Vrednotenje himernih razgrajevalcev proteina TAK 1 na človeški epitelijski celični liniji raka dojk MDA-MB 231

Tot, Zoja (author), Gobec, Martina (mentor), Strašek Benedik, Nika (comentor)

S TGF-β aktivirana kinaza 1 (TAK1) je eden izmed osrednjih regulatorjev celične smrti, ki se aktivira z različnimi znotraj- in zunajceličnimi dražljaji. TAK1 je nujen člen v signalni poti tumor nekrotizirajočega faktorja (TNF), interlevkina 1 (IL-1) in Toll-u podobnih receptorjev (TLR). Njegovo neuravnano delovanje tako predstavlja pomemben člen pri nekaterih patologijah. Študije kažejo, da je TAK1 potencialna terapevtska tarča pri vnetnih, rakavih in avtoimunskih obolenjih, saj sodeluje pri imunskem odzivu, preživetju celic in tudi pri njihovi presnovi. Ker na tržišču še ni registriranega zaviralca proteina TAK1, je bil namen magistrske naloge ovrednotiti nabor novih spojin kot morebitnih modulatorjev delovanja TAK1 in tako doprinesti k razvoju na tem področju. Za samo analizo smo uporabili himerne razgrajevalce (angl. Proteolysis Targeting Chimeras – PROTAC). To so molekule, ki so sestavljene iz treh delov, in sicer: liganda za izbrano ubikvitin ligazo E3, ustreznega vmesnika in liganda, ki se veže na tarčni protein. Zaradi svojih prednosti, postajajo molekule PROTAC pomembna alternativa klasičnim zaviralcem. Kot ligazo E3 smo izbrali tri različne ligande, in sicer: von Hippel-Lindau (VHL), cereblon (CRBN) in apoptozo zavirajoči protein (IAP). Vse poskuse smo izvajali na celični liniji raka dojke MDA-MB 231. Z encimskim testom smo najprej potrdili, da sintetizirane molekule PROTAC ohranijo afiniteto do tarčnega proteina TAK1. Sledil je test metabolne aktivnosti, kjer smo se želeli prepričati, da molekule PROTAC ne delujejo citotoksično. Viden vpliv na celično viabilnost smo opazili pri spojinah, ki vsebujejo ligand IAP. V nadaljevanju smo vrednotili, ali molekule PROTAC povzročijo razgradnjo proteina TAK1. Ugotovili smo, da so najbolj obetavni himerni razgrajevalci proteina TAK1 tisti, ki so osnovani na ligandih IAP. V prihodnje bodo potrebna dodatna biološka vrednotenja ter strukturne optimizacije, ki bodo opredelile bolj učinkovite himerne molekule.

• Language: Slovenian
• Keywords: Protein TAK1, himerni razgrajevalci, celična linija MDA-MB 231, prenos western.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151695
• Publication date in RUL: 17.10.2023

Secondary language

• Language: English
• Title: Evaluation of chimeric TAK 1 protein degraders of on the MDA-MB 231 epithelial human breast cancer cell line

Abstract

TGF-β-activated kinase 1 (TAK1) is one of the central regulators of cell death, activated by a variety of intra-cellular and extracellular stimuli. TAK1 serves as a critical element in the tumour necrosis factor (TNF), interleukin 1 (IL-1) and Toll-like receptors (TLR) signalling, thus its unbalanced function represents an essential component in some pathologies. Several studies suggest that TAK1 is potentially a therapeutic target in inflammatory, cancerous and autoimmune diseases, as it is implicated in the immune response, cell survival and also in cell metabolism. Since there is no TAK1 inhibitor on the market yet, the aim of this thesis is to identify and evaluate a series of new compounds as potential modulators of TAK1 activity thus contributing to the development in this field. Proteolysis targeting chimeras (PROTACs), which were used for the analysis, are molecules composed of three components: a ligand for the selected E3 ubiquitin ligase, a suitable PROTAC linker and a ligand that binds to the target protein. Due to their advantages, PROTAC molecules represent an important alternative to conventional inhibitors. Three different ligands for E3 ligases have been selected in our master's thesis: von Hippel–Lindau (VHL), cereblon (CRBN) and inhibitor of apoptosis (IAP) proteins. All experiments were carried out on the MDA-MB 231 breast cancer cell line. Using an enzyme-linked immunosorbent assay, we initially verified that the synthesised PROTAC molecules have retained the affinity for the TAK1. Next, we conducted a metabolic assay to ensure that the synthesised PROTAC molecules do not exhibit cytotoxic effects. A noticeable effect on cell viability was observed for compounds containing the IAP ligand. We further evaluated whether PROTAC molecules induce degradation of TAK1 protein. We concluded that the most promising chimeric degraders of TAK1 protein are IAP ligand-based ones. However, further biological evaluations and structure optimizations will be required in the future to identify more efficient chimeric molecules.

• Keywords: TAK1 protein, chimeric degraders, MDA-MB 231 cell line, western blot.