Razvoj dušikovih in žveplovih heterocikličnih zaviralcev metalo-β-laktamaz

Gričar, Simona (author), Kljun, Jakob (mentor)

Uporaba antibiotikov v kliniki je v zadnjih 100 letih bistveno vplivala na moderno medicino. Žal pa so bakterije sposobne razviti mehanizme, ki onemogočajo delovanje antibiotikov, in na ta način povzročijo odpornost, ki postaja vse večji problem v zdravstvu. Eden izmed možnih pristopov v boju proti odpornosti je zaviranje oz. inhibicija delovanja encimov, ki lahko antibiotike inaktivirajo. Pomembna skupina takšnih encimov so β-laktamaze, ki hidrolizirajo β-laktamske antibiotike, kot je npr. penicilin. Naša raziskava se osredotoča na zaviranje metalo-β-laktamaz, pri katerih v katalitičnem procesu hidrolize β-laktamov v aktivnem mestu sodelujejo cinkovi ioni. Raziskovalni cilj je bil sintetizirati dušikove in žveplove heterociklične spojine, ki bi lahko zavirale aktivnost metalo-β-laktamaz, specifično metaloencima NDM-1 (ang. New Delhi metallo-β-lactamase 1), ki je sposoben hidrolizirati skoraj vse β-laktamske antibiotike, vključno s karbapenemi. Zato smo izbrali tri skupine heterocikličnih spojin: 1,2,4-triazolidin-3-tione, 1,3,4-tiadiazole in pirimidin-2-tione. 1,2,4-Triazolidin-3-tioni in 1,3,4-tiadiazoli so bili sintetizirani v reakcijah ciklizacije tiosemikarbazidov s ketoni, pirimidin-2-tioni pa iz tiosečnine in enaminonov. Spojine so bile okarakterizirane s standardnimi fizikalno-kemijskimi tehnikami jedrske magnetne resonančne spektroskopije, masne spektrometrije, infrardeče spektroskopije in elementne analize. Za nekatere spojine smo pridobili tudi monokristale in izvedli rentgensko analizo. Možnosti vezave sintetiziranih spojin na NDM-1 smo preverili in silico z metodo molekulskega umeščanja v programu GOLD. Želeli smo ugotoviti, ali izbrane spojine zavirajo delovanje NDM-1 in vitro. Zato smo izrazili NDM-1 in postavili encimski test, s katerim smo določili zaviralno aktivnost izbranih spojin. Z nekaj izbranimi spojinami smo želeli pripraviti tudi kristal kompleksa NDM-1 z vezanim ligandom, zato smo preizkusili številne pogoje kristalizacij, najuspešnejšega optimizirali in pridobili dovolj kakovosten kristal za rentgensko analizo. Ta je pokazala, da sta v odprtini aktivnega mesta najverjetneje dve molekuli spojine 3b (4-(pirazin-2-il)pirimidin-2-tion) in da je ena od njiju je preko žveplovega atoma vezana na Zn$^{2+}$ v aktivnem mestu.

• Language: Slovenian
• Keywords: protimikrobna odpornost, zaviranje, NDM-1, 1, 2, 4-triazolidin-3-tion, 1, 3, 4-tiadiazol, pirimidin-2-tion
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-174871
• COBISS.SI-ID: 254404867
• Publication date in RUL: 10.10.2025

Secondary language

• Language: English
• Title: Development of nitrogen and sulfur heterocyclic inhibitors of metallo-β-lactamases

Abstract

The use of antibiotics in clinical practice contributed enormously to the field of modern medicine. Unfortunately, bacteria can develop various mechanisms that prevent antibiotics from working, causing antibiotic resistance, which is becoming an increasing problem in healthcare worldwide. One of the possible approaches in the fight against antibiotic resistance is the inhibition of enzymes that inactivate antibiotics. An important group of such enzymes are β-lactamases, which hydrolyze β-lactam antibiotics, e.g. penicillin. Our research focuses on the inhibition of metallo-β-lactamases, which are interesting from the point of bioinorganic chemistry because zinc ions in their active site are involved in the catalytic reaction mechanism of hydrolysis. Our aim was to synthesize nitrogen- and sulfur-containing heterocyclic compounds as potential inhibitors of metallo-β-lactamases, specifically New Delhi metallo-β-lactamase 1 (NDM-1), which is able to hydrolyze almost all β-lactam antibiotics, including carbapenems. We selected three types of compounds: 1,2,4-triazolidine-3-thiones, 1,3,4-thiadiazoles and pyrimidine-2-thiones. 1,2,4-Triazolidine-3-thiones and 1,3,4-thiadiazoles were synthesized by cyclization of thiosemicarbazides and ketones, while pyrimidine-2-thiones were prepared from thiourea and enaminones. The compounds were analyzed using standard physicochemical techniques: nuclear magnetic resonance spectroscopy, mass spectrometry, infrared spectroscopy and elemental analysis. Single crystals were obtained for some of the compounds and used for X-ray structure analysis. The binding possibilities of the synthesized compounds to NDM-1 were investigated with molecular docking using GOLD software. We expressed NDM-1 and established an enzymatic assay to determine the inhibitory effect of the selected compounds on NDM-1. We wanted to prepare crystals of a complex of NDM-1 with a bound ligand, so we tested a range of crystallization conditions, optimized the most successful one, and obtained a crystal of NDM-1 with compound 3b (4-(pyrazin-2-yl)pyrimidine-2-thione). It was shown that there are probably two 3b molecules in the active site aperture, while only one of the 3b molecules binds coordinatively to the catalytic Zn$^{2+}$.

• Keywords: antimicrobial resistance, inhibition, NDM-1, 1, 2, 4-triazolidine-3-thione, 1, 3, 4-thiadiazole, pyrimidine-2-thione