Sinteza N-fenilindolamidnih zaviralcev proteina Hsp90

Korošec, Tajda (avtor), Zidar, Nace (mentor)

Proteini toplotnega šoka (ang. heat shock proteins, Hsp) so skupina funkcionalno povezanih proteinov, ki so odgovorni za stabilizacijo in pravilno zvijanje proteinov. Njihova naloga je zaviranje denaturacije in agregacije proteinov v celici ter stabilizacija razvite polipeptidne verige pri izpostavljenosti celičnemu stresu. Zaradi tega so zelo pomembni tudi pri rasti in preživetju rakavih celic, kjer sta sinteza proteinov in celični stres povečana. To je razlog, da so zaviralci Hsp potencialne protirakave učinkovine. Hsp90 je 90 kDa velik, najpogostejši in povsod navzoč protein toplotnega šoka. Je molekularni spremljevalni protein, ki je odgovoren za pravilno zvijanje več kot dvesto proteinov, med drugim tudi mnogih onkoproteinov, vključno s kinazami in transkripcijskimi dejavniki, ki imajo ključen pomen pri maligni transformaciji. Novobiocin, kumarinski antibiotik, je prvi odkriti zaviralec C-končne domene Hsp90. Na žalost se je izkazalo, da imajo novobiocin in drugi kumarinski antibiotiki dokaj nizko aktivnost v celicah in zato omejen terapevtski potencial. Vseeno pa predstavljajo novobiocin in njegovi analogi dobro osnovo za razvoj analogov z izboljšano zaviralno aktivnostjo. V sklopu magistrske naloge smo sintetizirali ter fizikalno-kemijsko in biološko ovrednotili substituirane N-fenilindolamide kot zaviralce proteina Hsp90. Izhajali smo iz znanih zaviralcev Hsp90 in s strukturnimi spremembami poskušali izboljšati njihovo delovanje. Ohranili smo osnovni skelet, ki je ključen za delovanje. Pirolni obroč na levi strani molekule smo zamenjali z različno substituiranimi indoli, verigo na desni strani molekule pa smo podaljšali. S spreminjanjem strukture smo pripravili tri nove spojine, ki se od znanih zaviralcev Hsp90 razlikujejo v velikosti, polarnost in možnosti tvorbe interakcij z vezavnim mestom na tarči. Končne sintetizirane spojine smo poslali na biološka testiranja, da bi ugotovili ali se spojine vežejo na N-končno domeno dveh izoform proteina Hsp90, Hsp90N alfa in Hsp90N beta. Za testiranje smo uporabili metodo FTSA (ang. fluorescence-based thermal shift assay), pri kateri merimo razliko v termični stabilnosti proteina Hsp90 ob prisotnosti in brez zaviralca. Naše končne spojine (15, 16 in 18) se ne vežejo na N-končno domeno izoform alfa in beta Hsp90 pri koncentraciji 200 µM, torej niso aktivne. Na podlagi rezultatov v preglednici I lahko sklepamo, da so naše spojine prevelike za vezavo v N-končno domeno Hsp90.

• Jezik: Slovenski jezik
• Ključne besede: protein toplotnega šoka, Hsp90, šaperon, rak
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-139567
• Datum objave v RUL: 05.09.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The synthesis of N-phenylindolamide-based Hsp90 inhibitors

Izvleček

Heat shock proteins (Hsp) are a group of functionally related proteins that are responsible for stabilizing and for proper folding proteins. Their main roles are to inhibit the denaturation and aggregation of proteins in the cell and to stabilize the developed polypeptide chain upon exposure to cellular stress. Because of this, they are very important in the growth and survival of cancer cells, where protein synthesis and cellular stress are increased. Therefore, Hsp inhibitors are potential anticancer agents. Hsp90 is a 90 kDa large, most abundant, and ubiquitous heat shock protein. It is a molecular chaperone responsible for the proper folding of more than two hundred proteins, among others many oncoproteins, including kinases and transcription factors, which are crucial in malignant transformation. Novobiocin, a coumarin antibiotic, was the first discovered Hsp90 C-terminal domain inhibitor. Unfortunately, novobiocin and other coumarin antibiotics have shown to have relatively low cell activity and therefore limited therapeutic potential. However, novobiocin and its analogues provide a good basis for the development of analogues with improved inhibitory activity. The aim of the master’s thesis was to synthesize and evaluate the physicochemical and biological properties of substituted N-phenylindolamides as Hsp90 inhibitors. We started from the known Hsp90 inhibitors and tried to improve their activity with structural changes. We preserved the basic scaffold, which is crucial for the activity. The pyrrole ring on the left side of the molecule was replaced with various substituted indoles, and the chain on the right side of the molecule was extended. By changing the structure, we synthesized three new compounds that differ from known Hsp90 inhibitors in size, polarity, and the possibility of making interactions with the binding site on the target. The final synthesized compounds were biologically tested to determine whether they bind to the N-terminal domain of the two isoforms of the Hsp90 protein, Hsp90N alpha and Hsp90N beta. Compounds’ binding to Hsp90 was determined by the fluorescence-based thermal shift assay (FTSA), which determines the thermal stability of the free and ligand-bound protein. Our final compounds (15, 16 and 18) do not bind to the N-terminal domain of the isoform alpha and beta Hsp90 at a concentration of 200 µM, therefore they are not active. Based on the results in Table I, we can conclude that our compounds are too large to bind to the N-terminal domain of Hsp90.

• Ključne besede: heat shock protein, Hsp90, chaperone, cancer