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.
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