My thesis project concerned the synthesis, purification and characterization of Sigma Receptors (subtype S1R and S2R) and translocator protein TSPO bivalent ligands, two proteins overexpressed in different cancer cells. The designed molecules are multitarget chemical entities as pharmacological tools to study the entanglement of selected proteins in the molecular pathways involved in the onset and proliferation of different tumours. Sigma receptors, expressed in the CNS and in peripheral tissues, are considered promising therapeutic targets for the treatment of various pathologies, as they are involved in cell growth processes and inhibition of apoptosis. The TSPO protein is a transmembrane protein located on the outer mitochondrial membrane and mainly expressed in glial cells, but also overexpressed in glioblastoma, prostate, breast and colon cancer cells. It plays an important role in the transport of cholesterol and in the activation of the mitochondrial apoptosis process. Considering the multifactorial nature of cancer, the idea of simultaneously modulating Sigma receptors and TSPO protein arose to develop a new therapeutic approach with higher efficacy and/or selectivity. Bivalent ligands that I have prepared in my thesis work will therefore be useful to verify the feasibility of this approach. These molecules are constituted by the conjugation through linkers - of different length and nature - of modulators of the receptors Sigma 1 and Sigma 2 (RC106) and TSPO (PK-11195). RC-106 was identified by the MedChemLab research group as a ligand with good affinity towards both Sigma receptors and promising antiproliferative activity on different tumor cells. PK11195 is a TSPO ligand with high affinity which radiolabelled form is clinically used in PET analyses. Eight bivalent ligands (ACPKRC1 – ACPKRC8) were therefore designed, which synthesis required the use of analogues of the above-mentioned model compounds, suitable for derivatization via linkers: PK1AC, a commercially available derivative of PK11195, and PV172 and PV174, hydroxylated derivatives of RC106 (as outlined in the graphical abstract). The latter were synthesized by scaling and optimizing protocols previously tested at the MedChemLab, in sufficient amounts for the synthesis of the entire library. Subsequently I dealt with the synthesis of bivalent ligands starting from these building blocks, exploiting different approaches for their conjugation to the different linkers: amidation reactions, Steglich esterification and click chemistry. Once the full series of bivalent ligands has been obtained, their purity was determined by HPLC and their identity confirmed by NMR analysis and mass spectrometry. Where necessary, a second purification was performed after conventional flash chromatography to ensure a purity suitable for biological investigations. Currently, the entire library has been obtained and characterized from a physicochemical point of view, except for compound ACPKRC6 (whose purification is ongoing). The compounds obtained in sufficient quantities and purity have instead begun the phase of preliminary biological investigations, which include the evaluation of interaction with the selected targets and the therapeutic effect on various tumor cell lines. The results will serve to provide the proof of concept of the therapeutic potential, in the oncological field, deriving from the simultaneous modulation of the Sigma Receptors and the TSPO protein.
Il mio progetto di tesi ha previsto la sintesi, la purificazione e la caratterizzazione di ligandi dimerici dei recettori Sigma (sottotipo recettoriale S1R e S2R) e della proteina Traslocatrice TSPO, due proteine sovraespresse in diverse cellule tumorali. Le molecole oggetto della mia tesi sono entità chimiche multitarget che sono state progettate come tools farmacologici per studiare la correlazione delle proteine selezionate nei pathways molecolari coinvolti nell’insorgenza e proliferazione di diversi tumori. I recettori Sigma, espressi nel SNC e nei tessuti periferici, sono considerati promettenti target terapeutici per la cura di diverse patologie, in quanto coinvolti in processi di crescita cellulare e inibizione dell’apoptosi. Il secondo target dei ligandi bivalenti è la proteina TSPO, una proteina transmembrana collocata sulla membrana mitocondriale esterna e principalmente espressa nelle cellule gliali, ma anche sovraespressa in cellule tumorali di glioblastoma, prostata, mammella e colon. Essa gioca un ruolo fondamentale nel trasporto del colesterolo e attivazione del processo di apoptosi mitocondriale. Considerando la natura multifattoriale del cancro, è nata l’idea di modulare simultaneamente i recettori Sigma e la proteina TSPO per sviluppare un nuovo approccio terapeutico con maggiore efficacia e/o selettività. I ligandi bivalenti che ho preparato nel mio lavoro di tesi saranno quindi utili per verificare la fattibilità di questo approccio. Tali molecole sono costituite dalla coniugazione mediante linker – di diversa lunghezza e natura – di modulatori dei recettori Sigma 1 e Sigma 2 (RC106) e TSPO (PK-11195). RC-106 è stato identificato dal gruppo di ricerca del MedChemLab quale ligando dotato di buona affinità verso entrambi i Recettori Sigma e promettente attività antiproliferativa su differenti cellule tumorali. PK11195 è un ligando TSPO dotato di elevata affinità e la cui forma radiomarcata è clinicamente utilizzata nelle analisi PET. Sono stati quindi disegnati otto ligandi bivalenti (ACPKRC1 – ACPKRC8), la cui sintesi ha previsto l’uso di analoghi dei composti modello sopracitati, adatti alla derivatizzazione tramite linker: PK1AC, derivato di PK11195 commercialmente disponibile, e PV172 e PV174, derivati ossidrilati di RC106 (come schematizzato nel graphical abstract). Questi ultimi sono stati sintetizzati scalando e ottimizzando protocolli precedentemente sperimentati presso il MedChemLab, in modo da averne in quantità sufficiente per la sintesi dell’intera libreria. Successivamente mi sono occupata della sintesi dei ligandi bivalenti a partire da questi building block, sfruttando diversi approcci per la loro coniugazione ai diversi linker: reazioni di ammidazione, esterificazione di Steglich e click chemistry. Ottenuta l’intera serie di ligandi bivalenti, la loro purezza è stata determinata tramite HPLC e la loro identità è stata confermata tramite spettroscopia NMR e spettrometria di massa. Laddove necessario, è stata eseguita una seconda purificazione, dopo la convenzionale cromatografia flash, per garantire un grado di purezza adatto alle investigazioni biologiche. Attualmente, l’intera libreria è stata ottenuta e caratterizzata da un punto di vista chimico-fisico, ad eccezione del composto ACPKRC6 (la cui purificazione è ancora in corso d’opera). I composti ottenuti in quantità e purezza sufficiente hanno invece iniziato la fase di indagini biologiche preliminari, che comprendono la valutazione dell’interazione con i target e dell’effetto terapeutico su diverse linee cellulari tumorali. I risultati serviranno a fornire la proof of concept del potenziale terapeutico, in campo oncologico, derivante della modulazione simultanea dei Recettori Sigma e della proteina TSPO.
Identificazione di potenziali agenti antitumorali. Preparazione e caratterizzazione di ligandi bivalenti dei recettori Sigma e della proteina TSPO.
LIPANI, ALESSANDRA
2022/2023
Abstract
My thesis project concerned the synthesis, purification and characterization of Sigma Receptors (subtype S1R and S2R) and translocator protein TSPO bivalent ligands, two proteins overexpressed in different cancer cells. The designed molecules are multitarget chemical entities as pharmacological tools to study the entanglement of selected proteins in the molecular pathways involved in the onset and proliferation of different tumours. Sigma receptors, expressed in the CNS and in peripheral tissues, are considered promising therapeutic targets for the treatment of various pathologies, as they are involved in cell growth processes and inhibition of apoptosis. The TSPO protein is a transmembrane protein located on the outer mitochondrial membrane and mainly expressed in glial cells, but also overexpressed in glioblastoma, prostate, breast and colon cancer cells. It plays an important role in the transport of cholesterol and in the activation of the mitochondrial apoptosis process. Considering the multifactorial nature of cancer, the idea of simultaneously modulating Sigma receptors and TSPO protein arose to develop a new therapeutic approach with higher efficacy and/or selectivity. Bivalent ligands that I have prepared in my thesis work will therefore be useful to verify the feasibility of this approach. These molecules are constituted by the conjugation through linkers - of different length and nature - of modulators of the receptors Sigma 1 and Sigma 2 (RC106) and TSPO (PK-11195). RC-106 was identified by the MedChemLab research group as a ligand with good affinity towards both Sigma receptors and promising antiproliferative activity on different tumor cells. PK11195 is a TSPO ligand with high affinity which radiolabelled form is clinically used in PET analyses. Eight bivalent ligands (ACPKRC1 – ACPKRC8) were therefore designed, which synthesis required the use of analogues of the above-mentioned model compounds, suitable for derivatization via linkers: PK1AC, a commercially available derivative of PK11195, and PV172 and PV174, hydroxylated derivatives of RC106 (as outlined in the graphical abstract). The latter were synthesized by scaling and optimizing protocols previously tested at the MedChemLab, in sufficient amounts for the synthesis of the entire library. Subsequently I dealt with the synthesis of bivalent ligands starting from these building blocks, exploiting different approaches for their conjugation to the different linkers: amidation reactions, Steglich esterification and click chemistry. Once the full series of bivalent ligands has been obtained, their purity was determined by HPLC and their identity confirmed by NMR analysis and mass spectrometry. Where necessary, a second purification was performed after conventional flash chromatography to ensure a purity suitable for biological investigations. Currently, the entire library has been obtained and characterized from a physicochemical point of view, except for compound ACPKRC6 (whose purification is ongoing). The compounds obtained in sufficient quantities and purity have instead begun the phase of preliminary biological investigations, which include the evaluation of interaction with the selected targets and the therapeutic effect on various tumor cell lines. The results will serve to provide the proof of concept of the therapeutic potential, in the oncological field, deriving from the simultaneous modulation of the Sigma Receptors and the TSPO protein.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.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/16213