Design, synthesis and evaluation of DNA gyrase B inhibitors based on the benzothiazole scaffoldGjorgjieva, Marina (Avtor) Kikelj, Danijel (Mentor) Peterlin-Mašič, Lucija (Komentor) protibakterijske učinkovinebakterijska rezistencaDNA-girazainhibitorjinačrtovanjesintezavrednotenjedisertacijeAntibacterial resistance has become a huge problem lately. The big euphoria from the middle of the previous century with the discovery of the antibiotics did not last for long. Soon after the discovery of the first antibiotics, researchers realized that the fight with the microbes would not be so easy. The fast evolution of microorganisms and their ability to adapt to different external influences led to the development of resistance against most known and clinically used antibacterial drugs. Despite the increased need of new antibacterial compounds, not many new antibacterials reached the market in the last 20 years. DNA gyrase is a well-established and an attractive target in the search for new antibacterial compounds, with an important role in the process of DNA replication. DNA gyrase is an ATPase and the ATP binding site of the enzyme offers an opportunity for designing new antibacterial compounds with a mechanism of action that until now has not been enough exploited. Well-known structure of fragments of DNA gyrase offers great opportunity for structure-based design of new antibacterials, while many discovered inhibitors of DNA gyrase with different scaffolds and good inhibitory potential offer opportunity for ligand-based design of antibacterial compounds with an inhibitory activity on DNA gyrase. Dual targeting is an attractive approach in the design of new biologically active compounds, especially in the design of new antibacterial compounds, since the possibility for developing bacterial resistance is lower if two bacterial targets are inhibited simultaneously. Topoisomerase IV is a “twin” enzyme of DNA gyrase with a similar structure and function. Structural similarity between these two enzymes give an exceptional opportunity for designing dual targeting antibacterial compounds. Clinically used quinolones are a good example of successful dual inhibitors of DNA gyrase and topoisomerase IV, targeting their GyrA and ParC subunits. Lately, more attention has been paid to the less used part as antibacterial target of these enzymes, GyrB and ParE subunits, where the ATP-binding site is situated. Within the framework of the doctoral dissertation we designed, synthesized and biologically evaluated compounds with benzothiazole scaffold as potential inhibitors of DNA gyrase. Design of our compounds was based on modification of recently discovered inhibitors of DNA gyrase based on 4,5,6,7-tetrahydrobenzo-[d]thiazole scaffold in our research group. In this context we replaced the 4,5,6,7-tetrahydrobenzo-[d]thiazole scaffold with benzo[d]thiazole scaffold, performed similar structural modification as were done in the case of 4,5,6,7-tetrahydrobenzo-[d]thiazole compounds and studied the influence of the structural modification on the inhibitory activity. Our benzothiazole compounds were evaluated for inhibitory activities against DNA gyrase, isolated from Staphylococcus aureus and from Escherichia coli. Since topoisomerase IV is an enzyme that has similar structure, novel benzothiazole compounds were also evaluated for inhibitory activities against topoisomerase IV isolated also from S. aureus and E. coli. Most of our compounds exhibited potent in vitro inhibitory activities against E. coli DNA gyrase and some of the compounds also showed inhibitory activities against S. aureus DNA gyrase. The best inhibitor of E. coli gyrase was exhibited by compound 2-((6-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzo[d]thiazol-2-yl)amino)-2-oxoacetic acid (13) with an IC50 of 33 nM. Certain compounds showed dual inhibition against gyrase and topoisomerase IV isolated from both bacterial types. The best dual inhibition was observed by compound 2-((2-(4,5-dichloro-1H-pyrrole-2-carboxamido)benzo[d]thiazol-6-yl)amino)-2-oxoacetic acid (27) (IC50 values for E. coli DNA gyrase, S. aureus DNA gyrase, E. coli topoisomerase IV and S. aureus topoisomerase IV are 0.087, 0.51, 1.8 and 1.7 μM, respectively). Molecular docking studies showed that benzothiazole compounds bind to the ATP-binding site of the gyrase, which was further confirmed with a crystal structure of compound 2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzothiazol-6-yl)amino)-2-oxoacetic acid (24) bound to the ATP binding site of the gyrase. Our benzothiazole-based inhibitors of DNA gyrase and topoisomerase IV were biologically evaluated for antibacterial activity against Gram-positive (S. aureus ATCC 25923, Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853). In general, most of the compounds did not show significant antibacterial activity against all tested bacterial strains. Notable activities showed only compounds ethyl 2-((6-(4,5-dichloro-1H-pyrrole-2-carboxamido)benzo[d]thiazol-2-yl)amino)-2-oxoacetate (11) and 3-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido) benzo[d]thiazol-6-yl)amino)-3-oxopropanoic acid (25) against Gram-positive E. faecalis with 47 % and 59 % inhibition of the bacterial growth of E. faecalis at a 50 μM concentration after 24 h incubation, respectively. Compound 25 was found as potential ligand of AcrAB-tolC efflux pump, which could explain its weak antibacterial activity against Gram-negative bacterial strains (12 % inhibition of the growth of E. coli after a 24 h incubation and 11 % inhibition of the growth of P. aeruginosa after 24 h incubation). Furthermore, in a larger screening project, benzothiazole inhibitors of DNA gyrase, synthesized within the framework of this doctoral dissertation, were found to possess potent antiviral activity against influenza viruses (Influenza A/H1N1, Influenza A/H3N2 and Influenza B). Considering the importance of Hsp90 chaperon in the replication of virus influenza and also the similarities in the structure of the ATP binding sites of Hsp90 and Gyrase B (members of the GHKL group of enzymes with resembles in the ATP binding sites) we hypothesized that the antiviral activity of benzothiazole compounds is probably due to the inhibition of Hsp90. In a microscale thermophoresis assay, eleven of our compounds showed good binding to Hsp90 with Kd in the range of 0.26-51 μM. Compound 27 has the best Kd value (0.26 μM) which is highly comparable with the already known Hsp90 inhibitor, 17-desmethoxy-17-N,N-dimethylaminoethylamino geldanamycin (17-DMAG) (Kd = 0.27 μM). This study provided an excellent series of compounds with dual inhibition of both DNA gyrase and topoisomerase IV from E. coli and S. aureus. The crystal structure of compound 24 in the ATP-binding place of DNA gyrase B gives also opportunities for further optimization of these compounds in order to improve their inhibitory activities. The finding that compounds have antiviral activity against virus influenza with inhibition of Hsp90 host chaperone, gives a good starting point for designing and developing compounds with dual action that will combine both antibacterial and antiviral activity in the same molecule.[M. Gjorgjieva]20162023-01-12 07:29:17Doktorska disertacija143774UDK: 615.281.9(043.3)COBISS_ID: 286920960sl