The spread of antibiotic resistance is a growing problem in both healthcare and pharmaceutical science. To mitigate the problem, we need to search for new antimicrobial compounds, especially those that act on novel cell targets. In the first part of this thesis, we screened a selection of compounds from the compound library of the Chair of Organic Chemistry (FKKT UL). We aimed to determine which of these molecules were bioactive – in particular, which ones inhibited the bacterial growth (we worked with the laboratory strain Escherichia coli DH5α). We focused primarily on 2 pyridone derivatives, as the pyridone scaffold has been utilised in drug discovery. After completing the initial screening tests, we identified two promising compounds: N,N-diethyl-6-oxo-1,4-diphenyl-1,6-dihydropyridine-3-carboxamide and 4 (3-chlorophenyl)-1-(2-methoxyethyl)-5-(piperidine-1-carbonyl)pyridin-2(1H)-one. In the following experiments we focused on these two compounds and several structurally similar molecules. To determine the minimal inhibitory concentration of the first above-mentioned compound, we prepared a series of dilutions of this compound and observed how it affected the growth of the bacterial culture of E. coli DH5α in a liquid growth medium. We also performed disk diffusion tests with six of the observed compounds and generated growth curves of E. coli DH5α in the presence of the selected compounds.
To identify novel targets, we searched for interactions between a small molecule and a biomacromolecule. When attempting to identify a target of an observed compound, we often use methods of affinity chromatography. Therefore, in the second part of this thesis, we preformed affinity chromatography using two different columns with immobilized related pyrazole compounds – one with an immobilised ligand with a benzene ring (4 (2 aminoethyl)-1-phenyl-1H-pyrazole-5-ol) and one with a cyclohexyl group (4 (2 aminoethyl) -1-cyclohexyl-1H-pyrazol-5-ol). As the mobile phase we used a cell lysate of wt E. coli MG1655. Then we analysed the obtained samples using SDS-PAGE and compared the results of these experiments with the results of two sets of affinity chromatography experiments preformed previously. One of them was obtained using the same columns but different cells (human cell line U937), and the other with a column on which the immobilised ligand was 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrazol-5-ol (which does not include a pyridone, benzene or cyclohexane ring, but rather a pyridine ring) and the mobile phase was a cell lysate of E. coli DH5α.
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