Načrtovanje in sinteza na harminu temelječih heterobifunkcionalnih razgrajevalcev monoamin oksidaze A

Kranjc, Aljaž (avtor), Knez, Damijan (mentor)

Monoamin oksidaza (MAO) je encim, ki v človeškem telesu sodeluje pri procesih oksidacije tako endogenih kot tudi eksogenih aminov. Najdemo ga v dveh izooblikah – MAO-A in MAO-B, ki se obe nahajata v membranah mitohondrijev, razlikujeta pa se predvsem po substratih, ki jih oksidirata. MAO-A sodeluje pri razgradnji živčnega prenašalca serotonina zaradi česar predstavlja tarčo pri zdravljenju oziroma zaviranju nevrodegenerativnih bolezni, depresije ter motenj pozornosti, nedavne raziskave pa so potrdile tudi njeno vpletenost v nekatere oblike raka. MAO-A je viden dejavnik pri širjenju rakavih celic preko t.i. perinevralne invazije, ki je pomemben proces metastaziranja raka prostate, zaviralci MAO-A pa so na mišjih modelih raka prostate učinkovito zavrli ta proces. Vidno novo in perspektivno skupino učinkovin predstavljajo heterobifunkcionalni razgrajevalci – molekule PROTAC, ki za izničenje učinka proteina preko njegove razgradnje izkoristijo ubikvitin-proteasomski sistem. V magistrskem delu smo sintetizirali ter biokemijsko ovrednotili pet razgrajevalcev usmerjenih proti encimu MAO-A. Na harmin, ligand za tarčni MAO-A, smo preko različnih distančnikov pripeli ligand za ligazo E3 von Hippel-Lindau. Kot distančnike smo uporabili alkilne verige, etilenglikolne verige ter piperidin oziroma piperidinmetanol. Distančnike smo na harmin pripeli preko reakcije O-alkiliranja – Williamsonove sinteze etrov - po SN2 mehanizmu nukleofilne substitucije, sledila je alkalna hidroliza ali acidoliza zaščitene karboksilne skupine, v zadnji stopnji pa še reakcija tvorbe amidne vezi z uporabo sklopitvenega reagenta HATU. Po čiščenju in karakterizaciji smo za pet spojin izvedli še biokemijsko testiranje, kjer smo preverili, kako učinkovito in selektivno zavirajo MAO-A. Vse spojine so selektivno zavirale MAO-A, izooblike MAO-B niso zavirale, najmočnejši zaviralec pa je bila spojina 34 (IC50 = 34,0 ± 2,7 nM). V splošnem lahko trdimo, da je za močnejšo zaviralno aktivnost spojin bolj ugodna linearna alkilna veriga, manj ugodna pa je uporaba volumsko večjih piperidinskih distančnikov ter etilenglikolnih analogov s približno enako razdaljo med harminom ter ligandom za ligazo von Hippel-Lindau. Opažanja lahko povežemo s strukturnimi značilnostmi aktivnega mesta MAO-A, ki je relativno hidrofobno ter pri izhodu iz aktivnega mesta tudi precej ozko. V nadaljnjih stopnjah vrednotenja bi bilo treba za izbrane ligande na ustreznem celičnem modelu bolezni, bodisi nevrodegenerativne bodisi onkološke, preveriti njihovo sposobnost znotrajcelične razgradnje MAO-A.

• Jezik: Slovenski jezik
• Ključne besede: monoamin oksidaza A, heterobifunkcionalni razgrajevalci, nevrodegenerativne bolezni, harmin, rak prostate
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-164483
• Datum objave v RUL: 26.10.2024

Sekundarni jezik

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

Izvleček

Monoamine oxidase (MAO) is an enzyme involved in the oxidation of both endogenous and exogenous amines in the human body. MAO is found in two isoforms – MAO-A and MAO-B, both of which are located in the membranes of mitochondria, but differ mainly in the substrates they oxidize. MAO-A is involved in the degradation of the neurotransmitter serotonin, making it an important target in the treatment or inhibition of neurodegenerative diseases, depression and attention deficit disorders. Recent research has also confirmed its involvement in some cancers. MAO-A is an important factor in the spread of cancer cells via the so-called perineural invasion, an important process in the metastasis of prostate cancer. MAO-A inhibitors have effectively inhibited this process in mouse models of prostate cancer. An important new and promising group of active substances are heterobifunctional degraders – PROTAC molecules, which exploit the ubiquitin-proteasome system to counteract the effect of a protein via its degradation. In this thesis, we synthesized and biochemically evaluated five ligands – potential degraders that target MAO-A. The ligand for the E3 von Hippel-Lindau ligase was attached to harmine, the ligand for the target MAO-A, via different linkers – alkyl chains, ethylene glycole chains and piperidine or piperedine-4-methanol. The linker was attached to harmine via an O-alkylation, i.e., Williamson synthesis of ethers, by the S2N nucleophilic substitution mechanism, followed by alkaline hydrolysis or hydrolysis of the protected carboxyl group, and in the last step an amide bond formation using the coupling reagent HATU. Following purification and characterization, five compounds were subjected to biochemical testing to verify how efficiently and selectively they inhibit MAO-A. All compounds selectively inhibited MAO-A over MAO-B, and the most potent inhibitor was 34 (IC50 = 34.0 ± 2.7 nM). Arguably, linear alkyl chain is favorable for the potent inhibition of MAO-A, whereas the use of bulkier piperidine spacers and ethylene glycol analogues with approximately the same distance between the harmine and the ligand for the von Hippel-Lindau ligase is unfavorable. The observations can be related to the structural characteristics of the MAO-A active site, which is relatively hydrophobic and also rather narrow at its entrance. In further evaluation steps, selected ligands should be tested for their intracellular MAO-A degradation in a suitable neurodegenerative or oncological cellular disease model.

• Ključne besede: monoamine oxidase A, heterobifunctional degraders, neurodegenerative diseases, harmine, prostate cancer