Načrtovanje, sinteza in biološko vrednotenje substratov encima ALDH1A3 za selektivno ciljanje matičnih rakavih celic

Matjašec, Nuša (avtor), Hrast Rambaher, Martina (mentor), Josa Culleré, Laia (komentor)

V zadnjih desetletjih je velik napredek na področju rakavih matičnih celic omogočil boljši vpogled v patofiziologijo rakavih obolenj. Čeprav ta subpopulacija celic predstavlja manjšino malignih celic v tumorju, imajo njihove lastnosti samoobnavljanja in diferenciacije pomembno vlogo pri razumevanju ponovnega nastanka tumorja, pojava metastaz in kemorezistence. Pomembna značilnost matičnih rakavih celic, zlasti pri raku dojke, je prekomerna ekspresija encima ALDH1A3, ki oksidira aldehide v ustrezne kisline ob prisotnosti kofaktorja NADH. Ta mehanizem se uporablja za selektivno označevanje in prepoznavanje matičnih rakavih celic z uporabo fluorescenčnega substrata Aldefluor. Namen tega dela je bil raziskati nov pristop za zdravljenje raka dojk s sintezo substratov ALDH1A3, povezanih z zaviralcem HDAC1 SAHA, ki predstavlja farmakološko aktivni del spojine. Mehanizem temelji na kopičenju oksidirane spojine po presnovi z ALDH1A3, kar omogoča selektivno ciljanje rakavih matičnih celic preko sledeče inhibicije HDAC1. Sintezo substratov ALDH1A3 smo zasnovali na strukturi Aldefluora, ki je bila modificirana z benzaldehidom in dodatno alkilno skupino. Za biološko vrednotenje smo uporabili dva različna pristopa z rekombinantnim ALDH1A3. Prva metoda je temeljila na detekciji tvorjenega NAD+, katerega nismo zaznali pri nobeni spojini. Zato smo se osredotočili na detekcijo kislinskega produkta s pomočjo HPLC in dokazali oksidacijo sintetiziranih spojin. Sledila je sinteza konjugata SAHA z azidno skupino, ki je bil nato povezan s substratom preko klik reakcije, da smo dobili končno spojino. S pomočjo rekombinantnega HDAC1 smo izmerili zaviralni učinek konjugata SAHA in potrdili biološko aktivnost spojine. Za overdnotenje končne spojine so potrebne dodatne optimizacije v sinteznem postopku ter metodah biološkega vrednotenja. Rezultati magistrske naloge služijo kot izhodišče za nadaljno raziskovanje novega pristopa terapije proti rakavim matičnim celicam.

• Jezik: Slovenski jezik
• Ključne besede: matične rakave celice, ALDH1A3 encim, selektivno ciljanje, združevanje molekul, SAHA
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-151187
• Datum objave v RUL: 30.09.2023

MATJAŠEC, Nuša, 2023, Načrtovanje, sinteza in biološko vrednotenje substratov encima ALDH1A3 za selektivno ciljanje matičnih rakavih celic [na spletu]. Magistrsko delo. [Dostopano 5 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=151187

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design, synthesis and biological evaluation of ALDH1A3 substrates for the selective targeting of cancer stem cells

Izvleček

Cancer treatment remains to be one of the biggest challenges in medical research, especially due to chemoresistance, metastasis, and tumour relapse. As conventional therapies do not provide the desired outcome, various novel approaches for tumour eradication are being investigated. The discovery and research on CSCs have shown great promise for the development of new potential therapies, consequently, different approaches for the selective pharmacological targeting of CSCs are being investigated. One notable characteristic of CSCs, particularly in breast cancer, is the overexpression of the ALDH1A3 enzyme, which oxidises aldehydes to corresponding acids. This mechanism is used to selectively mark and identify CSCs using the fluorescent substrate Aldefluor. The present study aimed to investigate a novel approach for breast cancer treatment, through the synthesis of ALDH1A3 substrates linked to the HDAC1 inhibitor SAHA, which represents the pharmacologically active part of the compound. Upon its metabolism by upregulated ALDH1A3, the drug is expected to accumulate inside the CSCs, resulting in a selectively higher concentration of the agent and thereby leading to their eradication. The synthesis of the ALDH1A3 substrates was based on the Aldefluor structure, which was modified with a benzaldehyde and an additional alkyne group. Following this, a SAHA conjugate incorporating an azide group was designed and subsequently coupled with the substrate to yield the final molecule. Several bioassays were conducted to evaluate the biological activity of the ALDH1A3 substrates and to measure the inhibitory effect of the SAHA conjugate on HDAC1. Results confirmed the biological activity of both compounds separately, although further modifications of the assays are required to evaluate the final compound. The research findings show great potential and represent a further step toward the development of treatment strategies targeting CSCs.

• Ključne besede: Cancer stem cells (CSC), ALDH1A3 enzyme, selective targeting, molecular conjugation, SAHA