Preučevanje odnosa med strukturo in delovanjem pri substituiranih derivatih nitroksolina

Činč Ćurić, Laura (author), Sosič, Izidor (mentor)

Katepsin B je človeška lizosomska peptidaza, ki se normalno pojavlja v telesu. Posebnost katepsina B je t.i. zaporna zanka, ki mu omogoča endo- in eksopeptidazno aktivnost. Njegova fiziološka vloga je razgradnja proteinov v lizosomu, poleg tega pa so odkrili tudi bolj specifične funkcije, ki jih opravlja npr. v sekretornih celicah. Prekomerno povečana aktivnost katepsina B lahko vodi v procese tumorske progresije. V tumorskem tkivu je bilo pokazano, da katepsin B razgrajuje zunajcelični matriks in omogoča razrast tumorja ter angiogenezo, povečana koncentracija in aktivnost katepsina B pa predstavljata tudi pomemben diagnostični in prognostični tumorski kazalec. Ker je njegova aktivnost najbolj izražena na obrobju tumorja, ga lahko zaviramo z majhnimi sinteznimi molekulami, ki lahko tvorijo reverzibilne ali ireverzibilne interakcije v aktivnem mestu encima. V dosedanjih raziskavah je bilo pri zaviralcih katepsina B precej pozornosti namenjeno nitroksolinu (8-hidroksi-5-nitrokinolinu), sicer znani protibakterijski učinkovini. V več študijah je bilo namreč dokazano, da učinkovito zavira endopeptidazno aktivnost katepsina B. V magistrski nalogi smo sintetizirali različne derivate nitroksolina ter izbrane spojine biokemijsko ovrednotili na izoliranem katepsinu B, da bi preverili njihovo zaviralno aktivnost. Derivati so se razlikovali po substituentih na obročnem mestu 2 pri nitroksolinskem skeletu, poleg tega smo nitro skupino na mestu 5 zamenjali s klorom, da bi preverili vpliv tovrstne substitucije na zaviralno aktivnost spojin. Nekaj modifikacij izhodnega nitroksolina smo izvedli tudi na mestu 7. Ugotovili smo, da je spojina 3 imela enak mehanizem zaviranja kot nitroksolin. Kljub slabšemu zaviranju endopeptidazne aktivnosti v primerjavi z nitroksolinom je spojina 3 izkazovala boljše zaviranje eksopeptidazne aktivnosti od nitroksolina. Z enako afiniteto kot nitroksolin je endopeptidazno aktivnost zavirala spojina 2, in sicer z mehanizmom akompetitivnega zaviranja. Spojina 2 je tudi izkazovala boljše zaviranje eksopeptidazne aktivnosti od nitroksolina. Pridobljeni rezultati predstavljajo nov kamen v mozaiku do sedaj pripravljenih zaviralcev katepsina B s kinolinskim skeletom in hkrati tvorijo temelje za pripravo dodatno optimiziranih zaviralcev.

• Language: Slovenian
• Keywords: derivati nitroksolina, katepsin B, napredovanje tumorja, nitriranje, selektivni zaviralci
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-114883
• Publication date in RUL: 24.03.2020

Secondary language

• Language: English
• Title: Study of structure-activity relationship in substituted nitroxoline derivatives

Abstract

Cathepsin B (catB) is a human lysosomal protease that is normally present in the human cells. Its unique feature is the presence of the so called occluding loop, which enables catB to possess both endopeptidase and exopeptidase activities. Its physiological role is mainly the degradation of proteins in the lysosome; however, specific functions were discovered. On the other hand, increased expression and activity of catB can lead to tumor progression. In transformed and cancer cells it adopts a more peripheral distribution in the cytoplasm and is associated with the plasma membrane. There, catB can degrade the extracellular matrix and promotes angiogenesis. CatB was also coined as a diagnostic and prognostic tumour marker. It was shown previously that catB can be inhibited with small synthetic molecules, which can form reversible or irreversible bonds within the active site. In several recent studies, nitroxoline (8-hydroxy-5-nitroquinoline), a well-known antimicrobial agent, was given a lot of attention as a reversible catB inhibitor. Namely, it was shown that it successfully inhibits endo- and exopeptidase activities of catB. In this thesis various derivatives of nitroxoline were synthesized, characterized, and biochemically evaluated on the isolated cathepsin B to establish their inhibitory activities. The derivatives prepared were modified at the position 2 of nitroxoline and in addition, the nitro group was substituted with chlorine to establish the importance of nitro group for catB inhibition. A few derivatives with substitution at the position 7 of nitroxoline were also synthesized. The biochemical evaluation showed that only compound 3 exhibited the same mechanism of inhibition as nitroxoline. Although the inhibition of endopeptidase activity was weaker, compound 3 inhibited the exopeptidase activity of catB to a greater extent than nitroxoline. Compound 2 showed the same inhibition of the endopeptidase activity as nitroxoline, but with a different mechanism of inhibition, i.e. uncompetitive. Compound 2 also showed greater exopeptidase activity inhibition in comparison with nitroxoline. The results obtained in this thesis represent a step forward in establishing a clear structure-activity relationship for this structural class of catB inhibitors. The data is a basis for further work in this field, which could lead to even more potent and selective catB-targeting compounds.

• Keywords: cathepsin B, nitration, nitroxoline, selective inhibitors, tumor progression