Sinteza in biološko vrednotenje na pomalidomidu temelječih heterobifunkcionalnih razgrajevalcev butirilholin esteraze

Črešnar, Tina (avtor), Pišlar, Anja (mentor), Knez, Damijan (komentor)

Pri patogenezi Alzheimerjeve bolezni se je med prvimi uveljavila holinergična hipoteza, kjer je kot vzrok upada kognitivnih sposobnosti v ospredju znižanje holinergičnega prenosa. Pri tem imata ključno vlogo holin esterazi, acetilholin esteraza (AChE) ter butirilholin esteraza (BChE), odgovorni za razgradnjo acetilholina v sinaptični špranji. Encima sta si strukturno precej podobna, le da aktivno mesto BChE omogoča vezavo večjih substratov, kar lahko izkoristimo pri načrtovanju selektivnih zaviralcev le-te. Učinkovit farmakološki pristop utišanja biološke funkcije proteinov je tudi tarčna razgradnja proteinov s tehnologijo razgrajevalcev PROTAC, ki izkorišča z ubikvitinom posredovano proteasomsko razgradnjo proteinov. Molekulo PROTAC sestavljajo ligand za ubikvitin ligazo E3, distančnik in ligand, ki veže tarčni protein. Najpogosteje uporabljena ubikvitin ligaza E3 je cereblon, ki kot ligand prepozna talidomid ter njegova analoga lenalidomid in pomalidomid. Pri sintezi molekul PROTAC je kombinacija dolžine, hidrofobnosti, rigidnosti distančnika kot tudi mesta pritrditve na ligande ključnega pomena za uspešno delovanje razgrajevalcev. V magistrski nalogi smo pripravili različno dolge alkilne in etilenglikolne distančnike, ki smo jih z nukleofilno substitucijo pripeli na osnovni ligand – zaviralec BChE, na drug konec pa na pomalidomid – ligand za ligazo E3 cereblon. Končnim spojinam smo določili rezidualno aktivnost in zaviralno koncentracijo – vrednost IC50, s čimer smo ovrednotili njihovo zmožnost selektivnega zaviranja encimske aktivnosti človeške BChE. Iz rezultatov smo sklepali, da pri obeh encimih (AChE ter BChE) dolžina distančnika nima bistvenega vpliva na zaviralno aktivnost, malenkost bolj učinkoviti zaviralci so bile spojine z alkilni distančniki, kar lahko razložimo s hidrofobno strukturo aktivnega mesta BChE. V zadnji stopnji smo ovrednotili, kako uspešno pripravljeni himerni razgrajevalci, molekule PROTAC, razgradijo humano BChE v celični liniji SH-SY5Y. Najbolj učinkovito sta ravni izražanja BChE v celicah SH-SY5Y znižala razgrajevalca 35 (po 6 h, pri koncentraciji 0,1 µM) in 32 (po 24 h, pri koncentraciji 1 µM). Nekatere spojine PROTAC, sintetizirane v okviru magistrske naloge, so izkazale delno učinkovitost pri razgradnji tarčnega proteina BChE, kar nakazuje na potencial nadaljnjega raziskovanja spojin PROTAC in morebitne klinične uporabnosti pri patogenezi Alzheimerjeve bolezni.

• Jezik: Slovenski jezik
• Ključne besede: butirilholin esteraza, himerni razgrajevalci, pomalidomid
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-160575
• Datum objave v RUL: 31.08.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis and biological evaluation of pomalidomide-based heterobifunctional butyrylcholinesterase degraders

Izvleček

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.

• Ključne besede: butyrylcholinesterase, proteolysis targeting chimeras, pomalidomide