This thesis project consisted the design and synthesis of bivalent ligands, which are directed towards three proteins overexpressed in cancer: Sigma1 and Sigma2 Receptors (respectively S1R and S2R) and Translocator Protein TSPO. These bivalent ligands will be evaluated in vitro to better understand correlations between the pathways the proteins are involved in and to evaluate potential therapeutic effects from their simultaneous modulation. The general chemical structure of bivalent ligands is characterized by model ligands RC-106 and PK11195 pharmacophores, connected through a linker. RC-106 was discovered by the LabMedChem research group and defined a pan-SR ligand for its good binding affinity to both SRs. Its cytotoxic activity was assessed on prostate, glioblastoma, pancreas and breast cancer cell lines. PK11195 is a high affinity TSPO ligand and its radiolabeled form is clinically used as a PET tracer. RC-106 and PK11195 chemical structures do not possess functional groups feasible for direct derivatization; because of this, their derivatives were chosen: PV172, PV174 and PK1AC. Six bivalent ligands were designed; three constituted of PV172 and PK1AC (labelled as PVPK1, PVPK2, PVPK3) and three of PV174 and PK1AC (labelled as PVPK4, PVPK5, PVPK6). Afterwards, in silico evaluations were performed, to validate the bivalent ligand approach. Semi-rigid docking of RC-106, PVPK1 and PVPK4 revealed good docking scores on S2R crystal. In addition, PVPK1 was docked on TSPO crystal structure too. These results demonstrated that the fundamental interactions with the proteins should not be compromised with the addition of a linker in the positions selected and that the derivatization on both naphthyl (PV172) and benzyl (PV174) should be useful for preliminary SAR studies. A synthetic route for PVPK1, PVPK2, PVPK3 was successfully optimized, which identities were confirmed by 1H NMR and UHPLC-MS analysis and they will be subjected to in vitro evaluations. Therefore, the synthetic routes applied for the first three bivalent ligands are efficient and can be translated on PV174 derivatives too. To date, PVPK6 was synthetized and its characterization is still ongoing. Once the series is completed, the products will be subjected to opportune biological investigation, evaluating the interaction with the targets and their therapeutic effect on different cancer cell lines.
Il mio progetto di tesi ha previsto il disegno e la sintesi di ligandi bivalenti, diretti verso tre proteine sovraespresse in diverse cellule tumorali: i Recettori Sigma1 e Sigma2 (S1R e S2R), e la Proteina Traslocatrice TSPO. Tali ligandi bivalenti verranno valutati in vitro, per comprendere meglio la correlazione tra i pathway in cui sono coinvolte le tre proteine selezionate e valutare i potenziali effetti terapeutici dovuti alla loro simultanea modulazione. La struttura generale dei ligandi bivalenti disegnati è costituita dall’unione, tramite un opportuno linker, dei farmacofori dei composti modello RC -106 e PK11195. RC-106 è stato identificato dal gruppo di ricerca del MedChemLab quale ligando pan-Sigma dotato di buona affinità verso entrambi i Recettori Sigma. L’attività citotossica è stata dimostrata su linee cellulari tumorali di prostata, glioblastoma, pancreas e mammella. PK11195 è un ligando TSPO dotato di elevata affinità, e la cui forma radiomarcata è clinicamente utilizzata come tracciante PET. Le strutture chimiche di RC-106 e PK11195 non presentano gruppi funzionali che ne consentono direttamente la derivatizzazione. Per questo motivo, sono stati impiegati i loro derivati PV172, PV174 e PK1AC. Sono stati disegnati in totale sei ligandi bivalenti: tre costituiti da PV172 e PK1AC (nominati PVPK1, PVPK2, PVPK3) e tre da PV174 e PK1AC (nominati PVPK4, PVPK5, PVPK6). Dopodiché, sono state effettuate valutazioni in silico, per validare l’approccio dei ligandi bivalenti. Il docking semi-rigido di RC-106, PVPK1 e PVPK4 ha dato come risultato dei buoni docking scores sul cristallo di S2R. Di PVPK1 è stato effettuato il docking anche sulla struttura cristallizzata di TSPO. I risultati in silico hanno evidenziato che la presenza di un linker nelle posizioni selezionate non sembra compromettere le interazioni fondamentali con le proteine e quindi che i derivati sia sulla porzione naftilica (PV172) sia sulla porzione benzilica (PV174) possono essere utili per studi SAR preliminari. Disegnati i nuovi composti, è stata messa a punto con successo una via di sintesi che ha consentito di ottenere PVPK1, PVPK2, PVPK3, le cui identità sono state confermate dall’analisi 1H NMR e UHPLC-MS, e che verranno quindi sottoposti ai saggi in vitro. Le vie sintetiche che sono state applicate per i primi tre ligandi bivalenti sono risultate efficaci e potranno essere traslate sui derivati da PV174. Ad oggi è stato sintetizzato PVPK6, la cui caratterizzazione è attualmente in corso. Una volta completata la serie, i prodotti ottenuti saranno sottoposti ad opportuna investigazione biologica, misurando l’interazione con i target e valutando il potenziale effetto terapeutico in diverse linee cellulari tumorali.
Modulazione dei Recettori Sigma e della Proteina Traslocatrice TSPO in pathways oncogenici: disegno e sintesi di ligandi bivalenti
GIORGI, MARGHERITA
2021/2022
Abstract
This thesis project consisted the design and synthesis of bivalent ligands, which are directed towards three proteins overexpressed in cancer: Sigma1 and Sigma2 Receptors (respectively S1R and S2R) and Translocator Protein TSPO. These bivalent ligands will be evaluated in vitro to better understand correlations between the pathways the proteins are involved in and to evaluate potential therapeutic effects from their simultaneous modulation. The general chemical structure of bivalent ligands is characterized by model ligands RC-106 and PK11195 pharmacophores, connected through a linker. RC-106 was discovered by the LabMedChem research group and defined a pan-SR ligand for its good binding affinity to both SRs. Its cytotoxic activity was assessed on prostate, glioblastoma, pancreas and breast cancer cell lines. PK11195 is a high affinity TSPO ligand and its radiolabeled form is clinically used as a PET tracer. RC-106 and PK11195 chemical structures do not possess functional groups feasible for direct derivatization; because of this, their derivatives were chosen: PV172, PV174 and PK1AC. Six bivalent ligands were designed; three constituted of PV172 and PK1AC (labelled as PVPK1, PVPK2, PVPK3) and three of PV174 and PK1AC (labelled as PVPK4, PVPK5, PVPK6). Afterwards, in silico evaluations were performed, to validate the bivalent ligand approach. Semi-rigid docking of RC-106, PVPK1 and PVPK4 revealed good docking scores on S2R crystal. In addition, PVPK1 was docked on TSPO crystal structure too. These results demonstrated that the fundamental interactions with the proteins should not be compromised with the addition of a linker in the positions selected and that the derivatization on both naphthyl (PV172) and benzyl (PV174) should be useful for preliminary SAR studies. A synthetic route for PVPK1, PVPK2, PVPK3 was successfully optimized, which identities were confirmed by 1H NMR and UHPLC-MS analysis and they will be subjected to in vitro evaluations. Therefore, the synthetic routes applied for the first three bivalent ligands are efficient and can be translated on PV174 derivatives too. To date, PVPK6 was synthetized and its characterization is still ongoing. Once the series is completed, the products will be subjected to opportune biological investigation, evaluating the interaction with the targets and their therapeutic effect on different cancer cell lines.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14571