Synthesis and biochemical evaluation of nonpeptidic reversible and irreversible immunoproteasome inhibitors

Evkariontski proteasom je središče ubikvitin-proteasomskega sistema, ki je odgovoren za ohranjanje homeostaze proteinov ter regulacijo celičnih procesov. Ločimo dve glavni obliki, in sicer konstitutivni proteasom, ki se izraža v vseh evkariontskih celicah, ter imunoproteasom, ki se izraža v hematopoetskih celicah ter v vseh evkariontskih celicah med akutnimi imunskimi in stresnimi odgovori. Zaviralci obeh oblik proteasoma so perspektivne učinkovine za zdravljenje različnih bolezni, vključujoč nevrodegenerativne bolezni. Napredek v poznavanju imunoproteasoma ter njegove vloge pri različnih boleznih v zadnjih letih je vodil do napredka v načrtovanju in razvoju zaviralcev njegove aktivnosti. Na Fakulteti za farmacijo raziskujemo spojine psoralenskega tipa, katerih spojino vodnico smo odkrili z virtualnim rešetanjem ZINC knjižnice spojin. V doktorskem delu sta opisana sinteza in biokemijsko vrednotenje spojin psoralenskega tipa s spremembami na mestih 3 in 4\u27 psoralenskega skeleta. Sintetizirali smo derivate z izboljšano selektivnostjo do [beta]5i podenote imunoproteasoma in opisali smo posledično razširjeno razmerje med strukturo in aktivnostjo spojin psoralenskega tipa. Doktorsko delo opisuje tudi načrtovanje spojin, ki bi hkrati zavirale imunoproteasom, kelirale kovinske ione ter delovale antioksidativno. Pristop hkratnega ciljanja omenjenih tarč z namenom razvoja zdravljenja nevrodegenerativnih bolezni v literaturi še ni opisan. Razvili smo inovativno pot sinteze predhodno opisanega, selektivnega in reverzibilnega zaviralca imunoproteasoma (2-cikloheksil-N-(7-(4-feniltiazol-2-il)-kinolin-2-il)sukcinaminska kislina) ter sintezo njegovega analoga z 8-hidroksikinolinskim fragmentom kot strukturnim motivom, ki omogoča kelacijo kovinskih ionov. Čeprav je uvedba 8-hidroksikinolinskega fragmenta v strukturo znanega zaviralca vodila do izgube zaviralne aktivnosti napram imunoproteasomu, predstavljajo rezultati opisanega dela dobro izhodišče za nadaljnje načrtovanje in sintezo spojin, ki bi hkrati zavirale imunoproteasom, kelirale kovinske ione ter delovale antioksidativno.The eukaryotic proteasome is the centre of the ubiquitin-proteasome system, which is responsible for the regulation of different cellular processes and the protein homeostasis. We differentiate two major types of proteasomes. The constitutive proteasome is expressed in all eukaryotic cells while the immunoproteasome is expressed in haematopoietic cells and in other eukaryotic cells during acute stress and/or immune responses. Inhibitors of both proteasome types are being investigated as potential therapeutic approaches for various diseases, including neurodegenerative disorders. In recent years, advances in the understanding of the immunoproteasome and its pathophysiological functions enabled significant progress in the development of immunoproteasome inhibitors. At the Faculty of Pharmacy, compounds with a psoralen core are being investigated as selective immunoproteasome inhibitors. The initial lead compound was discovered through virtual screening of the ZINC compound library. This doctoral thesis describes the synthesis and biochemical evaluation of psoralen-based compounds with changes in the substituents at positions 3 and 4\u27 of the psoralen core. We synthesised derivatives with improved selectivity for the [beta]5i subunit of the immunoproteasome. The synthesised compounds also provided information necessary to further describe the structure-activity relationships of the psoralen-based immunoproteasome inhibitors. This doctoral thesis also includes the development of compounds with the aim of simultaneously inhibiting the immunoproteasome, chelating metal ions and acting as antioxidants. This type of multitarget approach to treat neurodegenerative disorders has not yet been described. We developed an innovative synthesis route for a known selective immunoproteasome inhibitor (2-cyclohexyl-4-oxo-4-((7-(4-phenylthiazol-2-yl)quinolin-2-yl)amino)butanoic acid) and the synthesis of its 8-hydroxyquinoline derivative. The 8-hydroxyquinoline structural motif was incorporated for its metal chelating properties. The incorporation of this motif led to loss of inhibitory activity against the immunoproteasome but the results of this work, nevertheless, represent a good starting point for further development of compounds with the described multitarget approach for the treatment of neurodegenerative diseases