20.500.12556/RUL-144354 Sinteza in biokemijsko vrednotenje novih kloracetamidnih kovalentnih zaviralcev encima MurA Synthesis and biochemical evaluation of new chloracetamide covalent MurA inhibitors Razvoj antibiotikov se zaradi vse večjega pojava bakterijske rezistence in posledično grožnje javnemu zdravju usmerja v razvoj alternativnih antibiotikov. Naraščajoča pojavnost lete je privedla do vse večjega interesa za načrten razvoj kovalentnih tarčnih zaviralcev, ki so v preteklosti zaradi reaktivne strukture in potencialne nespecifičnosti vezave, veljali za neselektivne in zato potencialno toksične. Ta trend so v nasprotno smer premaknila spoznanja, da kot kovalentni zaviralci delujejo že nekatere učinkovine, ki so na trgu že dalj časa. Najbolj znana tarčna kovalentna zaviralca sta namreč acetilsalicilna kislina, ki je na trgu že od leta 1899, ter penicilin, ki je na voljo od leta 1928. Peptidoglikan, ki tvori bakterijsko celično steno, je rigidna struktura sestavljena iz premreženih polimernih verig Nacetilmuraminske kisline in Nacetilglukozamina. Zaradi odsotnosti celične stene v človeških celicah predstavlja odlično tarčo za razvoj novih protibakterijskih učinkovin. S tem namenom smo iz prej pripravljene knjižnice fragmentov, ki so bili virtualno sidrani na aktivno mesto encima MurA, izbrali 14 spojin s potencialnim zaviralnim učinkom na omenjeni encim. Spojine smo pripravili z Naciliranjem kloracetilkloridne bojne glave na ogrodja z različnimi strukturami. Namen uporabe različnih ogrodij je bilo doseči čim večje število dodatnih nekovalentnih interakcij z aktivnim mestom encima, ki bi se izrazile kot nižja inhibitorna koncentracija. Te koncentracije bomo določili z uporabo kolorimetrične metode, kjer bomo s prostim fosfatom, ki nastaja ob normalnem delovanju encima, tvorili obarvan kompleks. Na podlagi pridobljenih absorbanc lahko določimo ali posamezna spojina zavira delovanje encima ter kakšna je koncentracija spojin, ki zavre 50 % delovanja encima (IC50). Ugotovili smo, da spojine 15 na encim učinkujejo zaviralno, z IC50 vrednostmi med 44,4 in 452,4 μmol/L, medtem ko spojina 6 ne izkazuje zaviralnih lastnosti. The ever increasing bacterial resistance to the antibiotic drugs of the present and consequently the risk of public safety is prompting the researchers to search for an alternative approach to the antibiotic design. . This resistance has led to the increased interest to design targeted covalent inhibitors, which have in the past been regarded as dangerous due to their reactive structure and nonspecific mode of action, thus deeming them a bad research starting point. This belief was changed when several of the well established drugs were proven to act as targeted covalent inhibitors. The most known of them being acetylsalicylic acid and penicillin, which are avalable since 1899 and 1928, respectivelly. Peptidoglycan that forms the bacterial cell wall is a rigid structure, formed by interlinked polymer chains consisting of an alternating monomers of Nacetylmuramic acid and Nacetylglucosamine. Due to it's absence in mammalian cells, this structure offers an excellent target for development of new antibacterial drugs. In this work, we synthesised 14 substances that were found to be potential MurA inhibitors using virtual anchoring on MurA's active site. The substances were synthesized using Nalkylation of a chloracetylchloride warhead using scaffolds that vary in structure. The aim was to acheive more noncovalent interactions between the molecule and the enzyme's active site, which would result in a lower IC50 value. This value was determined using a colorimetric method that forms a colored complex with the inorganic phosphate that is present when the enzyme is active. Using these values we will be able to determine if and how effectively each compound is able to affect the enzyme's performance. The synthesised compounds 15 show an inhibiting effect on the isolated E.coli MurA enzyme, with IC50 in the concentration range from 44,4 to 452,4 μmol/L, while compound 6 exhibits complete absence of inhibitory activity. Alternativni antibiotik bakterijska rezistenca kovalentni tarčni zaviralec MurA peptidoglikan. MurA targeted covalent inhibitor alternative antibiotic peptidoglycan bacterial resistance true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-02-16 07:45:24 2023-02-16 07:45:31 2023-02-17 03:34:27 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 95025 14086.pdf 14086.pdf 1 CA7B012550B2F99DE72F1B4DA4FEFDD7 9417f6ce4f4a61314081847dad7075e874dbab6dc2a5c8255fc61aecdefacb0a 2c2edd3e-adc5-11ed-9c94-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=166634 Fakulteta za farmacijo 0 0 0