Mycobacteria are intracellular pathogens that grow slowly and have a lipid-rich cell wall, which contribute to their high resistance to antimicrobial agents, together with efflux pumps, biofilms and granulomas that they form. In addition to tuberculosis, they can cause a variety of complex non-tuberculous infections. The treatment of infections caused by mycobacteria is lengthy and requires the use of combination therapy, which frequently includes broad-spectrum antibiotics, thereby increasing the risk of side effects and the development of resistant bacterial strains. The DNA gyrase essential enzyme is the only topoisomerase present in mycobacteria and represents a validated target for antimycobacterial agents. Based on the structural features of the ATP-binding site of the GyrB subunit, and previous studies at the Faculty of Pharmacy in Ljubljana, we have designed and synthesized four new compounds with the aim of developing selective DNA gyrase inhibitors specifically targeted against mycobacteria. The compounds were divided into a class with a 2-aminobenzothiazole ring and a class with a 2-aminothiazolo[4,5-b]pyridine ring. All the designed compounds had 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride bonded via an amide bond, but differed in the substituents at the C4 site of the benzothiazole or thiazolopyridine ring, where heterocycles containing nitrogen atoms were used. These substituents, as demonstrated in recent studies, contribute significantly to improving the accumulation of compounds within mycobacterial cells, increasing their selectivity of action. The inhibitory activity of the synthesized compounds against DNA gyrase from Escherichia coli was evaluated using enzymatic assays; all compounds demonstrated low nanomolar activity. All tested compounds were confirmed to be selective for their activity on the bacterial enzyme by human topoisomerase II assays. In this thesis, we review methods for the evaluation of in vitro and in vivo antibacterial activity of compounds on mycobacteria. Standard dilution and diffusion methods, as well as advanced methods taking into account the formation of biofilms and granulomas, are available for the evaluation of antibacterial activity. These approaches enable the assessment of compound penetration and efficacy against mycobacteria within these highly resistant structures. Among animal models, the Danio rerio zebrafish and the larvae of the Galleria mallonella honeycomb moth stand out. The in vitro antibacterial activity of our inhibitors against the Mycobacterium tuberculosis H37Rv wild strain was evaluated by the resazurin microtitre assay (REMA), which showed inhibitory activity of all tested compounds at a concentration of 10 µM.
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