Since the early days of antibiotic development, bacterial resistance has posed a major challenge and one of the greatest threats to modern medicine, highlighting the urgent need to discover novel antimicrobial compounds and their protein targets. Hydroxynaphthoic acid derivatives are biologically active small molecules that have demonstrated inhibitory activity primarily against Gram-positive bacterial strains. Their protein targets have been identified in E. coli and S. aureus as the flavodoxin-like enzymes WrbA, Ffp1, and Ffp2. The aim of this thesis was to extend previous studies on the inhibitory activity of hydroxynaphthoic acid derivatives against WrbA, Ffp1, and Ffp2, and to confirm the stability of these compounds in the enzymatic reaction and independently. In previous studies, 1-hydroxy-2-naphthoic acid exhibited inhibitory activity against both bacterial growth and the activity of the three enzymes. The determined inhibition constants were (314 53.1) M for WrbA, (12.2 1.75) M for Ffp1, and (22.9 1.79) M for Ffp2. The two investigated derivatives, 1-methoxy-2-naphthoic acid and methyl 1-hydroxy-2-naphthoate, had shown no apparent inhibitory effect on bacterial growth. However, inhibition assays demonstrated that both compounds inhibited the activity of WrbA, Ffp1, and Ffp2, although to a lesser extent than 1-hydroxy-2-naphthoic acid. For 1-methoxy-2-naphthoic acid, the IC50 values were (2020 1361) M for WrbA, (299.2 51.52) M for Ffp1, and (197.2 22.58) M for Ffp2. Methyl 1-hydroxy-2-naphthoate showed very weak inhibition; therefore, an IC50 value was determined only for Ffp2, for which it was (737.3 3232) M. These results suggest that both the carboxyl (-COOH) and hydroxyl (-OH) groups play a key role in the binding of hydroxynaphthoic acids to the enzyme and in their inhibitory activity. Since the compound with a modified carboxyl group was the least effective, this group likely plays a greater role in proper binding than the hydroxyl group, whose modification resulted in a smaller decrease in activity. HPLC analysis confirmed that all compounds were stable, supporting the interpretation of the inhibition assays and their further development as potential antimicrobial agents. The results also indicate that the compounds remain chemically unchanged during the enzymatic reaction and act as classical enzyme inhibitors rather than as cofactor or substrate analogues. Overall, the findings provide a valuable basis for further investigation of hydroxynaphthoic acid derivatives as potential antimicrobial agents.
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