magistrsko delo
Katja Malenšek (Author), Iztok Turel (Mentor), Ajda Taler-Verčič (Thesis defence commission member), Marko Novinec (Thesis defence commission member)

Abstract

Katepsini so skupina encimov, ki sodelujejo pri razgradnji proteinov s cepitvijo peptidnih vezi. Prisotni so pri vseh organizmih znotraj in izven celic ter tako vključeni v najrazličnejše fiziološke procese. V primeru napačne lokalizacije, odsotnosti ali nereguliranega delovanja igrajo pomembno vlogo tudi pri pojavu različnih bolezni in drugih patoloških stanj. Katepsine glede na katalitične aminokislinske ostanke, ki se nahajajo v aktivnem mestu, delimo na serinske, aspartatne in cisteinske proteaze. Pripadnik slednjih je tudi katepsin L. Gre za lizosomsko endopeptidazo, ki je v celici zadolžena za razgradnjo znotrajceličnih proteinov, zunaj pa igra pomembno vlogo pri resorpciji kosti in sproščanju ščitničnih hormonov. Encim povezujemo tudi s patološkimi procesi, kot so miofibrilna nekroza, miopatija, miokardna ishemija in proteinurija, ter razvoj in metastaziranje rakavih celic. V zadnjem času je pridobil posebno pozornost zaradi bolezni Covid-19, saj virusu, ki bolezen povzroča, s svojim delovanjem omogoča vstop v celico. Posledično katepsin L (kot tudi ostali katepsini) predstavlja pomembno tarčo za razvoj novih terapevtskih učinkovin. V okviru magistrske naloge smo se tako odločili preučiti interakcije med izbranimi ligandi in katepsinom L v prisotnosti cinkovih(II) ionov ter preveriti njihov vpliv na aktivnost encima. Vse izbrane spojine uvrščamo v skupine O,O-, N,O-, N,N- in N-ligandov z znano farmakološko uporabo, ki so že v preteklosti izkazale (potencialen) vpliv na encimsko aktivnost katepsinov. Preučili smo tudi vpliv sintetiziranih kompleksov, ki sem jih pripravila iz ligandov in različnih cinkovih(II) soli. V okviru študije sem izvedla teste stabilnosti in teste encimske aktivnosti. Teste stabilnosti sem izvedla z UV/VIS spektrofotometrijo, ki je pokazala in situ tvorbo kompleksa v primeru dodatka cinkove(II) soli pri kliokinolu, in 1H NMR, ki ni podala jasnejših zaključkov (o identiteti posamičnih zvrsti nalidiksinske kisline in izoniazida), razen tega, da so vse testirane zvrsti v pufrski raztopini skozi čas stabilne. V okviru testov encimske aktivnosti sem s fluorimetričnimi meritvami določala vpliv različnih zvrsti na encimsko aktivnost katepsina L. Teste sem izvedla v acetatnem pufru in pufru MES. Izbrani ligandi (z izjemo izoniazida) sami po sebi le delno inhibirajo encim, v primeru dodatka cinkovih(II) ionov v obliki soli pa pride do popolne inhibicije. Omenjeni trend je prisoten tako v različnih pufrih kot pri različnih vrstah uporabljenih cinkovih soli. Kljub temu pa še boljše inhibitorne lastnosti od (večine) kombinacij ligandov in cinkovih soli izkazujejo same cinkove(II) soli – predvsem ZnCl2 v acetatnem pufru – zaradi česar bi lahko sklepali, da ligandi s kompleksiranjem poslabšajo inhibitorne lastnosti cinkovih(II) ionov. Sodeč po vrednosti EC50 je najboljši inhibitor katepsina L kompleks kliokinola in Zn(OAc)2·2H2O v acetatnem pufru. Hillov koeficient je pri tem večji od 1, kar kaže na pozitivno kooperativnost vezave.

Keywords

cisteinski katepsini;regulacija encimske aktivnosti;inhibicija encimske aktivnosti;inhibitorji;cinkovi(II) ioni;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [K. Malenšek]
UDC: 577.15:546.47(043.2)
COBISS: 188000259 Link will open in a new window
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Downloads: 5
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Other data

Secondary language: English
Secondary title: The study of interactions between selected ligands and cathepsin L in the presence of zinc(II) ions
Secondary abstract: Cathepsins are a group of enzymes that are responsible for hydrolysis of peptide bonds and are a vital part of many physiological processes in all living organisms. On the other hand, their mislocalization, absence or unregulated activity can result in different pathological conditions. Based on the amino-acid residues in active site we can further divide cathepsins into three groups: serine, aspartyl or cysteine proteases. The representative of the latter group is cathepsin L, a lysosomal endopeptidase that is responsible for degradation of intracellular proteins, bone resorption and release of thyroid hormones. The malfunction of this enzyme can result in myofibril necrosis, myopathy, myocardial ischemia and proteinuria as well as in development and metastasis of cancer cells. Recently it was discovered that it enables SARS-CoV-2 to enter the cell, which is why cathepsin L (and other cathepsins) represents a potential target for new therapeutic agents. In Master thesis I studied the interactions between selected ligands and cathepsin L in the presence of zinc(II) ions and their effect on enzyme activity. All of the selected compounds are pharmacologically active O,O-, N,O-, N,N- and N-ligands that have already exhibited (potential) effect on cathepsin activity. The effect of metal complexes prepared from ligands and various zinc(II) salts was evaluated as well. Studies included stability tests and enzyme tests. Stability tests were performed by using UV/VIS spectrophotometry that indicated in situ formation of zinc(II)-clioquinol complex, and by 1H NMR spectroscopy that did not provide clear results (regarding the identity of species of nalidixic acid and isoniazid) besides the fact that all of the tested species are stable over time. Enzyme inhibition effect was studied by fluorimetric methods in MES and acetate buffer. Ligands (with the exception of isoniazid) exhibited only partial inhibition of enzyme, while total inhibition was observed when zinc(II) salts were added. The trend was observed in different buffers and zinc(II) salts. Even better inhibitory characteristics were displayed by zinc(II) salts, especially ZnCl2 in acetate buffer, which could indicate that by complexation, the ligands reduce the inhibitory characteristics of the zinc(II) ions. However, based on EC50 values the best inhibitor of cathepsin L turned out to be the synthesized complex of Zn(OAc)2·2H2O and clioquinol in acetate buffer. Hill's coefficient larger than 1 indicates positive cooperativeness of binding.
Secondary keywords: cathepsin L;enzyme activity inhibition;zinc(II) ions;Katepsini;Encimska aktivnost;Cink;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: 1 spletni vir (1 datoteka PDF (70 str.))
ID: 23066007