20.500.12556/RUL-106172 Vrednotenje zaviralcev imunoproteasoma z elektrofilnim oksatiazolonom Evaluation of immunoproteasome inhibitors with electrophilic oxathiazolone Imunoproteasom je visoko učinkovit proteazni kompleks, ki se pretežno nahaja v celicah imunskega sistema, kjer sodeluje pri antigenski predstavitvi, uravnavanju izločanja citokinov in diferenciaciji ter proliferaciji limfocitov T. Pod vplivom vnetnih dejavnikov se lahko njegov nastanek inducira tudi v vseh ostalih celicah, kjer se vse tri katalitično aktivne β podenote konstitutivnega proteasoma (β1, β2 in β5) zamenjajo z ustreznimi homolognimi podenotami imunoproteasoma (β1i, β2i in β5i). Njegovo povišano aktivnost povezujejo z nekaterimi vnetnimi, rakavimi in avtoimunskimi obolenji, zato imajo selektivni zaviralci imunoproteasoma velik terapevtski potencial pri zdravljenju teh obolenj. S tem namenom smo v sklopu magistrske naloge kot potencialne zaviralce imunoproteasoma vrednotili deset novih spojin osnovanih na elektrofilnem oksatiazolonu. Z metodami encimske kinetike smo najprej na β5i podenoti določili IC50 vrednosti. Slednje so bile za vse spojine v območju 0,10 – 0,43 µM, pri čemer so najnižjo IC50 izkazovale spojine 55, 172 in 176. Nadalje smo preverili selektivnost zaviralcev v primerjavi z drugimi katalitično aktivnim podenotam imunoproteasoma (β1i, β2i) in proteasoma (β1, β2, β5). Ugotovili smo, da je pri uporabi 10 µM koncentracije spojin, rezidualna aktivnost na vseh ostalih podenotah za spojine 55, 57 in 176 bila nad 60%, zato lahko sklenemo, da te spojine izkazujejo selektivno delovanje na β5i podenoto imunoproteasoma. Spojini 55 in 172 sta imeli najnižjo IC50 za β5i podenoto in sta hkrati dobro selektivni v primerjavi z ostalimi podenotami, zato smo se odločili, da ju še dodatno ovrednotimo. V prvem delu smo preverili ali spojini ohranita svoje zaviralne lastnosti tudi v heterogenih proteinskih sistemih. Zato smo pripravili celične lizate iz celic THP-1 in HeLa. Moč zaviranja na celičnih lizatih se je glede na izolirano β5i podenoto zmanjšala za približno 10-krat, kar nakazuje, da so v kompleksnejših sistemih prisotni tudi drugi proteini na katere se spojini vežeta oziroma jih zavirata. Nadalje smo preverili ali te spojine uspešno prehajajo celice v in vitro pogojih. Ugotovili smo, da je rezidualna aktivnost pri 25 µM koncentraciji za spojino 55 65%, za 172 pa 74%. To kaže na slabo permeabilnost spojine preko intaktne celične membrane in potrebo po optimizaciji strukture. Ne glede na to, smo na koncu preverili ali spojine posredujejo imunomodulatorne učinke na perifernih mononuklearnih celicah (PBMC) zdravih posameznikov. PBMC-je smo aktivirali z lipopolisaharidom in preverili ali spojini vplivata na izločanje vnetnih citokinov (IL-1β, TNFα, IL-8, IL-6, IL-10 in IL-12p70). Ugotovili smo, da statistično znižanje IL-6 in IL-10 povzroči le spojina 172. Podoben trend smo opazili tudi pri spojini 55, a ni bil statistično značilen. Spojine so pokazale potencial kot selektivni zaviralci β5i podenote imunoproteasoma, ki pa bi potrebovale nadaljnjo strukturno optimizacijo, ki bi omogočila boljšo prehodnost preko celične membrane. Immunoproteasom is a highly effective protease complex which is predominantly found in cells of the immune system, where it participates in antigen presentation, regulation of cytokine secretion, differentiation and proliferation of lymphocytes T. In non-hematopoietic cells its expression can be induced under the influence of proinflammatory cytokines or oxidative stress, where all three catalytically active β subunits of constitutive proteasome (β1, β2 in β5) are replaced by the corresponding homologous subunits of the immunoproteasome (β1i, β2i in β5i). Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases and various types of cancer. Therefore, immunoproteasome inhibition offers an attractive therapeutic approach in the treatment of these diseases. In the course of our research, ten new compounds based on electrophilic oxathiazolone were evaluated as potential inhibitors of immunoproteasome. With methods of enzyme kinetics, we firstly determined IC50 values for the ten compounds on the β5i subunit. The values were in the range of 0.10-0.43 μM, with the lowest IC50 values for compounds 55, 172 and 176. Furthermore, we tested the selectivity of all the inhibitors against other catalytically active subunits of immunoproteasome (β1i, β2i) and proteasome (β1 , β2, β5). We found that at 10 μM concentration, the residual activity of all other subunits was above 60% for coumpound 55, 57 and 176. Based on this, we can conclude that these compounds selectively inhibit the β5i subunit of the immunoproteasome. Compounds 55 and 172 had the lowest IC50 for the β5i subunit and at the same time exerted high selectivity towards other subunits, thus decided for further evaluation. In the first part, we checked whether the compounds retain their inhibitory properties in heterogeneous protein systems. For this purpose, cell lysates from THP-1 and HeLa cells were prepared. In cell lysates the IC50 values increased by about 10-fold relative to the isolated β5i subunit, suggesting that in complex systems the compounds bind to or inhibit other proteins. Further, we addressed the properties of cell permeability of the selected compounds in in vitro conditions. We demonstrate that the residual activity at 25 μM concentration was 65% and 74% for the compound 55 and 172 respectively. This indicates the poor permeability of the compounds via the intact cell membrane and the need for structure optimization. In the last segment of our work, we were interested whether the compounds possess any immunomodulatory effects on peripheral mononuclear cells (PBMCs) of healthy individuals. PBMCs were activated with lipopolysaccharide and the potential effect of the two compounds on secretion of inflammatory cytokines (IL-1β, TNFα, IL-8, IL-6, IL-10, and IL-12p70) was determined. We found that the statistical reduction in IL-6 and IL-10 was caused only by compound 172. A similar trend was also observed for compound 55, however it was not statistically significant. To conclude, the compounds show great potential as selective inhibitors of the β5i subunit of immunoproteasom, but further structural optimization would be required to improve permeability over the cell membrane. proteasom zaviralci imunoproteasoma oksatiazoloni encimska kinetika proteasome immunoproteasome inhibitors oxathiazolones enzyme kinetics true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2019-02-06 08:35:07 2019-02-06 08:35:19 2022-08-18 03:48:16 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 67011 4.pdf 4.pdf 1 52B5DE0CBACAAAFA86F4152BD2FD412B a4c6f1aae7ff5444885f77285d1bb69c776e9a34aee994f3adba21d233ecf68b 4b883720-a1b6-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=116912 Fakulteta za farmacijo 0 0 0