Načrtovanje in sinteza himernih razgrajevalcev kinaze TAK1

Herakovič, Lea (author), Sosič, Izidor (mentor), Strašek Benedik, Nika (co-mentor)

Vnetna bolezenska stanja so razširjena skupina akutno in kronično pogojenih bolezni. Na voljo imamo širok nabor zdravil, s katerimi v večini primerov vnetje ustavimo ali ustrezno nadziramo. Kljub temu ta zdravila izkazujejo določene neželene učinke, ki otežujejo njihovo dolgoročno rabo. Z namenom razvoja novih protivnetnih zdravil so v ospredju raziskave protein kinaz, novejših tarč, ki so udeležene v znotrajceličnem vnetnem signaliziranju in v poteh sinteze vnetnih mediatorjev. Z njihovim zaviranjem bi lahko na drugačen način omejili vnetni odziv telesa in bolj specifično ciljali prizadeti del organizma. S transformirajočim rastnim dejavnikom β-aktivirana kinaza 1 (TAK1) spada v razred protein kinaz in ima pomembno vlogo pri uravnavanju signalnih poti, povezanih z vnetnimi procesi in celičnim preživetjem. Prekomerno aktivna TAK1 poganja pozitivno povratno zanko nastanka vnetnih mediatorjev, zato je privlačna tarča za razvoj novih protivnetnih učinkovin. Takinib je trenutno najvidnejši predstavnik do sedaj znanih zaviralcev TAK1, ki do tarče izkazuje visoko afiniteto in zadovoljivo selektivnost. Kot klasičen zaviralec ima tudi nekaj splošnih pomanjkljivosti, ki lahko omejijo uporabnost molekule v in vivo okolju, kot so vzdrževanje višjih koncentracij učinkovine na mestu delovanja, delovanje po modelu zasedenosti in v stehiometričnem razmerju glede na tarčo. Z obetavnim pristopom tehnologije himernih razgrajevalcev ali molekul PROTAC (ang. proteolysis targeting chimeras) se tem pomanjkljivostim lahko izognemo. Gre za uporabo bifunkcionalnih molekul, sestavljenih iz liganda za tarčni protein, distančnika in liganda za izbrano ligazo E3, ki za svoje delovanje izkoriščajo ubikvitin proteasomski sistem za tarčno razgradnjo patološkega proteina – v našem primeru je to TAK1. Na osnovi derivata takiniba smo v okviru magistrske naloge pripravili tri molekule PROTAC, ki se med seboj razlikujejo po ligandu za izbrano ligazo E3 – to je bodisi ligand za CRBN, VHL ali IAP. Končne spojine smo biokemijsko vrednotili s pomočjo encimskih testov, njihovo sposobnost tarčne proteasomske razgradnje TAK1 pa smo ovrednotili na izbrani celični liniji MDA-MB-231 z metodo prenosa western. Ugotovili smo, da nekatere izmed njih izkazujejo dobro afiniteto do TAK1 in uspešno sprožijo njeno razgradnjo.

• Language: Slovenian
• Keywords: vnetje, TAK1, takinib, himerni razgrajevalci, ligaze E3
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-146916
• Publication date in RUL: 16.06.2023

Secondary language

• Language: English
• Title: Design and synthesis of TAK1 proteolysis targeting chimeras

Abstract

Inflammatory diseases are a wide group of acutely and chronically conditioned diseases. We have acess to a wide range of drugs, which we can use to stop or at least control inflammation. However, these drugs exhibit certain side effects that makes their long-term use questionable. This is why the research of protein kinases, newer targets that are involved in intracellular inflammatory pathways and synthesis of inflammatory mediators, is gaining more recognition. By inhibiting these targets we could limit the body's inflammatory response in an alternative way and target the affected part of the body more specifically. Transforming growth factor β-activated kinase 1 (TAK1) belongs to the class of protein kinases and plays an important role in regulating signaling pathways related to inflammatory processes and cell survival. Overactive TAK1 drives a positive feedback loop of inflammatory mediator production, making it an attractive target for the development of new anti-inflammatory agents. Takinib is currently the most prominent representative of TAK1 inhibitors known to date, showing favorable affinity and selectivity within the kinome. As a classical inhibitor, it also has some general disadvantages, such as the need to maintain high concentrations of the active substance at the site of action in vivo, relying on the occupancy driven model and in a stoichiometric ratio to the target. These shortcomings can be avoided with proteolysis targeting chimeras (PROTACs). This approach involves the use of bifunctional molecules consisting of a ligand for the target protein, a linker and a ligand for the selected E3 ligase which exploit the ubiquitin proteasome system for the targeted degradation of the pathological protein which in our case is TAK1. As part of the master's thesis, we prepared three PROTACs based on the takinib derivative, which differ from each other in terms of the ligand for the selected E3 ligase – these are either the ligand for CRBN, VHL or IAP. The final compounds were evaluated biochemically with enzymatic assays and on the selected cell line MDA-MB-231. We found that some of the prepared molecules showed a good affinity for TAK1 and successfully induced its degradation.

• Keywords: inflammation, TAK1, takinib, proteolysis targeting chimeras, ligases E3