20.500.12556/RUL-147031 Sinteza in biološko vrednotenje heterobifunkcionalnih razgrajevalcev proteinov iz družine IAP Synthesis and biological evaluation of heterobifunctional IAP protein degraders Tradicionalni pristop načrtovanja zdravilnih učinkovin je usmerjen v regulacijo aktivnosti proteinov z uporabo malih molekul z visoko afiniteto. Uporabnost takšnega pristopa omejuje potreba po ustreznem vezavnem mestu, ki ga okoli 80 % človeškega proteoma ne izkazuje in je posledično farmakološko nedostopen za takšne zaviralce. Rešitev lahko predstavlja nova metodologija tarčne razgradnje proteinov z uporabo heterobifunkcionalnih molekul (ang. proteolysis targeting chimeras - PROTACs), ki za svoje delovanje uporabljajo telesu endogeni mehanizem za razgradnjo proteinov. Te himerne spojine so sestavljene iz treh delov, in sicer iz liganda za ligazo E3, tarčnega proteina ter distančnika, ki ju povezuje. Njihova učinkovitost leži v mehanizmu in katalitični naravi delovanja, ki jim omogoča, da pri zelo nizkih koncentracijah s prehodnimi interakcijami s katerimkoli mestom na proteinu izzovejo njegovo razgradnjo. Zaradi tega so himerni razgrajevalci postali uporabno orodje pri farmakološki modulaciji proteoma, ki je težko dostopen preko klasičnega načrtovanja učinkovin. Narava delovanja takšnih razgrajevalcev je pritegnila tudi pozornost onkološkega zdravljenja, saj molekule PROTAC z razgradnjo proteina lažje zaobidejo določene mehanizme pridobitve odpornosti, ki predstavlja težavo mnogim zdravilnim učinkovinam na tem področju. Ena izmed pomembnih tarč v onkologiji so proteini, imenovani zaviralci apoptoze (IAP), ki so v celicah zadolženi za zaviranje celične smrti. Znanstveniki so odkrili, da se ti proteini prekomerno izražajo znotraj rakavih tkiv, kar je povezano s progresijo bolezni, slabo prognozo in nizkim odzivom na zdravljenje. Zaradi teh razlogov so proteini IAP postali zanimive tarče zdravljenja rakavih obolenj. V sklopu magistrskega dela smo sintetizirali himerni razgrajevalec, ki je vseboval med seboj povezana liganda za cereblon in IAP. Končna molekula heteroPROTAC je v testih na rakavi celični liniji multiplega mieloma MM.1S z metodo prenosa western izkazala koncentracijsko-odvisno selektivno razgradnjo predstavnika družine IAP, in sicer XIAP. Pri višjih koncentracijah smo pokazali tako razgradnjo vseh vrednotenih proteinov IAP kot tudi delno razgradnjo transkripcijskega faktorja IKZF3. Nivo ligaze cereblon je ostal tekom testiranja nespremenjen, kar priča o dejstvu, da je potekla le enostranska razgradnja proteinov IAP. Poleg metode western smo razgradnjo dokazali tudi s proteomiko, ki je pokazala statistično značilno znižanje nivoja proteina XIAP. Sintetizirana molekula himernega razgrajevalca predstavlja tako uporabno probo za raziskave vpletenosti proteinov IAP v patogenezo malignih obolenj kot tudi dobro izhodišče za nadaljnji razvoj terapevtikov. The traditional approach to development of therapeutics is aimed at regulating the activity of proteins with the use of small-molecule inhibitors. The limitation of such an approach is the need for an appropriate binding site, which around 80 % of the human proteome does not exhibit, thus making them undruggable with such inhibitors. A new method of targeted protein degradation could provide the solution with the use of heterobifunctional molecules (PROTACs) which hijack the body's endogeneous mechanism for protein degradation. These chimeric molecules are comprised of three parts: E3 ligase ligand, target protein ligand and a linker. The effectiveness lies in their mechanism of action and catalytic nature, which allows them to induce targeted protein degradation with only transient interactions with protein at low concentrations. For these reasons, chimeric degraders showed usefulness in the field of oncology as they can circumvent certain resistance mechanisms due to their mode of action. One of many important targets in oncology are inhibitor of apoptosis (IAP) proteins, which play a key role inhibiting cell apoptosis. Scientists have uncovered that these proteins are overly expressed inside cancerous tissue and are associated with bad prognosis, disease progression and low response to treatment. Therefore, IAP proteins represent interesting targets for potential cancer treatment. In the scope of this thesis, we synthesised a chimeric degrader which incorporates ligands for ligases cereblon and IAP that were connected via a linker. Our heteroPROTAC was tested on a multiple myeloma cell line MM.1S with the method of western blotting, and showed concentration-dependent selective degradation of XIAP, an important member of the IAP protein family. At higher concentrations, we observed elimination of all IAP proteins tested, as well as partial degradation of transcription factor IKZF3. The protein levels of ligase cereblon remained unaltered, confirming the unilateral degradation of IAP proteins. Protein depletion was also demonstrated with the use of global proteomic analysis, where a selective reduction of XIAP protein levels was shown. The synthesised degrader represents both a useful probe for future research of the involvement of IAP proteins in the pathogenesis of malignant diseases, as well as a good starting point for further therapeutic development. Himerni razgrajevalci tarčna razgradnja proteinov apoptoza cereblon protein IAP. Proteolysis-targeting chimeras targeted protein degradation apoptosis cereblon IAP protein true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-06-21 08:45:02 2023-06-21 08:45:18 2023-06-22 03:37:30 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 95251 14267.pdf 14267.pdf 1 DFA9E0EDD619BFBC714497C049BFD52B 7be663a83abbc548b492af074f26ba77669c985b734c92feb5b9e96408954a95 d0e9d925-0ffe-11ee-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=170377 Fakulteta za farmacijo 0 0 0