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Tradicionalni pristop načrtovanja zdravilnih učinkovin je usmerjen v regulacijo aktivnosti proteinov z uporabo malih molekul z visoko afiniteto. Uporabnost takšnega pristopa omejuje potreba po ustreznem vezavnem mestu, ki ga okoli 80 % človeškega proteoma ne izkazuje in je posledično farmakološko nedostopen za takšne zaviralce. Rešitev lahko predstavlja nova metodologija tarčne razgradnje proteinov z uporabo heterobifunkcionalnih molekul (ang. proteolysis targeting chimeras - PROTACs), ki za svoje delovanje uporabljajo telesu endogeni mehanizem za razgradnjo proteinov. Te himerne spojine so sestavljene iz treh delov, in sicer iz liganda za ligazo E3, tarčnega proteina ter distančnika, ki ju povezuje. Njihova učinkovitost leži v mehanizmu in katalitični naravi delovanja, ki jim omogoča, da pri zelo nizkih koncentracijah s prehodnimi interakcijami s katerimkoli mestom na proteinu izzovejo njegovo razgradnjo. Zaradi tega so himerni razgrajevalci postali uporabno orodje pri farmakološki modulaciji proteoma, ki je težko dostopen preko klasičnega načrtovanja učinkovin. Narava delovanja takšnih razgrajevalcev je pritegnila tudi pozornost onkološkega zdravljenja, saj molekule PROTAC z razgradnjo proteina lažje zaobidejo določene mehanizme pridobitve odpornosti, ki predstavlja težavo mnogim zdravilnim učinkovinam na tem področju. Ena izmed pomembnih tarč v onkologiji so proteini, imenovani zaviralci apoptoze (IAP), ki so v celicah zadolženi za zaviranje celične smrti. Znanstveniki so odkrili, da se ti proteini prekomerno izražajo znotraj rakavih tkiv, kar je povezano s progresijo bolezni, slabo prognozo in nizkim odzivom na zdravljenje. Zaradi teh razlogov so proteini IAP postali zanimive tarče zdravljenja rakavih obolenj. V sklopu magistrskega dela smo sintetizirali himerni razgrajevalec, ki je vseboval med seboj povezana liganda za cereblon in IAP. Končna molekula heteroPROTAC je v testih na rakavi celični liniji multiplega mieloma MM.1S z metodo prenosa western izkazala koncentracijsko-odvisno selektivno razgradnjo predstavnika družine IAP, in sicer XIAP. Pri višjih koncentracijah smo pokazali tako razgradnjo vseh vrednotenih proteinov IAP kot tudi delno razgradnjo transkripcijskega faktorja IKZF3. Nivo ligaze cereblon je ostal tekom testiranja nespremenjen, kar priča o dejstvu, da je potekla le enostranska razgradnja proteinov IAP. Poleg metode western smo razgradnjo dokazali tudi s proteomiko, ki je pokazala statistično značilno znižanje nivoja proteina XIAP. Sintetizirana molekula himernega razgrajevalca predstavlja tako uporabno probo za raziskave vpletenosti proteinov IAP v patogenezo malignih obolenj kot tudi dobro izhodišče za nadaljnji razvoj terapevtikov. The traditional approach to development of therapeutics is aimed at regulating the activity of proteins with the use of small-molecule inhibitors. The limitation of such an approach is the need for an appropriate binding site, which around 80 % of the human proteome does not exhibit, thus making them undruggable with such inhibitors. A new method of targeted protein degradation could provide the solution with the use of heterobifunctional molecules (PROTACs) which hijack the body's endogeneous mechanism for protein degradation. These chimeric molecules are comprised of three parts: E3 ligase ligand, target protein ligand and a linker. The effectiveness lies in their mechanism of action and catalytic nature, which allows them to induce targeted protein degradation with only transient interactions with protein at low concentrations. For these reasons, chimeric degraders showed usefulness in the field of oncology as they can circumvent certain resistance mechanisms due to their mode of action. One of many important targets in oncology are inhibitor of apoptosis (IAP) proteins, which play a key role inhibiting cell apoptosis. Scientists have uncovered that these proteins are overly expressed inside cancerous tissue and are associated with bad prognosis, disease progression and low response to treatment. Therefore, IAP proteins represent interesting targets for potential cancer treatment. In the scope of this thesis, we synthesised a chimeric degrader which incorporates ligands for ligases cereblon and IAP that were connected via a linker. Our heteroPROTAC was tested on a multiple myeloma cell line MM.1S with the method of western blotting, and showed concentration-dependent selective degradation of XIAP, an important member of the IAP protein family. At higher concentrations, we observed elimination of all IAP proteins tested, as well as partial degradation of transcription factor IKZF3. The protein levels of ligase cereblon remained unaltered, confirming the unilateral degradation of IAP proteins. Protein depletion was also demonstrated with the use of global proteomic analysis, where a selective reduction of XIAP protein levels was shown. The synthesised degrader represents both a useful probe for future research of the involvement of IAP proteins in the pathogenesis of malignant diseases, as well as a good starting point for further therapeutic development.

Razvoj udova često se koristi kao model za istraživanje načina na koji genski izražaj određuje razlike koje se između pojedinih vrsta pojavljuju tijekom normalnog i poremećenog oblikovanja udova. Usprkos mnogobrojnim istraživanjima provedenim na oblikovanju prstiju u pokusnih životinja, samo je nekoliko istraživanja analiziralo razvoj ljudskih udova ostavljajući tako ovaj problem nerazjašnjenim. U ovoj je studiji razvoj ljudskih udova analiziran u serijskim histološkim rezovima 16 ljudskih zametaka starosti od 4. do 10. razvojnih tjedana, pri čemu su korištene imunohistokemijske metode te metode dvostruke imunofluorescencije i elektronske mikroskopije. Statistički podaci analizirani su Mann– Whitney, Kruskal–Wallis i Dunnovims post hoc testom. Posebna pažnja posvećena je razvoju prstiju, naročito čimbenicima koji su uključeni u njihovo razdvajanje i izduživanje. Prostorni i vremenski izražaj istraživanih čimbenika važnih za kontrolu stanične proliferacije uključivali su biljega stanične proliferacije Ki-67 i antiapoptotsku bjelančevinu Bcl-2, kao i fibroblastne čimbenike rasta FGF8 i FGF2, njihov receptor FGFR1 te transkripcijske čimbenike MSX-1 i MSX-2. Procesi apoptoze analizirani su primjenom čimbenika kaspaze-3, AIF, BAX, kao i čimbenika povezanih s apoptozom p19 i RIP5. Apoptoza je dodatno dokazana TUNEL metodom. Tijekom ranog razvoja, ljudski udovi sastojali su se od mezenhimskog središta i površinskog ektoderma s apikalnim ektodermalnim grebenom (AEG). S napredovanjem razvoja, jaka proliferacijska aktivnost postepeno se smanjivala u mezenhimu (od 78% na 68%) i u epitelu (od 62% na 42%), dok je u hrskavicama prstiju proliferacija stalno bila niska (26-27%). Apoptotske stanice pozitivne na kaspazu-3 i AIF bile su svojstvene za mezenhim i AEG tijekom ranih faza razvoja, dok su tijekom razdvajanja prstiju bile prisutne i u mezenhimu između prstiju (interdigitalnom mezenhimu). Intenzivni izražaj Bcl-2 uočen je u AEG, mezenhimu neposredno ispod AEG i u hrskavičnim falangama, dok je izražaj BAX bio svojstven područjima s apoptozom. Na ultrastrukturnoj razini, proliferirajuće stanice su imale prepoznatljive mitotske figure, dok su apoptotske stanice pokazivale fragmentaciju jezgre. Makrofagi su uočeni oko apoptotskih stanica. Tijekom razvoja, pojačavao se izražaj svih fibroblastnih čimbenika rasta (osim FGF8) u mezenhimu i AEG-u, mezenhimu neposredno ispod grebena (zona rasta) i hondrocitima hrskavica udova. Dok je istovremeni citoplazmatski izražaj (ko-ekspresija) MSX-1 i MSX-2 nađen u epitelu i mezenhimu, p19 je pokazivao snažni citoplazmatski izražaj u neproliferirajućim stanicama. Jezgrin izražaj Ki-67 u proliferirajućim stanicama, a djelomično i čimbenika MSX-1 i MSX-2 uočen je u čitavoj osnovi udova. Snažni izražaj p19 i RIP5 bio je prisutan u AEG-u i mezenhimu udova. Rezultati naših istraživanja pokazuju da intenzivna stanična proliferacija omogućuje rast i izduživanje osnove udova, kao i različiti rast prstiju. Dva puta stanične smrti, kaspaza-3 ovisni i AIF-ovisni put, uključeni su u kontrolu veličine AEG-a i broja stanica u podležećem mezenhimu u najranijim razvojnim stadijima, kao i u proces odvajanja prstiju tijekom kasnijih faza razvoja udova. Prostorni i vremenski izražaj Bcl-2 i BAX u osnovi udova ukazuje na njihovu moguću ulogu u suzbijanju apoptoze i selektivnom poticanju rasta tijekom oblikovanja udova. Obrazac izražaja čimbenika p19 i RIP5 ukazuje na njihovu uključenost u kontroli staničnog starenja i smrti. Nasuprot istraživanjima na životinjama, izražaj FGFR1 u površinskom epitelu i p19 u čitavoj osnovi ljudskih udova mogli bi biti rezultat razlika u oblikovanju udova između različitih vrsta. Izražaj FGF2 i podređenog mu RIP5 gena, kao i transkripcijskih čimbenika MSX-1 i MSX-2 nije bio specifičan za čovjeka u odnosu na druge vrste. Temeljem prostornog i vremenskog izražaja istraživanih čimbenika tijekom razvoja ljudskih udova, naši rezultati upućuje na ulogu FGF i MSX gena u poticanju stanične proliferacije, rastu udova, izduživanju i odvajanju prstiju, te dodatno na ulogu MSX- 2 u kontroli vaskulogeneze. Kaskada međusobno orkestriranih genskih izražaja, uključujući i one analizirane u ovoj studiji, zajednički doprinosi složenom procesu razvoja udova., The limb development has been widely used as a model to investigate how gene expression controls interspecies differences appearing in normal limb morphology and in limb malformations. Despite numerous investigations performed on digit formation of experimental animals, only few investigations analyzed human limb development, thus remaining this problem largely unclear. In the present study, the development of human limbs was analyzed in serial histological sections of 16 normal human conceptuses between the 4th and 10th developmental weeks by using immunohistochemical and double immunofluorescence methods, and electron microscopy. Statistical data were analyzed by Mann–Whitney test, Kruskal–Wallis and Dunn’s post hoc test. Special attention was paid to development of fingers, in particular to factors involved in their separation and elongations. Spatiotemporal expression pattern of factors involved in control of cell proliferation included proliferation marker Ki-67 and antiapoptotic marker Bcl-2, as well as fibroblast growth factors FGF8 and FGF2, their receptor FGFR1, and transcription factors MSX-1 and MSX-2. Processes of apoptosis were analyzed by markers to caspase-3, AIF, BAX as well as to apoptosis associated factors19 and RIP5. Apoptosis was additionally conformed by TUNEL method. Initially, developing human limbs consisted of mesenchymal core and surface ectoderm with apical ectodermal ridge (AER). During progression of development, strong proliferation activity gradually decreased in the mesenchyme (from 78% to 68%) and in the epithelium (from 62% to 42%), while in the differentiating finger cartilages proliferation was constantly low (26–7%). Apoptotic caspase-3 and AIF—positive cells characterized mesenchyme and AER at earliest stages, while during digit separation they appeared in interdigital mesenchyme as well. Strong Bcl-2 expression was observed in AER, subridge mesenchyme and phalanges, while BAX expression characterized limb areas undergoing apoptosis. Ultrastructurally, proliferating cells showed mitotic figures, while apoptotic cells were characterized by nuclear fragmentation. Macrophages were observed around the apoptotic cells. Increasing expression of all analyzed growth factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordia. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme was observed in human developing limbs. Our data suggest that intense proliferation enables growth and elongation of human limb primordia, and differential growth of digits. Both caspase-3 and AIF–dependant pathways of cell death control the extent of AER and number of cells in the subridge mesenchyme at earliest developmental stages, as well as process of digit separation at later stages of limb development. Spatio-temporal co-expression of Bcl-2 and BAX indicates their role in suppression of apoptosis and selective stimulation of growth during human limb morphogenesis. Expression pattern of p19 and RIP5 indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordia might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and MSX genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX- 2 in control of vasculogenesis. The cascade of orchestrated gene expressions including the analyzed developmental factors, jointly contribute to the complex human limb development.

Aripiprazol (ARI) in olanzapin (OLA) sta pogosto uporabljena atipična antipsihotika s številnimi prednostmi pri zdravljenju shizofrenije in bipolarne motnje. Oba antipsihotika izkazujeta zaščitno delovanje, vendar so ob dolgotrajni terapiji zlasti problematične metabolne komplikacije, katerih vzroki so slabo poznani in bi jih potencialno lahko povezali z nepravilnim delovanjem jeter. Jetra so namreč osrednji organ presnove in detoksikacije obeh antipsihotikov, zato bi lahko motene celične funkcije bistveno pripomogle k razvoju opaženih učinkov. Namen dela je bil raziskati morebitne škodljive in zaščitne učinke ARI in OLA v celičnih modelih jeter. Proučili smo odziv celic na kratkotrajne in dolgotrajne tretmaje z izbranima antipsihotikoma ter vpliv zunanjega vira oksidativnega stresa. ARI je za jetrne celice bolj citotoksičen v primerjavi z OLA. OLA namreč ne izkazuje neposrednega citotoksičnega vpliva na jetrne celice niti po večtedenski izpostavitvi, medtem ko je učinek ARI takojšen, in sicer v obliki mitohondrijske hiperpolarizacije. Mitohondrijska hiperpolarizacija znatno upočasni celični metabolizem in delitev celic ter vodi v trajno povečan oksidativni stres. Prav ta pa bi lahko hkrati imel znaten prispevek k zaščitnemu delovanju ARI proti zunanjemu viru oksidativnega stresa, saj morajo celice vseskozi vzdrževati visok nivo antioksidativne obrambe. Zaščitni učinek ARI vsaj deloma poteka preko antioksidativnih encimov po dolgotrajni izpostavitvi in ni neposredno povezan z uravnavanjem apoptoze, avtofagije in proteasoma. ARI ščiti celice Fao in primarne hepatocite, medtem ko smo zaščitni učinek OLA opazili zgolj v kratkotrajnih poskusih na primarnih hepatocitih. Dolgotrajna izpostavitev celic antipsihotikoma je bistveno pripomogla k relevantnosti raziskave, saj je razkrila učinke, ki jih zgolj s kratkotrajnimi tretmaji ne bi uspeli zaznati. Predstavljen celični model kronične izpostavitve antipsihotikom torej bolje odraža delovanje učinkovin v ravnotežnem stanju in je potencialno uporaben tudi pri proučevanju drugih zdravil s predvidenimi dolgotrajnimi terapijami. Aripiprazole (ARI) and olanzapine (OLA) are commonly used atypical antipsychotics with many benefits in the treatment of schizophrenia and bipolar disorder. Despite of protective effects of both antipsychotics, there are metabolic complications, particularly during long-term therapy. The causes are poorly understood and could potentially be associated with liver dysfunction. The liver is the central organ of metabolism and detoxification of both antipsychotics. Impaired cellular functions could significantly contribute to the development of the observed effects. The purpose of this study was to investigate the potential adverse and protective effects of ARI and OLA in liver cell models. We studied the cellular response to short-term and long-term antipsychotic treatments exposed to acute oxidative stress. ARI is more cytotoxic to liver cells compared to OLA. OLA does not have a direct cytotoxic effect on liver cells even after several weeks of exposure, while the effect of ARI is immediate, in the form of mitochondrial hyperpolarization. Mitochondrial hyperpolarization significantly slows down cellular metabolism as well as cell division and leads to permanently elevated oxidative stress. The latter could be also linked with the protective role of ARI against external sources of oxidative stress, as cells must permanently maintain the high level of antioxidant defense. The protective effect of ARI is at least partially mediated by endogenous antioxidant enzymes after prolonged ARI exposure and it is not directly linked with the regulation of apoptosis, autophagy or proteasome activity. ARI protected Fao cells and primary hepatocytes, while the protective effect of OLA was observed only on primary hepatocytes during acute treatment. Long-term exposure to antipsychotics revealed effects that could not be detected with short-term treatments alone. Therefore, the proposed cellular model of chronic exposure to antipsychotics better reflects the action of drugs in the equilibrium and is also potentially useful to study the effects of other drugs used in long-term therapies.

Cisplatina se koristi za liječenje velikog broja različitih zloćudnih tumora. Učinkovitost liječenja ograničena je pojavom ozbiljnih neželjenih pojava i/ili zbog otpornosti stanica zloćudnih tumora na liječenje. U posljednjih 18 godina se za liječenje zloćudnih tumora koriste spojevi koji moduliraju epigenom. Epigenetičke promjene su uključene, barem djelomično, u razvoj neosjetljivosti stanica zloćudnih tumora na velik broj protutumorskih lijekova. Važna epigenetička modifikacija koja je uključena u nastanak neosjetljivosti je trimetilacija lizina na položaju 27 u histonu H3 (H3K27me3). Ovu represivnu oznaku uspostavlja enzim EZH2 koji se može selektivno inhibirati lijekom tazemetostatom. Uklanjanje ove oznake kataliziraju enzimi KDM6A i KDM6B koji se mogu inhibirati spojem GSK-J4. Cilj rada bio je istražiti učinak spojeva tazemetostat i GSK-J4, u odnosu na promjenu osjetljivosti stanica na cisplatinu. Model istraživanja bile su stanice podrijetlom od zloćudnih tumora glave i vrata (Detroit 562 i FaDu) i debelog crijeva (HT-29). Kombinacije ovih spojeva s cisplatinom nisu dovoljno, ili uopće, istražene na ovim modelima. Dodatno je istražen učinak ovih spojeva u odnosu na razinu glukoze u hranjivom mediju. Cisplatina je uzrokovala stanično specifičan odgovor izmjeren primjenom funkcionalnih staničnih testova (test vijabilnosti, test proliferacije). Linija stanica FaDu pokazala se najosjetljivijom na djelovanje cisplatine, Detroit 562 umjereno osjetljivom, dok su stanice HT29 bile najotpornije. Kombiniranje cisplatine s tazemetostatom nije dovelo do dodatnog povećanja učinka cisplatine na proliferaciju stanica FaDu i Detroit 562, dok je u stanicama zloćudnog tumora debelog crijeva, HT-29, tazemetostat antagnonizirao djelovanje cisplatine. Za razliku od tazemetostata, spoj GSK-J4 sinergistički je djelovao s cisplatinom u odnosu na smanjenje proliferacije stanica Detroit 562 i HT-29. Sinergistički učinak izostao je u stanicama FaDu. Učinci kombinacija spojeva nisu se značajno razlikovali u odnosu na koncentraciju glukoze u mediju. Pozadina opaženih promjena istražila se na molekularno-genetičkoj razini, mjerenjem transkripata i proteinskih produkata gena uključenih u regulaciju staničnog ciklusa. Iako je cisplatina uzrokovala porast transkripta CDKN1A (P21) u svim linijama, u stanicama Detroit 562 i FaDu (ali ne i HT-29,) došlo je do sniženja razine P21 u ukupnim proteinima i proteinima citoplazme. Tazemetostat je uzrokovao porast P21 u svim stanicama. Također, primjena tazemetostata, samostalno ili s cisplatinom, uzrokovala je porast transkripta i proteina TGFB1 u stanicama HT-29. Porast se poklapa s povećanom proliferacijom u ovim eksperimentalnim uvjetima. Ovaj rad poslužit će kao osnova za dodatna istraživanja kojima će se ispitati mogućnost inhibicije enzima KDM6A i KDM6B u kombinaciji s cisplatinom, u pretkliničkim modelima. Također, ovo istraživanje pokazalo je i potencijalno opasne učinke tazemetostata u slučaju primjene na bolesnicima koji imaju karcinom debelog crijeva. Cisplatin is used to treat a number of various malignant tumors. The effectiveness of the treatment is limited by the occurrence of serious side effects and/or the cancer cells develop resistance to the treatment. Over the last 18 years, epigenome modulating compounds have been used to treat cancer. Epigenetic changes are involved, at least in part, in the development of cancer cell resistance to a number of antitumor drugs. An important epigenetic mark which is involved in development of resistance is trimethylation of lysine at the position 27 in histone H3 (H3K27me3). This repressive mark is established by the enzyme EZH2, which can be selectively inhibited by the drug tazemetostat. The removal of this mark is catalyzed by the enzymes KDM6A and KDM6B, which can be inhibited by the compound GSK-J4. The aim of this work was exploration of the effect of tazemetostat and GSK-J4, on sensitivity of cancer cells to cisplatin. The research was conducted using cells originating from head and neck (Detroit 562 and FaDu) and colon malignant tumors (HT-29). Combinations of tazemetostat and GSK-J4 with cisplatin have not been sufficiently, if at all, investigated in these models. Additionally, the effect of these compounds in relation to the content of glucose in the nutrient medium was investigated. The cellular response to cisplatin was cell-type specific and was measured with functional cellular assays (viability assay, proliferation assay). FaDu cell line proved to be the most sensitive to cisplatin, Detroit 562 was moderately sensitive, while HT-29 cells were the most resistant. Combining cisplatin with tazemetostat did not increase the effect of cisplatin with respect to the rate of FaDu and Detroit 562 proliferation, while in colon cancer cells, HT-29, tazemetostat antagonized the effect of cisplatin. Contrary to tazemetostat, the compound GSKJ4 exerted synergistic effect with cisplatin, which was obvious through the reduced proliferation rate of Detroit 562 and HT-29 cells. This effect was absent in FaDu cell lines. The effects of combined treatments did not differ significantly regarding the concentration of glucose in the medium. The underlying causes of the changes observed were investigated at the molecular-genetic level, by measuring the transcriptional activity, as well as the protein level of the selected genes involved in the regulation of the cell cycle and their protein products. Cisplatin application was associated with an increase of CDKN1A (P21) transcription in all cell lines. However, in Detroit 562 and FaDu, it was joined with a decrease of the P21 protein in both the total proteins and in the cytoplasmic protein fraction. This decrease was not observed in HT-29 cells. Tazemetostat caused an increase of P21 in all cell lines. Also, administration of tazemetostat, whether alone or in combination with cisplatin, caused an increase of TGFB1 168 HT-29 cells, on both, transcriptional and protein level. This increase coincides with increased proliferation under these specific experimental conditions. This work will serve as the basis for additional research that will explore the possibility of combining inhibition of enzymes KDM6A and KDM6B with cisplatin application, in preclinical cancer models. This in vitro research showed the potentially dangerous side effects of tazemetostat should it be used in patients with colon cancer.

Rak je skupina sorodnih bolezni, katerim je skupna nenadzorovana delitev celic. Nastane zaradi nepopravljive spremembe v celičnem jedru kot posledica mutacije genov v spolnih ali telesnih celicah. Pri zdravljenju uporabljamo zdravila, ki zavirajo razmnoževanje rakavih celic ter povzročijo apoptozo. Na področju razvoja protirakavih učinkovin pomembno odkritje predstavljajo himerni razgrajevalci (molekule PROTAC), ki izkoriščajo v celici naravno prisoten sistem za odstranjevanje poškodovanih in nefunkcionalnih proteinov. Delovanje himernih razgrajevalcev temelji na farmakološkem modelu dogodka, pri katerem obvladujemo delovanje patoloških proteinov z zniževanjem njihove koncentracije. Prednost mehanizma delovanja je, da se po ubikvitinaciji tarče molekula PROTAC sprosti z obeh vezavnih mest ter tako lahko doseže skoraj popolno razgradnjo tudi v sub-stehiometričnih (katalitičnih) količinah. Človeški genom kodira za več kot 600 različnih ligaz E3, vendar jih je bila le peščica uspešno izkoriščenih s strani spojin PROTAC. Večina nedavno sintetiziranih himernih razgrajevalcev izkorišča cereblon kot ligazo E3, saj so ligandi zanjo sintezno dostopni, imajo dobro afiniteto vezave in ugodne fizikalno-kemijske lastnosti. Cereblon je beljakovina, sestavljena iz 442 aminokislin, in je primarna tarča imunomodulatornih zdravil imidnega strukturnega tipa. Vezava teh spojin okrepi protein-protein interakcijo s transkripcijskima faktorjema IKZF1 in IKZF3, posledica česar je njuna ubikvitinacija ter razgradnja, kar vodi v doseganje antiproliferativnega učinka pri multiplem mielomu. Večina molekul PROTAC, ki razgradnjo drugih tarčnih proteinov inducira preko cereblona, v strukturi vsebuje derivate talidomida, lenalidomida in pomalidomida. V sklopu magistrskega dela smo sintetizirali štiri nove ligande za cereblon, ki so potencialno uporabni kot orodja za preučevanje njegove biološke vloge ter kot gradniki himernih razgrajevalcev. Koncept zasnove novih ligandov je tvorba planarnega obroča, ki spominja na talidomid, in je sestavljen iz 4-aminobenzojske kisline z različnimi orto¬-substitucijami, povezane z glutarimidnim delom preko amidne vezi. Sinteza ligandov je temeljila na reakciji tvorbe amidne vezi in sledeče redukcije. V sodelovanju z Univerzitetno kliniko v Berlinu smo sintetiziranim spojinam določili njihovo afiniteto do cereblona. Rezultati biološkega vrednotenja so pokazali, da imata spojini s hidroksi- in metoksi-substitucijo podobno afiniteto do cereblona kot talidomid, medtem ko sta kloro- in metil-substituirana liganda izkazovala nekoliko nižjo afiniteto. Cancer represents a group of related disorders, characterised by uncontrolled cell division. The disease develops as a result of irreparable changes in the nucleus of the cell, i.e. gene mutations in sex cells or body cells. Cancer treatment involves the use of drugs to halt cell division and cause cell apoptosis. An important development for future cancer therapeutics was the discovery of targeted protein degradation by PROTAC molecules, which take advantage of the cell’s naturally occurring mechanisms for degradation of damaged proteins. PROTAC-based cancer treatment aims to control the activity of pathologically-relevant proteins by lowering their concentrations. An advantage of using PROTACs in cancer therapy lies in the fact that PROTACs dissociate from both of their binding sites, act as a catalyst, and thereby achieve an almost total protein degradation even in substoichiometric amounts. The human genome encodes for more than 600 different E3 ligases, however, only a number of them have been successfully applied in PROTAC design. Most of the recently synthesised PROTACs target the cereblon ligase. Ligands for this E3 ligase are synthetically accessible, have high binding affinity and favourable physico-chemical properties. Cereblon is a protein composed of 442 amino acids and is the primary target of immunomodulatory imide drugs. The binding of these drugs enhances the interaction with the IKZF1 and IKZF3 transcription factors, resulting in their ubiquitination and degradation, ultimately causing an antiproliferative effect in multiple myeloma. The majority of the PROTAC molecules which act via cereblon-induced degradation consist of thalidomide, lenalidomide or pomalidomide derivatives. In this master’s project, we have synthesised four new cereblon ligands, which are potentially useful as tools for studying the biological role of cereblon and as building blocks of PROTACs. Ligand design was based on the formation of a planar open ring, which resembles thalidomide and is composed of 4-aminobenzoic acid with different ortho-substituents, connected to the glutarimide component via an amide bond. Ligand synthesis was based on amide bond formation and subsequent reduction. In collaboration with the Charité clinical centre in Berlin, the binding affinities of the synthesised ligands for cereblon were determined. Results revealed that ligands with the hydroxy- and methoxy-substitution exhibit similar binding affinities as thalidomide, while the affinities of the chloro- and methyl-substituted ligands were slightly worse.

Tumor mokraćnog mjehura deseti je najčešće dijagnosticirani oblik tumora u svijetu s jako visokom stopom recidiva i niskom stopom preživljenja. Tradicionalne terapije najčešće imaju teške nuspojave, a pacijenti gotovo uvijek razviju otpornost na liječenje. Niklozamid je antiparazitski lijek koji se koristi za liječenje bolesti uzrokovanih trakavicama. Kod nekoliko tipova tumora pokazano je njegovo antitumorsko djelovanje induciranjem apoptoze i inhibicijom nekoliko signalnih puteva, kao što su WNT/CTNNB1, mTORC1, STAT3, NFκB, Notch i Hedgehog. Budući da su njegove nuspojave i utjecaj na organizam već dijelom istraženi, preusmjeravanje niklozamida prema liječenju tumora bilo bi kraće i jednostavnije od razvoja novog lijeka. Cilj mog diplomskog rada bio je ispitati mehanizme antitumorskog djelovanja niklozamida na stanice tumora mokraćnog mjehura; inducira li apoptozu i kakve učinke ima na staničnu proliferaciju. Metodom western blot analizirala sam ekspresiju proteina, a protočnom citometrijom apoptozu i stanični ciklus. Rezultati pokazuju da niklozamid inducira apoptozu i zaustavlja proliferaciju stanica tumora mokraćnog mjehura. Stoga, niklozamid u kombinaciji s postojećim lijekovima može imati potencijal za liječenje tumora mokraćnog mjehura. Bladder cancer is the 10th most common cancer worldwide, with a very high recurrence rate and low survival rate. Traditional therapies often cause unpleasant side effects, and patients develop drug resistance. Niclosamide is an antiparasitic medication that has been used for many years to treat tapeworm infestations. In several types of tumors, niclosamide has been reported to exhibit antitumor effects by inducing apoptosis and inhibiting several signaling pathways, such as WNT/CTNNB1, mTORC1, STAT3, NFκB, Notch, and Hedgehog. As its adverse effects have been partially investigated, repurposing niclosamide as a treatment for cancer would be less time-consuming and less complicated than the development of a new drug. The aim of my master thesis was to examine the mechanisms of the antitumor activity of niclosamide on bladder cancer cells; does it induce apoptosis and what effects does it have on cell proliferation. Protein expression was analyzed by western blotting, apoptosis analysis, and cell cycle analysis using flow cytometry. These results demonstrate that niclosamide induces apoptosis and inhibits the proliferation of bladder cancer cells. Therefore, niclosamide in combination with existing drugs may have the potential to treat bladder tumors.

Hepatociti so visoko specializirane polarizirane jetrne celice, v katerih se vrši večina za jetra specifičnih funkcij. Izolacija primarnih hepatocitov je skupek stresnih dejavnikov, med katerimi izstopa encimska razgradnja medceličnih stikov. Zaradi stresa ob izolaciji se v primarnih podganjih hepatocitih pojavi reverzibilna stresna prilagoditev, imenovana predapoptotski celični odziv na stres (PACOS), ki preko inaktivacije kaspaze 9 onemogoči proženje apoptoze po mitohondrijski poti ter nastopi takoj po izolaciji in izzveni do 6. dne v kulturi. Namen našega dela je bil proučiti PACOS ter njegov vpliv na delovanje hepatocitov in na odziv hepatocitov na dodatni stresni dejavnik (hipoksija in reoksigenacija). Zanimalo nas je tudi, ali so pri proučevanju PACOS sveže tkivne rezine jeter kontrola celičnim kulturam primarnih hepatocitov. V doktorskem delu smo pokazali, da primarni hepatociti v kulturi preživijo ter ohranijo nivo presnovne aktivnosti in sposobnost tvorbe sečnine. Potrdili smo stalno pojavnost PACOS v izoliranih primarnih hepatocitih. Raziskali smo proženje apoptoze v podganjih primarnih hepatocitih in dokazali, da v normalnih pogojih poteče po mitohondrijski poti. Sveže jetrne rezine so glede PACOS lahko kontrola primarnim hepatocitom, saj je v rezinah kaspazo 9 moč učinkovito aktivirati. Prvič smo pokazali, da sta posledici stresa pri izolaciji hepatocitov dva reverzibilna odziva na stres: indukcija endogenega antioksidativnega odziva in PACOS. Oba sproži povečano nastajanje ROS, ki tako omogoči preživetje in normalno delovanje celic. Poleg tega smo pokazali, da so na stres prilagojeni primarni hepatociti manj občutljivi na proženje apoptoze po hipoksiji/reoksigenaciji kot hepatociti pozne kulture, pri katerih sta PACOS in antioksidativni odziv izzvenela. Primarni hepatociti so relevanten in z nekaterimi omejitvami ustrezen model za temeljno proučevanje detoksikacije, metabolizma in potencialnih terapevtikov. Na podlagi rezultatov naše raziskave poudarjamo, da se na stres prilagojeni hepatociti na dodatne stresne dejavnike odzivajo drugače kot hepatociti in vitro ter in vivo, v katerih stresnih odzivov ni. Razumevanje mehanizmov ROS in odzivov na stres v primarnih hepatocitih je ključnega pomena za načrtovanje raziskav, za interpretacijo rezultatov, za prenos izsledkov na razmere in vivo in v končni fazi za razumevanje fiziologije in patofiziologije jeter ter oblikovanje posegov za preprečevanje in zdravljenje jetrne patologije. Hepatocytes are highly specialized polarized liver cells in which most liver-specific functions are performed. Isolation of primary hepatocytes is a set of stressors, among which the enzymatic degradation of intercellular junctions stands out. Due to stress upon isolation, a reversible stress adaptation called pre-apoptotic cell stress response (PACOS) occurs in primary rat hepatocytes and through inactivation of caspase-9 prevents apoptosis triggering through the intrinsic pathway. PACOS is present immediately after isolation and dissipates by day 6 in culture. The aim of our study was to examine/investigate PACOS, its impact on hepatocyte function, hepatocyte response to additional stressors (hypoxia and reoxygenation), and whether precision-cut liver slices can be used as a control to primary hepatocyte cell cultures in the PACOS study. In our work we demonstrated that primary hepatocytes survive in culture and maintain the level of metabolic activity and the ability of urea production. We confirmed the constant occurrence of PACOS in isolated primary hepatocytes. We investigated the induction of apoptosis in primary hepatocytes and demonstrated that it is normally activated through the intrinsic pathway. Precision-cut liver slices can serve as a control to primary hepatocytes with respect to PACOS, as caspase-9 can be efficiently activated in the slices. For the first time, we have shown that the consequences of stress in hepatocyte isolation are two reversible responses to stress: induction of an endogenous antioxidant response and PACOS. Both are triggered by increased ROS, which mediate cell survival and normal function. In addition, we showed that stress-adapted primary hepatocytes are less susceptible to triggering apoptosis after hypoxia/reoxygenation than hepatocytes in which PACOS and antioxidant response are no longer present. Primary hepatocytes are a relevant and, with some limitations, appropriate model to study detoxification, metabolism, and potential therapeutics. Based on the results of our study, we emphasize that stress-adapted hepatocytes respond differently to additional stressors than hepatocytes in vitro and in vivo, in which the stress responses are not induced. Understanding the mechanisms of ROS and stress responses in primary hepatocytes is crucial for planning research, interpreting the results, applying them in vivo and, ultimately, for understanding liver physiology, pathophysiology, and devising interventions for prevention and treatment of liver pathology.

This thesis principally focuses on the development of glycomimetic ligands with improved affinity to Siglec-8. Siglec-8 is a member of the Siglec family, I-type lectins that contain a sialic acid-binding domain and that play different roles in cell-cell interactions and cell signaling. Siglec-8 is a CD33- related protein expressed exclusively on mast cells and eosinophils, and weakly on basophils. Crosslinking of Siglec-8 with antibodies leads to apoptosis of eosinophils and inhibition of degranulation of mast cells. Apoptosis has also been induced when eosinophils were treated with a glycopolymer (6’-sulfo-sLex-polyacrylamide polymer). Therefore, Siglec-8 represents a very interesting new pharmacological target for the treatment of eosinophil- or mast cell-associated diseases, such as asthma, chronic rhinosinusitis, chronic urticaria, hypereosinophilic syndromes, mast cell and eosinophil malignancies, and eosinophilic gastrointestinal disorders. Recently, the NMR solution structure of the Siglec-8 lectin domain was solved in complex with its preferred ligand, the tetrasaccharide 6′-sulfo sialyl Lewisx (Neu5Acα2-3[6S]Galβ1-4[Fucα1-3]GlcNAc). The main interactions involve a salt bridge between Arg109 and the carboxylate of sialic acid and a second salt bridge between Arg56 and Gln59 and the sulfate at the 6-position of the galactose moiety. Interestingly, the sulfate plays a key role in both binding and specificity to Siglec-8. In addition, hydrogen bonds exist between hydroxyl groups 7, 8 and 9 of sialic acid and Tyr7, Ser118 and Gln122. In contrast, the fucose and glucosamine subunits show only minor interactions. This led to the suggestion that the disaccharide substructure 6-sulfo-Sia-Gal might represent the minimal binding epitope for Siglec-8. Ta doktorska naloga se osredotoča predvsem na razvoj glikomimetičnih ligandov z izboljšano afiniteto do Siglec-8. Siglec-8 je član družine Siglec, lektinov tipa I, ki vsebujejo domeno, ki veže sialično kislino, in imajo različne vloge pri interakcijah med celicami in celični signalizaciji. Siglec- 8 je CD33 sorodni protein, izražen izključno v tkivnih bazofilcih (mastocitih) in eozinofilcih, šibko pa v bazofilcih. Povezovanje Siglec-8 s protitelesi vodi do apoptoze eozinofilcev in zaviranja degranulacije mastocitov. Apoptoza se je pojavila tudi pri tretiranju eozinofilcev z glikopolimerom (6'-sulfo-sLex-poliakrilamidnim polimerom). Zato Siglec-8 predstavlja zelo zanimivo novo farmakološko tarčo za zdravljenje bolezni, povezanih z eozinofilci ali mastociti, kot so astma, kronični rinosinuzitis, kronična urtikarija, hipereozinofilni sindromi, malignosti mastocitov in eozinofilcev ter eozinofilne gastrointestinalne motnje. Pred kratkim so razrešili NMR strukturo lektinske domene Siglec-8 v kompleksu s svojim ligandom tetrasaharidnim 6'-sulfo sialil Lewisx (Neu5Acα2-3[6S]Galβ1-4[Fucα1-3]GlcNAc). Glavne interakcije vključujejo solni most med Arg109 in karboksilatom sialične kisline ter drugi solni most med Arg56 in Gln59 ter sulfatom na mestu 6 galaktoznega dela. Zanimivo je, da ima sulfat ključno vlogo pri vezavi in specifičnosti na Siglec-8. Poleg tega obstajajo vodikove vezi med hidroksilnimi skupinami 7, 8 in 9 sialične kisline ter Tyr7, Ser118 in Gln122. Nasprotno pa podenote fukoze in glukozamina tvorijo le šibkejše interakcije. To je pripeljalo do predloga, da bi disaharidna podstruktura 6-sulfo-Sia-Gal lahko predstavljala minimalni vezavni epitop za Siglec-8.

Protein science 32(2), e4563 (2023). doi:10.1002/pro.4563, Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+, known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis., Published by Protein Society, Bethesda, Md.

Človeška nevtrofilna elastaza (HNE) in proteinaza 3 (PR 3) sta serinski proteazi v primarnih granulah nevtrofilcev. S svojo proteolizno aktivnostjo sodelujeta v patogenezi kroničnih vnetnih stanj, uravnavanju imunskega sistema in uničevanju patogenov. Poleg tega imata zaradi neravnovesja med proteazami in antiproteazami pomembno vlogo pri patofiziologiji različnih bolezni, npr. kronični obstruktivni pljučni bolezni, cistični fibrozi in Wegnerjevi granulomatozi. Ravno zaradi njune vpletenosti pri tovrstnih obolenjih predstavljata validirani tarči za razvoj novih biološko aktivnih molekul in je iskanje njunih zaviralcev zelo pomembno. Namen magistrskega dela je bilo načrtovanje in sinteza novih piperazin-1-karbohidrazidnih zaviralcev encimov HNE in PR 3. Na osnovi predhodno sintetiziranih spojin smo v strukturi ohranili N-monosubstituirani piperazinski obroč in amidoksimsko skupino, medtem ko smo namesto naftilne skupine izbrali manjše lipofilne fragmente, s katerimi smo razširili kemijski prostor tovrstnih zaviralcev. V okviru magistrske naloge smo preverili še vpliv kisle karboksilne skupine, ki smo jo vgradili na mesto, kjer je bila v predhodnih spojinah amidoksimska skupina. V prvih dveh sinteznih stopnjah smo na 4-formilbenzonitril in metil 4-formilbenzoat pripeli z Boc zaščiten hidrazin in s sledečim katalitskim hidrogeniranjem dobili izhodni spojini za vse nadaljnje korake sinteze. Pri reakciji s trifosgenom in izbranim terciarnim aminom smo s pripenjanjem dveh piperazinskih derivatov tvorili azafenilalaninsko ogrodje. Temu je sledila odstranitev zaščitne skupine Boc in vezava lipofilnih fragmentov R3. Zadnjo stopnjo sinteze je predstavljala pretvorba ciano skupine v amidoksimsko oziroma hidroliza metilnega estra do karboksilne kisline. Deset novo sintetiziranih končnih spojin smo biokemijsko ovrednotili kot zaviralce proteaz z določanjem encimske kinetike, pri čemer smo uporabili pet serinskih proteaz (HNE, PR 3, Cat G, tripsin in α-kimotropsin). Vse spojine smo uspešno ovrednotili na zaviralno aktivnost HNE, PR 3, tripsina in α-kimotripsina, medtem ko s Cat G nismo ovrednotili dveh spojin. Ena izmed končnih spojin je zavirala nevtrofilne serinske proteaze, medtem ko so bile ostale spojine praktično neaktivne v izbranem testnem sistemu. Human neutrophil elastase (HNE) and proteinase 3 (PR 3) are serine proteases found in the primary granules of neutrophils. Their proteolytic activity contributes to chronic inflammatory conditions, immune regulation and pathogen destruction. In addition, due to the imbalance between proteases and antiproteases, they play important roles in the pathophysiology of various diseases, e.g. chronic obstructive pulmonary disease, cystic fibrosis, and Wegner's granulomatosis. It is their involvement in these diseases that makes them validated targets for the development of biologically active molecules and the search for their inhibitors is an important area. The aim of the MSc thesis was to design and synthesise novel piperazine-1-carbohydrazide inhibitors of HNE and PR 3 enzymes. Based on previously synthesised compounds, we retained the N-monosubstituted piperazine ring and the amidoxime group in the structure, while we replaced the naphthyl group with smaller lipophilic fragments to extend the chemical space of such inhibitors. In addition, the replacement of amidoxime with an acidic carboxylic group was evaluated. In the first two steps of our synthesis, a Boc-protected hydrazine was attached to 4-formylbenzonitrile and methyl 4-formylbenzoate and the subsequent catalytic hydrogenation afforded the starting compounds for all further synthetic steps. In the reaction with triphosgene and a selected tertiary amine, an azaphenylalanine scaffold was formed by the attachment of two piperazine derivatives. This was followed by removal of the Boc protecting group and introduction of lipophilic R3 fragments. The final step of the synthesis was the conversion of the nitrile to an amidoxime group or the hydrolysis of the methyl ester to carboxylic acid. The ten newly synthesised final compounds were biochemically evaluated as protease inhibitors by determining enzyme kinetics using five serine proteases (HNE, PR 3 and Cat G, trypsin and α-chymotrypsin. All compounds were successfully evaluated as HNE, PR 3, trypsin and α-chymotrypsin inhibitors, while two compounds were not evaluated on Cat G. One of the final compounds inhibited neutrophil serine proteases, while the other compounds showed poor ability to inhibit neutrophil serine proteases and were practically inactive in the selected test system.

Apoptoza ima izreden pomen pri odstranjevanju poškodovanih in rakavih celic, njena odsotnost ali oslabljeno delovanje pa lahko vodi v različna nevrodegenerativna, avtoimuna ali rakava bolezenska stanja. Eden ključnih proteinov, ki zavira apoptozo in tako spodbuja celično preživetje, je Bcl-2. Za zaviranje proteina Bcl-2 so bile načrtovane številne spojine, vendar je večina izkazovala slabo topnost, neugodno farmakokinetiko in toksične učinke. Za enega izmed dobro toleriranih zaviralcev Bcl-2 iz skupine BH3-mimetikov se je izkazala peroralno uporabna spojina S55746, ki inducira apoptozo že v zelo nizkih koncentracijah. Kljub nedvoumni uporabnosti pa nizkomolekularni zaviralci niso sposobni ciljati velikega dela človeškega proteoma, ki je farmakološko težko dostopen, prav tako pa se lahko pri terapiji s protirakavimi zdravili hitro razvije rezistenca. Novo terapevtsko alternativo predstavlja tarčna razgradnja proteinov, ki izkorišča celici lastne mehanizme za razgradnjo proteinov. V tej smeri se je v zadnjih 20 letih pojavilo veliko zanimanje za razvoj t.i. himernih molekul oz. proteolysis-targeting chimeras (PROTACs), ki proteolitično razgradnjo dosežejo preko ubikvitin-proteasomskega sistema. Uspešno delovanje molekul PROTAC je odvisno od ustrezne izbire vseh treh delov molekule: liganda, ki se veže na tarčni protein, liganda za ligazo E3 in vmesnika, ki liganda med seboj povezuje. Tovrstne molekule so zmožne ciljati proteine, ki so bili do sedaj izven farmakološkega dosega, zaradi svojega katalitičnega mehanizma delovanja pa dosegajo željen učinek že v zelo nizkih koncentracijah. Sintetizirali smo ligand za Bcl-2 na osnovi spojine S55746 z modifikacijo strukture, tako da smo omogočili pripenjanje vmesnika. S ciljem doseči čim višji izkoristek smo ključno stopnjo sinteze optimizirali z ustrezno kombinacijo spremenljivk (tip in količina katalizatorja, čas poteka reakcije). Pripravljen ligand za Bcl-2 smo vgradili v molekulo PROTAC tako, da smo ga povezali s rigidnim piperidinskim vmesnikom in ligandom za cereblon kot ligazo E3. Molekulo PROTAC smo testirali na celični liniji HeLa in ugotovili, da ne inducira razgradnje proteina Bcl-2. Apoptosis is of utmost importance in eliminating damaged and cancerous cells. Absence or impaired function of apoptosis can lead to various neurodegenerative, autoimmune, or cancerous diseases. One of the key proteins that inhibits apoptosis and thus promotes cell survival is Bcl-2. Many Bcl-2 inhibitors have been designed, but most showed poor solubility, unfavorable pharmacokinetics, and toxic effects. One of the well-tolerated BH3-mimetics proved to be the orally bioavailable compound S55746, which induces apoptosis even in low concentrations. However, such approaches for traditional inhibitor design are inadequate for targeting the vast majority of the human proteome which is pharmacologically difficult to access. Additionally, resistance to anticancer drug therapy can rapidly develop. Targeted protein degradation represents a new therapeutic alternative, which utilizes the cell's own endogenous mechanisms for inducing protein degradation. The development of chimeric molecules or proteolysis-targeted chimeras (PROTACs), which achieve proteolytic degradation by hijacking the ubiquitin-proteasome system has garnered immense interest in the last 20 years. The successful operation of PROTAC molecules depends on the appropriate selection of all three parts of the molecule: the ligand that binds to the protein of interest, the ligand for the E3 ligase, and the linker that connects the two ligands. These molecules are able to target proteins that have long been out of pharmacological reach and can achieve the desired effect even in very low concentrations due to their catalytic mechanism of action. We synthesized the Bcl-2 ligand based on the compound S55746, modified in such a way that enabled linker attachment. To achieve the highest possible yield, a crucial synthetic step was optimized with an appropriate combination of variables (type and amount of catalyst, reaction time). The ligand for Bcl-2 was incorporated into a PROTAC by linking it with a rigid piperidine linker and the ligand for cereblon as the E3 ligase. The PROTAC molecule was tested on the HeLa cell line, and it was found that it did not induce degradation of the Bcl-2 protein.

Prodigiosins (PGs) are a class of bacterial secondary metabolites with remarkable biological activities and colour. In this study, optimised fermentative production of prodigiosin (PG) using waste processed meat as a substrate has been achieved to levels of 83.1 ± 3.0 mg/L from a commercially available Serratia marcescens ATCC 27117 strain within 12 h. Methods were established for the reliable PG extraction from both the bacterial cell pellet and the culture supernatant, while gravitation column chromatography was used to obtain pure bacterial PG. The structure of the isolated PG was optimised by environmentally acceptable oxidative bromination reactions, obtaining mono- and dibrominated derivatives (PG-Br and PG-Br2). Chemical structures were confirmed by structural characterisation using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), showing that PG-Br is a mixture of two monobrominated isomers in approximately equal ratios, while PG-Br2 was afforded as a pure derivative. PG and its brominated derivatives (Br-derivatives) showed anticancer potential with half-maximal inhibitory concentration (IC50) values ranging from 0.62 to 17.00 μg/mL on four tested cancer cell lines (A549 lung, A375 skin, MDA-MB-231 breast, HCT116 colon) and an induction of early apoptosis, but low selectivity against healthy cell lines (MRC-5 lung fibroblasts and HaCaT skin keratinocytes). All three PG, PG-Br and PG-Br2 compounds did not affect roundworms (Caenorhabditis elegans) at concentrations up to 50 μg/mL. However, an improved toxicity profile of Br-derivatives in comparison to the parent PG was observed in vivo using zebrafish (Danio rerio) model system, when 10 μg/mL applied at 6 h post fertilisation caused death rate of 100, 30 and 0% by PG, PG-Br and PG-Br2, respectively, which is a significant finding for further structural optimisations of bacterial PGs. Prodigiozini (PG) so razred obarvanih sekundarnih bakterijskih metabolitov z izjemno biološko aktivnostjo. V doktorski raziskovalni nalogi smo za proizvodnjo prodigiozina uporabili komercialni sev bakterije Serratia marcescens ATCC 27117, kot substrat za fermentacijo pa odpadne produkte procesiranja mesa. Na ta način smo pridobili do 83.1 ± 3.0 prodigiozina v 12-ih urah fermentacije. V nadaljevanju smo vzpostavili metode za ekstrakcijo PS tako iz peleta bakterijskih celic kot tudi iz supernatanta bakterijske kulture. Za pridobitev čistega bakterijskega PG smo uporabili gravitacijsko kolonsko kromatografijo. Strukturo izoliranega PG smo optimizirali z okolju prijazno metodo oksidativnega bromiranja, pri čemer smo dobili mono- in di-bromirane derivate (PG-Br in PG-Br2). Strukturna karakterizacija bromiranih spojin s pomočjo jedrske magnetne resonance (NMR) in masne spektrometrije (MS) je pokazala, da je PG-Br zmes dveh mono-bromiranih izomer v približno enakih razmerjih, medtem ko je bil PG-Br2 pridobljen kot čisti derivat. PG in njegove bromirane derivate smo testirali na štirih rakavih celičnih linijah (A549 – pljučne celice, A375 – kožne celice, MDA-MB-231 – celice raka na prsih in HCT116 – črevesne celice). Potrdili smo antitumorske lastnosti PG in njegovih bromiranih derivatov s polovičnimi maksimalnimi inhibitornimi koncentracijami (IC50) v razponu od 0,62 do 17,00 μg/mL ter indukcijo zgodnje apoptoze, vendar hkrati tudi nizko selektivnost proti zdravim celičnim linijam (MRC-5 - pljučni fibroblasti in HaCaT - kožni keratinociti). Nobena od testiranih PG, PG-Br in PG-Br2 spojin ni imela opaznega vpliva na gliste Caenorhabditis elegans pri koncentracijah do 50 μg/mL. Ugotovili pa smo manjšo toksičnost bromiranih derivatov PG v primerjavi z matičnim PG v modelnem sistemu rib cebric (Danio rerio) in vivo. Pri cebricah, ki so bile 6 ur po oploditvi izpostavljene 10 μg/mL PG, PG-Br oziroma PG-Br2, smo ugotovili 100%, 30% in 0% smrtnost, kar je pomembna ugotovitev za nadaljnjo strukturno optimizacijo bakterijskih PG.

Frangula alnus (krušina) ima dugu istoriju korišćenja u tradicionalnoj medicini, a u ovom radu su ispitana njena određena nova svojstva, naročito antibiofilm aktivnost. Pripremljen je etil-acetatni ekstrakt kore krušine koji je hemijski okarakterisan te su ispitane njegova biološke aktivnosti. GC×GC/MS analiza je pokazala da je u ekstraktu dosta zastupljena 4-etoksi-benzoeva kiselina, , dok je LC-MS/MS analiza pokazala da je bogat fenolnim i flavonoidnim jedinjenjima među kojima je najdominantniji emodin koji je odabran za dalje testiranje uz ekstrakt. Pokazano je da ekstrakt ima snažnu antioksidantnu aktivnost za razliku od emodina. MIK vrednosti su pokazale da su svi testirani sojevi Staphylococcus aureus izuzetno osetljivi na delovanje ekstrakta i emodina, posebno klinički izolati. Rezultati ispitivanja antibiofilm aktivnosti su pokazali da ekstrakt i emodin imaju izuzetno snažan efekat na inhibiciju formiranja biofilmova sojeva S. aureus, ali da je efekat na već formirane biofilmove slabiji. Pokazano je da obe test supstance imaju inhibitorni efekat na koncentraciju ekstracelularnih polisaharida, da menjaju količinu eDNK u biofilmu kao i broj ćelija perzistera, ali i deluju na metabolizam ćelija. SEM i AFM mikroskopija su potvrdile antibiofilm efekat ukazujući na promene u morfologiji, strukturi i hrapavosti biofilmova. Dodatno, obe supstance su modulisale ekspresiju icaA, icaD, srrA, srrB gena i RNA III. Rezultati MTT testa su ukazali na jaku citotoksičnu aktivnost ekstrakta i emodina prema HepG2 i HCT 116 ćelijskim linijama, dok na imortalizovanoj MRC-5 liniji nije bilo efekta. Međutim, pokazano je da supstance remete ćelijski ciklus svih linija, kao i da indukuju apoptozu i nekrozu i remete njihov mitohondrijski membranski potencijal. Obe test supstance su ispoljile snažan genotoksični efekat na svim odabranim ćelijskim linijama. Frangula alnus (alder buckthorn) has a long history of use in traditional medicine, thus in this study its certain new properties, especially antibiofilm, were examined. Ethyl-acetate extract of buckthorn bark was prepared, chemically characterized and its biological activities were examined. GC×GC/MS analysis showed that 4-ethoxy-benzoic acid, is abundant in the extract, while LC-MS/MS analysis showed that extract is rich in phenolic and flavonoid compounds, with emodin as dominant one, which was selected for further testing along with extract. It was shown that extract has strong antioxidant activity unlike emodin. MIC values showed that all tested strains of Staphylococcus aureus were extremely sensitive to the action of the extract and emodin, especially clinical isolates. The results of the study of antibiofilm activity showed that the extract and emodin have an extraordinary inhibitory effect on biofilm formation of S. aureus strains, but that the effect on already formed biofilms is less pronounced. It was demonstrated that both test substances have inhibitory effect on the concentration of extracellular polysaccharides, alter the amount of eDNA in the biofilms as well as the number of persister cells, but also affect cell metabolism. SEM and AFM microscopy confirmed the antibiofilm effect, indicating changes in the morphology, structure and roughness of the biofilms. Additionally, both substances modulated the expression of icaA, icaD, srrA, srrB genes and RNA III. The results of the MTT test indicated strong cytotoxic activity of the extract and emodin against HepG2 and HCT 116 cell lines, while there was no effect on the healthy MRC-5 line. However, the substances disturbed the cell cycle of all cell lines, as well as induced apoptosis and necrosis and modulated mitochondrial membrane potential. Both test substances showed a strong genotoxic effect on all selected cell lines.

Ren (Armoracia rusticana, G. Gaertn, B. Mey. and Scherb.) je otporna višegodišnja biljka koja se uzgaja zbog specifičnog intenzivnog i ljutog ukusa. Cilj ovog rada bio je ispitivanje antitumorske i hepatoprotektivne aktivnosti ekstrakata i soka korena rena in vitro i in vivo. Primenom tečno-tečne ekstrakcije izolovane su i razdvojene polarne od nepolarnih komponenti, a HPLC metodom identifikovana su i kvantifikovana polifenolna jedinjenja u dobijenim ekstraktima i soku korena rena. Antiproliferativna aktivnost ekstrakata i soka korena rena ispitana je in vitro na tumorskim ćelijskim linijama: karcinoma grlića materice (HeLa), adenokarcinoma dojke (MCF7 i MDA-MB-231), adenokarcinoma debelog creva (HT-29), adenokarcinoma pluća (A549), adenokarcinoma prostate (PC-3), karcinoma kože (Hs 294T), karcinoma jetre (Hep G2), kao i na ćelijskim linijama karcinoma jetre pacova (H-4-II-E) i normalnim fetalnim ćelijskim linijama pluća (MPC-5) upotrebom Sulforodamin B testa. Mehanizam ćelijske smrti određen je detekcijom apoptoze i nekroze upotrebom Cell Death Detection ELISAPLUS kompleta. In vivo je ispitana antitumorska aktivnosti ekstrakata i soka korena rena na ćelijama Ehrlich-ovog ascitnog karcinoma (EAK) implantiranih NMRI miševima, kao i antioksidantna i hepatoprotektivna aktivnost kod intoksikacije jetre indukovane ugljen-tertrahloridom (CCL4) kod miševa. Ispitana je aktivnost osam ekstrakata korena rena i to iz pulpe: dihlormetanski (E1), hloroformski (E2), butanolni (E8) i vodeni (E7) i iz soka: dihlormetanski (E3), hloroformski (E4), butanolni (E6) i vodeni (E5) i sok korena rena (J9). Dihlormetanski ekstrakti korena rena imali su najveći sadržaj katehina, p-hidroksibenzoeve, siringinske i galne kiseline (pulpa, E1) i epikatehina (sok, E3). Hloroformski ekstrakt pulpe (E2) imao je visok sadržaj kvercetina i kemferola, dok je sok korena rena sadržao veće koncentracije katehina i galne kiseline. Rezultati su pokazali snažnu i neselektivnu antiproliferativnu aktivnost hloroformskih i dihlormetanskih ekstrakata i soka korena rena, sa najsnažnijim delovanjem na ćelijske linije jetre, dojke i pluća. Dobijene IC50 vrednosti bile su pri niskim koncentracijama (IC50 = 3,49-28,46 μg/mL) i visokim razblaženjima (IC50 = 418-1590). Sok (J9) i hloroformski ekstrakt soka rena (E4) pokazali su snažnu, nepoželjnu sposobnost indukcije nekroze. Ekstrakti i sok korena rena uticali su na povećanje oksidativnog stresa u ćelijama Ehrlich-ovog ascitnog karcinoma (EAK). Kod životinja koje su pretretirane sokom korena rena (J9) i posttretirane hloroformskim ekstraktom pulpe (E2) i dihlormetanskim ekstraktom soka (E3) postojalo je značajno povećanje aktivnosti superoksid dismutaze (SOD), ksantin oksidaze (XOD), glutation peroksidaze (GSHPx), glutation reduktaze (GR), intenziteta lipidne peroksidacije (LPx), kao i smanjenje aktivnosti katalaze (CAT) i nivoa glutationa (GSH) u ćelijama EAK. Efekti ekstrakata i soka korena rena na oksidativni stres kod hepatotoksičnog oštećenja jetre indukovanog ugljen-tetrahloridom bili su procenjeni merenjem parametara antioksidantne aktivnosti, kao i biohemijskih parametara funkcije jetre. Pretretmani sokom korena rena, hloroformskim ekstraktom pulpe (E2) i dihlormetanskim ekstraktom soka (E3), pre aplikacije CCl4, uticali su na značajno povećanje aktivnosti CAT, SOD, GR i nivoa GSH, kao i na značajno smanjenje aktivnosti XOD, GSHPx i intenziteta LPx, u odnosu na grupu životinja intoksikovanu CCl4-om. Takođe, pretretmani sa sokom korena rena, kao i hloroformskim i dihlormetanskim ekstraktima pre aplikacije CCl4 uticali su na značajno smanjenje aktivnosti ALT/AST i koncentracije hidroksiprolina, koje su bile povišene kod životinja nakon intoksikacije CCl4-om. Može se zaključiti da je detektovano snažno i neselektivno antiproliferativno dejstvo in vitro hloroformskih i dihlormetanskih ekstrakata i soka korena rena, sa nekrozom kao osnovnim mehanizmom indukovane ćelijske smrti. Rezultati dobijeni u in vivo ispitivanjima ukazali su da sok korena rena (J9), hloroformski ekstrakt pulpe (E2) i dihlormetanski ekstrakti soka (E3) ispoljavaju potencijalnu antitumorsku aktivnost prema ćelijama EAK, kao i potencijalnu antioksidantnu i hepatoprotektivnu aktivnost prevenirajući oštećenja jetre indukovana hepatotoksičnim CCl4-om., Horseradish (Armoracia rusticana, G. Gaertn, B. Mey. and Scherb.) is a hardy perennial herb, cultivated for its delicious, pungency and cooling taste. The aim of this study was to investigate antitumour and hepatoprptective activity of horseradish (Armoracia rusticana, Brasicaceae) root juice and extracts in vitro and in vivo. Liquid-liquid extraction of polar and non-polar compounds was used and polyphenolic compounds were identified and quantified by HPLC analysis. Antiproliferative activity was examinated in vitro on human cervix carcinoma (HeLa), breast adenocarcinoma (MCF7, MDA-MB-231), colon adenocarcinoma (HT-29), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), melanocyte carcinoma (Hs 294T), hepatocyte carcinoma (Hep G2), as well as rat hepatocyte carcinoma (H-4-II-E), and normal human fetal lung (MRC-5) cell line using Sulforhodamine B assay. The mechanism of cell-death in cell line was determinated using Cell Death Detection ELISAPLUS kit. The antiproliferative activity of horseradish root juice and extracts against Ehrlich ascites carcinoma (EAC) in Hannover National Medical Institute (Hann:NMRI) mice, as well as antioxidant and hepatoprotective activity against carbon tetrachloride (CCl4)-induced liver injury in mice were examinated in vivo. This investigation was performed by use of dichloromethane (E1), chloroform (E2), butanol (E8) and aqueous (E7) extracts of horseradish pulp; dichloromethane (E3), chloroform (E4), butanol (E6) and aqueous (E5) extracts of horseradish juice, and unaltered horseradish juice (J9). Dichloromethane extracts had the highest content of catechin, p-hydroxybenzoic, syringic and gallic acid (pulp, E1), and epicatechin (juice, E3). Choroform pulp extract (E2) contained quercetin and kaempferol while gallic acid and catechin were mostly found in the juice (J9). The results showed strong and non-selective antiproliferative activity of chloroform and dichloromethane extracts and root juice - highest being towards liver, breast and lung tissue cells. IC50 values of extracts and juice were obtained in low range of concentrations (IC50 = 3,49-28,46 μg/mL) and high range of dilutions (IC50 = 418-1590). High and unfavorable potential of horseradish juice (J9) and chloroform juice extract (E4) to induce necrotic cell death was detected. Both the extracts and juice caused an increase of oxidative stress in EAC cells. Animals pre-treated with horseradish root juice (J9) and post-treated with chloroform pulp (E2) or dichloromethane juice (E3) extracts showed increased activities of superoxide dismutase (SOD), xanthine oxidase (XOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and the intensity of lipid peroxidation (LPx). On the other hand, catalase (CAT) activity and the amount of glutathione (GSH) significantly decreased in EAC cells. Effects of horseradish root juice and extracts on CCl4-induced oxidative stress were evaluated by measuring stress parameters and biochemical parameters of liver function. Pre-treatment with horseradish root juice, pulp chloroform (E2) and dichloromethane juice (E3) extracts, before application of CCl4, led to a significant increase in CAT, SOD, GR and GSH levels, and significantly decreased XOD, GSHPx and LPx levels, bringing them closer to the values of the EAC control group. Also, pre-treatment with horseradish root juice and chloroform and dichloromethane extracts, before application of CCl4 led to significant decrease in ALT/AST activities and hydroxyproline concentration, which were elevated in CCl4-intoxicated animals. It could be conclude that strong and non-selective in vitro antiproliferative activity of chloroform and dichloromethane extracts and root juice of horseradish was detected, with necrosis as a main mechanism of induced cell death. Obtained results suggest that the horseradish root juice (J9), as well as pulp chloroform (E2) and dichloromethane juice (E3) extracts exert antitumour effects on Ehrlich ascites carcinoma (EAC) and antioxidant and hepatoprotective effects against CCl4-induced liver toxicity in vivo.

Ren (Armoracia rusticana, G. Gaertn, B. Mey. and Scherb.) je otporna višegodišnja biljka koja se uzgaja zbog specifičnog intenzivnog i ljutog ukusa. Cilj ovog rada bio je ispitivanje antitumorske i hepatoprotektivne aktivnosti ekstrakata i soka korena rena in vitro i in vivo. Primenom tečno-tečne ekstrakcije izolovane su i razdvojene polarne od nepolarnih komponenti, a HPLC metodom identifikovana su i kvantifikovana polifenolna jedinjenja u dobijenim ekstraktima i soku korena rena. Antiproliferativna aktivnost ekstrakata i soka korena rena ispitana je in vitro na tumorskim ćelijskim linijama: karcinoma grlića materice (HeLa), adenokarcinoma dojke (MCF7 i MDA-MB-231), adenokarcinoma debelog creva (HT-29), adenokarcinoma pluća (A549), adenokarcinoma prostate (PC-3), karcinoma kože (Hs 294T), karcinoma jetre (Hep G2), kao i na ćelijskim linijama karcinoma jetre pacova (H-4-II-E) i normalnim fetalnim ćelijskim linijama pluća (MPC-5) upotrebom Sulforodamin B testa. Mehanizam ćelijske smrti određen je detekcijom apoptoze i nekroze upotrebom Cell Death Detection ELISAPLUS kompleta. In vivo je ispitana antitumorska aktivnosti ekstrakata i soka korena rena na ćelijama Ehrlich-ovog ascitnog karcinoma (EAK) implantiranih NMRI miševima, kao i antioksidantna i hepatoprotektivna aktivnost kod intoksikacije jetre indukovane ugljen-tertrahloridom (CCL4) kod miševa. Ispitana je aktivnost osam ekstrakata korena rena i to iz pulpe: dihlormetanski (E1), hloroformski (E2), butanolni (E8) i vodeni (E7) i iz soka: dihlormetanski (E3), hloroformski (E4), butanolni (E6) i vodeni (E5) i sok korena rena (J9). Dihlormetanski ekstrakti korena rena imali su najveći sadržaj katehina, p-hidroksibenzoeve, siringinske i galne kiseline (pulpa, E1) i epikatehina (sok, E3). Hloroformski ekstrakt pulpe (E2) imao je visok sadržaj kvercetina i kemferola, dok je sok korena rena sadržao veće koncentracije katehina i galne kiseline. Rezultati su pokazali snažnu i neselektivnu antiproliferativnu aktivnost hloroformskih i dihlormetanskih ekstrakata i soka korena rena, sa najsnažnijim delovanjem na ćelijske linije jetre, dojke i pluća. Dobijene IC50 vrednosti bile su pri niskim koncentracijama (IC50 = 3,49-28,46 μg/mL) i visokim razblaženjima (IC50 = 418-1590). Sok (J9) i hloroformski ekstrakt soka rena (E4) pokazali su snažnu, nepoželjnu sposobnost indukcije nekroze. Ekstrakti i sok korena rena uticali su na povećanje oksidativnog stresa u ćelijama Ehrlich-ovog ascitnog karcinoma (EAK). Kod životinja koje su pretretirane sokom korena rena (J9) i posttretirane hloroformskim ekstraktom pulpe (E2) i dihlormetanskim ekstraktom soka (E3) postojalo je značajno povećanje aktivnosti superoksid dismutaze (SOD), ksantin oksidaze (XOD), glutation peroksidaze (GSHPx), glutation reduktaze (GR), intenziteta lipidne peroksidacije (LPx), kao i smanjenje aktivnosti katalaze (CAT) i nivoa glutationa (GSH) u ćelijama EAK. Efekti ekstrakata i soka korena rena na oksidativni stres kod hepatotoksičnog oštećenja jetre indukovanog ugljen-tetrahloridom bili su procenjeni merenjem parametara antioksidantne aktivnosti, kao i biohemijskih parametara funkcije jetre. Pretretmani sokom korena rena, hloroformskim ekstraktom pulpe (E2) i dihlormetanskim ekstraktom soka (E3), pre aplikacije CCl4, uticali su na značajno povećanje aktivnosti CAT, SOD, GR i nivoa GSH, kao i na značajno smanjenje aktivnosti XOD, GSHPx i intenziteta LPx, u odnosu na grupu životinja intoksikovanu CCl4-om. Takođe, pretretmani sa sokom korena rena, kao i hloroformskim i dihlormetanskim ekstraktima pre aplikacije CCl4 uticali su na značajno smanjenje aktivnosti ALT/AST i koncentracije hidroksiprolina, koje su bile povišene kod životinja nakon intoksikacije CCl4-om. Može se zaključiti da je detektovano snažno i neselektivno antiproliferativno dejstvo in vitro hloroformskih i dihlormetanskih ekstrakata i soka korena rena, sa nekrozom kao osnovnim mehanizmom indukovane ćelijske smrti. Rezultati dobijeni u in vivo ispitivanjima ukazali su da sok korena rena (J9), hloroformski ekstrakt pulpe (E2) i dihlormetanski ekstrakti soka (E3) ispoljavaju potencijalnu antitumorsku aktivnost prema ćelijama EAK, kao i potencijalnu antioksidantnu i hepatoprotektivnu aktivnost prevenirajući oštećenja jetre indukovana hepatotoksičnim CCl4-om. Horseradish (Armoracia rusticana, G. Gaertn, B. Mey. and Scherb.) is a hardy perennial herb, cultivated for its delicious, pungency and cooling taste. The aim of this study was to investigate antitumour and hepatoprptective activity of horseradish (Armoracia rusticana, Brasicaceae) root juice and extracts in vitro and in vivo. Liquid-liquid extraction of polar and non-polar compounds was used and polyphenolic compounds were identified and quantified by HPLC analysis. Antiproliferative activity was examinated in vitro on human cervix carcinoma (HeLa), breast adenocarcinoma (MCF7, MDA-MB-231), colon adenocarcinoma (HT-29), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), melanocyte carcinoma (Hs 294T), hepatocyte carcinoma (Hep G2), as well as rat hepatocyte carcinoma (H-4-II-E), and normal human fetal lung (MRC-5) cell line using Sulforhodamine B assay. The mechanism of cell-death in cell line was determinated using Cell Death Detection ELISAPLUS kit. The antiproliferative activity of horseradish root juice and extracts against Ehrlich ascites carcinoma (EAC) in Hannover National Medical Institute (Hann:NMRI) mice, as well as antioxidant and hepatoprotective activity against carbon tetrachloride (CCl4)-induced liver injury in mice were examinated in vivo. This investigation was performed by use of dichloromethane (E1), chloroform (E2), butanol (E8) and aqueous (E7) extracts of horseradish pulp; dichloromethane (E3), chloroform (E4), butanol (E6) and aqueous (E5) extracts of horseradish juice, and unaltered horseradish juice (J9). Dichloromethane extracts had the highest content of catechin, p-hydroxybenzoic, syringic and gallic acid (pulp, E1), and epicatechin (juice, E3). Choroform pulp extract (E2) contained quercetin and kaempferol while gallic acid and catechin were mostly found in the juice (J9). The results showed strong and non-selective antiproliferative activity of chloroform and dichloromethane extracts and root juice - highest being towards liver, breast and lung tissue cells. IC50 values of extracts and juice were obtained in low range of concentrations (IC50 = 3,49-28,46 μg/mL) and high range of dilutions (IC50 = 418-1590). High and unfavorable potential of horseradish juice (J9) and chloroform juice extract (E4) to induce necrotic cell death was detected. Both the extracts and juice caused an increase of oxidative stress in EAC cells. Animals pre-treated with horseradish root juice (J9) and post-treated with chloroform pulp (E2) or dichloromethane juice (E3) extracts showed increased activities of superoxide dismutase (SOD), xanthine oxidase (XOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and the intensity of lipid peroxidation (LPx). On the other hand, catalase (CAT) activity and the amount of glutathione (GSH) significantly decreased in EAC cells. Effects of horseradish root juice and extracts on CCl4-induced oxidative stress were evaluated by measuring stress parameters and biochemical parameters of liver function. Pre-treatment with horseradish root juice, pulp chloroform (E2) and dichloromethane juice (E3) extracts, before application of CCl4, led to a significant increase in CAT, SOD, GR and GSH levels, and significantly decreased XOD, GSHPx and LPx levels, bringing them closer to the values of the EAC control group. Also, pre-treatment with horseradish root juice and chloroform and dichloromethane extracts, before application of CCl4 led to significant decrease in ALT/AST activities and hydroxyproline concentration, which were elevated in CCl4-intoxicated animals. It could be conclude that strong and non-selective in vitro antiproliferative activity of chloroform and dichloromethane extracts and root juice of horseradish was detected, with necrosis as a main mechanism of induced cell death. Obtained results suggest that the horseradish root juice (J9), as well as pulp chloroform (E2) and dichloromethane juice (E3) extracts exert antitumour effects on Ehrlich ascites carcinoma (EAC) and antioxidant and hepatoprotective effects against CCl4-induced liver toxicity in vivo.

Preverjali smo, ali so morfološke spremembe pri apoptozi in feroptozi, vidne z vrstičnim elektronskim mikroskopom, dovolj značilne in jasne, da lahko zgolj na podlagi le-teh opredelimo tip smrti opazovane celice. Prav tako želimo ugotoviti, ali superparamagnetni železovi oksidni nanodelci (SPIONi) povzročijo celično smrt, ki ima molekularne in morfološke lastnosti, ki definirajo feroptozo. Celice HUVEC smo izpostavljali staurosporinu, ki je induktor apoptoze, RSL3, ki je induktor feroptoze ter SPIONom. Izvedli smo test citotoksičnosti, merili pa smo tudi vpliv staurosporina, RSL3 in SPIONov na specifično aktivnost acetilholinesteraze (AChE), stopnjo lipidne peroksidacije ter stopnjo apoptoze. Morfološke spremembe površine izpostavljenih celic smo opisali z vrstično elektronsko mikroskopijo. AChE se ni izkazala kot uporaben biomarker za ovrednotenje celične smrti, stopnja lipidne peroksidacije kot biomarker feroptoze pa je bila povišana pri tretiranju s RSL3 in SPIONi. Test stopnje apoptoze se je izkazal kot premalo specifičen, saj ni omogočal razločevanja med različnimi vrstami celične smrti. Staurosporin je na površini celic povzročil morfološke spremembe značilne za apoptozo, na celicah, izpostavljenih RSL3 so bile opazne morfološke značilnosti, ki jih literatura ne opisuje kot značilne za feroptozo. Tudi celice, izpostavljene SPIONom, niso imele za apoptozo in feroptozo-značilnih morfoloških značilnosti. Predvidevali smo, da SPIONi aktivirajo kombinacijo različnih mehanizmov celičnih smrti. The purpose of this master thesis was to find out if the morphological changes due to apoptosis in ferroptosis that we can observe with scanning electron microscope (SEM) are significantly different from eachother, which would show that we can use SEM as a method to differentiate between apoptosis in ferroptosis. Furthermore, we wanted to know if SPIONs cause a type of cell death that is morphologically and molecularly similar to ferroptosis. HUVEC cells were treated with inductor of apoptosis, staurosporin and inductor of ferroptosis, RSL. We also treated cells with SPIONs so we could determine what type of cell death they induced. We tested cells for cytotoxicity, AChE specific activity, lipid peroxidation and apoptosis. Treated cells were observed with SEM to describe morphological characteristics of their surface. AChE has not proven as a biomarker for a specific type of cell death. Biomarker for ferroptosis, lipid peroxidation, was, on the contrary, elevated in cells, treated with RSL3 and SPIONs. Test for apoptosis was not specific enough for differentiation between different cell deaths. Staurosporin induced morphological characteristics were specific for apoptosis, cells, treated with RSL on the other hand, were exhibiting morphological characteristic previously not described in the literature. Cells, treated with SPIONs, also had a unique morphology, not typical for apoptosis or ferroptosis

Obetaven terapevtski pristop pri razvoju zdravilnih učinkovin predstavlja modulacija aktivnosti proteinov v organizmu. Farmacevtska industrija se je v zadnjih 20 letih osredotočala predvsem na razvoj majhnih molekul, kamor spadajo zaviralci encimov, s katerimi lahko vplivamo na proteine. Majhne molekule delujejo po farmakološkem modelu zasedenosti, kar omejuje njihovo uporabo pri ciljanju farmakološko težje dostopnega dela proteoma. Poleg tega je za njihov maksimalen učinek pogosto potrebna visoka sistemska izpostavljenost, kar lahko vodi v pojav neželenih učinkov. Strategije, ki temeljijo na farmakološkem modelu dogodka, ponujajo bolj obetavne pristope za ciljanje tarčnih proteinov v primerjavi s tradicionalnimi nizkomolekularnimi zaviralci. Primer tovrstnega pristopa so molekule PROTAC, ki so sestavljene iz treh delov: liganda za vezavo na tarčni protein, liganda za vezavo na ligazo E3 in distančnika. Za svoje delovanje izkoriščajo ubikvitin proteasomski sistem, preko katerega inducirajo razgradnjo tarčnega proteina ter ga v celoti odstranijo. Molekule PROTAC so prvič opisali Crews in sodelavci leta 2001, do danes pa so bile razvite že številne spojine, ki ciljajo na več kot 50 različnih proteinov, mnogi izmed slednjih imajo pomembno vlogo pri razvoju raka, virusnih okužb, avtoimunskih in nevrodegenerativnih bolezni. Vseeno pa lahko kljub dobrim lastnostim molekul PROTAC njihov razvoj omejujejo težave pri doseganju ustrezne celične permeabilnosti. Kljub razcvetu področja pa metodologija PROTAC še zdaleč ni dosegla popolnega razvoja. Velik potencial kaže predvsem pri zdravljenju raka, ki je sposoben hitro mutirati in lahko povzroči rezistenco na klasične zaviralce. Ena izmed tarč, ki je pogosto udeležena v razvoju različnih oblik raka (multipli mielom, kronična limfocitna levkemija, akutna limfoblastna levkemija, rak dojke in pljučni rak), je antiapoptotični protein Bcl-2, saj njegovo prekomerno izražanje zavre apoptozo in pogosto vodi v pojav rezistence na zdravljenje s klasičnimi zaviralci. Ravno zato predstavlja obetavno tarčo za zdravljenje teh oblik bolezni prav z uporabo himernih razgrajevalcev. V sklopu magistrske naloge smo sintetizirali ligand za IAP (ligaza E3), ki smo ga z dvema različnima distančnikoma povezali z ligandom za Bcl-2 v končni molekuli PROTAC. Preliminarno vrednotenje na eni izmed celičnih linij pri izbranih pogojih testiranja je pokazalo, da razgradnje Bcl-2 nismo dosegli. V prihodnje bo smiselno te spojine vrednotiti še na drugih celičnih linijah, pri različnih koncentracijah in s spreminjanjem časa testiranja. A promising therapeutic approach in the development of medicinal agents is modulation of protein activity. In the last 20 years, the pharmaceutical industry has mainly focused on the development of small molecules, including enzyme inhibitors which can be used for target modulation. Small-molecule inhibitors exert their action through ''occupancy-driven'' model, which limits their use in targeting ''undruggable'' proteome. In addition, their maximum effect often requires a high systemic exposure which can lead to the occurrence of unwanted side effects. Strategies based on ''event-driven'' pharmacology offer more promising approaches for targeting proteins compared to traditional small-molecule inhibitors. An example of this approach are PROTACs (proteolysis-targeting chimeras), which consist of three parts: a ligand for binding to the target protein, a ligand for binding to the E3 ligase and a linker. These compounds exploit the ubiquitin proteasome pathway by inducing degradation of the target protein and thus completely removing it. PROTACs were first described by Crews and coworkers in 2001, and to date numerous compounds have been developed. These target more than 50 different proteins, many of which play an important role in the development of cancer, viral infections, autoimmune and neurodegenerative diseases. However, despite their great characteristics the use of PROTACs is mainly limited by their low cell permeability. The PROTACs methodology it is still far from fully developed. It shows a great potential especially in the treatment of cancer, as PROTACs are also capable of depleting rapidly mutatig and resistance-prone targets. One target that is often involved in the development of various forms of cancer (such as multiple myeloma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, breast cancer and lung cancer) is Bcl-2 protein. In cases of its overexpression apoptosis in blocked, which causes resistance to treatment with classical small-molecule inhibitors. Therefore, Bcl-2 represents an appropriate target for the treatment of Bcl-2 dependent cancers using PROTACs. In this work we synthesized a ligand for IAP (an E3 ligase), which we connected with two different linkers to a ligand for Bcl-2 to obtain the designed PROTAC molecules. Preliminary cell-based evaluations showed no Bcl-2 degradation under selected treatment conditions. Future cellular evaluation should encompass a wider variety of cancer cells and more treatment conditions such as prolonged treatment time and different concentration range.

Človeška nevtrofilna elastaza (HNE) je encim, ki sodi v skupino serinskih proteaz. V organizmu ima pomembno vlogo pri vzdrževanju homeostaze in regulaciji celičnih procesov. Povečana aktivnost HNE lahko povzroči poškodbe tkiva, ki so povezane z infekcijami in vnetji. Iz tega lahko sklepamo, da je HNE udeležena v raznih hujših kroničnih boleznih. Udeležena je tudi v regulaciji celične smrti, tako apoptoze kot nekroptoze. Namen magistrske naloge je bil na podlagi že sintetiziranih spojin sintetizirati novo serijo zaviralcev encima HNE. Pripravili smo derivate spojin, ki temeljijo na 3-substituiranem N-benzilpiperazin-1-karbohidrazidnem ogrodju, pred tem pa smo že pripravili eno končno spojino, ki je bila 4-substituirana. Od že sintetiziranih azafenilalaninskih derivatov smo ohranili amidoksimski fragment na fenilnem obroču, 2-naftoilno skupino na drugem dušiku hidrazinskega dela in ustrezen ciklični derivat (piperidinski oziroma piperazinski derivat). V šeststopenjski sintezni poti smo najprej 3-formilbenzonitril pretvorili v hidrazon, zaščiten z BOC zaščitno skupino. Tega smo katalitsko hidrogenirali in dobili hidrazin. Na prvi dušik spojine smo vezali ustrezni piperazinski oziroma piperidinski derivat. Nato smo odstranili zaščitno skupino BOC z drugega dušika in nanj pripeli naftoilno skupino. V zadnji stopnji pa smo nitrilno skupino pretvorili v amidoksim. Uspešno smo sintetizirali le dve od petih načrtovanih končnih spojin. Za ovrednotenje istovetnosti in čistosti intermediatov in končnih spojin smo uporabili tankoplastno kromatografijo, jedrsko magnetno resonanco, masno spektrometrijo, masno spektrometrijo visoke ločljivosti, infrardečo spektroskopijo in določitev temperature tališča. Vseh pet spojin predzadnje in eno spojino zadnje sintezne stopnje smo biokemijsko vrednotili z meritvijo encimske kinetike. Kot serinske proteaze smo poleg tripsina in α-kimotripsina želeli uporabiti še tri nevtrofilne serinske proteaze (HNE, katepsin G in proteinaza 3), vendar smo izmed teh spojine lahko uspešno biokemijsko vrednotili le na HNE. Najmočnejše zaviranje HNE dosežeta spojini predzadnje stopnje, ki imata na piperazinu vezan fenilni obroč s klorom oziroma fluorom na α-mestu. Tudi ostale spojine so pri nekoliko višjih vrednostih IC50 kazale sposobnost zaviranja. Spojine niso dosegle visoke selektivnosti med serinskimi proteazami. Human neutrophil elastase (HNE) is an enzyme that belongs to the serine protease family. It has an important role in human organism in the preservation of homeostasis and regulation of cell processes. Increased activity of HNE can induce tissue damage that may be linked to infections and inflammation. Therefore, it can be concluded, that HNE is involved in a variety of severe chronic diseases. It is also involved in the regulation of cell death, both apoptosis and necroptosis. The purpose of this master's thesis was to synthesize a new series of HNE inhibitors, based on previously synthesized compounds. We prepared derivatives of compounds that are based on a 3-substituted N-benzylpiperazine-1-carbohydrazide scaffold. A 4-substituted final compound was previously prepared. From already synthesized azaphenylalanine derivatives, we kept the amidoxime group on a phenyl ring, 2-naphtoyl group on the second nitrogen of hydrazine fragment and an adequate cyclic derivate (piperazine or piperidine). In the six-step synthetic pathway, 3-formylbenzonitrile was first converted into hydrazone, protected with a BOC group. Hydrazone was catalytically hydrogenated to hydrazine. The piperazine or piperidine derivative was attached to the first nitrogen. Subsequently, the BOC group was removed from the second nitrogen and the naphthoyl group was introduced. In the last step, the nitrile group was converted to amidoxime. We managed to synthesize only two of five final compounds. The identity and purity of all intermediates and final compounds were evaluated by thin layer chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, high-resolution mass spectrometry, infrared spectroscopy and melting point determination. All five compounds of the fifth and one compound of the sixth step were biochemically evaluated with enzyme kinetics measurements. Besides serine proteases trypsin and α-chymotrypsin, three additional neutrophil serine proteases (HNE, cathepsin G and proteinase 3) were planned to be used, but we were only able to make biochemical evaluations on the HNE enzyme. The strongest inhibition of HNE was shown by two compounds of the fifth step with phenyl ring, with chlorine or fluorine on α-position, bound to piperazine. The ability to inhibit HNE was also shown by other tested compounds at slightly higher IC50 values. High selectivity between serine proteases was not achieved.

Taxol (Paclitaxel), tetraciklički diterpen, je sekundarni metabolit biljnog porijekla s antitumorskim svojstvima kojeg sintetiziraju stabla roda Taxus. Prvi put je izoliran iz kore biljke Taxus baccata početkom druge polovice 20. stoljeća, skoro nijedan drugi lijek nije zaplijenio pažnju javnosti kao paclitaxel. Njegov uspjeh u tretmanu mnogo vrsta tumora je od velike važnosti. Veže se na βtubulin i ometa dinamiku mikrotubula inhibirajući njihovu depolimerizaciju, za razliku od ostalih agensa koji djeluju na mikrotubule, poput vinkristina i vinblastina koji onemogućuju polimerizaciju. Apoptozu stanica uzrokuje inhibicijom staničnog ciklusa, no i sekundarnim genetskim mehanizmima. Njegova ekstremno niska prirodna zastupljenost primorala je na traženje alternativnih puteva proizvodnje poput kulture stanica, bakterijskih kultura, endofita, kompletne sinteze i polusinteze. Taxol posjeduje vrlo kompleksnu biosintezu koja uključuje više od 19 koraka, i do danas nisu svi enzimi koji sudjeluju u potpunosti okarakterizirani. Uz to, glavni izazovi za buduća istraživanja su pronalazak ekonomski isplativog puta proizvodnje Taxola mikroorganizmima i povećanje biodostupnosti lijeka nanotehnologijom bez negativnih utjecaja na zdravlje pacijenata. Taxol (Paclitaxel), a tetracyclic diterpene, is a secondary metabolite of plant origin with antitumoral activity. It is synthesized by trees of the genus Taxus and it was first extracted from the bark of Taxus baccata in the 1960s, no other drug attracted so much media attention since. Its success in the treatment of a variety of cancers is of great importance. It binds to β-tubulin and disrupts the microtubular dynamics through inhibition of their depolymerization, unlike other microtubule targeting agents like vinblastine and vincristine which disable the polymerization. Cell apoptosis is caused by the arrest of the cell cycle but also by exerting effects on gene expression. Its extremely low natural occurence in the yew bark forced the search for alternative production sources such as harvesting it from cell culture, bacterial cultures, endophytes, total synthesis and semi-synthesis routes. Taxol has an extremely complicated biosynthesis pathway which has over 19 steps in total, and still today some of the enzymes involved have not been characterized. Besides that, main challenges for the future development and research include finding an economically viable microbial way of production, increasing bioavailability of the drug by nanotechnology without adverse effects on patients health.

Cancer was the leading cause of death in 2020 worldwide, consequently it exerts a huge burden on the health care system and on people’s lives. An early detection and correct treatment are crucial for curing cancer. Biomarkers are an effective tool used by clinicians to predict who will benefit from a certain treatment. One such potential biomarker is the protein TP53INP2, more specifically for the anticancer treatment TRAIL. TP53INP2 is an intrinsically disordered protein with many different roles in the cell. Protein levels of TP53INP2 are increased when kinases such as Checkpoint 1 kinase (Chk1), Casein kinase 2 (CK2), and Cyclin-dependent kinases 4 or 6 (CDK4,6) are inhibited. In this thesis it is shown that in the Ishikawa, SH-SY5Y and MDA-MB-231 cell lines TP53INP2 levels increase when treated with inhibitors Palbociclib (CDK4,6 inhibitor), Silmitasertib (CK2 inhibitor), or Chk1 inhibitor. Moreover, this increase in protein abundance is not due to an increase in gene expression, therefore phosphorylation probably marks TP53INP2 for degradation. TP53INP2 sensitizes cancer cells to synergistic treatment with TRAIL and kinase inhibitors of CDK4,6, CK2, and Chk1. The Ishikawa cells die more often by apoptosis when treated with the combined treatment (TRAIL and kinase inhibition) than when treated with TRAIL alone. However, the loss of TP53INP2 abrogated apoptosis, suggesting that the levels of TP53INP2 could be used as a biomarker for this combined treatment. Our results indicate that TP53INP2 is a potential biomarker for combination anticancer treatment with TRAIL and the above mentioned kinase inhibitors. Further research is needed to better understand the full importance of this protein in the fight against cancer. Rak je bil v letu 2020 najpogostejši vzrok smrti na svetu in tako predstavlja veliko breme za zdravstveni sistem in življenja ljudi. Zgodnje odkrivanje in pravilno zdravljenje sta ključna za ozdravitev teh bolezni. Biomarkerji so učinkovito orodje, ki ga zdravniki uporabljajo za napovedovanje, komu bo določeno zdravljenje koristilo. Eden od takšnih primerov je protein TP53INP2, ki je potencialni biomarker zlasti za protirakavo zdravilo TRAIL. TP53INP2 je intrinzično neurejen protein s številnimi različnimi vlogami v celici. Raven proteina TP53INP2 se poveča, kadar so kinaze, kot so kinaza Chk 1, kazein kinaza 2 (CK2) ter od ciklina odvisni kinazi 4 in 6 (CDK4,6), inhibirane. V tem magistrskem delu smo ugotovili, da se v celicah celičnh linij Ishikawa, SH-SY5Y in MDA-MB-231 raven proteina TP53INP2 poveča, če jih izpostavimo inhibitorjem Palbociclib (zaviralec CDK4,6), Silmitasertib (zaviralec CK2) oziroma inhibitorjem Chk1. Poleg tega to povečanje koncentracije proteina ni posledica povečanja izražanja gena, tako da fosforilacija verjetno označi TP53INP2, da se ta razgradi. TP53INP2 senzibilizira rakave celice tako, da te postanejo bolj občutljive za kombinirano sinergistično zdravljenje s TRAIL in inhibitorji kinaz CDK4,6, CK2 in Chk1. Celice Ishikawa v večji meri umirajo z apoptozo, če jih zdravimo s kombiniranim zdravljenjem (TRAIL in inhibitorji kinaz), kakor če jih zdravimo samo s TRAIL. Izguba TP53INP2 pa je odpravila apoptozo, kar nakazuje, da bi lahko raven proteina TP53INP2 uporabili kot biomarker za to kombinirano zdravljenje. Naši rezultati kažejo, da je TP53INP2 potencialni biomarker za kombinirano protirakavo zdravljenje s TRAIL in prej omenjenimi zaviralci kinaz.

Exposure of cells to high voltage electric pulses increases transiently membrane permeability through membrane electroporation. Electroporation can be reversible and is used in gene transfer and enhanced drug delivery but can also lead to cell death. Electroporation resulting in cell death (termed as irreversible electroporation) has been successfully used as a new non-thermal ablation method of soft tissue such as tumours or arrhythmogenic heart tissue. Even though the mechanisms of cell death can influence the outcome of electroporation-based treatments due to use of different electric pulse parameters and conditions, these are not elucidated yet. We review the mechanisms of cell death after electroporation reported in literature, cell injuries that may lead to cell death after electroporation and membrane repair mechanisms involved. The knowledge of membrane repair and cell death mechanisms after cell exposure to electric pulses, targets of electric field in cells need to be identified to optimize existing and develop of new electroporation-based techniques used in medicine, biotechnology, and food technology.