Protein kinase C (PKC) represents a family of serine/threonine kinases regulating fundamental cellular processes including cell proliferation, differentiation and apoptosis, and therefore they are involved in several pathologies such as cancer, Alzheimer’s disease and cardiac fibrosis. Targeting the regulatory C1 domain offers an alternative approach to modulating PKC activity, with the potential for greater selectivity compared than traditional ATP-competitive inhibitors. Previous work by Professor Jari Yli-Kauhaluoma (University of Helsinki) and his team of researchers identified dialkyl 5-(hydroxymethyl)isophthalates (HMIs) as compounds targeting the C1 domain of PKC with antiproliferative, neuroprotective, and antifibrotic effects. However, their high lipophilicity (cLogP 6-7) negatively affects their drug-like properties, thereby limiting their potential as drug candidates. To overcome this limitation, computational studies including molecular docking and membrane-ligand dynamics simulations, identified the indole scaffold as a promising template for reducing lipophilicity while preserving favourable orientation for the binding to the C1 domain. Building upon these findings, this thesis aimed to develop simplified synthetic routes to access novel hydroxymethyl indole derivatives and to investigate their pharmacological activities. An efficient two-step synthesis was successfully established for the preparation of the target indole derivatives. Although purification of the final compounds presented a significant challenge, three compounds were successfully isolated and subjected to biological evaluation. The competitive radioligand displacement assay revealed modest binding affinity towards the C1 domain of PKCα and, consistent with these findings, the compounds exhibited no cytotoxic effects in DU145 human prostate cancer cell. Overall, this work provides a viable synthetic framework for the preparation of novel hydroxymethyl indole derivatives and identifies three compounds with modest C1-domain binding affinity. Nevertheless, these novel compounds represent a promising starting point for the development of new C1 domain-binding compound providing a basis for future structural optimization and the refinement of purification methodologies.
Le protein chinasi C (PKC) rappresentano una famiglia di serina/treonina chinasi coinvolte nella regolazione di processi cellulari fondamentali quali proliferazione cellulare, differenziamento e apoptosi e, per questo motivo, sono implicate in diverse patologie quali cancro, malattie di Alzheimer e fibrosi cardiaca. Il targeting del dominio regolatorio C1 costituisce un approccio alternativo alla modulazione dell’attività di PKC, con il potenziale vantaggio di una maggiore selettività rispetto ai tradizionali inibitori ATP-competitivi. Studi precedenti condotti dal gruppo del Professor Jari Yli-Kauhaluoma presso l’Università di Helsinki hanno identificato i dialchil 5-(idrossimetil)isoftalati (HMIs) come composti in grado di legarsi al dominio C1 della PKC, mostrando attività antiproliferative, neuroprotettive e antifibrotiche. Tuttavia, l’elevata lipofilia di queste molecole (cLogP 6–7) ne compromette le proprietà farmacocinetiche limitandone il potenziale sviluppo come candidati farmaci. Per superare questa limitazione, studi computazionali, tra cui docking molecolare e simulazioni di dinamica molecolare ligando-membrana, hanno individuato lo scaffold indolico come una struttura alternativa per ridurre la lipofilia mantenendo al contempo un orientamento favorevole per il legame al dominio C1. Sulla base di questi risultati, il presente lavoro di tesi ha avuto come obiettivo lo sviluppo di strategie sintetiche semplificate per la preparazione di nuovi derivati indolici idrossimetilati e la successiva valutazione delle loro proprietà farmacologiche. É stata sviluppata con successo una procedura sintetica in due passaggi per l‘ottenimento dei composti target e ,nonostante la purificazione dei prodotti finali abbia rappresentato una sfida significativa, tre derivati sono stati isolati con successo e sottoposti a valutazione biologica. I saggi di spiazzamento competitivo del radioligando hanno evidenziato una moderata affinità di legame verso il dominio C1 di PKCα e, coerentemente con tali risultati, i composti non hanno mostrato effetti citotossici in cellule di carcinoma prostatico umano DU145. Nel complesso, questo studio ha permesso di sviluppare una strategia sintetica per la preparazione di nuovi derivati indolici idrossimetilati in grado di legare moderatamente il dominio C1 di PKC. Tali composti rappresentano un promettente punto di partenza per lo sviluppo di nuovi ligandi del dominio C1 e pongono le basi per future ottimizzazioni strutturali e per il perfezionamento delle metologie di purificazione
Esplorazione del nucleo indolico per la modulazione allosterica delle protein chinasi C: sintesi e valutazione biologica di nuovi composti.
CIANFANO, MARCO
2025/2026
Abstract
Protein kinase C (PKC) represents a family of serine/threonine kinases regulating fundamental cellular processes including cell proliferation, differentiation and apoptosis, and therefore they are involved in several pathologies such as cancer, Alzheimer’s disease and cardiac fibrosis. Targeting the regulatory C1 domain offers an alternative approach to modulating PKC activity, with the potential for greater selectivity compared than traditional ATP-competitive inhibitors. Previous work by Professor Jari Yli-Kauhaluoma (University of Helsinki) and his team of researchers identified dialkyl 5-(hydroxymethyl)isophthalates (HMIs) as compounds targeting the C1 domain of PKC with antiproliferative, neuroprotective, and antifibrotic effects. However, their high lipophilicity (cLogP 6-7) negatively affects their drug-like properties, thereby limiting their potential as drug candidates. To overcome this limitation, computational studies including molecular docking and membrane-ligand dynamics simulations, identified the indole scaffold as a promising template for reducing lipophilicity while preserving favourable orientation for the binding to the C1 domain. Building upon these findings, this thesis aimed to develop simplified synthetic routes to access novel hydroxymethyl indole derivatives and to investigate their pharmacological activities. An efficient two-step synthesis was successfully established for the preparation of the target indole derivatives. Although purification of the final compounds presented a significant challenge, three compounds were successfully isolated and subjected to biological evaluation. The competitive radioligand displacement assay revealed modest binding affinity towards the C1 domain of PKCα and, consistent with these findings, the compounds exhibited no cytotoxic effects in DU145 human prostate cancer cell. Overall, this work provides a viable synthetic framework for the preparation of novel hydroxymethyl indole derivatives and identifies three compounds with modest C1-domain binding affinity. Nevertheless, these novel compounds represent a promising starting point for the development of new C1 domain-binding compound providing a basis for future structural optimization and the refinement of purification methodologies.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/37193