Identifikacija proteinskih tarč derivata pirazola v bakteriji Escherichia coli

Mikulič Vernik, Nika (avtor), Novinec, Marko (mentor)

Odkrivanje spojin s protimikrobnimi lastnostmi je odločilnega pomena za preprečevanje rastoče odpornosti proti antibiotikom. Za iskanje zdravil farmacevtska industrija uporablja pristop iskanja ligandov, ki vežejo (in s tem inhibirajo) tarčni protein. Da bi zaznali tudi interakcije z drugimi celičnimi komponentami in se s tem izognili spojinam s potencialno toksičnim vplivom, v našem laboratoriju uporabljamo pristop iskanja proteinskih tarč malih molekul. V diplomskem delu smo želeli določiti proteinske tarče male molekule 4-(2-aminoetil)-1-(piridin-2-il)-1H-pirzol-5-ol, za katero smo ugotovili, da zavira rast laboratorijskega seva DH5α bakterije Escherichia coli. Spojino smo imobilizirali na agarozni nosilec in pripravljeno kolono uporabili za afinitetno kromatografijo, pri kateri smo iz lizata E. coli izolirali interagirajoče proteine. Eluirane frakcije smo nato analizirali z NaDS-PAGE in poslali na analizo z masno spektrometrijo. Ugotovili smo, da dve intenzivni lisi na gelu predstavljata treonin dehidrogenazo in Fe–S podenoto sukcinat dehidrogenaze. Glede na literaturo je možen mehanizem bakteriostatičnega delovanja tarčne spojine inhibicija treonin dehidrogenaze, ki tako ni sposobna sinteze signalnih molekul. Inhibicija sukcinat dehidrogenaze verjetno ne vpliva na rast bakterije E. coli.

• Jezik: Slovenski jezik
• Ključne besede: kemoterapevtiki, malomolekulski ligand, afinitetna kromatografija, masna spektrometrija, medcelična signalizacija
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-118724
• COBISS.SI-ID: 27183363
• Datum objave v RUL: 31.08.2020

MIKULIČ VERNIK, Nika, 2020, Identifikacija proteinskih tarč derivata pirazola v bakteriji Escherichia coli [na spletu]. Diplomsko delo. [Dostopano 3 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=118724

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Identification of protein targets of a pyrazole derivative in Escherichia coli

Izvleček

The discovery of compounds with antimicrobial properties is crucial to prevent the growing resistance to antibiotics. To search for drugs, the pharmaceutical industry utilises the approach of finding ligands that bind (and thus inhibit) the target protein. In order to detect interactions with other cellular components and thus avoid compounds with potentially toxic side effects, we used a small molecule to protein targeting approach in our laboratory. In this thesis, we aimed to identify the protein targets of the small molecule 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrzol-5-ol, which was shown to inhibit the growth of Escherichia coli laboratory strain DH5α. The compound was immobilized on agarose beads and the prepared column was used for affinity chromatography with which we isolated interacting proteins from the E. coli lysate. Eluted fractions were analyzed by SDS-PAGE and sent for protein identification by mass spectrometry. Two prominent bands on the gel were identified as threonine dehydrogenase and the Fe-S subunit of succinate dehydrogenase. According to the literature, the possible mechanism for bacteriostatic action of the compound is inhibition of threonine dehydrogenase which is involved in the biosynthesis of signalling molecules. Inhibition of succinate dehydrogenase is unlikely to affect E. coli growth.

• Ključne besede: chemotherapeutics, low molecule weight ligand, affinity chromatography, mass spectrometry, quorum sensing