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Kloniranje skrajšane različice zapisa za dihidroorotat dehidrogenazo iz organizma Plasmodium falciparum in in silico študija inhibicije proteina z izbranimi inhibitorji
MEDVED, TADEJ (Author), Klemenčič, Marina (Mentor) More about this mentor... This link opens in a new window

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Abstract
Malarija trenutno še vedno spada med najbolj zaskrbljujoče nalezljive bolezni po svetu. Vsako leto terja več kot 400.000 smrtnih žrtev, blažji primeri bolezni pa negativno vplivajo tudi na prebivalstvo najbolj prizadetih držav. Hudo obliko malarije povzroča protozoj Plasmodium falciparum, katerega določeni sevi so v zadnjih letih razvili odpornost proti uveljavljenim protimalarijskim zdravilom. Posledično je razvoj novih, vrstno specifičnih zdravil postal zelo aktualen. Ena bolj obetavnih tarč za inhibicijo je biosintezna pot pirimidinskih nukleotidov, v kateri hitrost omejujočo reakcijo izvaja dihidroorotat dehidrogenaza. Do sedaj so raziskovalci že razvili več skupin organskih molekul, ki ta encim inhibirajo, in določili njihovo kristalno strukturo v kompleksu s tem encimom. Ena od teh je skupina bicikličnih pirazolidinonov, katerih kristalna struktura pa še ni bila razrešena. V tem diplomskem delu smo želeli pripraviti konstrukt z zapisom za rekombinantno dihidroorotat dehidrogenazo Plasmodium falciparum, uporaben za ekspresijo tega proteina in za določanje njegove kristalne strukture v kompleksu s tremi najmočnejšimi pirazolidinonskimi inhibitorji. Nato smo hoteli z in silico simulacijami sklepati na način vezave. Pripravili smo konstrukt z zapisom za rekombinanten encim z delecijo strukturno neurejene zanke, ki ne vpliva na vezavo inhibitorjev, a encimu preprečuje tvorbo kristalov. Delecijo smo uspešno opravili z mutagenezo z delno prekrivajočima oligonukleotidoma, nastali konstrukt pa je tudi pripravljen za vključitev v ekspresijski vektor z ligacijo lepljivih koncev. Simulacijo vezave oziroma umestitev 3D-struktur inhibitorjev smo opravili s programom AutoDock Vina. Umestitev je bila zastavljena z delno fleksibilnostjo vezavnega mesta, zaradi česar smo dobili bolj informativne rezultate kot prejšnja umestitvena študija. Ti rezultati so tudi bolj v skladu z obstoječimi kinetičnimi podatki. Rezultati simulacije nakazujejo, da se ti pirazolidinoni opirajo predvsem na hidrofobne interakcije z ostanki vezavnega mesta in se ne vežejo tako globoko kot druge skupine inhibitorjev, poleg tega pa tudi ne nastanejo klasične vodikove vezi. Vezavo naj bi tudi olajšale interakcije π-elektronskega oblaka fenilne skupine z različnimi stranskimi verigami, odvisno od položaja te skupine na pirazolidinonskem obroču.

Language:Slovenian
Keywords:dihidroorotat dehidrogenaza, Plasmodium falciparum, pirazolidinoni, umestitev
Work type:Bachelor thesis/paper (mb11)
Tipology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2020
COBISS.SI-ID:28392707 This link opens in a new window
Views:229
Downloads:103
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Secondary language

Language:English
Title:Cloning of the truncated variant of dihydroorotate dehydrogenase from Plasmodium falciparum and an in silico study of inhibition with chosen inhibitors
Abstract:
Malaria currently remains one of the most worrisome global infectious diseases, causing more than 400.000 deaths annually, with non-lethal infections still having a stark negative impact on the populations of countries where the disease is widespread. Severe malaria is predominantly caused by the protozoan Plasmodium falciparum, with strains of the organism resistant to established antimalarial drugs appearing in recent years. This development strengthened the incentive to discover novel, species-selective drugs to inhibit the growth of Plasmodium falciparum. One of the most promising targets for potential inhibitors is the biosynthetic pathway for pyrimidine nucleotides, whose rate-limiting step is catalysed by dihydroorotate dehydrogenase. To date, researchers have identified several classes of potential small-molecule dihydroorotate dehydrogenase inhibitors as well as solved the crystal structures of some of these inhibitors in complex with the enzyme. A recently discovered inhibitor class are the bicyclic pyrazolidinones, whose crystal structures with the enzyme have yet to be elucidated. The aim of our research was to prepare a construct for the expression of a recombinant variant of Plasmodium falciparum dihydroorotate dehydrogenase capable of forming diffraction-quality crystals as was previously described. This would aid future research aiming to study the crystal structures of the three most promising pyrazolidinone inhibitors bound to the enzyme. The recombinant protein contains a deletion of a disordered loop that would otherwise prevent crystallisation of the protein. The construct was successfully prepared through mutagenesis by overlap extension PCR and can further be ligated into an expression vector of choice. We also performed an in silico docking study using AutoDock Vina to predict the binding modes of these pyrazolidinones. The 3D-structures of the inhibitors were docked into a partially flexible binding pocket. As a result, we obtained more informative data compared to a recent similar docking study. Our results were also more in line with existing kinetic data. We postulate that the binding of pyrazolidinones relies predominantly upon hydrophobic interactions with the binding pocket, with little possibility for classic hydrogen bond formation, which distinguishes this inhibitor class from most of the other inhibitor types. The results also suggest that the substituted phenyl group, present on each of the docked inhibitors, plays a non-negligible part in lowering their binding energy by π-stacking and similar interactions.

Keywords:dihydroorotate dehydrogenase, Plasmodium falciparum, pyrazolidinones, docking

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