Načrtovanje in sinteza na pomalidomidu temelječih himernih razgrajevalcev monoamin oksidaze A

Maffi, Katarina (avtor), Knez, Damijan (mentor), Pišlar, Anja (komentor)

Tehnologija usmerjene razgradnje proteinov s himernimi razgrajevalci kaže velik potencial za modulacijo aktivnosti proteinov, ki običajnim nizkomolekularnim zaviralcem predstavljajo težavno tarčo. Veliko takih proteinov igra vlogo pri nastanku nevrodegenerativnih bolezni, kot sta Alzheimerjeva in Parkinsonova bolezen. Prednost omenjene tehnologije je, da ena molekula razgrajevalca povzroči razgradnjo več molekul tarčnega proteina, zaradi česar bi bil za terapevtski učinke potreben nižji odmerek zdravila. Izoobliki encima monoamin oksidaza; monoamin oksidaza A (MAO-A) in monoamin oksidaza B (MAO-B) igrata večplastno vlogo pri delovanju nevronov in pri različnih boleznih možganov, sintezni in naravni zaviralci teh izooblik pa kažejo nevroprotektivne učinke. Številne študije potrjujejo učinkovitost zaviralcev monoamin oksidaze pri lajšanju simptomov bolezni in zdravljenju motenj razpoloženja, nevrodegenerativnih boleznih ter tudi nekaterih vrst raka. V sklopu magistrske naloge smo načrtovali in sintetizirali na pomalidomidu temelječe himerne razgrajevalce encima MAO-A. Molekulo razgrajevalca sestavljata molekuli harmina (ligand za humano (h)MAO-A) in pomalidomida (ligand za ligazo E3), povezani z distančniki različnih struktur. Pri sintezi smo izhajali iz harmina, ki smo mu pod kislimi pogoji demetilirali metoksi skupino, dobljeni fenol pa alkilirali s predhodno pripravljenim distančnikom, ki je na eni strani vseboval terc-butiloksikarbonil zaščiteno amino skupino, na drugi strani pa elektrofilni alkil bromid. Sledila je acidolitična odstranitev zaščitne skupine in reakcija primarnega amina s 4-fluorotalidomidom. Po opisanem postopku smo sintetizirali štiri molekule, ki so vsebovale distančnike različnih dolžin (heksil, dietilenglikol, trietilenglikol in tetraetilenglikol), pri čemer analitski podatki za trietilenglikolni derivat niso bili v skladu s pričakovano strukturo. Rezultati biokemijskega testa na izoliranem encimu hMAO-A so pokazali, da so trije derivati zavirali encimsko aktivnost hMAO-A z IC50 vrednostmi v nanomolarnem območju, trietilenglikolni analog pa je bil neaktiven. Za derivate, ki so zavirali hMAO-A in se torej vežejo v njegovo aktivno mesto, smo na nevroblastomski celični liniji SH-SY5Y preverili, ali spojine inducirajo razgradnjo hMAO-A. Preliminarni rezultati so pokazali, da je bila razgradnja hMAO-A pod uporabljenimi eksperimentalnimi pogoji žal neučinkovita.

• Jezik: Slovenski jezik
• Ključne besede: himerni razgrajevalci, monoamin oksidaza, nevrodegenerativne bolezni, harmin, pomalidomid
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-143235
• Datum objave v RUL: 09.12.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design and synthesis of pomalidomide-based monoamine oxidase A proteolysis targeting chimeras

Izvleček

Targeted protein degradation technology, including proteolysis-targeting chimeras (PROTACs), offers great potential for modulating the activity of proteins that are difficult targets for conventional small molecules. Many of these proteins play key roles in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's. The advantage of this technology is that a single PROTAC molecule causes the degradation of multiple molecules of the target protein requiring a lower dose of drug. The isoforms of monoamine oxidase; monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) play a multifaceted role in neuronal function and healthy brain. In addition, synthetic and natural inhibitors of these two isoforms have neuroprotective effects. Numerous studies confirm their efficacy in alleviating disease symptoms and progression of mood disorders, neurodegenerative disorders, and certain cancers. As part of our master’s thesis, we designed and synthesised pomalidomide-based MAO-A proteolysis-targeting chimeras. The PROTAC molecule consists of harmine (ligand for human (h)MAO-A) and pomalidomide (ligand for E3 ligase) connected by linkers of different structures. The synthesis was based on harmine demethylated at the methoxy group under acidic conditions. The resulting phenol was alkylated with a previously prepared linker containing tert-butyloxycarbonyl protected amine moiety on one side and an electrophilic alkyl bromide on the other side of the molecule. Subsequently, the protecting group was removed by acidolysis and the amine was reacted with 4-fluorothalidomide. Four molecules with linkers of different lengths (hexyl, diethylene glycol, triethylene glycol, and tetraethylene glycol) were synthesised by the described route, ant the analytical data for the putative triethylene glycol derivative were not in accordance with the expected structure. The results of the biochemical assay on isolated hMAO-A showed that three derivatives inhibited the enzymatic activity of hMAO-A with IC50 values in the nanomolar range, whereas the triethylene glycol analogue was inactive. For the three derivatives that inhibited hMAO-A and therefore bound to the active site, we tested whether they induced hMAO-A degradation in the neuroblastoma SH-SY5Y cell line. According to the preliminary results, unfortunately, the degradation of hMAO-A was not effective.

• Ključne besede: proteolysis-targeting chimeras, monoamine oxidase, neurodegenerative diseases, harmine, pomalidomide