diplomsko delo
Lara Hrvatin (Author), Iztok Turel (Mentor)

Abstract

Katepsin K spada v družino papainu podobnih človeških cisteinskih proteaz s katalitično diado v obliki ionskega para Cys−His+. Izraža se predvsem v osteoklastih in ima ključno vlogo pri procesu preoblikovanja kosti zaradi sposobnosti cepitve kolagena tipa I in II ter drugih proteinov zunajceličnega matriksa. Regulacija proteolitične aktivnosti je pomembna za normalno delovanje organizma. Prekomerna aktivnost katepsina K je povezana z osteoporozo, osteoartritisom in revmatoidnim artritisom, zato inhibitorji, ki bi povrnili optimalno aktivnost, predstavljajo potencialna zdravila za zgoraj naštete bolezni. Bigvanidi tvorijo stabilne komplekse s kovinskimi ioni kot N,N–bidenatni ligandi. Posebej zanimiv bigvanid v kontekstu inhibicije cisteinskih katepsinov je fenformin (N-feniletilbigvanid), ki se je včasih uporabljal kot zdravilo za diabetes tipa II. S primerjavo struktur sintetičnega substrata proteaz benzoil–L–arginin amid (BAA) in fenformina v kompleksu s kovinskim ionom lahko sklepamo, da se fenilna skupina fenformina veže na vezavno mesto S2 na encimu, ki pomembno vpliva na moč vezave substrata pri cisteinskih katepsinih, kovinski ion pa interagira s katalitično diado. V diplomskem delu smo določili vpliv cinkovih(II) ionov in svinčevih(II) ionov v kombinaciji z bigvanidom fenforminom na aktivnost katepsina K. Z merjenem začetnih hitrosti hidrolize fluorogenega substrata Z-FR↓AMC smo določili vrednost EC50 za fenformin in vpliv dodatka fenformina na EC50 vrednosti cinkovih(II) in svinčevih(II) ionov. Ugotovili smo, da je fenformin hiperbolični inhibitor katepsina K in poveča inhibitorni učinek svinčevih(II) ionov. Pri inhibicij s cinkovimi(II) ioni pa dodatek fenformina nima bistvenega vpliva. Analizirali smo tudi prileganje fenformina, kovinskih ionov Pb2+ in Zn2+ ter njunih kompleksov s fenforminom na katepsin K z uporabo programa GaudiMM. Rezultati prileganja napovedujejo vezavo fenilne skupine fenformina na vezavno mesto S2 in interakcijo kovinskih ionov s Cys25 iz katalitične diade.

Keywords

cisteinski katepsini;katepsin K;kovinski ioni;cinkov(II) ion;svinčev(II) ion;bigvanidni ligandi;inhibicija encimov;molekulska umestitev;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [L. Hrvatin]
UDC: 577.15:546.47:546.815(043.2)
COBISS: 54411011 Link will open in a new window
Views: 507
Downloads: 111
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Other data

Secondary language: English
Secondary title: Effects of biguanide-metal ion complexes on activity of cathepsin K
Secondary abstract: Cathepsin K is a papain-like human cysteine peptidase with a catalytic diad consisting of a Cys−His+ ion pair. The enzyme is predominantly expressed in osteoclasts and is a crucial peptidase involved in bone remodelling due to its capability to cleave collagen types I and II besides other proteins in the extracellular matrix. Regulation of proteolytic activity is important for normal functioning of an organism. Excessive activity of cathepsin K has been associated with osteoporosis, osteoarthritis and rheumatoid arthritis. Thus, inhibitors which would reduce the proteolytic activity to its optimum are potential candidates for therapeutic use. Biguanides form stable complexes with metal ions as N,N-bidenate ligands. Phenformin (N-phenylethylbiguanide), a formerly used drug for diabetes type II, is especially interesting in the context of cysteine cathepsins inhibition. Structural comparison of benzyl-L-arginine amide (BAA), a synthetic protease substrate, and phenformin-metal ion complex suggests that the phenyl group binds to S2 substrate biding site, a major determinant of substrate preference for cysteine cathepsins, and metal ion interacts with the catalytic diad. In this work we studied the effects of zinc(II) and lead(II) ions in combination with biguanide phenformin on the activity of cathepsin K. We determined the EC50 value for phenformin and how the addition of phenformin influences the EC50 values for zinc(II) and lead(II) ions by measuring initial velocity of hydrolysis of fluorogenic substrate Z-FR↓AMC. Our results show that phenformin is a hyperbolic inhibitor of cathepsin K and it increases the inhibitory potency of lead(II) ions. The same effect was not observed with zinc(II) ions. We also analysed the binding of phenformin, Zn2+, Pb2+ and their complexes on cathepsin K by using GaudiMM programme. Docking results predict biding of the phenyl moiety into the S2 biding site and interaction of metal ions with Cys25 from the catalytic diad.
Secondary keywords: cathepsin K;metal ions;biguanide ligands;inhibition;molecular docking;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Pages: 40 str.
ID: 12625091