20.500.12556/RUL-182206 Razvoj metode tekočinske kromatografije, sklopljene z masno spektrometrijo, za ugotavljanje znotrajcelične koncentracije izbranih zaviralcev ß-N-acetilglukozaminil transferaze Development of a liquid chromatography–mass spectrometry-based method for determining the intracellular concentration of selected β-N-acetylglucosaminyl transferase inhibitors Znotrajcelične koncentracije učinkovine so ključni parameter pri oceni njene farmakološke učinkovitosti. Odražajo količino, ki doseže svojo tarčo znotraj celice. Na prehod učinkovine skozi celično membrano vpliva vrsta dejavnikov kot so lastnosti celične membrane, prisotnost transportnih mehanizmov, intracelularna vezava in presnovna stabilnost spojine. Zato merjenje zgolj zunajcelične koncentracije ne omogoča natančne ocene dejanskega vnosa spojine v celico. Za določanje znotrajceličnih koncentracij je potrebna občutljiva in selektivna analitska tehnika, saj so količine učinkovine v celičnih lizatih pogosto zelo nizke, matrica pa kompleksna in vsebuje številne endogene spojine, ki lahko motijo analizo. Najpogosteje uporabljena tehnika za določanje znotrajceličnih koncentracij učinkovin je tekočinska kromatografija visoke ločljivosti (HPLC), sklopljena z masno spektrometrijo (MS), ki omogoča občutljivo, selektivno in zanesljivo analizo širokega spektra spojin.V sklopu magistrske naloge smo razvili LC-MS analizno metodo za določanje znotrajceličnih koncentracij spojine Osmi-4b in njenih šestih analogov, ki delujejo kot zaviralci encima O-β-N-acetilglukozaminil transferaze (OGT). Spojine imajo skupen heterociklični skelet, razlikujejo pa se po substituentu na dušikovem atomu amidne skupine. OGT sodeluje v procesu O-GlcNAc glikozilacije, pri katerem se na serinske ali treoninske ostanke proteinov dodaja N-acetilglukozamin (GlcNAc). Zaviranje OGT ima potencialno terapevtsko vrednost pri Alzheimerjevi bolezni, nekaterih vrstah raka in drugih boleznih, povezanih s spremenjeno glikozilacijo. Pri razvoju analizne metode smo za vsako spojino s testiranjem izbranih verifikacijskih parametrov potrdili ustreznost metode za določanje znotrajceličnih koncentracij. Nato smo analizirali celične lizate iz linije AMO-1 po inkubaciji z izbranimi spojinami. Rezultati so pokazali, da so znotrajcelične koncentracije izbranih spojin sorazmerne z vrednostmi logD pri pH 7,5. Najnižje znotrajcelične koncentracije smo določili za polarnejše spojine DY-38 in DY-39, medtem ko je DY-34 pokazala podobno porazdelitev kot Osmi-4b. Najvišje koncentracije so bile določene pri spojinah AVS-18 in AVS-20, ki so presegale vrednosti Osmi-4b. Intracellular concentrations represent a key parameter in assessing pharmacological efficacy. They reflect the amount of active compound that reaches its target within the cell. The passage of an active compound across the cellular membrane is influenced by various factors including the properties of the cell membrane, the presence of transport mechanisms, intracellular binding, and the compound’s metabolic stability. Consequently, measurement of extracellular concentrations alone does not provide an accurate estimate of the active compound’s true intracellular uptake. Determining intracellular concentrations requires a sensitive and selective analytical technique, as the amounts of the active compound in cell lysates are often very low, and the matrix is complex, containing numerous endogenous compounds that can interfere with the analysis. The most used technique for measuring intracellular concentrations of active compounds is high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS), which allows for sensitive, selective, and reliable analysis of a wide range of compounds. In the scope of this master’s thesis, we developed an LC-MS analytical method for determining intracellular concentrations of the compound Osmi-4b and its six analogues, which act as inhibitors of the enzyme O-β-N-acetylglucosaminyl transferase (OGT). These compounds share a common heterocyclic scaffold but differ in the substituent (-R) on the nitrogen atom of the amide group. OGT is involved in the process of O-GlcNAc glycosylation, in which N-acetylglucosamine (GlcNAc) is added to serine or threonine residues of proteins. Inhibiting OGT has potential therapeutic value for Alzheimer’s disease, certain types of cancer, and other disorders associated with altered glycosylation. During the development of the analytical method, we validated its suitability for determining intracellular concentrations for each compound by testing selected verification parameters. Subsequently, we analyzed cell lysates from the AMO-1 cell line after incubation with the selected compounds. The results showed that the intracellular concentrations of the selected compounds were proportional to their logD values at pH 7.5. The lowest intracellular concentrations were observed for the more polar compounds DY-38 and DY-39, while DY-34 exhibited a distribution similar to that of Osmi-4b. The highest concentrations were measured for AVS-18 and AVS-20, which exceeded the values of Osmi-4b. tekočinska kromatografija visoke ločljivosti HPLC masna spektrometrija znotrajcelična koncentracija zaviralci OGT encim OGT high-performance liquid chromatography HPLC mass spectrometry intracelullar concentrations OGT inhibitors enzyme OGT true false false V. Zlatevska Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-04-30 14:28:43 2026-04-30 14:29:01 2026-05-01 03:56:21 0000-00-00 00:00:00 2026 0 Ljubljana 2026 Ljubljana 1 spletni vir (1 datoteka PDF (IX, 69 str.)) 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 615:543(043.2) 272579587 Fakulteta za farmacijo 0 0 0