The technology of heterobifunctional degraders – PROTACs, also nammed chimeric degraders, represents an advanced approach to targeted protein degradation, which utilizes the ubiquitin-proteasome system for the selective removal of pathological proteins. This approach enables selective targeted degradation of proteins, which could be useful in the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's. Butyrylcholineesterase (BChE) is an enzyme hydrolyzing acetylcholine, an important neurotransmitter in the central and peripheral nervous system. It is present in various tissues, including liver, brain and blood plasma, and although its physiological function has not been studied in as much detail as that of the related acetylcholinesterase (AChE), BChE is gaining attention for its role in the pathology of Alzheimer's disease. Indeed, research shows that inhibition of BChE improves cholinergic transmission and cognitive functions in patients with this disease, which opens up possibilities for the development of novel therapies. Several BChE inhibitors are described in the literature, however, chimeric degraders that selectively degrade BChE are yet unknown.
In the course of the thesis, we synthesized BChE degraders, which contain a ligand for BChE connected to E3 ligase von Hippl-Lindau ligand via ethylene glycol and alkyl linkers. A cycloheptylindole-based, nanomolar BChE inhibitor was used as the BChE recognition motif, which enables the attachment of the linker via the central amine. When bound to BChE active site, this linker is pointing outside the active site cavity. The synthesis included the preparation of protected linker, N-alkylation of the secondary amine vithe previously prepared linker, deprotection of the ester and, in the last step, the formation of the amide bond using a coupling reagent. For a total of seven synthesized heterobifunctional ligands, we first determined their inhibitory potencies using recombinant human AChE and hBChE. Compound 31 with an octyl linker is the most potent BChE inhibitor, with lowest IC50 value of 0.005345 ± 0.00040 µM. The results also showed that derivatives with a longer lipophilic alkyl chain inhibit BChE more potently than shorter analogues or analogues with an ethylene glycol linker. This observation can be attributed to the relatively lipophilic character of the BChE active site. In the final step, we also evaluated the effectiveness of BChE degradation using SH-SY5Y neuroblastoma cell line. The levels of BChE in the cell line was most effectively reduced by hexylamide 29 at a concentration of 1 µM and following 24 h exposure, while some additional analogues also reduced BChE levels, albeit to a lesser extent. Heterobifunctional ligands prepared within this thesis, in particular derivative 29, represent a good starting point for further studies and development of in vivo active BChE degraders.
|
