Evaluation of bacterial topoisomerase inhibitors\u27 selectivity

Zaradi bakterijske odpornosti proti znanim protibakterijskim učinkovinam je potreben razvoj novih protibakterijskih spojin s ciljanjem novih tarč in z novimi mehanizmi delovanja, ki delujejo proti odpornim bakterijskim sevom. V razvoju protibakterijskih učinkovin so vzpostavljene in validirane tarče bakterijske topoizomeraze tipa IIA, in sicer heterotetramerna encima DNA-giraza in topoizomeraza IV. DNA-giraza je sestavljena iz dveh podenot A (GyrA) in dveh podenot B (GyrB) in topoizomeraza IV iz dveh podenot E (ParE) in dveh podenot C (ParC). Encima sta strukturno in funkcionalno podobna, zato je razvoj zaviralcev z dvojnim delovanjem potencialno lahko dostopna možnost. Na Katedri za farmacevtsko kemijo Fakultete za farmacijo je bilo nedavno odkritih več novih strukturnih tipov ATP-kompetitivnih zaviralcev bakterijskih topoizomeraz kot potencialnih protibakterijskih učinkovin. Na podlagi teh strukturnih tipov je bilo načrtovanih in sintetiziranih več zaviralcev, ki so zbrani v DNA-girazni knjižnici. Testirali smo 80 spojin iz te knjižnice z uporabo biokemijskega testa in ovrednotili njihovo zaviralno aktivnost na encimih DNA-giraza in topoizomeraza IV iz bakterij Escherichia coli in Staphylococcus aureus ter primerjali selektivnost spojin kot potencialnih zaviralcev z ozirom na tip topoizomeraz in bakterijski sev. Za oceno biokemijskega testa smo ovrednotili ponovljivost rezultatov med neodvisnimi testiranji. Najbolj ponovljive rezultate neodvisnih meritev rezidualnih aktivnosti in napovedanih IC50 vrednosti iz presejalnih testov smo dobili na encimu DNA-giraza iz E. coli. Prav tako smo primerjali določene IC50 vrednosti v neodvisnih ponovitvah testa in ugotovili, da je povprečni relativni standardni odklon 50 % na encimu DNA-giraza iz bakterije E. coli, medtem ko smo imeli premalo podatkov za oceno ponovljivosti na encimih DNA-giraza iz S. aureus in topoizomeraza IV iz obeh bakterij. Za spojine, ki smo jih testirali, smo ugotovili, da so najbolj aktivne na encimu DNA-giraza iz bakterije E. coli, nivo selektivnosti napram ostalim testiranim topoizomerazam pa je bil odvisen predvsem od centralnega skeleta strukture zaviralcev. Najaktivnejše spojine na tem encimu so imele IC50 vrednost nižjo od 10 nM. Na vseh testiranih bakterijskih topoizomerazah so se za najbolj zaviralno aktivne izkazale spojine 9, 32 in 34, ki so zato perspektivne za nadaljnji razvoj. Najmanj selektivna glede na testirane encime je bila spojina 59 iz skupine fenilpirolamidov.Due to bacterial resistance to known antibacterial agents, the development of antibacterial compounds with new targets and mechanisms of action with activity against resistant bacterial strains is needed. Established and validated targets in antibacterial drug discovery are bacterial topoisomerases type IIA namely heterotetrameric proteins DNA gyrase and topoisomerase IV. DNA gyrase consists of two A subunits (GyrA) and two B subunits (GyrB) and topoisomerase IV consists of two C subunits (ParC) and two E subunits (ParE). Enzymes are structurally and functionally similar, which potentially allows accessibility to dual targeting inhibitors. Several new structural types of ATP-competitive inhibitors of bacterial topoisomerases have recently been discovered at the Department of Pharmaceutical Chemistry at the Faculty of Pharmacy as potential antibacterial agents. Based on these structural types, several inhibitors were designed and synthesized, which are collected in a DNA gyrase library. We tested 80 compounds from this library using biochemical test and evaluated the inhibitory activity of the compounds on the DNA-gyrase enzyme and the topoisomerase IV from Escherichia coli and Staphylococcus aureus bacteria and compared the selectivity of the compounds as potential inhibitors with respect to the type of topoisomerase and bacterial strain. To evaluate the biochemical assay, we evaluated the reproducibility of the results during independent tests. The most reproducible results of independent measurements of residual activity and predicted IC50 values from screening tests were obtained on the E. coli DNA gyrase enzyme. We also compared the IC50 values in independent test iterations and found out that an average relative standard deviation is 50 % on the E. coli DNA-gyrase enzyme while there was insufficient data to evaluate the reproducibility on S. aureus DNA-gyrase enzyme and topoisomerase IV enzymes from both bacteria. We found out that the tested compounds are most active on the E. coli DNA-gyrase enzyme, with the level of their selectivity against other tested topoisomerases depending mainly on the central scaffold of the structure of inhibitors. The most active compounds on this enzyme had an IC50 value of less than 10 nM. Compounds 9, 32 and 34 showed the most active inhibition on all tested topoisomerases, and are therefore promising for further development. The least selective according to tested enzymes was the compound 59 from the phenylpyrrolamide group