Tamara Knific (Avtor), Stanislav Gobec (Mentor), Izidor Sosič (Komentor)

Povzetek

V zadnjih 30 letih so številne študije natančno okarakterizirale strukturo in fiziološko vlogo ubikvitin proteasomskega sistema. Gre za sofisticiran 2,5 MDa velik proteinski sistem imenovan 26S proteasom, ki je sestavljen iz proteolitičnega osrednjega 20S jedrnega dela in dveh terminalnih regulatornih 19S delov. Proteasom je multikatalitični encimski kompleks, ki regulira številne ključne fiziološke in patološke celične procese preko selektivne razgradnje proteinov. Identifikacija in odstranitev napačno zvitih, poškodovanih in toksičnih proteinov je ključna za regulacijo in vzdrževanje celične homeostaze. Substrati za proteasom so tudi številni proteini, ki so del karcinogeneze in so ključnega pomena za preživetje rakave celice. Inhibicija proteasoma v rakavih celicah tako vodi v akumulacijo proapoptotičnih tarčnih proteinov ter indukcijo celične smrti. Področje onkologije daje velik poudarek na identifikaciji novih in za raka specifičnih molekularnih tarč. Eden izmed takšnih tarčnih sistemov je tudi imunoproteasom, natančno reguliran, visoko specifičen sistem, odgovoren za procesiranje številnih proteinov znotraj celice. Odkritje, da so tumorske celice veliko bolj občutljive na delovanje zaviralcev proteasoma kot ostale celice, je usmerilo razvoj v načrtovanje in sintezo številnih terapevtsko uspešnih zaviralcev. S poznavanjem strukture in delovanja predstavlja danes ubikvitin proteasomski sistem obetavno tarčo v terapiji številnih rakavih obolenj. V okviru magistrske naloge smo uspešno sintetizirali več potencialnih zaviralcev imunoproteasoma. Vse pripravljene spojine imajo kot osnovo psoralenski skelet, na katerega je na mestu 4 vezana metilna skupina. Z namenom raziskati kemijski prostor smo na izbrana mesta na skeletu uvajali različne substituente. Sintezo načrtovanih spojin smo izvajali v več zaporednih reakcijah. Najprej smo iz resorcinola ter dietil 2-acetilsukcinata ali dietil 2-acetilpentandionata sintetizirali osnovni 7-hidroksikumarinski obroč. Sledila je uvedba derivatov 2-bromoacetofenona na hidroksi skupino. Nato smo izvedli reakcijo kondenzacije, pri čemer je prišlo do ciklizacije in nastanka psoralenskega strukturnega fragmenta. Na C-terminalni del smo nazadnje uvajali še elektrofilne fragmente, in sicer 1-hidroksipirolidin-2,5-dion, 2-(metilamino)acetonitril ali 2-aminoacetonitril, s čimer smo preko kovalentnih interakcij s katalitično aminokislino v aktivnem mestu želeli doseči močnejšo vezavo sintetiziranih spojin na imunoproteasom. Na Katedri za klinično biokemijo na Fakulteti za farmacijo so nato sintetizirane spojine še biokemijsko ovrednotili. Določevali so rezidualno aktivnost kimotripsinske podenote imunoproteasoma po dodatku spojine. Po pridobljenih podatkih ima največje zaviralno delovanje spojina z oznako 5c. Na podlagi rezultatov lahko sklepamo, da je ključnega pomena tvorba aktiviranega estra, ki lahko tvori kovalentno interakcijo s treoninom v aktivnem mestu imunoproteasoma. Pretvorba C-terminalne karboksilne kisline v amid ter uvedba N-cianometilamidov kot elektrofilov ne vodita v boljše zaviralno delovanje. Prav tako vezava broma na para mesto fenilnega obroča ne pripomore k povečanju afinitete za vezavo na imunoproteasom.

Ključne besede

homeostaza;imunoproteasom;psoralen;cianoderivati;kovalentni zaviralci;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [T. Knific]
UDK: 616-006+577.152.34(043.3)
COBISS: 3703921 Povezava se bo odprla v novem oknu
Št. ogledov: 338
Št. prenosov: 57
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Design and synthesis of psoralene-based inhibitors of the immunoproteasome
Sekundarni povzetek: In the past 30 years there were several studies that have thoroughly characterized the structure and the physiological function of the ubiquitin proteasome system. This is a sophisticated 2.5 MDa protein complex named 26S proteasome, which consists of 20S core particle and two terminal 19S regulatory particles. The proteasome is a complex multicatalytic enzymatic system that regulates vital physiological and pathological cellular processes through selective breakdown of the proteins. Identification and removal of misfolded, damaged and toxic proteins is crucial for the regulation and maintenance of cell homeostasis. Proteins that are involved in the processes of carcinogenesis and cancer cell survival are also substrates for the proteasome. Its inhibition in cancer cells therefore leads to accumulation of pro-apoptotic proteins and induction of cell death. The cancer drug discovery puts a significant emphasis on the identification of new and cancer-specific molecular targets. One of the most recently discovered and very promising targets is also the immunoproteasome, tightly regulated, highly-specific system that is responsible for cellular protein turnover. The discovery that cancer cells are more susceptive to proteasome inhibition than other cells had a strong impact on the discovery and synthesis of novel therapeutically useful inhibitors. The knowledge and recent findings associated with the structure and the functions of the ubiquitin proteasome system make this enzyme machinery a promising target in cancer treatment. Within this masters degree, we successfully synthesized several potential imunoproteasome inhibitors. The prepared compounds had a psoralen core scaffold with methyl group bound at the position 4. With the aim to explore the chemical space, we introduced several carefully planned substituents on the main psoralen structure. The synthesis of compounds was performed in several consecutive reaction steps. Firstly, we synthesized the 7-hydroxycoumarin ring by reacting diethyl 2-acethylsuccinate or diethyl 2-acetylpentanedionate with resorcinol. Then, different 2-bromoacetophenones were introduced to properly modify the hydroxyl group. Afterwards, the condensation reaction was carried out, leading to the formation of the core psoralen ring. Finally, we introduced three different electrophilic moieties at the C-terminus, such as 1- hydroxypyrrolidine-2,5-dione, 2-aminoacetonitrile or 2-(methylamino)acetonitrile. The purpose of this last step was to achieve covalent bonding with the catalytic amino acid in the immunoproteasome active site. All compounds were biochemically evaluated at the Department of Clinical Biochemistry. The residual activities of the chymotrypsin-like imunoproteasome subunit after the addition of compounds were determined. Based on the data, compund 5c showed the most potent inhibitory activity. On the basis of results from inhibition assays, we can also conclude that the transformation of the C-terminal carboxylic acid into an activated ester is crucial for improving inhibition; most probably because of the formation of covalent bonds within the active site of the immunoproteasome. Introduction of an amide bond and Ncianomethylamides as electrophiles did not improve inhibitory effect. In addition, bromine at the para position of the phenyl fragment led to diminished affinity for binding into the active site of the immunoproteasome.
Sekundarne ključne besede: Onkologija;
Vrsta dela (COBISS): Magistrsko delo/naloga
Komentar na gradivo: Univ. Ljubljana, Fak. za farmacijo
Strani: IV, 56 f.
ID: 12053354