To date, many different antibiotics have been developed and discovered, but their overuse in both medicine and industry has contributed to the development of bacterial resistance. One of the most promising strategies for finding new antimicrobial agents is based on small molecules that inhibit the activity of specific bacterial targets. For example, such molecules are derivatives of hydroxynaphthoic acid, which have been shown in previous studies to be very promising small molecules for inhibiting the growth of gram-positive bacteria, such as Staphylococcus aureus. These derivatives also bind to the Escherichia coli-derived enzyme WrbA and affect its activity.
In this thesis, we aimed to charaterise in more detail the effect of selected hydroxynaphthoic acid derivatives on the WrbA enzyme and the homologous enzymes Ffp1 and Ffp2, which originate from Staphylococcus aureus. In the first part, we determined the Michaelis constants for the substrate NADH and potassium ferricyanide for all three enzymes. Since we found in the literature that these enzymes can also use NADPH as a substrate, we decided to determine the Michaelis constants for it as well. From comparisons of the constants obtained for the WrbA enzyme (Km, NADH with a value of (135 ± 23) µM, Km, NADPH with a value of (483 ± 109) µM), we demonstrated that all three enzymes have a much lower affinity for NADPH than for NADH. We then determined the pH profiles of the enzymes and found that WrbA and Ffp1 have optimal activity at pH 5,0, while Ffp2 is optimally active over a broader pH range of 6,0 to 8,0. Using mass spectrometry, we showed that in the solution, the homodimeric and homotetrameric forms of WrbA and Ffp1 are in equilibrium, while Ffp2 forms only homodimers in the solution.
In the central part of the thesis, we examined the inhibitory effects of five hydroxynaphthoic acid derivatives. Among them, 2-hydroxy-1-naphthoic acid showed the strogest inhibitory effect on the enzymes, with the following inhibition constants: EC50, WrbA equals (4,4 ± 2,7) µM, EC50, Ffp1 equals (5,0 ± 0,6) µM and EC50, Ffp2 equals (6,1± 0,3) µM. The enzyme Ffp1 was particularly sensitive to all the tested compounds. Based on the results, we evaluated 2-hydroxy-1-naphthalic acid as the most promising compound for further development. We also performed an HPLC analysis of 1-hydroxy-2-naphthalic acid, which showed that it decomposes in the presence of NaClO and p-benzoquinone, an important finding for understanding both its utility and the utility of other derivatives in biological systems. The results obtained represent a starting point for further research into small molecules that could act as new antimicrobial agents.
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