magistrsko delo
Nika Mikulič Vernik (Avtor), Marko Novinec (Mentor), Miha Pavšič (Član komisije za zagovor), Ajda Taler-Verčič (Član komisije za zagovor)

Povzetek

Zaradi naraščajoče odpornosti mikroorganizmov proti antibiotikom se mnoge raziskovalne skupine trudijo identificirati biološke makromolekule in metabolne poti znotraj celic mikroorganizmov, ki bi lahko delovale kot tarče za novo odkrite antibiotike. Predhodno smo pokazali, da derivat pirazola 4-(2-aminoetil)-1-(piridin-2-il)-1H-pirazol-5-ol zavira rast bakterije E. coli in se veže na proteina L-treonin dehidrogenaza in sukcinat dehidrogenaza v celičnem lizatu. L-treonin dehidrogenaza, ki nima človeškega homologa, sodeluje pri metabolizmu aminokislin in medcelični komunikaciji, celičnih procesih, ki pri prokariontih in evkariontih potekata zelo različno. Katalizira začetni korak pretvorbe aminokisline L-treonin v L-glicin, prav tako pa L-treonin lahko služi kot izhodna spojina za sintezo analogov avtoinduktorja-3, pomembnega za zaznavanje kvoruma. Sukcinat dehidrogenaza je encim citratnega cikla in je za razliko od L-treonin dehidrogenaze prisotna pri E. coli in pri človeku, njena inhibicija lahko vpliva torej tudi na celice gostiteljskega organizma. Namen magistrske naloge je bil podrobneje okarakterizirati interakcije derivata pirazola z obema encimoma. V ta namen smo pripravili rekombinantno L-treonin dehidrogenazo in topni podenoti A in B sukcinat dehidrogenaze v E. coli. Naš primarni cilj je bil ugotoviti, ali spojina deluje inhibitorno na encim L-treonin dehidrogenaza. Aktivnost L-treonin dehidrogenaze v prisotnosti derivata pirazola smo spremljali z merjenjem fluorescence nastajanja produkta NADH. Vezavo male molekule na proteine L-treonin dehidrogenazo ter podenoti A in B sukcinat dehidrogenaze pa smo preverjali z uporabo izotermne titracijske kalorimetrije (ITC). Z uporabo ITC smo ugotovili, da se derivat pirazola veže na obe citosolni podenoti sukcinat dehidrogenaze in na L-treonin dehidrogenazo z afinitetami v mikromolarnem območju. Prav tako je derivat pirazola inhibiral delovanje L-treonin dehidrogenaze predvsem pri koncentracijah substrata, nižjih od Km. Iz tega lahko sklepamo, da gre za linearni kompetitivni mehanizem inhibicije L-treonin dehidrogenaze. Uporabljen derivat pirazola glede na naše rezultate ne deluje tako, kot bi moral delovati antibiotik. Zaradi vezave na sukcinat dehidrogenazo lahko deluje citotoksično za celice gostitelja. Spojina prav tako verjetno deluje kot kelator kovinskih ionov, kar pomeni, da lahko posredno inhibira tudi mnoge druge bakterijske in gostiteljske encime. S testiranjem delovanja analogov spojine bi postopno lahko odkrili molekule z ožjim naborom bakterijskih proteinskih tarč in močnejšo inhibicijo L-treonin dehidrogenaze.

Ključne besede

derivati pirazola;L-treonin dehidrogenaza;sukcinat dehidrogenaza;interakcije protein-mala molekula;protimikrobno delovanje;magistrska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [N. Mikulič Vernik]
UDK: 547.77:577.15(043.2)
COBISS: 142853891 Povezava se bo odprla v novem oknu
Št. ogledov: 13
Št. prenosov: 4
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Characterisation of interactions between a pyrazole derivative and L-threonine dehydrogenase and succinate dehydrogenase of Escherichia coli
Sekundarni povzetek: Due to the increasing resistance of microorganisms against antibiotics, many research groups are trying to identify novel biological macromolecules and metabolic pathways that could serve as targets for newly discovered antibiotics. Two of the cellular processes, that proceed differently in prokaryotes than in eukaryotes, are amino acid metabolism and cellular communication. The protein L-threonine dehydrogenase is involved in both; it starts the conversion of the amino acid L-threonine to L-glycine, but L-threonine may also lead to the synthesis of autoinducer-3 analogues, important for quorum sensing. Unlike L-threonine dehydrogenase, succinate dehydrogenase is present in E. coli and humans, so its inhibition might also affect the host cell. We have previously shown that a pyrazole derivative 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrazole-5-ol inhibits the growth of bacterium Escherichia coli and binds to the proteins L-threonine dehydrogenase and succinate dehydrogenase. The enzyme L-threonine dehydrogenase has no human counterpart and is involved in amino acid metabolism and cellular communication. Both cellular processes operate differently in prokaryotes than in eukaryotes. L-threonine dehydrogenase initiates the conversion of the amino acid L-threonine to L-glycine or its conversion to autoinducer-3 analogues, which are important for quorum sensing. In contrast, succinate dehydrogenase, an enzyme of the citric acid cycle. Is found in E. coli and in humans, so its inhibition could also affect the host cell. In this master’s’ thesis, our aim was to better characterize interactions between the pyrazole derivative and the two dehydrogenases. To this end, we overexpressed and purified recombinant L-threonine dehydrogenase and soluble subunits A and B of succinate dehydrogenase in E. coli. Our primary goal was to determine whether the compound had an inhibitory effect on L-threonine dehydrogenase. Inhibition of L-threonine dehydrogenase in the presence of the pyrazole derivative was monitored by measuring the fluorescence of NADH formed during the reaction. Binding of the compound to L-threonine dehydrogenase and to subunits A and B of succinate dehydrogenase was detected by isothermal titration calorimetry (ITC). We found that the pyrazole derivative weakly binds both domains of succinate dehydrogenase and L-threonine dehydrogenase. It also inhibits the activity of L-threonine dehydrogenase, especially when the substrate concentration was lower than Km. This suggests a linear competitive mechanism of inhibition. According to our results, the pyrazole derivative we used does not act as an antibiotic compound. Due to its binding to succinate dehydrogenase, it may have a cytotoxic effect on the host cell. The compound also likely acts as a metal ion chelator, meaning that it could potentially inhibit many other enzymes in bacterial and host cells. Nevertheless, by developing novel analogues of the compound, it may be possible to discover molecules with a narrower range of bacterial protein targets and stronger inhibition of L-threonine dehydrogenase.
Sekundarne ključne besede: pyrazole derivative;L-threonine dehydrogenase;succinate dehydrogenase;protein-small molecule interactions;antimicrobial activity;Pirazoli;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 1000377
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani: 62 str.
ID: 18068463