20.500.12556/RUL-186593 Načrtovanje, sinteza in vrednotenje karbamoil fluoridov kot zaviralcev serinske hidrolaze FphI iz Staphylococcus aureus Design, synthesis, and evaluation of carbamoyl fluoride inhibitors of serine hydrolase FphI from Staphylococcus aureus Protimikrobna odpornost bakterije Staphylococcus aureus predstavlja enega največjih izzivov sodobne medicine in spodbuja razvoj novih terapevtskih pristopov, usmerjenih proti bakterijskim virulentnim dejavnikom. Med obetavne tarče sodi serinska hidrolaza FphI, za katero specifični zaviralci z nizko molekulsko maso doslej še niso bili opisani. V magistrski nalogi smo sintetizirali knjižnico karbamoil fluoridov in ovrednotili njihovo zaviralno aktivnost na encimu FphI. Biokemijsko vrednotenje je omogočilo identifikacijo več visoko aktivnih zaviralcev z aktivnostjo v nanomolarnem območju. Kinetične študije so potrdile njihov dvostopenjski kovalentni mehanizem delovanja, masna spektrometrija in rentgenska kristalografija pa sta potrdili kovalentno vezavo na katalitični Ser94 ter razkrili način vezave v aktivnem mestu encima. Molekulsko modeliranje in simulacije molekulske dinamike so pokazale, da je za učinkovito zaviranje ključna tvorba kovalentne vezi med karbamoil fluoridno skupino in katalitičnim serinskim ostankom, medtem ko nekovalentne interakcije same po sebi ne zagotavljajo stabilne vezave ligandov. Izbrane spojine niso imele izrazite protibakterijske aktivnosti. Rezultati magistrske naloge predstavljajo prve opisane kovalentne zaviralce encima FphI ter pomembno prispevajo k razumevanju njegovega mehanizma delovanja in biološke vloge. Razvite spojine predstavljajo obetavno izhodišče za nadaljnji razvoj selektivnih zaviralcev FphI in kemijskih sond za preučevanje biološke vloge tega encima, ne pa za razvoj novih antibiotikov proti bakteriji Staphylococcus aureus. The increasing antimicrobial resistance of Staphylococcus aureus represents one of the greatest challenges in modern medicine and has driven the development of novel therapeutic strategies targeting bacterial virulence factors. One of the promising targets is serine hydrolase FphI, for which no specific small-molecule inhibitors have been reported to date. In this master's thesis, a focused library of carbamoyl fluorides was synthesized and evaluated for their inhibitory activity against FphI. Biochemical evaluation identified several highly potent inhibitors with nanomolar activity. Kinetic studies confirmed their two-step covalent mechanism of inhibition, while mass spectrometry and X-ray crystallography verified covalent modification of the catalytic Ser94 residue and revealed the binding mode of the inhibitors within the enzyme active site. Molecular modelling and molecular dynamics simulations demonstrated that the formation of a covalent bond between the carbamoyl fluoride warhead and the catalytic serine residue is essential for effective inhibition, whereas non-covalent interactions alone are insufficient to ensure stable ligand binding. The selected compounds did not exhibit significant antibacterial activity. The results of this master's thesis describe the first covalent inhibitors of FphI and provide important insights into its inhibition mechanism and biological role. The developed compounds represent promising starting points for the further development of selective FphI inhibitors and chemical probes for investigating the biological role of this enzyme, but not for development of novel antibiotics against Staphylococcus aureus. Staphylococcus aureus FphI serinske hidrolaze karbamoil fluoridi kovalentni zaviralci Staphylococcus aureus FphI serine hydrolases carbamoyl fluorides covalent inhibitors true false false [L. Prestreši] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-03 08:47:35 2026-09-03 08:47:42 2026-09-07 15:28:31 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 615.31(043.2) 128023 290274307 33137.pdf 33137.pdf 1 70F80B5F79DC23D318D7FABB7386E56C 47bb5a4fcca09ecbd662f895fc0680814985bd6a84337e0e687f67fe50684bd4 eda9e22f-a762-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245869 Fakulteta za farmacijo 0 0 0