In the pathogenesis of Alzheimer’s disease, the cholinergic hypothesis was one of the earliest theories to explain the disease, in which a decrease in cholinergic transmission is considered to be the main cause of cognitive decline. Cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are the main enzymes responsible for the degradation of acetylcholine in the synaptic cleft. They are structurally quite similar, with the exception that the active site of BChE allows the binding of larger substrates, which may be useful in the design of selective BChE inhibitors. PROTAC technology, which utilizes the ubiquitin-proteasome system, has been shown to be an effective pharmacological approach for targeted protein degradation to silence the biological activity of proteins. PROTAC molecules consist of an E3 ubiquitin ligase ligand, a linker and a ligand that binds the protein of interest. The most commonly used ubiquitin ligase E3 is cereblon, which binds thalidomide and its analogs lenalidomide and pomalidomide. When synthesizing PROTACs, the right combination of length, hydrophobicity and rigidity of the linker as well as the binding of the two ligands is crucial.
In the master's thesis, we synthesized alkyl and ethylene glycol linkers of different lengths, which were attached to BChE inhibitor by nucleophilic substitution, and to pomalidomide, a ligand for E3 ligase cereblon. Residual activities and inhibitory potencies, IC50 values, were determined for final compounds, thereby establishing their ability to selectively inhibit the enzymatic activity of BChE. From the results, we concluded that for both enzymes (AChE and BChE), the length of the linker has no significant effect on the inhibitory potency, slightly more potent were inhibitors with alkyl linkers. The latter can be explained by the hydrophobic structure of the BChE’s active site. In the last step, we evaluated efficiency of chimeric degraders, PROTAC molecules, to degrade human BChE in the SH-SY5Y cell line. BChE levels were most effectively reduced by the degraders 35 (after 6 hours, at a concentration of 0.1 µM) and 32 (after 24 hours, at a concentration of 1 µM). In the scope of the master’s thesis, some synthesized PROTAC compounds have shown partial efficacy in the degradation of the target protein BChE, indicating the potential for further investigation and clinical use in the pathogenesis of Alzheimer's disease.
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