Karakterizacija vpliva aminokislinskih derivatov sukcinimida na aktivnost katepsina L

Obaha, Ana (author), Novinec, Marko (mentor)

Človeški katepsin L je papainu podobna cisteinska endopeptidaza izražena v večini celic. Ima pomembno vlogo pri razgradnji proteinov, predstavitvi antigenov in regulaciji celičnega cikla. Namen naše raziskave je bil identificirati alosterične inhibitorje katepsina L. Slednji imajo prednost pred ortosteričnimi, saj zagotavljajo večjo specifičnost vezave. Kot tarčno alosterično mesto smo si izbrali tisto, kamor se dokazano vežejo alosterični efektorji na katepsinu K, ki sodi v družino katepsinu L podobnih peptidaz. Kot potencialne inhibitorje smo testirali spojine, ki so bile sintetizirane kot možni inhibitorji katepsina K in S. Testiranja smo izvedli z meritvijo aktivnosti encima s fluorogenim substratom Z-LR-AMC. Identificirali smo 12 linearnih in 5 hiperboličnih inhibitorjev. Da pa bi preverili, ali se spojina res veže v alosterično mesto, smo naredili mutanto, ki ima pet aminokislinskih ostankov v alosteričnem mestu zamenjanih s tistimi iz sorodnega katepsina V. Kot najboljši inhibitor s hiperboličnim mehanizmom delovanja se je izkazala spojina 2. Z logistično enačbo s štirimi parametri smo določili faktor EC50 670 ± 300 µM. Spojina zmanjša aktivnost encima za 30 %. Afiniteta vezave se pri mutiranih oblikah encima spremeni, zato lahko trdimo, da se veže v tarčno alosterično mesto.

• Language: Slovenian
• Keywords: peptidaza, inhibicija, alosterična regulacija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-109289
• COBISS.SI-ID: 1538301635
• Publication date in RUL: 29.08.2019

Secondary language

• Language: English

Abstract

Human cathepsin L is a papain-like cysteine endopeptidase expressed in most cells. It plays crucial roles in protein degradation, antigen presentation and regulation of the cell cycle. Our research is focused on identifying allosteric inhibitors of cathepsin L. As our target allosteric site we choose a site where allosteric effectors bind on the related cathepsin K. We characterized the effects of potential allosteric inhibitors from a library of amino acid derivatives of succinimide that were synthesized as potential inhibitors of cathepsins K and S. By measuring the activity of cathepsin L with the synthetic fluorogenic substrate Z-LR-AMC we identified 12 linear inhibitors and 5 hyperbolic inhibitors. To test whether the inhibitors bind to the allosteric site we produced a mutant variant of cathepsin L with five amino acids in target allosteric site substituted with those from the closely related cathepsin V. The best hyperbolic inhibitor was compound 2 which lowered the activity of the enzyme by 30 % and had an EC50 value of 670 ± 300 µM for wild-type cathepsin L. The affinity of binding on mutant enzyme of chatepsin L is different so compound 2 most likely binds to allosteric site.

• Keywords: peptidase, inhibition, allosteric regulation