diplomsko delo
Sonja Gabrijelčič (Author), Marko Novinec (Mentor)

Abstract

Širjenje bakterijske odpornosti na antibiotike je v zdravstvu in znanosti vedno večji problem. Za reševanje tega problema je ključno odkrivanje novih spojin s protimikrobnimi lastnostmi, predvsem takih, ki vplivajo na nove celične tarče. V prvem delu diplomske naloge smo zato presejali del knjižnice spojin, sintetiziranih na Katedri za organsko kemijo FKKT UL. Iskali smo biološko učinkovite male molekule – specifično tiste, ki zavrejo rast bakterij (za delo smo uporabili laboratorijski sev bakterije Escherichia coli DH5α). Osredotočili smo se predvsem na derivate 2 piridona, saj je piridonsko ogrodje farmacevtsko pomembno pri iskanju novih zdravilnih učinkovin. Po osnovnih presejalnih testih smo se osredotočili na bolj natančno karakterizacijo protibakterijskih lastnosti N,N-dietil-6-okso-1,4-difenil-1,6-dihidropiridin-3-karboksiamida in 4-(3-klorofenil)-1-(2-metoksietil)-5-(piperidin-1-karbonil)piridin-2(1H)-ona ter strukturno sorodnih spojin. Za oceno minimalne inhibitorne koncentracije prve spojine smo opazovali odvisnost protibakterijskih lastnosti od koncentracije – pripravili smo serijo redčitev spojine, ki smo jo dodajali bakterijski kulturi E. coli DH5α v tekočem gojišču. Izvedli smo tudi difuzijske antibiograme šestih strukturno sorodnih spojin in pripravili rastne krivulje E. coli DH5α v tekočem gojišču LB, v katero smo dodali izbrane spojine. Pri iskanju novih tarč iščemo interakcijski par malomolekulske učinkovine in biološke makromolekule. Če izhajamo iz znanih spojin in iščemo njihove tarče, pogosto uporabljamo tehnike afinitetne kromatografije. Zato smo v drugem delu diplomske naloge izvedli afinitetni kromatografiji na kolonah z vezanima ligandoma z benzenovim (4-(2-aminoetil)-5-hidroksi-1-fenil-1H-pirazol dihidroklorid) oziroma cikloheksilnim (4-(2-aminoetil)-5-hidroksi-1-cikloheksil-1H-pirazol dihidroklorid) obročem. Na kolono smo nanašali celični lizat bakterijske kulture E. coli MG1655 (divji tip). Dobljene vzorce smo analizirali z NaDS-PAGE in rezultate primerjali z dvema skupinama rezultatov, ki so bili pridobljeni iz predhodnih eksperimentov. Opazovali smo razliko med vzorci lis po elektroforezni analizi vzorcev afinitetnih kromatografij, izvedenih v okviru te diplomske naloge, afinitetnih kromatografij, izvedenih z istimi kolonami, vendar z lizatom človeških celic celične linije U937, ter afinitetnih kromatografij, izvedenih s kolono z vezanim ligandom 4-(2-aminoetil)-1-(piridin-2-il)-1H-pirazol-5-ol (ki namesto piridonskega, benzenovega ali cikloheksilnega obroča vsebuje piridinski obroč) in lizatom celic bakterije E. coli DH5α.

Keywords

antibiotiki;malomolekulske učinkovine;proteinske tarče;interakcije protein - mala molekula;difuzijski antibiogram;afinitetna kromatografija;piridoni;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [S. Gabrijelčič]
UDC: 547.824(043.2)
COBISS: 74874883 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Assessment of antibacterial properties of selected 2-pyridone derivatives
Secondary abstract: 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.
Secondary keywords: antibiotics;small-molecule drug;disk diffusion test;protein-small molecule interactions;affinity chromatography;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 40 str.
ID: 13253954