Razvoj novih N-fenilpirolamidov kot zaviralcev DNA-giraze in topoizomeraze IVBenedetto Tiz, Davide (Avtor) Zidar, Nace (Mentor) Kikelj, Danijel (Komentor) The greatest turning point in the moderrn treatment of infections is represented by the discovery of penicillin by Alexander Fleming in 1928. In the following decades, many other antibiotic classes have been discovered. With the high rate of use, sometimes even misuse of antibiotics, however, the degree of resistance to these agents increased, which complicates the treatment of infections that were previously manageable. Thus, the discovery of novel antibacterials is considered essential. We can fight antibacterial resistance by either searching for new ways of suppressing the bacterial growth or by preventing the emerging of resistance. This doctoral thesis focuses on the first mentioned strategy. The thesis describes the design, synthesis and evaluation of novel compounds endowed with inhibitory activities against bacterial topoisomerases - DNA gyrase and topoisomerase IV - enzymes critical for introducing topological changes to the DNA molecule and thus for the normal function of bacterial cells. DNA gyrase has important roles in the process of DNA replication and is a well-established and attractive target. DNA gyrase is an ATPase enzyme and its ATP binding site offers an opportunity for designing new antibacterial compounds with mechanism of action that until now has not been exploited enough. Well-known structure of DNA gyrase offers good opprotunities for structure-based design of new antibacterials, whereas many discovered small-molecule inhibitors of DNA gyrase with different scaffolds and good inhibitory properties can be used for ligand-based design of new antibacterials. The simultaneous inhibition of numerous bacterial targets to reduce target-based resistance has become an appealing strategy in medicinal chemistry. Topoisomerase IV possesses a similar structure and function as DNA gyrase. The similarity between these two enzymes provides the opportunity for designing dual targeting antibacterial compounds. Clinically used fluoroquinolones are well-known examples of dual inhibitors of topoisomerase IV and DNA gyrase, targeting their ParC and GyrA subunits. Lately, increased attention has been devoted to the GyrB and ParE subunits of these enzymes, where the ATP-binding site is located. In the present doctoral dissertation we designed, synthesized and biologically evaluated compounds with N-phenylpyrrolamide scaffold as potential inhibitors of GyrB and ParE. Beginning with the X-ray crystal structure of complexes of known inhibitors in the active site of GyrB, optimized N-phenyl-4,5-dibromopyrrolamides and N-phenyl-3,4-dichloro-5-methylpyrrolamides were prepared. We introduced structural modifications to investigate the optimal chemical space and the influence of such modifications on the inhibitory activity. Moreover, special focus was given on modifications that would increase the accumulation of inhibitors into the bacterial cells, such as the isosteric substitution of the terminal –COOH- group with less acidic groups or heterocycles. The prepared compounds were assayed for their inhibitory activities against DNA gyrase from Escherichia coli and Staphylococcus aureus. Since topoisomerase IV is an enzyme that has similar structure to DNA gyrase, novel N-phenylpyrrolamides were also evaluated against topoisomerase IV isolated also from S. aureus and E. coli. Selected compounds exhibited potent in vitro IC50 values against E. coli DNA gyrase and some of the compounds also showed good IC50 values against S. aureus DNA gyrase. The best inhibitors of E. coli DNA gyrase were zwitterionic compound 86 with excellent IC50 value of 6.3 nM and compound 9d with an IC50 value of 6.9 nM. Some compounds showed dual inhibition against DNA gyrase and topoisomerase IV. The best dual inhibitior was compound 9c, for which IC50 values on E. coli DNA gyrase, S. aureus DNA gyrase, E. coli topoisomerase IV and S. aureus topoisomerase IV were 9.9 nM, 99 nM, 9100 nM and 689 nM, respectively. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes) were chosen for the evaluation of our prepared inhbitors. Additionally, we evaluated certain compounds against wild type E. coli strain and two E. coli mutant strains, JD17464 and JW5503. E. coli JD17464 is an lpxC deletion mutant with impaired outer membrane, while E. coli JW5503 is a tolC deletion mutant with defective efflux pump. Five derivatives (24, 25, 43b, 50 and 57) blocked the growth of E. faecalis by more than 85% at 50 μM concentration. In line with our expectations, the hydrazide derivative 24 displayed worse minimum inhibitory concentration (MIC = 12.5 μM) than the corresponding heterociclic compound 25 (MIC = 6.25 μM). The lower polarity of the heterocyclic ring in 25 compared to the more polar hydrazide of 24 is believed to facilitate the entry of this compound into the bacterial cells. The growth of E. faecium was inhibited by more than 65% by six compounds (7c, 8c, 25, 57, 76 and 87), and S. aureus was inhibited by more than 95% by five compounds (50, 57, 85, 86 and 87) at 50 μM concentration. Of notable interest was compound 57, with excellent MIC values of 1.56 μM against E. faecalis and E. faecium and of 0.78 μM against S. aureus. One compound (compound 87) showed significant inhibition of wild type E. coli (growth inhibition was 100%) and eight derivatives (7c, 10a, 10b, 18, 39, 43b, 50 and 61) showed good activities against E. coli tolC deletion mutant with defective efflux pump. These results suggest that active transporters are likely the main justification for the weaker activity of these compounds against Gram-negative bacteria. This work describing the discovery of new N-phenylpyrrolamides as potent inhibitors of DNA gyrase and topoisomerase IV with good antibacterial activities against Gram-positive bacteria (markedly compound 57) and Gram-negative bacteria (compound 87) represents a good starting point which, combined with a broader understanding of the requirements for the entry of small molecules into bacteria, will allow us to obtain brad-spectrum antibacterial agents in the future.[D. Benedetto Tiz]20192022-06-10 10:30:30Doktorska disertacija137318sl