20.500.12556/RUL-121043 Načrtovanje in sinteza derivatov psoralen-3-ocetne kisline kot zaviralcev imunoproteasoma Design and synthesis of psoralen-3-acetic acid derivatives as immunoproteasome inhibitors Ubikvitin proteasomski sistem je ogromen celični sistem, ki je odgovoren za razgradnjo kratkoživečih, poškodovanih in nepravilno zvitih citoplazemskih proteinov. Sestavljen je iz ubikvitinaznega sistema, ki prepozna tarčne proteine, ter iz proteasoma, v katerem poteka razgradnja proteinov. Imunoproteasom predstavlja različico standardnega proteasoma. V velikih količinah se izraža v celicah imunskega sistema, v ostalih celicah pa pod vplivom oksidativnega stresa ter provnetnih citokinov. Ob njegovem izražanju se tri katalitične podenote: kaspazi podobna, tripsinu podobna ter kimotripsinu podobna podenota sintetizirajo ter zamenjajo podenote standardnega proteasoma β1, β2 in β5. Dobro raziskana naloga imunoproteasoma je tvorba peptidov s hidrofobnim C-terminalnim koncem, ki se nato vežejo na molekule poglavitnega kompleksa tkivne skladnosti I. Vse več raziskav pa se osredotoča tudi na njegovo neimunsko vlogo. Povečano izražanje in aktivnost katalitičnih podenot imunoproteasoma se pojavlja tudi pri določenih bolezenskih stanjih, kot so rak, kronična vnetna črevesna bolezen ter nevrodegenerativne bolezni, zato postaja imunoproteasom zanimiv kot farmakološka tarča. V okviru našega dela smo uspešno načrtovali, sintetizirali ter okarakterizirali dvanajst strukturno novih spojin z zaviralnim delovanjem na kimotripsinu podobni podenoti imunoproteasoma. Vse sintetizirane spojine v svoji osnovni strukturi vsebujejo psoralenski skelet, na katerega smo na mestih 3 in 4' uvajali strukturne spremembe. Sinteza končnih produktov je potekala v več zaporednih korakih. Najprej smo iz resorcinola in dietil 2-acetilsukcinata pripravili osnovni 7-hidroksikumarinski skelet ter alfa bromirali izbrane heterociklične derivate ketona. Nato smo na hidroksi skupino 7-hidroksikumarina z reakcijo nukleofilne substitucije uvajali alfa bromirane ketone, v naslednji stopnji pa smo spojine ciklizirali do derivatov psoralen-3-ocetne kisline z reakcijo kondenzacije. Na koncu smo sintetizirali aktivirane estre ter N-cianometilamide, ki lahko tvorijo kovalentno interakcijo s treoninom v aktivnem mestu imunoproteasoma. Za ovrednotenje zaviralnega delovanja so sintetiziranim produktom na Katedri za klinično biokemijo določili še rezidualno aktivnost kimotripsinu podobne podenote imunoproteasoma ob prisotnosti sintetiziranih zaviralcev. Ugotovili smo, da imajo najboljše zaviralno delovanje spojine z 2,5-dimetiltiofenskim heteroaromatskim sistemom na 4'- psoralenskem obroču in aktiviranim estrom v obliki sukcinimida kot elektrofilnim The ubiquitin proteasome system is the major cellular system for the regulated degradation of short-lived, damaged or defectively folded cytoplasmic proteins. It consists of the ubiquitination system, which selects and targets proteins, and the proteasome that performs the degradation. The immunoproteasome is a variant form of the standard proteasome. Immune system cells express a higher level of immunoproteasome, while in other cells, oxidative stress and proinflammatory cytokines are the stimuli that lead to elevated production of immunoproteasome. Upon induction, the immunoproteasome catalytic subunits: caspase-like, trypsin-like and chymotrypsin-like subunit, are synthesized and incorporated, replacing their standard proteasome counterparts β1, β2 and β5, respectively. Although the immunoproteasome is thought to be specialized in the generation of major histocompatibility complex class I antigenic peptides with hydrophobic C-terminal residues, increasing evidence indicates additional non-immune functions. Elevated levels of immunoproteasome catalytic subunits have been also observed in certain disease states, such as cancer, inflammatory bowel disease and neurodegenerative diseases. For all these reasons immunoproteasome is becoming an interesting pharmacological target. In the presented work, we successfully designed, synthesized and structurally characterized twelve new compounds with inhibitory activity on the chymotrypsin-like catalytic subunit of the immunoproteasome. All of the synthesized compounds were based on a psoralen scaffold, on which structural changes were introduced at the positions 3 and 4'. The synthesis of final products was performed in several consecutive reaction steps. First, we synthesized 7-hydroxycoumarin ring by reacting resorcinol with diethyl 2- acetylsuccinate and concurrently performed the alpha bromination of selected heterocyclic ketone derivates. Then, the alpha brominated ketones were introduced to properly modify the hydroxyl group of the 7-hydroxycoumarin ring. In the next stage, the condensation reaction was performed leading to the formation of the psoralen-3-acetic acid derivatives. Finally, the carboxylic acid of the prepared compounds was transformed into activated esters and N-cyanomethylamides. These can form a covalent interaction with the threonine in the active site of the immunoproteasome. At the Department of the Clinical Biochemistry, the inhibitory activity of the synthesized products was evaluated. This was performed by determination of the residual activity of the chymotrypsin-like activity of the immunoproteasome in the presence of inhibitors. We have discovered that compounds with 2,5-dimethylthiophene heteroaromatic system at the position 4' of the psoralen ring and an activated succinimidyl ester show the best inhibitory activity. The most potent inhibitor in the presented series was compound, which possessed both above-mentioned fragments in its structure. homeostaza proteasom imunoproteasom zaviralci imunoproteasoma psoralenski skelet homeostasis proteasome immunoproteasome immunoproteasome inhibitors psoralens true false false [N. Erdani] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-29 11:08:00 2020-09-29 11:08:00 2022-09-01 04:01:17 0000-00-00 00:00:00 2015 0 Ljubljana 2015 Ljubljana IV, 58 f. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 615.2:616-097 3942513 MAG_Erdani_Natasa_2015.pdf MAG_Erdani_Natasa_2015.pdf 1 E8C3E0674363F35FC19E5B4F5CE77B65 e41ea788b343f52f7a04e7877a8484a52bd3cf905ff94d32622ef24e2209fda5 35e98eb8-a1ba-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=135893 Fakulteta za farmacijo 0 0 0