoperta di molecole in grado di interagire con essa.
Il mio lavoro di tesi ha previsto una fase iniziale di self-docking per determinare quale dei 12 programmi di docking presenti nel mio laboratorio, fosse il migliore per prevedere il corretto binding mode del ligando. I programmi usati sono VINA, che si è dimostrato il migliore con un valore di RMSD intorno ai 2Å, AutoDock, GLAMDOCK, PLANTS, FRED, DOCK6, GLIDE (con le funzione Standard Precision ed Extra Precision) e GOLD (con le funzioni ASP, PLP, ChemScore e GoldScore). Qui è stato preso come riferimento il cristallo 4YOE.
di 1’500’000 composti. La strategia ha previsto la scelta di tutte quelle molecole aventi un peso molecolare maggiore o uguale a 350 Da, dove, questo valore, è stato preso come riferimento dall’ RNA.
In seguito è stato eseguito uno screening receptor-based mediante l’utilizzo della piattaforma FLAP. Qui è stato possibile stabilire la presenza di cinque tasche di legame e valutare dove avvenisse il legame con il ligando naturale. In seguito, basandomi sulle informazioni ricavate dal self-docking, ho effettuato il docking delle 180 molecole ottenute da FLAP.
Con la successiva valutazione della ligand efficency, ho selezionate 20 molecole che ho sottoposto a simulazione di dinamica molecolare con la piattaforma di AMBER14.
Gli studi di dinamica molecolare su queste molecole hanno portato come risultato la scelta di un sola possibile molecola che potrà essere acquistata e sottoposta ad opportuni saggi enzimatici. Qualora i risultati di quest’ultimi fossero positivi, potrà essere utilizzata come nuovo lead per lo sviluppo di potenti ligandi della proteina NONO.
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RINGRAZIAMENTI
…. e dopo anni di duro lavoro eccomi qui a scrivere i ringraziamenti in fondo alla tesi. Che dire, è stato un percorso lungo, faticoso, caratterizzato da momenti di estrema felicità alternati a momenti di sconforto.
Ho incontrato diverse difficoltà, ma alla fine la costanza e la perseveranza mi hanno ripagata.
Con il passare degli anni, le persone che mi sono state vicine e che hanno vissuto con me tutte le emozioni, sono state tante. Alcune ci sono dall’ inizio di tutto, altre le ho incontrate a metà strada ed altre ancora verso la fine. E ad ognuna di loro devo dire grazie.
Il primo ringraziamento va sicuramente a voi, babbo e mamma, che mi avete supportata e sopportata in ogni momento, avete gioito con me e mi avete presa per i capelli tutte le volte che volevo rinunciare. Avete creduto in me quando nemmeno io lo facevo.
Poi ci sei te, Luca, che fin dall’ inizio hai capito l’importanza che davo ai miei studi e mi hai appoggiata in ogni momento, consolandomi quando le cose non andavano come speravo, e amplificando tutte le cose positive.
Grazie anche a Dotty e Tiziano che mi hanno accolto in laboratorio e fatta sentire a mio agio.
E No, non mi sono dimenticata… grazie anche a VOI, amiche e amici, a voi che avete condiviso sia nelle aule di lezione che al di fuori tutto quanto… e che siete diventati amici al di fuori dell’ UNIPI. Grazie a Irene e Giove, per le risate e le pause pranzo