20.500.12556/RUL-185025 Optimizacija protibakterijskega delovanja in varnosti novih zaviralcev bakterijske topoizomeraze tipa II z uvedbo novih DNA interkalirajočih skupin raziskovalni podatki, obravnavani v doktorskem delu Optimization of antibacterial activity and safety of novel bacterial topoisomerase type II inhibitors by introducing new DNA intercalating moieties research data underlying the doctoral dissertation Bakterijske topoizomeraze tipa II so dobro uveljavljene in raziskane tarče za protibakterijsko terapijo. To doktorsko delo se osredotoča na načrtovanje, sintezo in biološko vrednotenje novih zaviralcev bakterijskih topoizomeraz (angl. novel bacterial topoisomerase inhibitors, NBTIs). Delo združuje in silico pristope, farmacevtsko kemijo ter širok nabor in vitro in in vivo bioloških testov za ovrednotenje novih NBTI-jev. Vsem spojinam smo določili protibakterijsko delovanje na panelu grampozitivnih (npr. Staphylococcus aureus) in gramnegativnih (npr. Escherichia coli) bakterij, vezavo na hERG kalijeve kanale ter rezidualno aktivnost na bakterijske in humane topoizomeraze. Za najbolj perspektivne molekule smo tudi določili zaviralno aktivnost na tarčne encime, baktericidno kinetiko in aktivnost proti biofilmom, ter ovrednotili njihovo varnost na embrijih cebric. Učinkovitost novih spojin in vivo smo ovrednotili na modelu cebric okuženih z na meticilin odporno S. aureus (MRSA). S sistematično optimizacijo levega dela molekule (angl. left-hand side moiety, LHS) je bila razvita nova serija NBTI spojin z razširjenim protibakterijskim delovanjem in izboljšano varnostjo. Nekatere optimizirane spojine so pokazale močno protibakterijsko delovanje proti širokemu spektru bakterij (MIK za S. aureus nad 0,004 µg/mL in MIK za E. coli nad 0,125 µg/mL). Vrednotenje vezave na kalijeve kanale hERG je pokazalo, da nekatere spojine izkazujejo zmerno, vendar izboljšano zaviranje hERG (IC50, hERG ≈ 5–15 µM). Izbrane spojine so pokazale tudi minimalno toksičnost v embrijih cebric ter dokazale svojo učinkovitost in vivo v modelu z MRSA okuženimi embriji cebric. Na podlagi podatkov iz literature smo razvili tudi kvantitativni modeli odnosa med strukturo in aktivnostjo (QSAR) in farmakoforni modeli, ki opisujejo NBTI spojine z eksperimentalno določenimi in vitro aktivnostmi proti hERG. Ti modeli so bili nato uporabljeni za napovedovanje vezave na hERG (kvantitativno in kvalitativno) ter ovrednoteni z novo sintetizirano serijo spojin. Več NBTI spojin smo uporabili v kombinaciji z različnimi zaviralci izlivnih črpalk (angl. efflux pump inhibitors, EPI). Rezultati uporabe metode šahovnice so pokazali, da lahko nekateri EPI povečajo protibakterijsko aktivnost NBTI spojin. Za konec pa smo razvili tudi in silico napovedni model, zgrajen na razmerju med MIK pri divjem tipu E. coli in MIK pri E. coli z nedelujočimi iztočnimi črpalkami. Rezultati nakazujejo, da je napovedovanje substratov izlivnih črpalk izvedljivo, vendar je za doseganje statistično zanesljive napovedne moči modelov potrebno bistveno povečati obseg razpoložljivih eksperimentalnih podatkov. Optimizirani analogi NBTI, ki smo jih razvili tekom te doktorske naloge, predstavljajo osrednji dosežek doktorata in pomembno prispevajo k razširitvi področja zaviralcev bakterijskih topoizomeraz tipa II. To delo predstavlja dobro izhodišče za nadaljnje raziskave, usmerjene v dodatno izboljšanje in širjenje kemijskega prostora protibakterijskih učinkovin. Bacterial type II topoisomerases are well-established and extensively studied targets in antibacterial chemotherapy. This doctoral dissertation focuses on the design, synthesis, and biological evaluation of novel bacterial topoisomerase inhibitors (NBTIs). The research combined in silico approaches, synthetic organic chemistry, and a broad range of in vitro and in vivo biological assays. The antibacterial activity against a wide panel of Gram-positive (e.g., Staphylococcus aures) and Gram-negative bacteria (e.g., Escherichia coli), hERG potassium channel binding, and the residual activity against topoisomerases (both bacterial and human) was determined for all new compounds. The antibacterial potency (IC50) against the target enzymes, bactericidal kinetics, and antibiofilm properties, as well as safety on zebrafish embryos were determined for some of the most promising compounds. Their in vivo efficacy was also determined in a methicillin-resistant Staphylococcus aureus (MRSA) infection model in zebrafish embryos. Through systematic optimization of the left-hand side (LHS) moiety, a new series of NBTIs with a broadened antibacterial spectrum and enhanced selective toxicity was developed. Several optimized compounds demonstrated strong antibacterial activity against a broad spectrum of Gram-positive and Gram-negative bacteria (MICS. aureus as low as 0.004 µg/mL and MICE. coli as low as 0.125 µg/mL). Evaluation of hERG potassium channel binding revealed that some compounds exhibited moderate, but improved hERG inhibition (IC50, hERG ≈ 5 – 15 µM). Importantly, selected compounds showed minimal or no toxicity in zebrafish embryos and demonstrated in vivo efficacy in MRSA-infected zebrafish embryos. Additionally, Quantitative Structure–Activity Relationship (QSAR) and pharmacophore models were derived based on the data available in the literature, describing NBTIs with experimentally determined in vitro inhibitory activities against hERG. These models were subsequently used for the prediction of hERG binding (both quantitative and qualitative) and were validated by the newly synthetized series of compounds. Several NBTIs were also used in a checkerboard assay with different efflux pump inhibitors (EPIs). This experiment demonstrated that EPIs can enhance the antibacterial activity of NBTIs. Lastly, an in silico predictive model, constructed by using the ratio between the MIC of wild-type E. coli and the MIC of E. coli with non-functioning efflux pumps, demonstrated that while prediction of efflux pump substrates is feasible, it requires a lot more data for developing statistically sound models. The newly optimized NBTI analogs, representing the central achievement of this research, make a significant contribution to the field of bacterial type II topoisomerase inhibitors and support the development of novel antibacterial agents. Furthermore, this work provides a strong foundation for future studies aimed at further improving and expanding the chemical space of antibacterial agents. bakterijska odpornost protibakterijske učinkovine topoizomerazni zaviralci novi zaviralci bakterijskih topoizomeraz NBTI bacterial resistence antibacterial compounds topoisomerase inhibitors novel bacterial topoisomerase inhibitors NBTI true false false Slovenski jezik Angleški jezik Neznano 2026-07-21 10:19:13 2026-07-21 20:28:03 2026-07-22 04:04:16 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-07-21 README.docx README.docx README 1 1EE52E713C4C8E0CBAA03A8D4BF18231 457ba88a0b544f9293767a4b1b2f2b98183dbfc71cc247ad3bf56202a2275080 5cb1b674-84dd-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243039 1_In_silico_data.zip 1_In_silico_data.zip 1 In silico data 1 E053F670B40DD45FF4D521D1C2F0AB23 628e4a3264e014a49d8f593f7a0be84492e137e383551247cdc81224bad4e9c1 e1b2428c-84dd-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243041 2_Chemical_data.zip 2_Chemical_data.zip 2 Chemical data 1 58DE03A97C09DFE3AD5FDFC803DA6CF4 f0d745521bd25aaf0a330e7a52df297364ff16ac1e10a373f108f5be194bdc6b dcab6bd4-84de-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243043 3_Biological_data.zip 3_Biological_data.zip 3 Biological data 1 B4595DFCF691F96719CBB8100409DC75 c688e96c90fef3f1740db39d5bfc88b85a312eaecdede5f08890f0c08e636b4a 46c3d964-84df-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243044 https://doi.org/10.1016/j.ejmech.2026.118993 Članek / Article 0 4c4d4d8d-84dd-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243036 Fakulteta za farmacijo Poskus: Laboratorij 0 0 0