Načrtovanje, sinteza in vrednotenje himernih molekul za inducirano razgradnjo proteinov, vpletenih v patogenezo malignih obolenj

Bricelj, Aleša (author), Sosič, Izidor (mentor), Anderluh, Marko (comentor)

Velika večina proteinov, vpletenih v patogenezo malignih obolenj, spada v del proteoma, ki je težko dostopen za modulacijo z majhnimi molekulami. Za ciljanje tovrstnih proteinov, ki nimajo jasno definiranega vezavnega mesta, potrebujemo alternativne farmakološke modalitete. V zadnjih letih je veliko pozornosti posvečene področju tarčne razgradnje proteinov, ki temelji na t.i farmakološkemu modelu dogodka, saj delovanje tovrstnih učinkovin sproži zmanjšanje celičnih nivojev tarčnega proteina in s tem odpravi njegovo biološko funkcijo. Med najbolj perspektivnimi pristopi so se izkazale himerne molekule PROTAC (proteolysis-targeting chimera), ki preko ubikvitin-proteasomskega sistema sprožijo razgradnjo tarčnega proteina. Sestavljene so iz liganda za vezavo na tarčni protein, distančnika in liganda za vezavo na ligazo E3. Vezava obeh ligandov vodi v tvorbo ternarnega kompleksa tarčni protein-PROTAC-ligaza E3, sledi ubikvitinacija proteina in njegova razgradnja s proteasomom. Zaradi številnih prednosti spojin PROTAC je področje himernih razgrajevalcev trenutno v središču pozornosti farmacevtske kemije, vendar njihovo načrtovanje spremljajo določeni izzivi in težave. V disertaciji poročamo o naših poskusih implementacije metodologije PROTAC na nabor tarč, ki so na različne načine vpletene v patogenezo malignih obolenj. Za modulacijo delovanja proteinov CDK4/6, ki regulirata celični cikel, smo iz znanega neselektivnega zaviralca pripravili tako dualne razgrajevalce CDK4/6 kot tudi selektivne razgrajevalce CDK6. Opisali smo tudi poskus priprave himernih spojin za odstranitev proteinov, vpletenih v intrinzično pot apoptoze. Pripravljene spojine, ki ciljajo Bcl-2, sicer niso dosegle selektivne razgradnje, vendar so kljub temu izkazovale ugodne učinke na celično viabilnost. Nasprotno pa je serija spojin, ki smo jo načrtovali za proapoptotski protein BAX, inducirala določeno stopnjo razgradnje, vendar ni izkazovala ugodnih celičnih učinkov. Uspešnejši smo bili pri razgradnji proapoptotskih proteinov družine IAP, kjer smo opisali tako spojine za selektivno razgradnjo XIAP kot tudi pan-IAP razgrajevalce. Modulirati smo poskusili tudi encim OGT, ki je odgovoren za posttranslacijsko modifikacijo proteinov, vendar pripravljene himerne spojine niso bile učinkovite. Poleg dela na specifičnih tarčah smo se posvetili tudi optimizaciji ligandov za ligaze E3, predvsem za cereblon, kar predstavlja pomemben doprinos k širšemu področju molekul PROTAC.

• Language: Slovenian
• Keywords: tarčna razgradnja proteinov, himerni razgrajevalci
• Work type: Doctoral dissertation
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151041
• Publication date in RUL: 28.09.2023

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of chimeric molecules for induced degradation of proteins involved in pathogenesis of malignancies

Abstract

Most of the proteins involved in the pathogenesis of malignancies are part of the undruggable proteome. To modulate the biological activity of proteins without a defined binding pocket, alternative pharmacological modalities are needed. The targeted protein degradation approach has garnered a lot of attention in recent years, due to the many advantages of this event-driven pharmacological model. Among the most promising new approaches are proteolysis-targeting chimeras (PROTACs). These chimeric molecules consist of a protein of interest binder, a linker, and an E3 ligase ligand. After binding, a ternary complex is formed, leading to ubiquitination and proteasomal degradation of the target protein via the ubiquitin-proteasome system. The many advantages of PROTAC-mediated degradation have catapulted the field to the forefront of drug design; however, there are numerous challenges to implementing this approach. Herein we report our attempts at utilizing the PROTAC methodology to target a panel of proteins involved in the pathogenesis of malignancies. In order to modulate the activity of the cell cycle regulators CDK4/6, we have transformed a non-selective inhibitor into a dual CDK4/6 and even a selective CDK6 degrader. We also focused on proteins that serve as regulators of the intrinsic pathway of apoptosis. A series of PROTACs, targeting Bcl-2, failed to selectively degrade the protein, yet showed the desired antiproliferative activity when tested on haematological cell lines. On the other hand, BAX-targeting PROTACs caused marginal degradation, but failed to salvage chemically-induced apoptosis. We were more successful in degrading proapoptotic members of the IAP protein family, where implementation of the heteroPROTAC approach resulted in both pan-IAP and selective XIAP degradation. Exploring the possibility to degrade OGT, an enzyme responsible for post-translational modification of proteins, led to inactive compounds. In addition to working on specific protein degraders, we dedicated a significant portion of our work on the optimization of E3 ligase ligands, especially for cereblon, the results of which will serve the broader PROTAC field for the development of the next generation of degraders.

• Keywords: targeted protein degradation, proteolysis-targeting chimeras