In today's world, the microbial resistance to antibiotics represents one of the greatest threats to public health. Therefore, numerous research groups place the search for new antibiotics and their targets among their priority tasks. Pyrazole and its derivatives have proven antimicrobial activity. The derivative 4-(2-aminoethyl)-1-(pyridin-2-yl)-1H-pyrazol-5-ol inhibits the growth of the bacterium Escherichia coli by binding to the protein L-threonine dehydrogenase (TDH) and inhibiting its activity. The latter is involved in intercellular communication and amino acid metabolism, playing a key role in the breakdown of L-threonine into glycine. Since no human homologs are known, it is a good candidate for a new antibiotic target. The aim of the MSc thesis was to test the inhibitory activity of selected pyrazole derivatives on TDH from E. coli and to assess their ability to inhibit the growth of four bacterial species: Escherichia coli, Bacillus thuringiensis, Pseudomonas aeruginosa, and Staphylococcus aureus using antibiograms. Since it is known that chelators can bind Zn2+ ions from the TDH structure and thus inhibit its activity, we also tested the ability of selected pyrazole derivatives to chelate Zn2+ by measuring their absorption spectra. TDH requires the cofactor NAD+, which is converted to NADH during the catalysed reaction, which can be monitored fluorimetrically. We prepared recombinant TDH and analysed its activity in the presence of twelve pyrazole derivatives. We found that ten of them act as either linear or hyperbolic inhibitor. Among the linear ones, the derivative I2 has the highest affinity; therefore, we analysed its mechanism of action and concluded that it is a competitive inhibitor. Using antibiograms, we found that all pyrazole derivatives tested, except the IC derivative, inhibited the growth of at least one strain tested. However, the derivatives that inhibit the growth of E. coli best act as weak inhibitors of the tested TDH target, and are therefore likely to act on other bacterial targets as well. Measurement of absorption spectra showed that most derivatives chelate zinc ions and are therefore not suitable antibiotic candidates. Although the derivative I2 does not chelate zinc ions and acts as a relatively good TDH inhibitor, its in vivo activity is not sufficiently effective for use as an antibiotic. Among the tested pyrazole derivatives, we did not find a compound with potential as a new antibiotic. Therefore, future research needs to be expanded to include a larger number of derivatives, and focus on finding other targets as well. Nevertheless, during the research, we characterised the activity of the TDH enzyme in greater detail and made two important discoveries: (I) the binding of Thr is accompanied by a homotropic cooperative effect, (II) zinc ions have a regulatory effect on TDH, as they inhibit its activity with an EC50 value of around 55 µM.
|
