magistrsko delo
Tadej Medved (Author), Marina Klemenčič (Mentor), Marko Dolinar (Thesis defence commission member), Marko Novinec (Thesis defence commission member)

Abstract

Malarija je še vedno ena izmed najbolj razširjenih nalezljivih bolezni na svetu, ki letno prizadene več kot 200 milijonov ljudi. Povzročajo jo protozoji iz rodu Plasmodium, izmed katerih je najbolj smrtonosna vrsta Plasmodium falciparum. Kljub širokem repertoarju protimalarijskih zdravil izkoreninjenje bolezni napreduje počasi, predvsem zaradi sprotnega pridobivanja odpornosti vrst Plasmodium proti uveljavljenim učinkovinam. Zaradi tega je nastala pobuda za sprotni razvoj novih, bolj učinkovitih in specifičnih zdravil, ki ciljajo različne tarče v plazmodijevem življenjskem ciklu. Med temi tarčami je encim dihidroorotat dehidrogenaza, ki v biosintezni poti pirimidinov katalizira pretvorbo L-dihidroorotata v orotat in je ključen za uspešno razmnoževanje plazmodijev. Z uporabo empiričnih metod in računalniškega načrtovanja so raziskovalci do zdaj sintetizirali že veliko selektivnih inhibitorjev tega encima z raznolikimi heterocikličnimi ogrodji. Tri novejše serije takih spojin so biciklični pirazolidinoni, 5-hidroksipirazoli in 3-hidroksipirazoli, katerih tarča je dihidroorotat dehidrogenaza iz vrste Plasmodium falciparum (PfDHODH). V magistrskem delu smo hoteli okarakterizirati zgornje tri serije spojin bodisi kinetično z encimskim testom bodisi strukturno s kokristalizacijo s tarčnim encimom. Obema skupinama hidroksipirazolov smo z uporabo kolorimetričnega encimskega testa želeli določiti moč inhibicije PfDHODH. Pokazali smo, da testirani hidroksipirazoli kvečjemu le šibko inhibirajo encim že pri sorazmerno visoki koncentraciji inhibitorja. Najboljšo inhibitorno aktivnost so pokazali 3-hidroksipirazoli, ki so močneje inhibirali encim kot etilmalonatni inhibitor, iz katerega so bili izpeljani. Biciklične pirazolidinone, katerih kinetični parametri so že znani, pa smo hoteli kokristalizirati v kompleksu s PfDHODH, da bi lahko pridobili strukturne podatke o vezavi teh spojin na encim. Izrazili smo rekombinantno, N-končno skrajšano obliko PfDHODH z dodatno delecijo strukturno neurejene zanke za izboljšano kristalizacijo. Protein smo očistili v treh stopnjah, pripravili komplekse s tremi bicikličnimi pirazolidinoni in jih poskusili kokristalizirati iz sedečih kapljic. Nepričakovano je dodatek teh spojin povzročil agregacijo rekombinantnega PfDHODH, kar je vplivalo na rezultat kristalizacije. Dobili smo en kristal kompleksa z enim od testiranih bicikličnih pirazolidinonov, a kristal ni bil dovolj kvaliteten za difrakcijsko analizo, je pa dal informacijo o ugodnem pogoju za tvorbo kristalov.

Keywords

malarija;Plasmodium falciparum;dihidroorotat dehidrogenaza;hidroksipirazoli;biciklični pirazolidinoni;kristalizacija;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [T. Medved]
UDC: 577.15:547.77(043.2)
COBISS: 131905283 Link will open in a new window
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Downloads: 27
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Other data

Secondary language: English
Secondary title: Characterization of select bicyclic pyrazolidinones and hydroxypyrazoles as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase
Secondary abstract: Malaria remains one of the most widespread infectious diseases, affecting over 200 million people around the world each year. The disease is caused by protozoans of the genus Plasmodium, among which Plasmodium falciparum is by far the deadliest. Despite the extensive repertoire of antimalarial drugs and therapies, elimination of malaria is progressing slowly, due in large part to the rapid development of resistance to established antimalarials in Plasmodium species. Consequently, efforts have been made to develop more efficacious and selective antimalarial drugs with various targets in the Plasmodium life cycle. One such target is the dihydroorotate dehydrogenase enzyme, which catalyzes the conversion of L-dihydroorotate to orotate in the rate-limiting step of pyrimidine biosynthesis and is crucial to plasmodial replication. Through either empirical methods or computational design, many selective inhibitors of dihydroorotate dehydrogenase with a wide variety of heterocyclic backbones have already been synthesized. Three recent examples are a series of bicyclic pyrazolidinones, 5-hydroxpyrazoles and 3-hydroxypyrazoles targeting Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). In this thesis, we aimed to characterize these three inhibitor series, either kinetically via enzyme assay, or structurally via cocrystallization with the target enzyme. First, we desired to determine the inhibitory strength of the hydroxypyrazole compounds using a colorimetric assay. We showed that, at relatively high inhibitor concentration, both groups of hydroxypyrazoles have weak inhibitory activity at most. The 3-hydroxypyrazoles were the more efficacious of the two series while also exhibiting greater inhibition of PfDHODH than the known ethyl malonate inhibitor that guided their design. Second, we aimed to crystallize the bicyclic pyrazolidinone compounds, whose kinetic parameters have already been determined, in complex with PfDHODH to obtain structural data on their binding modes. Through protein expression, we prepared an N-terminally truncated variant of PfDHODH with an additionally deleted disordered loop to improve crystallization. We purified the protein in three stages, complexed it with the chosen inhibitors and attempted to cocrystallize it from a sitting drop. Unexpectedly, incubation of the PfDHODH variant with the compounds caused it to aggregate, affecting the result of the crystallization. We managed to grow a single crystal with one of the tested bicyclic pyrazolidinones, however it was less than diffraction-quality. Nonetheless, we gained information on the specific conditions required for crystallization of this complex to occur.
Secondary keywords: dihydroorotate dehydrogenase;hydroxypyrazoles;bicyclic pyrazolidinones;crystallization;Encimski inhibitorji;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 67 str.
ID: 16525500