20.500.12556/RUL-182559 Načrtovanje in sinteza pirolidin-, piperidin- in indolinkarbonil fluoridov ter vrednotenje njihove zaviralne aktivnosti na kaspazi-1 Design and synthesis of pyrrolidine-, piperidine- and indolinecarbonyl fluorides and evaluation of their inhibitory activity on caspase-1 Kaspaza-1 je vnetna cisteinska proteaza, ključna za proteolitično aktivacijo provnetnih citokinov interlevkina 1β (IL-1β) ter interlevkina 18 (IL-18), zato predstavlja pomembno tarčo pri preprečevanju patološkega vnetja ter nevrovnetja. Kovalentni zaviralci z ustrezno izbrano elektrofilno bojno glavo lahko, v primerjavi z nekovalentnimi zaviralci, dosežejo dolgotrajnejše zaviranje encima, pri čemer je njihova selektivnost in jakost zaviranja odvisna od pravilne umestitve liganda v aktivnem mestu. V sklopu magistrske naloge smo načrtovali in sintetizirali zaviralce s karbamoil fluoridno bojno glavo, ki smo jim sistematično spreminjali velikost in naravo substituentov ter preučevali vpliv strukturnih modifikacij na zaviranje kaspaze-1. Produkte smo izolirali z normalno- in reverzno-fazno kromatografijo, njihovo identiteto pa potrdili z masno spektrometrijo in jedrsko magnetno resonanco. Za devet končnih spojin, karbamoil fluoride 13–18, 42, 47 in 50, smo z biokemijskim testiranjem in vitro na človeški rekombinantni kaspazi-1 ovrednotili vpliv strukturnih sprememb na aktivnost encima; rezultate smo izrazili kot rezidualno aktivnost kaspaze-1 (RA) pri koncentracijah spojin 10 µM in 100 µM. Za razumevanje vezave in interakcij med ligandom in kaspazo-1 smo izvedli tudi molekulsko sidranje. Biokemijsko vrednotenje je pokazalo, da je med pripravljenimi spojinami kaspazo-1 najmočneje zaviral derivat indolina 42 (RA = 10,5 % pri 100 µM), sledil pa mu je indolin 50 (RA = 33,9 % pri 100 µM). Analogi pirolidina ter piperidina 13–18 so bili praktično neaktivni, kar nakazuje, da odsotnost fenilnega obroča indolina v derivatih pirolidina in piperidina omejuje učinkovito približanje bojne glave katalitičnemu cisteinu Cys285. Pri sidranju smo pri izbranih spojinah 42, 47 in 50 opazili odstopanja od pričakovanih vezavnih poz in umeščanja funkcionalnih skupin v vezavna žepa S1 in S4. Molekulsko sidranje tako ni v celoti pojasnilo odnosa med strukturo in zaviralno aktivnostjo spojin, zato bi bilo v nadaljnjih korakih smiselno izvesti tudi molekulsko dinamiko, ki bi omogočila vpogled v dinamiko vezavnih interakcij. Rezultati naloge tako podpirajo izhodiščno predpostavko, da je za jakost zaviranja kovalentnega zaviralca poleg izbire elektrofilne bojne glave ključen tudi prispevek nekovalentne vezave in prileganje molekule aktivnemu mestu kaspaze-1. Caspase-1 is an inflammatory cysteine protease that is essential for the proteolytic activation of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), and therefore represents an important target aimed at preventing pathological inflammation and neuroinflammation. Covalent inhibitors can, in comparison with non-covalent inhibitors, achieve prolonged enzyme inhibition when equipped with an appropriately selected electrophilic warhead. Their selectivity and efficacy depend on the proper positioning of the ligand within the active site. In this master’s thesis, we designed and synthesized a series of inhibitors bearing carbamoyl fluoride warheads, systematically varying the size and nature of substituents, and investigating their impact on the inhibition of caspase-1. Reaction products were isolated using normal-phase and reversed-phase chromatography, and their identity was confirmed by mass spectrometry and nuclear magnetic resonance. For nine final compounds, carbamoyl fluorides 13–18, 42, 47 and 50, an in vitro biochemical assay using human, recombinant caspase-1 was used to evaluate how structural modifications affected the inhibitory activity. Results were expressed as residual caspase-1 activity (RA) at compound concentrations of 100 µM and 10 µM. To gain deeper insight into binding and interactions between ligand and caspase-1, molecular docking was also performed. Biochemical evaluation showed that among the tested compounds, indoline derivative 42 was the most potent inhibitor of caspase-1 (RA = 10.5% at 100 µM), followed by indoline 50 (RA = 33.9% at 100 µM). The pyrrolidine and piperidine analogues 13–18 were practically inactive, suggesting that compared to indoline derivatives, the absence of phenyl moiety limits the effective positioning of the warhead toward the catalytic cysteine Cys285. Docking of selected compounds 42, 47 and 50 showed deviations from the expected binding poses and the positioning of functional groups within the S1 and S4 binding pockets were observed. Molecular docking therefore did not fully elucidate the relationship between the structural features and the inhibitory activity of the compounds. In subsequent studies, it would be appropriate to perform molecular dynamics simulations, which could provide insight into the dynamics of the binding interactions. These findings support the initial premise that, in addition to the choice of electrophilic warhead, appropriate noncovalent anchoring and complementarity of the remainder of the molecular scaffold are critical determinants of covalent inhibitor performance. kaspaza-1 kovalentni zaviralci karbamoil fluorid bojna glava caspase-1 covalent inhibitors carbamoyl fluoride warhead true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-05-16 08:45:06 2026-05-16 08:45:19 2026-05-17 04:39:11 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 125337 32184.pdf 32184.pdf 1 C5E45DE6DEB6E7CD45AA1A3FC5922DF1 35f73767a1b7357c9f24b17b0c1b2b28bfa88ae6ee5c3a1b65bd0e1bd8057095 7fbf0c7c-50f2-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233934 Fakulteta za farmacijo 0 0 0