20.500.12556/RUL-164572 Proučevanje antifolatnega delovanja izbranih zdravilnih učinkovin in vitro The study of antifolate activity of selected medical compounds in vitro Folat je esencialen vodotopen vitamin B9, ki je ključen za razvoj in rast celic. Sodeluje pri reakcijah prenosa enega ogljika, ki so ključne za biosintezo purinov in timidina, homeostazo aminokislin (glicin, serin, homocistein in metionin), epigenetsko vzdrževanje in obrambo pred oksidativnim stresom. Potreba po folatu je med nosečnostjo povečana, saj je folat nujen za normalen razvoj in rast fetusa, placente in materničnih tkiv. Nizek folatni status nosečnice je povezan z resnimi prirojenimi napakami med katerimi so tudi napake nevralne cevi (NTDs - Neural Tube Defects). Zaprtje nevralne cevi je ključni dogodek v procesu embriogeneze, ki se ponavadi zaključi v 28. dneh po oploditvi. Napake v procesu nevrulacije rezultirajo v resnih prirojenih napakah, ki jih imenujemo napake nevralne cevi. Te imajo multifaktorsko etiologijo, ki vključuje medsebojne delovanje genetskih in okoljskih dejavnikov. Uveljavljeno je, da dodatek folne kisline zmanjšuje pojavnost okvar nevralne cevi, kar je privedlo do številnih javnozdravstvenih kampanj in ukrepov. Znižane ravni folata so lahko posledica prehranjevanja, genetskih predispozicij ali uporabe zdravil, ki vplivajo na folatni status. Zdravilom, ki vplivajo na metabolizem folatov in homocisteina, se morajo ženske v rodni dobi in nosečnice izogibati. Antifolatno delovanje je glavni mehanizem delovanja zdravil kot sta metotreksat in pemetreksed, poleg svojega primarnega delovanja pa lahko nekatera zdravila tudi posredno, nezaželjeno vplivajo na metabolizem folatov (nap. fenitoin, metformin). Cilj te magistrske naloge je bil poiskati učinkovine, ki še nimajo znanega antifolatnega delovanja med pogosto predpisanimi že registriranimi zdravilnimi učinkovinami (ZU). Delo je zajemalo določanje metabolne aktivnosti, analiziranje celičnega cikla in testiranje apoptotičnosti celic, ki smo jih gojili v mediju s folno kislino (+FA) in brez nje (-FA), pod vplivom spojin z domnevnim antifolatnim delovanjem. Med naborom ZU, ki smo jih dobili z virtualnim rešetanjem na osnovi tarče, to sta bila DHFR in MTHFR, in liganda, za katere smo uporabili že znane antifolate kot naprimer metotreksat, smo z analizo delovanja le teh na celičnih modelih SH-SY5Y, ugotovili naslednje: metotreksat (MTX) in pemetreksed (PMX), ki sta znana antifolata, izkazujeta višjo toksičnost v mediju brez folne kisline, v okolju pomanjkanja folata je metabolna aktivnost celic znižana, povzročata zastoj v S fazi celičnega cikla, še bolj v okolju pomanjkanja folata, kot v normalnih pogojih. Ugotovili smo, da fusidna kislina (FUS) izkazuje antifolatno delovanje, saj pri vseh izvedenih testiranjih deluje podobno kot MTX in PMX. Folate is an essential water-soluble vitamin B9 that is crucial for the development and growth of cells. It is involved in one-carbon transfer reactions that are key for the biosynthesis (of purines and thymidine), amino acid homeostasis (glycine, serine, homocysteine, and methionine), epigenetic maintenance, and defense against oxidative stress. The need for folate increases during pregnancy, as folate is necessary for the normal development and growth of the fetus, placenta, and maternal tissues. Low folate status in pregnant women is associated with serious congenital defects, including neural tube defects (NTDs). The closure of the neural tube is a critical event in the process of embryogenesis, typically completed within 28 days after fertilization. Errors in the process of neurulation result in serious congenital defects known as neural tube defects. These have a multifactorial etiology, involving the interaction of genetic and environmental factors. It is established that folic acid supplementation reduces the incidence of neural tube defects, leading to numerous public health campaigns and measures. Lowered folate levels can be due to diet, genetic predispositions, or the use of medications that affect folate status. Women of childbearing age and pregnant women should avoid medications that affect folate and homocysteine metabolism. Antifolate activity is the primary mechanism of action for drugs like methotrexate and pemetrexed; however, some medications can also indirectly and undesirably affect folate metabolism (e.g., phenytoin, metformin). The aim of this master's thesis was to identify compounds, among frequently prescribed already registered active pharmaceutical ingredients (APIs), that do not have known antifolate activity. The work involved determining metabolic activity, analyzing the cell cycle, and testing the apoptosis of cells cultured in a medium with folic acid (+FA) and without it (-FA), under the influence of compounds with suspected antifolate activity. Among the set of APIs obtained through virtual screening based on target, which were DHFR and MTHFR, and the ligands, we used already known antifolates such as methotrexate, we analyzed their effects on SH-SY5Y cell models and found the following: methotrexate (MTX) and pemetrexed (PMX), which are known antifolates, show greater toxicity in a folic acid-free medium; in a folate-deficient environment, the metabolic activity of the cells is reduced, causing a cell cycle arrest in the S phase, more so in a folate-deficient environment than in normal conditions. We discovered that fusidic acid (FUS) exhibits antifolate activity, as it behaves similarly to MTX and PMX in all conducted tests. Antifolati napake nevralne cevi metabolizem folatov fusidna kislina Antifolates neural tube defects folate metabolism fusidic acid true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-10-31 08:45:04 2024-10-31 08:45:17 2024-11-22 14:54:53 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 114269 25494.pdf 25494.pdf 1 0154DADF4B38BCB14F1D8F86E765487E 485a4893e0fc9e2a8cb6b6adaad3c595f79621212857cc5e81ff780e7dcd7c16 c12758ca-975b-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=195096 Fakulteta za farmacijo 0 0 0